Temporal Dynamics of Abundance and Composition of Nitrogen-Fixing Communities across Agricultural Soils

PubMed Central

Pereira e Silva, Michele C.; Schloter-Hai, Brigitte; Schloter, Michael; van Elsas, Jan Dirk; Salles, Joana Falcão

2013-01-01

Background Despite the fact that the fixation of nitrogen is one of the most significant nutrient processes in the terrestrial ecosystem, a thorough study of the spatial and temporal patterns in the abundance and distribution of N-fixing communities has been missing so far. Methodology/Principal Findings In order to understand the dynamics of diazotrophic communities and their resilience to external changes, we quantified the abundance and characterized the bacterial community structures based on the nifH gene, using real-time PCR, PCR-DGGE and 454-pyrosequencing, across four representative Dutch soils during one growing season. In general, higher nifH gene copy numbers were observed in soils with higher pH than in those with lower pH, but lower numbers were related to increased nitrate and ammonium levels. Results from nifH gene pyrosequencing confirmed the observed PCR-DGGE patterns, which indicated that the N fixers are highly dynamic across time, shifting around 60%. Forward selection on CCA analysis identified N availability as the main driver of these variations, as well as of the evenness of the communities, leading to very unequal communities. Moreover, deep sequencing of the nifH gene revealed that sandy soils (B and D) had the lowest percentage of shared OTUs across time, compared with clayey soils (G and K), indicating the presence of a community under constant change. Cosmopolitan nifH species (present throughout the season) were affiliated with Bradyrhizobium , Azospirillum and Methylocistis, whereas other species increased their abundances progressively over time, when appropriate conditions were met, as was notably the case for Paenibacilus and Burkholderia. Conclusions Our study provides the first in-depth pyrosequencing analysis of the N-fixing community at both spatial and temporal scales, providing insights into the cosmopolitan and specific portions of the nitrogen fixing bacterial communities in soil. PMID:24058578

Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules.

PubMed

Kim, Suk Kyeong; Kim, Dong-Lim; Han, Hye Seung; Kim, Wan Seop; Kim, Seung Ja; Moon, Won Jin; Oh, Seo Young; Hwang, Tae Sook

2008-06-01

Fine-needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant and of guiding therapeutic intervention in thyroid nodules. However, 10% to 30% of cases with indeterminate cytology in FNAB need other diagnostic tools to refine diagnosis. We compared the pyrosequencing method with the conventional direct DNA sequencing analysis and investigated the usefulness of preoperative BRAF mutation analysis as an adjunct diagnostic tool with routine FNAB. A total of 103 surgically confirmed patients' FNA slides were recruited and DNA was extracted after atypical cells were scraped from the slides. BRAF mutation was analyzed by pyrosequencing and direct DNA sequencing. Sixty-three (77.8%) of 81 histopathologically diagnosed malignant nodules revealed positive BRAF mutation on pyrosequencing analysis. In detail, 63 (84.0%) of 75 papillary thyroid carcinoma (PTC) samples showed positive BRAF mutation, whereas 3 follicular thyroid carcinomas, 1 anaplastic carcinoma, 1 medullary thyroid carcinoma, and 1 metastatic lung carcinoma did not show BRAF mutation. None of 22 benign nodules had BRAF mutation in both pyrosequencing and direct DNA sequencing. Out of 27 thyroid nodules classified as 'indeterminate' on cytologic examination preoperatively, 21 (77.8%) cases turned out to be malignant: 18 PTCs (including 2 follicular variant types) and 3 follicular thyroid carcinomas. Among these, 13 (61.9%) classic PTCs had BRAF mutation. None of 6 benign nodules, including 3 follicular adenomas and 3 nodular hyperplasias, had BRAF mutation. Among 63 PTCs with positive BRAF mutation detected by pyrosequencing analysis, 3 cases did not show BRAF mutation by direct DNA sequencing. Although it was not statistically significant, pyrosequencing was superior to direct DNA sequencing in detecting the BRAF mutation of thyroid nodules (P=0.25). Detecting BRAF mutation by pyrosequencing is more sensitive, faster, and less expensive than direct DNA sequencing and is proposed as an adjunct diagnostic tool in evaluating thyroid nodules of indeterminate cytology.

Deep-branching Novel Lineages and High Diversity of Haptophytes in the Skagerrak (Norway) Uncovered by 454 Pyrosequencing

PubMed Central

Egge, Elianne S; Eikrem, Wenche; Edvardsen, Bente

2015-01-01

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high-throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano- and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta-specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep-branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity. PMID:25099994

Characterization of Bacterial Communities in Venous Insufficiency Wounds by Use of Conventional Culture and Molecular Diagnostic Methods▿

PubMed Central

Tuttle, Marie S.; Mostow, Eliot; Mukherjee, Pranab; Hu, Fen Z.; Melton-Kreft, Rachael; Ehrlich, Garth D.; Dowd, Scot E.; Ghannoum, Mahmoud A.

2011-01-01

Microbial infections delay wound healing, but the effect of the composition of the wound microbiome on healing parameters is unknown. To better understand bacterial communities in chronic wounds, we analyzed debridement samples from lower-extremity venous insufficiency ulcers using the following: conventional anaerobic and aerobic bacterial cultures; the Ibis T5000 universal biosensor (Abbott Molecular); and 16S 454 FLX titanium series pyrosequencing (Roche). Wound debridement samples were obtained from 10 patients monitored clinically for at least 6 months, at which point 5 of the 10 sampled wounds had healed. Pyrosequencing data revealed significantly higher bacterial abundance and diversity in wounds that had not healed at 6 months. Additionally, Actinomycetales was increased in wounds that had not healed, and Pseudomonadaceae was increased in wounds that had healed by the 6-month follow-up. Baseline wound surface area, duration, or analysis by Ibis or conventional culture did not reveal significant differences between wounds that healed after 6 months and those that did not. Thus, pyrosequencing identified distinctive baseline characteristics of wounds that did not heal by the 6-month follow-up, furthering our understanding of potentially unique microbiome characteristics of chronic wounds. PMID:21880958

Combining flow cytometry and 16S rRNA gene pyrosequencing: a promising approach for drinking water monitoring and characterization.

PubMed

Prest, E I; El-Chakhtoura, J; Hammes, F; Saikaly, P E; van Loosdrecht, M C M; Vrouwenvelder, J S

2014-10-15

The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Rapid phylogenetic dissection of prokaryotic community structure in tidal flat using pyrosequencing.

PubMed

Kim, Bong-Soo; Kim, Byung Kwon; Lee, Jae-Hak; Kim, Myungjin; Lim, Young Woon; Chun, Jongsik

2008-08-01

Dissection of prokaryotic community structure is prerequisite to understand their ecological roles. Various methods are available for such a purpose which amplification and sequencing of 16S rRNA genes gained its popularity. However, conventional methods based on Sanger sequencing technique require cloning process prior to sequencing, and are expensive and labor-intensive. We investigated prokaryotic community structure in tidal flat sediments, Korea, using pyrosequencing and a subsequent automated bioinformatic pipeline for the rapid and accurate taxonomic assignment of each amplicon. The combination of pyrosequencing and bioinformatic analysis showed that bacterial and archaeal communities were more diverse than previously reported in clone library studies. Pyrosequencing analysis revealed 21 bacterial divisions and 37 candidate divisions. Proteobacteria was the most abundant division in the bacterial community, of which Gamma-and Delta-Proteobacteria were the most abundant. Similarly, 4 archaeal divisions were found in tidal flat sediments. Euryarchaeota was the most abundant division in the archaeal sequences, which were further divided into 8 classes and 11 unclassified euryarchaeota groups. The system developed here provides a simple, in-depth and automated way of dissecting a prokaryotic community structure without extensive pretreatment such as cloning.

Determination of multiple-clone infection at allelic dimorphism site of Plasmodium vivax merozoite surface protein-1 in the Republic of Korea by pyrosequencing assay.

PubMed

Dinzouna-Boutamba, Sylvatrie-Danne; Lee, Sanghyun; Son, Ui-Han; Yun, Hae Soo; Joo, So-Young; Jeong, Sookwan; Rhee, Man Hee; Kwak, Dongmi; Xuan, Xuenan; Hong, Yeonchul; Chung, Dong-Il; Goo, Youn-Kyoung

2017-12-01

Allelic diversity leading to multiple gene polymorphisms of vivax malaria parasites has been shown to greatly contribute to antigenic variation and drug resistance, increasing the potential for multiple-clone infections within the host. Therefore, to identify multiple-clone infections and the predominant haplotype of Plasmodium vivax in a South Korean population, P. vivax merozoite surface protein-1 (PvMSP-1) was analyzed by pyrosequencing. Pyrosequencing of 156 vivax malaria-infected samples yielded 97 (62.18%) output pyrograms showing two main types of peak patterns of the dimorphic allele for threonine and alanine (T1476A). Most of the samples evaluated (88.66%) carried multiple-clone infections (wild- and mutant-types), whereas 11.34% of the same population carried only the mutant-type (1476A). In addition, each allele showed a high frequency of guanine (G) base substitution at both the first and third positions (86.07% and 81.13%, respectively) of the nucleotide combinations. Pyrosequencing of the PvMSP-1 42-kDa fragment revealed a heterogeneous parasite population, with the mutant-type dominant compared to the wild-type. Understanding the genetic diversity and multiple-clone infection rates may lead to improvements in vivax malaria prevention and strategic control plans. Further studies are needed to improve the efficacy of the pyrosequencing assay with large sample sizes and additional nucleotide positions. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of bacterial communities of conventional and A-stage activated sludge systems

PubMed Central

Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Lotti, Tommaso; Garcia-Ruiz, Maria-Jesus; Osorio, Francisco; Gonzalez-Lopez, Jesus; van Loosdrecht, Mark C. M.

2016-01-01

The bacterial community structure of 10 different wastewater treatment systems and their influents has been investigated through pyrosequencing, yielding a total of 283486 reads. These bioreactors had different technological configurations: conventional activated sludge (CAS) systems and very highly loaded A-stage systems. A-stage processes are proposed as the first step in an energy producing municipal wastewater treatment process. Pyrosequencing analysis indicated that bacterial community structure of all influents was similar. Also the bacterial community of all CAS bioreactors was similar. Bacterial community structure of A-stage bioreactors showed a more case-specific pattern. A core of genera was consistently found for all influents, all CAS bioreactors and all A-stage bioreactors, respectively, showing that different geographical locations in The Netherlands and Spain did not affect the functional bacterial communities in these technologies. The ecological roles of these bacteria were discussed. Influents and A-stage bioreactors shared several core genera, while none of these were shared with CAS bioreactors communities. This difference is thought to reside in the different operational conditions of the two technologies. This study shows that bacterial community structure of CAS and A-stage bioreactors are mostly driven by solids retention time (SRT) and hydraulic retention time (HRT), as suggested by multivariate redundancy analysis. PMID:26728449

Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples.

PubMed

Pires, Ana C C; Cleary, Daniel F R; Almeida, Adelaide; Cunha, Angela; Dealtry, Simone; Mendonça-Hagler, Leda C S; Smalla, Kornelia; Gomes, Newton C M

2012-08-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.

Denaturing Gradient Gel Electrophoresis and Barcoded Pyrosequencing Reveal Unprecedented Archaeal Diversity in Mangrove Sediment and Rhizosphere Samples

PubMed Central

Pires, Ana C. C.; Cleary, Daniel F. R.; Almeida, Adelaide; Cunha, Ângela; Dealtry, Simone; Mendonça-Hagler, Leda C. S.; Smalla, Kornelia

2012-01-01

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages. PMID:22660713

Bacterial Diversity and Community Structure of Supragingival Plaques in Adults with Dental Health or Caries Revealed by 16S Pyrosequencing

PubMed Central

Xiao, Cuicui; Ran, Shujun; Huang, Zhengwei; Liang, Jingping

2016-01-01

Dental caries has a polymicrobial etiology within the complex oral microbial ecosystem. However, the overall diversity and structure of supragingival plaque microbiota in adult dental health and caries are not well understood. Here, 160 supragingival plaque samples from patients with dental health and different severities of dental caries were collected for bacterial genomic DNA extraction, pyrosequencing by amplification of the 16S rDNA V1–V3 hypervariable regions, and bioinformatic analysis. High-quality sequences (2,261,700) clustered into 10,365 operational taxonomic units (OTUs; 97% identity), representing 453 independent species belonging to 122 genera, 66 families, 34 orders, 21 classes, and 12 phyla. All groups shared 7522 OTUs, indicating the presence of a core plaque microbiome. α diversity analysis showed that the microbial diversity in healthy plaques exceeded that of dental caries, with the diversity decreasing gradually with the severity of caries. The dominant phyla of plaque microbiota included Bacteroidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria, and TM7. The dominant genera included Capnocytophaga, Prevotella, Actinomyces, Corynebacterium, Neisseria, Streptococcus, Rothia, and Leptotrichia. β diversity analysis showed that the plaque microbial community structure was similar in all groups. Using LEfSe analysis, 25 differentially abundant taxa were identified as potential biomarkers. Key genera (27) that potentially contributed to the differential distributions of plaque microbiota between groups were identified by PLS-DA analysis. Finally, co-occurrence network analysis and function predictions were performed. Treatment strategies directed toward modulating microbial interactions and their functional output should be further developed. PMID:27499752

Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia.

PubMed

Wongwilaiwalin, Sarunyou; Laothanachareon, Thanaporn; Mhuantong, Wuttichai; Tangphatsornruang, Sithichoke; Eurwilaichitr, Lily; Igarashi, Yasuo; Champreda, Verawat

2013-10-01

Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and β-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.

Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4

PubMed Central

Gancz, Ady Y; Kistler, Amy L; Greninger, Alexander L; Farnoushi, Yigal; Mechani, Sara; Perl, Shmuel; Berkowitz, Asaf; Perez, Noa; Clubb, Susan; DeRisi, Joseph L; Ganem, Don; Lublin, Avishai

2009-01-01

Background Proventricular dilatation disease (PDD) is a fatal disorder of psittacine birds worldwide. The disease is characterized by lymphoplasmacytic infiltration of the central and peripheral nervous systems, leading to gastrointestinal motility and/or central nervous system dysfunction. Recently, we detected a significant association between avian bornavirus (ABV) infection and clinical signs of PDD in psittacines. However, it remains unclear whether ABV infection actually causes PDD. To address this question, we examined the impact of ABV inoculation on the cockatiel (Nymphicus hollandicus). Results Five cockatiels were inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) avian bornavirus 4 (ABV4) (+) case (n = 3 inoculees) or from a PDD(-) ABV(-) control (n = 2 inoculees). The control birds remained free of clinical or pathological signs of PDD, and tested ABV(-) by RT-PCR and immunohistochemistry (IHC). In contrast, all three cockatiels inoculated with ABV4(+) brain homogenate developed gross and microscopic PDD lesions, and two exhibited overt clinical signs. In numerous tissues, ABV RT-PCR and sequence analysis demonstrated the presence of ABV4 RNA nearly identical to that in the inoculum. ABV was detected in the central nervous system of the three ABV-inoculees by IHC. Pyrosequencing to investigate the viral flora in the ABV4(+) inoculum uncovered 7 unique reads sharing 73–100% nucleotide sequence identity with previously identified ABV sequences and 24 reads sharing 40–89% amino acid sequence identity with viruses in the Retroviridae and Astroviridae families. Of these candidate viral species, only ABV RNA was recovered from tissues of the inoculated birds. Conclusion In this study, the clinical and pathological manifestations of PDD were induced by inoculation of cockatiels with brain homogenates containing avian bornavirus 4. By using high throughput pyrosequencing an in-depth view of the viral content of the inoculum was achieved, revealing that of 3 candidate virus families detected, only the presence of ABV RNA correlated with the development of PDD. This study provides evidence of a causal association between ABV4 infection and PDD in cockatiels. PMID:19589169

Random Amplification and Pyrosequencing for Identification of Novel Viral Genome Sequences

PubMed Central

Hang, Jun; Forshey, Brett M.; Kochel, Tadeusz J.; Li, Tao; Solórzano, Víctor Fiestas; Halsey, Eric S.; Kuschner, Robert A.

2012-01-01

ssRNA viruses have high levels of genomic divergence, which can lead to difficulty in genomic characterization of new viruses using traditional PCR amplification and sequencing methods. In this study, random reverse transcription, anchored random PCR amplification, and high-throughput pyrosequencing were used to identify orthobunyavirus sequences from total RNA extracted from viral cultures of acute febrile illness specimens. Draft genome sequence for the orthobunyavirus L segment was assembled and sequentially extended using de novo assembly contigs from pyrosequencing reads and orthobunyavirus sequences in GenBank as guidance. Accuracy and continuous coverage were achieved by mapping all reads to the L segment draft sequence. Subsequently, RT-PCR and Sanger sequencing were used to complete the genome sequence. The complete L segment was found to be 6936 bases in length, encoding a 2248-aa putative RNA polymerase. The identified L segment was distinct from previously published South American orthobunyaviruses, sharing 63% and 54% identity at the nucleotide and amino acid level, respectively, with the complete Oropouche virus L segment and 73% and 81% identity at the nucleotide and amino acid level, respectively, with a partial Caraparu virus L segment. The result demonstrated the effectiveness of a sequence-independent amplification and next-generation sequencing approach for obtaining complete viral genomes from total nucleic acid extracts and its use in pathogen discovery. PMID:22468136

Shifts of microbial community structure in soils of a photovoltaic plant observed using tag-encoded pyrosequencing of 16S rRNA.

PubMed

Wu, Shijin; Li, Yuan; Wang, Penghua; Zhong, Li; Qiu, Lequan; Chen, Jianmeng

2016-04-01

The environmental risk of fluoride and chloride pollution is pronounced in soils adjacent to solar photovoltaic sites. The elevated levels of fluoride and chloride in these soils have had significant impacts on the population size and overall biological activity of the soil microbial communities. The microbial community also plays an essential role in remediation of these soils. Questions remain as to how the fluoride and chloride contamination and subsequent remediation at these sites have impacted the population structure of the soil microbial communities. We analyzed the microbial communities in soils collected from close to a solar photovoltaic enterprise by pyrosequencing of the 16S rRNA tag. In addition, we used multivariate statistics to identity the relationships shared between sequence diversity and heterogeneity in the soil environment. The overall microbial communities were surprisingly diverse, harboring a wide variety of taxa and sharing significant correlations with different degrees of fluoride and chloride contamination. The contaminated soils harbored abundant bacteria that were probably resistant to the high acidity, high fluoride and chloride concentration, and high osmotic pressure environment. The dominant genera were Sphingomonas, Subgroup_6_norank, Clostridium sensu stricto, Nitrospira, Rhizomicrobium, and Acidithiobacillus. The results of this study provide new information regarding a previously uncharacterized ecosystem and show the value of high-throughput sequencing in the study of complex ecosystems.

Transcriptome Analysis of the Entomopathogenic Oomycete Lagenidium giganteum Reveals Putative Virulence Factors

PubMed Central

Quiroz Velasquez, Paula F.; Abiff, Sumayyah K.; Fins, Katrina C.; Conway, Quincy B.; Salazar, Norma C.; Delgado, Ana Paula; Dawes, Jhanelle K.; Douma, Lauren G.

2014-01-01

A combination of 454 pyrosequencing and Sanger sequencing was used to sample and characterize the transcriptome of the entomopathogenic oomycete Lagenidium giganteum. More than 50,000 high-throughput reads were annotated through homology searches. Several selected reads served as seeds for the amplification and sequencing of full-length transcripts. Phylogenetic analyses inferred from full-length cellulose synthase alignments revealed that L giganteum is nested within the peronosporalean galaxy and as such appears to have evolved from a phytopathogenic ancestor. In agreement with the phylogeny reconstructions, full-length L. giganteum oomycete effector orthologs, corresponding to the cellulose-binding elicitor lectin (CBEL), crinkler (CRN), and elicitin proteins, were characterized by domain organizations similar to those of pathogenicity factors of plant-pathogenic oomycetes. Importantly, the L. giganteum effectors provide a basis for detailing the roles of canonical CRN, CBEL, and elicitin proteins in the infectious process of an oomycete known principally as an animal pathogen. Finally, phylogenetic analyses and genome mining identified members of glycoside hydrolase family 5 subfamily 27 (GH5_27) as putative virulence factors active on the host insect cuticle, based in part on the fact that GH5_27 genes are shared by entomopathogenic oomycetes and fungi but are underrepresented in nonentomopathogenic genomes. The genomic resources gathered from the L. giganteum transcriptome analysis strongly suggest that filamentous entomopathogens (oomycetes and fungi) exhibit convergent evolution: they have evolved independently from plant-associated microbes, have retained genes indicative of plant associations, and may share similar cores of virulence factors, such as GH5_27 enzymes, that are absent from the genomes of their plant-pathogenic relatives. PMID:25107973

Pyrosequencing reveals changes in soil bacterial communities after conversion of Yungas forests to agriculture.

PubMed

Montecchia, Marcela S; Tosi, Micaela; Soria, Marcelo A; Vogrig, Jimena A; Sydorenko, Oksana; Correa, Olga S

2015-01-01

The Southern Andean Yungas in Northwest Argentina constitute one of the main biodiversity hotspots in the world. Considerable changes in land use have taken place in this ecoregion, predominantly related to forest conversion to croplands, inducing losses in above-ground biodiversity and with potential impact on soil microbial communities. In this study, we used high-throughput pyrosequencing of the 16S ribosomal RNA gene to assess whether land-use change and time under agriculture affect the composition and diversity of soil bacterial communities. We selected two areas dedicated to sugarcane and soybean production, comprising both short- and long-term agricultural sites, and used the adjacent native forest soils as a reference. Land-use change altered the composition of bacterial communities, with differences between productive areas despite the similarities between both forests. At the phylum level, only Verrucomicrobia and Firmicutes changed in abundance after deforestation for sugarcane and soybean cropping, respectively. In cultivated soils, Verrucomicrobia decreased sharply (~80%), while Firmicutes were more abundant. Despite the fact that local diversity was increased in sugarcane systems and was not altered by soybean cropping, phylogenetic beta diversity declined along both chronosequences, evidencing a homogenization of soil bacterial communities over time. In spite of the detected alteration in composition and diversity, we found a core microbiome resistant to the disturbances caused by the conversion of forests to cultivated lands and few or none exclusive OTUs for each land-use type. The overall changes in the relative abundance of copiotrophic and oligotrophic taxa may have an impact in soil ecosystem functionality. However, communities with many taxa in common may also share many functional attributes, allowing to maintain at least some soil ecosystem services after forest conversion to croplands.

Pyrosequencing Reveals Changes in Soil Bacterial Communities after Conversion of Yungas Forests to Agriculture

PubMed Central

Montecchia, Marcela S.; Tosi, Micaela; Soria, Marcelo A.; Vogrig, Jimena A.; Sydorenko, Oksana; Correa, Olga S.

2015-01-01

The Southern Andean Yungas in Northwest Argentina constitute one of the main biodiversity hotspots in the world. Considerable changes in land use have taken place in this ecoregion, predominantly related to forest conversion to croplands, inducing losses in above-ground biodiversity and with potential impact on soil microbial communities. In this study, we used high-throughput pyrosequencing of the 16S ribosomal RNA gene to assess whether land-use change and time under agriculture affect the composition and diversity of soil bacterial communities. We selected two areas dedicated to sugarcane and soybean production, comprising both short- and long-term agricultural sites, and used the adjacent native forest soils as a reference. Land-use change altered the composition of bacterial communities, with differences between productive areas despite the similarities between both forests. At the phylum level, only Verrucomicrobia and Firmicutes changed in abundance after deforestation for sugarcane and soybean cropping, respectively. In cultivated soils, Verrucomicrobia decreased sharply (~80%), while Firmicutes were more abundant. Despite the fact that local diversity was increased in sugarcane systems and was not altered by soybean cropping, phylogenetic beta diversity declined along both chronosequences, evidencing a homogenization of soil bacterial communities over time. In spite of the detected alteration in composition and diversity, we found a core microbiome resistant to the disturbances caused by the conversion of forests to cultivated lands and few or none exclusive OTUs for each land-use type. The overall changes in the relative abundance of copiotrophic and oligotrophic taxa may have an impact in soil ecosystem functionality. However, communities with many taxa in common may also share many functional attributes, allowing to maintain at least some soil ecosystem services after forest conversion to croplands. PMID:25793893

Pyrosequencing the Bemisia tabaci Transcriptome Reveals a Highly Diverse Bacterial Community and a Robust System for Insecticide Resistance

PubMed Central

Wu, Qing-jun; Wang, Shao-li; Yang, Xin; Yang, Ni-na; Li, Ru-mei; Jiao, Xiao-guo; Pan, Hui-peng; Liu, Bai-ming; Su, Qi; Xu, Bao-yun; Hu, Song-nian; Zhou, Xu-guo; Zhang, You-jun

2012-01-01

Background Bemisia tabaci (Gennadius) is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. Methodology and Principal Findings Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45%) unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10–5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. Conclusions This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the B. tabaci complex. Moreover, current pyrosequencing effort greatly enriched the existing whitefly EST database, and makes RNAseq a viable option for future genomic analysis. PMID:22558125

Prevalence of the genus Cladosporium on the integument of leaf-cutting ants characterized by 454 pyrosequencing.

PubMed

Duarte, A P M; Ferro, M; Rodrigues, A; Bacci, M; Nagamoto, N S; Forti, L C; Pagnocca, F C

2016-09-01

The relationship of attine ants with their mutualistic fungus and other microorganisms has been studied during the last two centuries. However, previous studies about the diversity of fungi in the ants' microenvironment are based mostly on culture-dependent approaches, lacking a broad characterization of the fungal ant-associated community. Here, we analysed the fungal diversity found on the integument of Atta capiguara and Atta laevigata alate ants using 454 pyrosequencing. We obtained 35,453 ITS reads grouped into 99 molecular operational taxonomic units (MOTUs). Data analysis revealed that A. capiguara drones had the highest diversity of MOTUs. Besides the occurrence of several uncultured fungi, the mycobiota analysis revealed that the most abundant taxa were the Cladosporium-complex, Cryptococcus laurentii and Epicoccum sp. Taxa in the genus Cladosporium were predominant in all samples, comprising 67.9 % of all reads. The remarkable presence of the genus Cladosporium on the integument of leaf-cutting ants alates from distinct ant species suggests that this fungus is favored in this microenvironment.

Shedding light on the microbial community of the macropod foregut using 454-amplicon pyrosequencing.

PubMed

Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y H; Maguire, Anita J; Kienzle, Marco; Klieve, Athol V

2013-01-01

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as 'shared' OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.

Gorilla gorilla gorilla gut: a potential reservoir of pathogenic bacteria as revealed using culturomics and molecular tools.

PubMed

Bittar, Fadi; Keita, Mamadou B; Lagier, Jean-Christophe; Peeters, Martine; Delaporte, Eric; Raoult, Didier

2014-11-24

Wild apes are considered to be the most serious reservoir and source of zoonoses. However, little data are available about the gut microbiota and pathogenic bacteria in gorillas. For this propose, a total of 48 fecal samples obtained from 21 Gorilla gorilla gorilla individuals (as revealed via microsatellite analysis) were screened for human bacterial pathogens using culturomics and molecular techniques. By applying culturomics to one index gorilla and using specific media supplemented by plants, we tested 12,800 colonies and identified 147 different bacterial species, including 5 new species. Many opportunistic pathogens were isolated, including 8 frequently associated with human diseases; Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus and Clostridium botulinum. The genus Treponema accounted for 27.4% of the total reads identified at the genus level via 454 pyrosequencing. Using specific real-time PCR on 48 gorilla fecal samples, in addition to classical human pathogens, we also observed the fastidious bacteria Bartonella spp. Borrelia spp., Coxiella burnetii and Tropheryma whipplei in the gorilla population. We estimated that the prevalence of these pathogens vary between 4.76% and 85.7%. Therefore, gorillas share many bacterial pathogens with humans suggesting that they could be a reservoir for their emergence.

Gorilla gorilla gorilla gut: a potential reservoir of pathogenic bacteria as revealed using culturomics and molecular tools

PubMed Central

Bittar, Fadi; Keita, Mamadou B.; Lagier, Jean-Christophe; Peeters, Martine; Delaporte, Eric; Raoult, Didier

2014-01-01

Wild apes are considered to be the most serious reservoir and source of zoonoses. However, little data are available about the gut microbiota and pathogenic bacteria in gorillas. For this propose, a total of 48 fecal samples obtained from 21 Gorilla gorilla gorilla individuals (as revealed via microsatellite analysis) were screened for human bacterial pathogens using culturomics and molecular techniques. By applying culturomics to one index gorilla and using specific media supplemented by plants, we tested 12,800 colonies and identified 147 different bacterial species, including 5 new species. Many opportunistic pathogens were isolated, including 8 frequently associated with human diseases; Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus and Clostridium botulinum. The genus Treponema accounted for 27.4% of the total reads identified at the genus level via 454 pyrosequencing. Using specific real-time PCR on 48 gorilla fecal samples, in addition to classical human pathogens, we also observed the fastidious bacteria Bartonella spp. Borrelia spp., Coxiella burnetii and Tropheryma whipplei in the gorilla population. We estimated that the prevalence of these pathogens vary between 4.76% and 85.7%. Therefore, gorillas share many bacterial pathogens with humans suggesting that they could be a reservoir for their emergence. PMID:25417711

Metagenome Analyses of Corroded Concrete Wastewater Pipe Biofilms Reveals a Complex Microbial System

EPA Science Inventory

Analysis of whole-metagenome pyrosequencing data and 16S rRNA gene clone libraries was used to determine microbial composition and functional genes associated with biomass harvested from crown (top) and invert (bottom) sections of a corroded wastewater pipe. Taxonomic and functio...

Use of pyrosequencing and denaturing gradient gel electrophoresis to examine the effects of probiotics and essential oil blends on digestive microflora in broilers under mixed Eimeria infection.

PubMed

Hume, Michael E; Barbosa, Nei A; Dowd, Scot E; Sakomura, Nilva K; Nalian, Armen G; Martynova-Van Kley, Alexandra; Oviedo-Rondón, Edgar O

2011-11-01

A protective digestive microflora helps prevent and reduce broiler infection and colonization by enteropathogens. In the current experiment, broilers fed diets supplemented with probiotics and essential oil (EO) blends were infected with a standard mixed Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities (MCs). Eight treatment groups included four controls (uninfected-unmedicated [UU], unmedicated-infected, the antibiotic BMD plus the ionophore Coban as positive control, and the ionophore as negative control), and four treatments (probiotics BC-30 and Calsporin; and EO, Crina Poultry Plus, and Crina PoultryAF). Day-old broilers were raised to 14 days in floor pens on used litter and then were moved to Petersime batteries and inoculated at 15 days with mixed Eimeria spp. Ileal and cecal samples were collected at 14 days and 7 days postinfection. Digesta DNA was subjected to pyrosequencing for sequencing of individual cecal bacteria and denaturing gradient gel electrophoresis (DGGE) for determination of changes in ileal and cecal MC according to percentage similarity coefficient (%SC). Pyrosequencing is very sensitive detecting shifts in individual bacterial sequences, whereas DGGE is able to detect gross shifts in entire MC. These combined techniques offer versatility toward identifying feed additive and mild Eimeria infection modulation of broiler MC. Pyrosequencing detected 147 bacterial species sequences. Additionally, pyrosequencing revealed the presence of relatively low levels of the potential human enteropathogens Campylobacter sp. and four Shigella spp. as well as the potential poultry pathogen Clostridiun perfringens. Pre- and postinfection changes in ileal (56%SC) and cecal (78.5%SC) DGGE profiles resulted from the coccidia infection and with increased broiler age. Probiotics and EO changed MC from those seen in UU ilea and ceca. Results potentially reflect the performance enhancement above expectations in comparison to broilers not given the probiotics or the specific EO blends as feed supplements.

MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.

PubMed

Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd

2016-01-01

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

Pyrosequencing analysis of the microbial diversity of airag, khoormog and tarag, traditional fermented dairy products of mongolia.

PubMed

Oki, Kaihei; Dugersuren, Jamyan; Demberel, Shirchin; Watanabe, Koichi

2014-01-01

Here, we used pyrosequencing to obtain a detailed analysis of the microbial diversities of traditional fermented dairy products of Mongolia. From 22 Airag (fermented mare's milk), 5 Khoormog (fermented camel's milk) and 26 Tarag (fermented milk of cows, goats and yaks) samples collected in the Mongolian provinces of Arhangai, Bulgan, Dundgobi, Tov, Uburhangai and Umnugobi, we obtained a total of 81 operational taxonomic units, which were assigned to 15 families, 21 genera and 41 species in 3 phyla. The genus Lactobacillus is a core bacterial component of Mongolian fermented milks, and Lactobacillus helveticus, Lactobacillus kefiranofaciens and Lactobacillus delbrueckii were the predominant species of lactic acid bacteria (LAB) in the Airag, Khoormog and Tarag samples, respectively. By using this pyrosequencing approach, we successfully detected most LAB species that have been isolated as well as seven LAB species that have not been found in our previous culture-based study. A subsequent analysis of the principal components of the samples revealed that L. delbrueckii, L. helveticus, L. kefiranofaciens and Streptococcus thermophilus were the main factors influencing the microbial diversity of these Mongolian traditional fermented dairy products and that this diversity correlated with the animal species from which the milk was sourced.

Analysis of bacterial xylose isomerase gene diversity using gene-targeted metagenomics.

PubMed

Nurdiani, Dini; Ito, Michihiro; Maruyama, Toru; Terahara, Takeshi; Mori, Tetsushi; Ugawa, Shin; Takeyama, Haruko

2015-08-01

Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating <90% identity with known XIs in the database accounted for 89% of the total xylA phylotypes. The differences among xylA members and compositions within each soil sample were significantly smaller than they were between different soils based on a UniFrac distance analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications. Copyright © 2015. Published by Elsevier B.V.

Impact of enrofloxacin on the human intestinal microbiota revealed by comparative molecular analysis.

PubMed

Kim, Bong-Soo; Kim, Jong Nam; Yoon, Seok-Hwan; Chun, Jongsik; Cerniglia, Carl E

2012-06-01

The indigenous human intestinal microbiota could be disrupted by residues of antibiotics in foods as well as therapeutically administered antibiotics to humans. These disruptions may lead to adverse health outcomes. To observe the possible impact of residues of antibiotics at concentrations below therapeutic levels on human intestinal microbiota, we performed studies using in vitro cultures of fecal suspensions from three individuals with 10 different concentrations (0, 0.1, 0.5, 1, 5, 10, 15, 25, 50 and 150 μg/ml) of the fluoroquinolone, enrofloxacin. The bacterial communities of the control and enrofloxacin dosed fecal samples were analyzed by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. In addition, changes of functional gene expression were analyzed by a pyrosequencing-based random whole-community mRNA sequencing method. Although each individual had a unique microbial composition, the communities of all individuals were affected by enrofloxacin. The proportions of two phyla, namely, Bacteroidetes and Proteobacteria, were significantly reduced with increasing concentrations of enrofloxacin exposure, while the proportion of Firmicutes increased. Principal Coordinate Analysis (PCoA) using the Fast UniFrac indicated that the community structures of intestinal microbiota were shifted by enrofloxacin. Most of the mRNA transcripts and the anti-microbial drug resistance genes increased with increasing concentrations of enrofloxacin. 16S rRNA gene pyrosequencing of control and enrofloxacin treated fecal suspensions provided valuable information of affected bacterial taxa down to the species level, and the community transcriptomic analyses using mRNA revealed the functional gene expression responses of the changed bacterial communities by enrofloxacin. Published by Elsevier Ltd.

Bacterial Population in Intestines of the Black Tiger Shrimp (Penaeus monodon) under Different Growth Stages

PubMed Central

Rungrassamee, Wanilada; Klanchui, Amornpan; Chaiyapechara, Sage; Maibunkaew, Sawarot; Tangphatsornruang, Sithichoke; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

2013-01-01

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities. PMID:23577162

Bacterial population in intestines of the black tiger shrimp (Penaeus monodon) under different growth stages.

PubMed

Rungrassamee, Wanilada; Klanchui, Amornpan; Chaiyapechara, Sage; Maibunkaew, Sawarot; Tangphatsornruang, Sithichoke; Jiravanichpaisal, Pikul; Karoonuthaisiri, Nitsara

2013-01-01

Intestinal bacterial communities in aquaculture have been drawn to attention due to potential benefit to their hosts. To identify core intestinal bacteria in the black tiger shrimp (Penaeus monodon), bacterial populations of disease-free shrimp were characterized from intestines of four developmental stages (15-day-old post larvae (PL15), 1- (J1), 2- (J2), and 3-month-old (J3) juveniles) using pyrosequencing, real-time PCR and denaturing gradient gel electrophoresis (DGGE) approaches. A total of 25,121 pyrosequencing reads (reading length = 442±24 bases) were obtained, which were categorized by barcode for PL15 (7,045 sequences), J1 (3,055 sequences), J2 (13,130 sequences) and J3 (1,890 sequences). Bacteria in the phyla Bacteroides, Firmicutes and Proteobacteria were found in intestines at all four growth stages. There were 88, 14, 27, and 20 bacterial genera associated with the intestinal tract of PL15, J1, J2 and J3, respectively. Pyrosequencing analysis revealed that Proteobacteria (class Gammaproteobacteria) was a dominant bacteria group with a relative abundance of 89% for PL15 and 99% for J1, J2 and J3. Real-time PCR assay also confirmed that Gammaproteobacteria had the highest relative abundance in intestines from all growth stages. Intestinal bacterial communities from the three juvenile stages were more similar to each other than that of the PL shrimp based on PCA analyses of pyrosequencing results and their DGGE profiles. This study provides descriptive bacterial communities associated to the black tiger shrimp intestines during these growth development stages in rearing facilities.

Sharing of Diverse Mycorrhizal and Root-Endophytic Fungi among Plant Species in an Oak-Dominated Cool–Temperate Forest

PubMed Central

Toju, Hirokazu; Yamamoto, Satoshi; Sato, Hirotoshi; Tanabe, Akifumi S.

2013-01-01

Most terrestrial plants interact with diverse clades of mycorrhizal and root-endophytic fungi in their roots. Through belowground plant–fungal interactions, dominant plants can benefit by interacting with host-specific mutualistic fungi and proliferate in a community based on positive plant–mutualistic fungal feedback. On the other hand, subordinate plant species may persist in the community by sharing other sets (functional groups) of fungal symbionts with each other. Therefore, revealing how diverse clades of root-associated fungi are differentially hosted by dominant and subordinate plant species is essential for understanding plant community structure and dynamics. Based on 454-pyrosequencing, we determined the community composition of root-associated fungi on 36 co-occurring plant species in an oak-dominated forest in northern Japan and statistically evaluated the host preference phenotypes of diverse mycorrhizal and root-endophytic fungi. An analysis of 278 fungal taxa indicated that an ectomycorrhizal basidiomycete fungus in the genus Lactarius and a possibly endophytic ascomycete fungus in the order Helotiales significantly favored the dominant oak (Quercus) species. In contrast, arbuscular mycorrhizal fungi were generally shared among subordinate plant species. Although fungi with host preferences contributed to the compartmentalization of belowground plant–fungal associations, diverse clades of ectomycorrhizal fungi and possible root endophytes were associated not only with the dominant Quercus but also with the remaining plant species. Our findings suggest that dominant-ectomycorrhizal and subordinate plant species can host different subsets of root-associated fungi, and diverse clades of generalist fungi can counterbalance the compartmentalization of plant–fungal associations. Such insights into the overall structure of belowground plant–fungal associations will help us understand the mechanisms that facilitate the coexistence of plant species in natural communities. PMID:24250752

Transcriptomic analysis reveals numerous diverse protein kinases and transcription factors involved in desiccation tolerance in the resurrection plant Myrothamnus flabellifolia

USDA-ARS?s Scientific Manuscript database

The woody resurrection plant Myrothamnus flabellifolia has remarkable tolerance to desiccation. Pyro-sequencing technology permitted us to analyze the transcriptome of M. flabellifolia during both dehydration and rehydration. We identified a total of 8287 and 8542 differentially transcribed genes du...

Pyrosequencing reveals the predominance of Pseudomonadaceae in gut microbiome of a Gall Midge

USDA-ARS?s Scientific Manuscript database

Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654...

Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing.

PubMed

Milanović, Vesna; Osimani, Andrea; Taccari, Manuela; Garofalo, Cristiana; Butta, Alessandro; Clementi, Francesca; Aquilanti, Lucia

2017-07-01

The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.

[Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].

PubMed

Sun, Shumei; Zhou, Hao; Zhou, Bin; Hu, Ziyou; Hou, Jinlin; Sun, Jian

2012-05-01

To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity. The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, P<0.05). Nested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, P<0.01). The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies (HBV DNA ≤3log10 copies/ml), where the sensitivity was 78%, significantly lower than that of pyrosequencing (100%, P<0.01). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. Compared with nested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

Pyrosequencing assessment of rhizosphere fungal communities from a soybean field.

PubMed

Sugiyama, Akifumi; Ueda, Yoshikatsu; Takase, Hisabumi; Yazaki, Kazufumi

2014-10-01

Soil fungal communities play essential roles in soil ecosystems, affecting plant growth and health. Rhizosphere bacterial communities have been shown to undergo dynamic changes during plant growth. This study utilized 454 pyrosequencing to analyze rhizosphere fungal communities during soybean growth. Members of the Ascomycota and Basiodiomycota dominated in all soils. There were no statistically significant changes at the phylum level among growth stages or between bulk and rhizosphere soils. In contrast, the relative abundance of small numbers of operational taxonomic units, 4 during growth and 28 between bulk and rhizosphere soils, differed significantly. Clustering analysis revealed that rhizosphere fungal communities were different from bulk fungal communities during growth stages of soybeans. Taken together, these results suggest that in contrast to rhizosphere bacterial communities, most constituents of rhizosphere fungal communities remained stable during soybean growth.

A role for the endometrial microbiome in dysfunctional menstrual bleeding.

PubMed

Pelzer, Elise S; Willner, Dana; Buttini, Melissa; Huygens, Flavia

2018-06-01

This study aimed to characterise the microbial community within the endometrial cavity and endocervix in women with menorrhagia or dysmenorrhea. Paired endocervical and endometrial biopsy samples were collected from women undergoing operative hysteroscopy and/or laparoscopy. Samples were cohorted based on pathology, indications for surgery, and histological dating of the endometrium. Samples were interrogated for the presence of microbial DNA using a two-step next generation sequencing technology approach to exploit the V5-V8 regions of the 16S rRNA gene. Pyrosequencing revealed that the endocervix and endometrium share a minor microbial community, but that each site harbours a separate and distinct microbial population (p = 0.024). This was also the case for women with menorrhagia and dysmenorrhea (p = 0.017). Lactobacillus spp. were the most abundant microbial taxa present in 50% of the cohorts, and across all endocervical groups. Members of the genera Prevotella, Fusobacterium and Jonquetella were the most abundant taxa identified in samples collected from nulliparous women. It can be concluded that the female upper genital tract is not sterile. Microbial community profiling revealed differences in the endometrial microbial community profiles for: (1) the endocervix compared to the endometrium, and (2), women with menorrhagia versus dysmenorrhea. The distinct microbial community profiles in these women may offer insight into the pathology and clinical management of dysfunctional menstrual bleeding.

Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing.

PubMed

Ye, Lin; Zhang, Tong

2011-09-01

This study applied 454 high-throughput pyrosequencing to analyze potentially pathogenic bacteria in activated sludge from 14 municipal wastewater treatment plants (WWTPs) across four countries (China, U.S., Canada, and Singapore), plus the influent and effluent of one of the 14 WWTPs. A total of 370,870 16S rRNA gene sequences with average length of 207 bps were obtained and all of them were assigned to corresponding taxonomic ranks by using RDP classifier and MEGAN. It was found that the most abundant potentially pathogenic bacteria in the WWTPs were affiliated with the genera of Aeromonas and Clostridium. Aeromonas veronii, Aeromonas hydrophila, and Clostridium perfringens were species most similar to the potentially pathogenic bacteria found in this study. Some sequences highly similar (>99%) to Corynebacterium diphtheriae were found in the influent and activated sludge samples from a saline WWTP. Overall, the percentage of the sequences closely related (>99%) to known pathogenic bacteria sequences was about 0.16% of the total sequences. Additionally, a platform-independent Java application (BAND) was developed for graphical visualization of the data of microbial abundance generated by high-throughput pyrosequencing. The approach demonstrated in this study could examine most of the potentially pathogenic bacteria simultaneously instead of one-by-one detection by other methods.

Pyrosequencing Analysis of the Microbial Diversity of Airag, Khoormog and Tarag, Traditional Fermented Dairy Products of Mongolia

PubMed Central

OKI, Kaihei; DUGERSUREN, Jamyan; DEMBEREL, Shirchin; WATANABE, Koichi

2014-01-01

Here, we used pyrosequencing to obtain a detailed analysis of the microbial diversities of traditional fermented dairy products of Mongolia. From 22 Airag (fermented mare’s milk), 5 Khoormog (fermented camel’s milk) and 26 Tarag (fermented milk of cows, goats and yaks) samples collected in the Mongolian provinces of Arhangai, Bulgan, Dundgobi, Tov, Uburhangai and Umnugobi, we obtained a total of 81 operational taxonomic units, which were assigned to 15 families, 21 genera and 41 species in 3 phyla. The genus Lactobacillus is a core bacterial component of Mongolian fermented milks, and Lactobacillus helveticus, Lactobacillus kefiranofaciens and Lactobacillus delbrueckii were the predominant species of lactic acid bacteria (LAB) in the Airag, Khoormog and Tarag samples, respectively. By using this pyrosequencing approach, we successfully detected most LAB species that have been isolated as well as seven LAB species that have not been found in our previous culture-based study. A subsequent analysis of the principal components of the samples revealed that L. delbrueckii, L. helveticus, L. kefiranofaciens and Streptococcus thermophilus were the main factors influencing the microbial diversity of these Mongolian traditional fermented dairy products and that this diversity correlated with the animal species from which the milk was sourced. PMID:25003019

DNA Methylation Pyrosequencing Assay Is Applicable for the Assessment of Epigenetic Active Environmental or Clinical Relevant Chemicals

PubMed Central

Florea, Ana-Maria

2013-01-01

Exposure of cells and organisms to stressors might result in epigenetic changes. Here it is shown that investigation of DNA methylation using pyrosequencing is an alternative for in vitro and in vivo toxicological testing of epigenetic effects induced by chemicals and drugs. An in vitro evaluation of global and CpG site specific DNA methylation upon treatment of cells with chemicals/drugs is shown. Bisulfite genomic sequencing of methylation controls showed high methylation of LINE1 in methylation positive control and low methylation in the negative controls. The CpG sites within the LINE1 element are methylated at different levels. In vitro cell cultures show a methylation level ranging from 56% to 49%. Cultures of drug resistant tumor cells show significant hypomethylation as compared with the originating nonresistant tumor cells. The in vitro testing of epigenetically active chemicals (5-methyl-2'-deoxycytidine and trichostatin A) revealed a significant change of LINE1 methylation status upon treatment, while specific CpG sites were more prone to demethylation than others (focal methylation). In conclusion, DNA methylation using pyrosequencing might be used not only for testing epigenetic toxins/drugs but also in risk assessment of drugs, food, and environmental relevant pollutants. PMID:24093099

[Effect of X-ray micro-computed tomography on the metabolic activity and diversity of soil microbial communities in two Chinese soils].

PubMed

Zu, Qianhui; Fang, Huan; Zhou, Hu; Zhang, Jianwei; Peng, Xinhua; Lin, Xiangui; Feng, Youzhi

2016-01-04

X-ray micro-computed tomography (micro-CT) technology, as used in the in situ and nondestructive analysis of soil physical structure, provides the opportunity of associating soil physical and biological assays. Due to the high heterogeneity of the soil matrix, X-ray micro-CT scanning and soil microbial assays should be conducted on the same soil sample. This raises the question whether X-ray micro-CT influences microbial function and diversity of the sample soil to be analyzed. To address this question, we used plate counting, microcalorimetry and pyrosequencing approaches to evaluate the effect of X-ray--at doses typically used in micro-CT--on soil microorganisms in a typical soil of North China Plain, Fluvo-aquic soil and in a typical soil of subtropical China, Ultisol soil, respectively. In both soils radiation decreased the number of viable soil bacteria and disturbed their thermogenic profiles. At DNA level, pyrosequencing revealed that alpha diversities of two soils biota were influenced in opposite ways, while beta diversity was not affected although the relative abundances of some guilds were changed. These findings indicate that the metabolically active aspects of soil biota are not compatible with X-ray micro-CT; while the beta molecular diversity based on pyrosequencing could be compatible.

454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters.

PubMed

Sundberg, Carina; Al-Soud, Waleed A; Larsson, Madeleine; Alm, Erik; Yekta, Sepehr S; Svensson, Bo H; Sørensen, Søren J; Karlsson, Anna

2013-09-01

The microbial community of 21 full-scale biogas reactors was examined using 454 pyrosequencing of 16S rRNA gene sequences. These reactors included seven (six mesophilic and one thermophilic) digesting sewage sludge (SS) and 14 (ten mesophilic and four thermophilic) codigesting (CD) various combinations of wastes from slaughterhouses, restaurants, households, etc. The pyrosequencing generated more than 160,000 sequences representing 11 phyla, 23 classes, and 95 genera of Bacteria and Archaea. The bacterial community was always both more abundant and more diverse than the archaeal community. At the phylum level, the foremost populations in the SS reactors included Actinobacteria, Proteobacteria, Chloroflexi, Spirochetes, and Euryarchaeota, while Firmicutes was the most prevalent in the CD reactors. The main bacterial class in all reactors was Clostridia. Acetoclastic methanogens were detected in the SS, but not in the CD reactors. Their absence suggests that methane formation from acetate takes place mainly via syntrophic acetate oxidation in the CD reactors. A principal component analysis of the communities at genus level revealed three clusters: SS reactors, mesophilic CD reactors (including one thermophilic CD and one SS), and thermophilic CD reactors. Thus, the microbial composition was mainly governed by the substrate differences and the process temperature. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Pyrosequencing reveals diverse microbial community associated with the zoanthid Palythoa australiae from the South China Sea.

PubMed

Sun, Wei; Zhang, Fengli; He, Liming; Li, Zhiyong

2014-05-01

Diverse sessile organisms inhabit the coral reef ecosystems, including corals, sponges, and sea anemones. In the past decades, scleractinian corals (Cnidaria, Anthozoa, Scleractinia) and their associated microorganisms have attracted much attention. Zoanthids (Cnidaria, Anthozoa, Zoanthidea) are commonly found in coral reefs. However, little is known about the community structure of zoanthid-associated microbiota. In this study, the microbial community associated with the zoanthid Palythoa australiae in the South China Sea was investigated by 454 pyrosequencing. As a result, 2,353 bacterial, 583 archaeal, and 36 eukaryotic microbial ribotypes were detected, respectively. A total of 22 bacterial phyla (16 formally described phyla and six candidate phyla) were recovered. Proteobacteria was the most abundant group, followed by Chloroflexi and Actinobacteria. High-abundance Rhizobiales and diverse Chloroflexi were observed in the bacterial community. The archaeal population was composed of Crenarchaeota and Euryarchaeota, with Marine Group I as the dominant lineage. In particular, Candidatus Nitrosopumilus dominated the archaeal community. Besides bacteria and archaea, the zoanthid harbored eukaryotic microorganisms including fungi and algae though their diversity was very low. This study provided the first insights into the microbial community associated with P. australiae by 454 pyrosequencing, consequently laid a basis for the understanding of the association of P. australiae-microbes symbioses.

Transcriptomics of the Bed Bug (Cimex lectularius)

PubMed Central

Rajarapu, Swapna P.; Jones, Susan C.; Mittapalli, Omprakash

2011-01-01

Background Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance. Methodology and Principal Findings Using 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database. Conclusions To our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies. PMID:21283830

Molecular analysis of the bacterial microbiome in the forestomach fluid from the dromedary camel (Camelus dromedarius).

PubMed

Bhatt, Vaibhav D; Dande, Suchitra S; Patil, Nitin V; Joshi, Chaitanya G

2013-04-01

Rumen microorganisms play an important role in ruminant digestion and absorption of nutrients and have great potential applications in the field of rumen adjusting, food fermentation and biomass utilization etc. In order to investigate the composition of microorganisms in the rumen of camel (Camelus dromedarius), this study delves in the microbial diversity by culture-independent approach. It includes comparison of rumen samples investigated in the present study to other currently available metagenomes to reveal potential differences in rumen microbial systems. Pyrosequencing based metagenomics was applied to analyze phylogenetic and metabolic profiles by MG-RAST, a web based tool. Pyrosequencing of camel rumen sample yielded 8,979,755 nucleotides assembled to 41,905 sequence reads with an average read length of 214 nucleotides. Taxonomic analysis of metagenomic reads indicated Bacteroidetes (55.5 %), Firmicutes (22.7 %) and Proteobacteria (9.2 %) phyla as predominant camel rumen taxa. At a finer phylogenetic resolution, Bacteroides species dominated the camel rumen metagenome. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism were the most abundant SEED subsystem representing 17 and 13 % of camel metagenome, respectively. A high taxonomic and functional similarity of camel rumen was found with the cow metagenome which is not surprising given the fact that both are mammalian herbivores with similar digestive tract structures and functions. Combined pyrosequencing approach and subsystems-based annotations available in the SEED database allowed us access to understand the metabolic potential of these microbiomes. Altogether, these data suggest that agricultural and animal husbandry practices can impose significant selective pressures on the rumen microbiota regardless of rumen type. The present study provides a baseline for understanding the complexity of camel rumen microbial ecology while also highlighting striking similarities and differences when compared to other animal gastrointestinal environments.

Microbiome Analysis of Stool Samples from African Americans with Colon Polyps

PubMed Central

Brim, Hassan; Yooseph, Shibu; Zoetendal, Erwin G.; Lee, Edward; Torralbo, Manolito; Laiyemo, Adeyinka O.; Shokrani, Babak; Nelson, Karen; Ashktorab, Hassan

2013-01-01

Background Colonic polyps are common tumors occurring in ~50% of Western populations with ~10% risk of malignant progression. Dietary agents have been considered the primary environmental exposure to promote colorectal cancer (CRC) development. However, the colonic mucosa is permanently in contact with the microbiota and its metabolic products including toxins that also have the potential to trigger oncogenic transformation. Aim To analyze fecal DNA for microbiota composition and functional potential in African Americans with pre-neoplastic lesions. Materials & Methods We analyzed the bacterial composition of stool samples from 6 healthy individuals and 6 patients with colon polyps using 16S ribosomal RNA-based phylogenetic microarray; the Human intestinal Tract Chip (HITChip) and 16S rRNA gene barcoded 454 pyrosequencing. The functional potential was determined by sequence-based metagenomics using 454 pyrosequencing. Results Fecal microbiota profiling of samples from the healthy and polyp patients using both a phylogenetic microarraying (HITChip) and barcoded 454 pyrosequencing generated similar results. A distinction between both sets of samples was only obtained when the analysis was performed at the sub-genus level. Most of the species leading to the dissociation were from the Bacteroides group. The metagenomic analysis did not reveal major differences in bacterial gene prevalence/abundances between the two groups even when the analysis and comparisons were restricted to available Bacteroides genomes. Conclusion This study reveals that at the pre-neoplastic stages, there is a trend showing microbiota changes between healthy and colon polyp patients at the sub-genus level. These differences were not reflected at the genome/functions levels. Bacteria and associated functions within the Bacteroides group need to be further analyzed and dissected to pinpoint potential actors in the early colon oncogenic transformation in a large sample size. PMID:24376500

Phylogenetic Diversity and Metabolic Potential Revealed in a Glacier Ice Metagenome▿ †

PubMed Central

Simon, Carola; Wiezer, Arnim; Strittmatter, Axel W.; Daniel, Rolf

2009-01-01

The largest part of the Earth's microbial biomass is stored in cold environments, which represent almost untapped reservoirs of novel species, processes, and genes. In this study, the first metagenomic survey of the metabolic potential and phylogenetic diversity of a microbial assemblage present in glacial ice is presented. DNA was isolated from glacial ice of the Northern Schneeferner, Germany. Pyrosequencing of this DNA yielded 1,076,539 reads (239.7 Mbp). The phylogenetic composition of the prokaryotic community was assessed by evaluation of a pyrosequencing-derived data set and sequencing of 16S rRNA genes. The Proteobacteria (mainly Betaproteobacteria), Bacteroidetes, and Actinobacteria were the predominant phylogenetic groups. In addition, isolation of psychrophilic microorganisms was performed, and 13 different bacterial isolates were recovered. Analysis of the 16S rRNA gene sequences of the isolates revealed that all were affiliated to the predominant groups. As expected for microorganisms residing in a low-nutrient environment, a high metabolic versatility with respect to degradation of organic substrates was detected by analysis of the pyrosequencing-derived data set. The presence of autotrophic microorganisms was indicated by identification of genes typical for different ways of carbon fixation. In accordance with the results of the phylogenetic studies, in which mainly aerobic and facultative aerobic bacteria were detected, genes typical for central metabolism of aerobes were found. Nevertheless, the capability of growth under anaerobic conditions was indicated by genes involved in dissimilatory nitrate/nitrite reduction. Numerous characteristics for metabolic adaptations associated with a psychrophilic lifestyle, such as formation of cryoprotectants and maintenance of membrane fluidity by the incorporation of unsaturated fatty acids, were detected. Thus, analysis of the glacial metagenome provided insights into the microbial life in frozen habitats on Earth, thereby possibly shedding light onto microbial life in analogous extraterrestrial environments. PMID:19801459

Phylogenetic Characterization of Fecal Microbial Communities of Dogs Fed Diets with or without Supplemental Dietary Fiber Using 454 Pyrosequencing

PubMed Central

Middelbos, Ingmar S.; Vester Boler, Brittany M.; Qu, Ani; White, Bryan A.; Swanson, Kelly S.; Fahey, George C.

2010-01-01

Background Dogs suffer from many of the same maladies as humans that may be affected by the gut microbiome, but knowledge of the canine microbiome is incomplete. This work aimed to use 16S rDNA tag pyrosequencing to phylogenetically characterize hindgut microbiome in dogs and determine how consumption of dietary fiber affects community structure. Principal Findings Six healthy adult dogs were used in a crossover design. A control diet without supplemental fiber and a beet pulp-supplemented (7.5%) diet were fed. Fecal DNA was extracted and the V3 hypervariable region of the microbial 16S rDNA gene amplified using primers suitable for 454-pyrosequencing. Microbial diversity was assessed on random 2000-sequence subsamples of individual and pooled DNA samples by diet. Our dataset comprised 77,771 reads with an average length of 141 nt. Individual samples contained approximately 129 OTU, with Fusobacteria (23 – 40% of reads), Firmicutes (14 – 28% of reads) and Bacteroidetes (31 – 34% of reads) being co-dominant phyla. Feeding dietary fiber generally decreased Fusobacteria and increased Firmicutes, but these changes were not equally apparent in all dogs. UniFrac analysis revealed that structure of the gut microbiome was affected by diet and Firmicutes appeared to play a strong role in by-diet clustering. Conclusions Our data suggest three co-dominant bacterial phyla in the canine hindgut. Furthermore, a relatively small amount of dietary fiber changed the structure of the gut microbiome detectably. Our data are among the first to characterize the healthy canine gut microbiome using pyrosequencing and provide a basis for studies focused on devising dietary interventions for microbiome-associated diseases. PMID:20339542

Pyrosequencing-based quantitative measurement of CALR mutation allele burdens and their clinical implications in patients with myeloproliferative neoplasms.

PubMed

Oh, Yejin; Song, Ik-Chan; Kim, Jimyung; Kwon, Gye Cheol; Koo, Sun Hoe; Kim, Seon Young

2018-05-01

We developed a pyrosequencing-based method for the quantification of CALR mutations and compared the results using Sanger sequencing, fragment length analysis (FLA), digital-droplet PCR (ddPCR), and next-generation sequencing (NGS). Method validation studies were performed using cloned plasmid controls. Samples from 24 patients with myeloproliferative neoplasms were evaluated. Among the 24 patients, 15 had CALR mutations (7 type 1, 2 type 2, and 6 other mutations). The type 1 or type 2 mutation-positive results from pyrosequencing exhibited 100% concordance with the Sanger sequencing results. One novel CALR mutation was not detected by pyrosequencing. The CALR mutation allele burdens measured by pyrosequencing were slightly lower than those measured by FLA but slightly higher than the results obtained using ddPCR. Pyrosequencing exhibited high correlations with both methods. The mutation allele burdens estimated by NGS were significantly lower than those measured by pyrosequencing. An increased CALR mutation allele burden was associated with overt primary myelofibrosis. Patients with >70% mutation allele burdens in myeloid cells had a significantly longer time from diagnosis (P = 0.007), more bone marrow fibrosis (P = 0.010), and lower hemoglobin (P = 0.007). Pyrosequencing was a useful rapid sequencing method to determine the burden of CALR mutations. Copyright © 2018 Elsevier B.V. All rights reserved.

Multiple approaches to characterize the microbial community in a thermophilic anaerobic digester running on swine manure: a case study.

PubMed

Tuan, Nguyen Ngoc; Chang, Yi-Chia; Yu, Chang-Ping; Huang, Shir-Ly

2014-01-01

In this study, the first survey of microbial community in thermophilic anaerobic digester using swine manure as sole feedstock was performed by multiple approaches including denaturing gradient gel electrophoresis (DGGE), clone library and pyrosequencing techniques. The integrated analysis of 21 DGGE bands, 126 clones and 8506 pyrosequencing read sequences revealed that Clostridia from the phylum Firmicutes account for the most dominant Bacteria. In addition, our analysis also identified additional taxa that were missed by the previous researches, including members of the bacterial phyla Synergistetes, Planctomycetes, Armatimonadetes, Chloroflexi and Nitrospira which might also play a role in thermophilic anaerobic digester. Most archaeal 16S rRNA sequences could be assigned to the order Methanobacteriales instead of Methanomicrobiales comparing to previous studies. In addition, this study reported that the member of Methanothermobacter genus was firstly found in thermophilic anaerobic digester. Copyright © 2014 Elsevier GmbH. All rights reserved.

Composition of soil microbiome along elevation gradients in southwestern highlands of Saudi Arabia.

PubMed

Yasir, Muhammad; Azhar, Esam I; Khan, Imran; Bibi, Fehmida; Baabdullah, Rnda; Al-Zahrani, Ibrahim A; Al-Ghamdi, Ahmed K

2015-03-14

Saudi Arabia is mostly barren except the southwestern highlands that are susceptible to environmental changes, a hotspot for biodiversity, but poorly studied for microbial diversity and composition. In this study, 454-pyrosequencing of 16S rRNA gene hypervariable region V6 was used to analyze soil bacterial community along elevation gradients of the southwestern highlands. In general, lower percentage of total soil organic matter (SOM) and nitrogen were detected in the analyzed soil samples. Total 33 different phyla were identified across the samples, including dominant phyla Proteobacteria, Actinobacteria and Acidobacteria. Representative OTUs were grouped into 329 and 508 different taxa at family and genus level taxonomic classification, respectively. The identified OTUs unique to each sample were very low irrespective of the altitude. Jackknifed principal coordinates analysis (PCoA) revealed, overall differences in the bacterial community were more related to the quantity of specific OTUs than to their diversity among the studied samples. Bacterial diversity and soil physicochemical properties did not show consistent changes along the elevation gradients. The large number of OTUs shared between the studied samples suggest the presence of a core soil bacterial community in the southwestern highlands of Saudi Arabia.

Bacterial and diazotrophic diversities of endophytes in Dendrobium catenatum determined through barcoded pyrosequencing.

PubMed

Li, Ou; Xiao, Rong; Sun, Lihua; Guan, Chenglin; Kong, Dedong; Hu, Xiufang

2017-01-01

As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ) to 96.08% (YN), and Delftia (10.39-38.42%), Burkholderia (2.71-15.98%), Escherichia/Shigella (4.90-25.12%), Pseudomonas (2.68-30.72%) and Sphingomonas (1.83-2.05%) dominated in four planting bases. Pseudomonas (17.94-22.06%), Escherichia/Shigella (6.59-11.59%), Delftia (9.65-22.14%) and Burkholderia (3.12-11.05%) dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples.

High-Throughput Amplicon Sequencing Reveals Distinct Communities within a Corroding Concrete Sewer System

PubMed Central

Dennis, Paul G.; Keller, Jurg; Tyson, Gene W.

2012-01-01

Microbially induced concrete corrosion (MICC) is an important problem in sewers. Here, small-subunit (SSU) rRNA gene amplicon pyrosequencing was used to characterize MICC communities. Microbial community composition differed between wall- and ceiling-associated MICC layers. Acidithiobacillus spp. were present at low abundances, and the communities were dominated by other sulfur-oxidizing-associated lineages. PMID:22843532

Metagenomic Analyses Reveal the Involvement of Syntrophic Consortia in Methanol/Electricity Conversion in Microbial Fuel Cells

PubMed Central

Yamamuro, Ayaka; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

2014-01-01

Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m−2 (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors. PMID:24852573

Metagenomic analyses reveal the involvement of syntrophic consortia in methanol/electricity conversion in microbial fuel cells.

PubMed

Yamamuro, Ayaka; Kouzuma, Atsushi; Abe, Takashi; Watanabe, Kazuya

2014-01-01

Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m(-2) (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.

Rhizosphere bacteriome of the medicinal plant Sapindus saponaria L. revealed by pyrosequencing.

PubMed

Garcia, A; Polonio, J C; Polli, A D; Santos, C M; Rhoden, S A; Quecine, M C; Azevedo, J L; Pamphile, J A

2016-11-03

Sapindus saponaria L. of Sapindaceae family is popularly known as soldier soap and is found in Central and South America. A study of such medicinal plants might reveal a more complex diversity of microorganisms as compared to non-medicinal plants, considering their metabolic potential and the chemical communication between their natural microbiota. Rhizosphere is a highly diverse microbial habitat with respect to both the diversity of species and the size of the community. Rhizosphere bacteriome associated with medicinal plant S. saponaria is still poorly known. The objective of this study was to assess the rhizosphere microbiome of the medicinal plant S. saponaria using pyrosequencing, a culture-independent approach that is increasingly being used to estimate the number of bacterial species present in different environments. In their rhizosphere microbiome, 26 phyla were identified from 5089 sequences of 16S rRNA gene, with a predominance of Actinobacteria (33.54%), Acidobacteria (22.62%), and Proteobacteria (24.72%). The rarefaction curve showed a linear increase, with 2660 operational taxonomic units at 3% distance sequence dissimilarity, indicating that the rhizosphere microbiome associated with S. saponaria was highly diverse with groups of bacteria important for soil management, which could be further exploited for agricultural and biotechnological purposes.

Pyrosequencing analysis of microbial communities reveals dominant cosmopolitan phylotypes in deep-sea sediments of the eastern Mediterranean Sea.

PubMed

Polymenakou, Paraskevi N; Christakis, Christos A; Mandalakis, Manolis; Oulas, Anastasis

2015-06-01

The deep eastern basin of the Mediterranean Sea is considered to be one of the world's most oligotrophic areas in the world. Here we performed pyrosequenicng analysis of bacterial and archaeal communities in oxic nutrient-poor sediments collected from the eastern Mediterranean at 1025-4393 m depth. Microbial communities were surveyed by targeting the hypervariable V5-V6 regions of the 16S ribosomal RNA gene using bar-coded pyrosequencing. With a total of 13,194 operational taxonomic units (OTUs) or phylotypes at 97% sequence similarities, the phylogenetic affiliation of microbes was assigned to 23 bacterial and 2 archaeal known phyla, 23 candidate divisions at the phylum level and distributed into 186 families. It was further revealed that the microbial consortia inhabiting all sampling sites were highly diverse, but dominated by phylotypes closely related to members of the genus Pseudomonas and Marine Group I archaea. Such pronounced and widespread enrichment probably manifests the cosmopolitan character of these species and raises questions about their metabolic adaptation to the physical stressors and low nutrient availability of the deep eastern Mediterranean Sea. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Pyrosequencing reveals regional differences in fruit-associated fungal communities

PubMed Central

Taylor, Michael W; Tsai, Peter; Anfang, Nicole; Ross, Howard A; Goddard, Matthew R

2014-01-01

We know relatively little of the distribution of microbial communities generally. Significant work has examined a range of bacterial communities, but the distribution of microbial eukaryotes is less well characterized. Humans have an ancient association with grape vines (Vitis vinifera) and have been making wine since the dawn of civilization, and fungi drive this natural process. While the molecular biology of certain fungi naturally associated with vines and wines is well characterized, complementary investigations into the ecology of fungi associated with fruiting plants is largely lacking. DNA sequencing technologies allow the direct estimation of microbial diversity from a given sample, avoiding culture-based biases. Here, we use deep community pyrosequencing approaches, targeted at the 26S rRNA gene, to examine the richness and composition of fungal communities associated with grapevines and test for geographical community structure among four major regions in New Zealand (NZ). We find over 200 taxa using this approach, which is 10-fold more than previously recovered using culture-based methods. Our analyses allow us to reject the null hypothesis of homogeneity in fungal species richness and community composition across NZ and reveal significant differences between major areas. PMID:24650123

Anthropogenic impact on diazotrophic diversity in the mangrove rhizosphere revealed by nifH pyrosequencing

PubMed Central

Jing, Hongmei; Xia, Xiaomin; Liu, Hongbin; Zhou, Zhi; Wu, Chen; Nagarajan, Sanjay

2015-01-01

Diazotrophs in the mangrove rhizosphere play a major role in providing new nitrogen to the mangrove ecosystem and their composition and activity are strongly influenced by anthropogenic activity and ecological conditions. In this study, the diversity of the diazotroph communities in the rhizosphere sediment of five tropical mangrove sites with different levels of pollution along the north and south coastline of Singapore were studied by pyrosequencing of the nifH gene. Bioinformatics analysis revealed that in all the studied locations, the diazotroph communities comprised mainly of members of the diazotrophic cluster I and cluster III. The detected cluster III diazotrophs, which were composed entirely of sulfate-reducing bacteria, were more abundant in the less polluted locations. The metabolic capacities of these diazotrophs indicate the potential for bioremediation and resiliency of the ecosystem to anthropogenic impact. In heavily polluted locations, the diazotrophic community structures were markedly different and the diversity of species was significantly reduced when compared with those in a pristine location. This, together with the increased abundance of Marinobacterium, which is a bioindicator of pollution, suggests that anthropogenic activity has a negative impact on the genetic diversity of diazotrophs in the mangrove rhizosphere. PMID:26539189

Analysis of Bacterial Diversity During Acetic Acid Fermentation of Tianjin Duliu Aged Vinegar by 454 Pyrosequencing.

PubMed

Peng, Qian; Yang, Yanping; Guo, Yanyun; Han, Ye

2015-08-01

The vinegar pei harbors complex bacterial communities. Prior studies revealing the bacterial diversity involved were mainly conducted by culture-dependent methods and PCR-DGGE. In this study, 454 pyrosequencing was used to investigate the bacterial communities in vinegar pei during the acetic acid fermentation (AAF) of Tianjin Duliu aged vinegar (TDAV). The results showed that there were 7 phyla and 24 families existing in the vinegar pei, with 2 phyla (Firmicutes, Protebacteria) and 4 families (Lactobacillaceae, Acetobacteracae, Enterobacteriaceae, Chloroplast) predominating. The genus-level identification revealed that 9 genera were the relatively stable, consistent components in different stages of AAF, including the most abundant genus Lactobacillus followed by Acetobacter and Serratia. Additionally, the bacterial community in the early fermentation stage was more complex than those in the later stages, indicating that the accumulation of organic acids provided an appropriate environment to filter unwanted bacteria and to accelerate the growth of required ones. This study provided basic information of bacterial patterns in vinegar pei and relevant changes during AAF of TDAV, and could be used as references in the following study on the implementation of starter culture as well as the improvement of AAF process.

Anthropogenic impact on diazotrophic diversity in the mangrove rhizosphere revealed by nifH pyrosequencing.

PubMed

Jing, Hongmei; Xia, Xiaomin; Liu, Hongbin; Zhou, Zhi; Wu, Chen; Nagarajan, Sanjay

2015-01-01

Diazotrophs in the mangrove rhizosphere play a major role in providing new nitrogen to the mangrove ecosystem and their composition and activity are strongly influenced by anthropogenic activity and ecological conditions. In this study, the diversity of the diazotroph communities in the rhizosphere sediment of five tropical mangrove sites with different levels of pollution along the north and south coastline of Singapore were studied by pyrosequencing of the nifH gene. Bioinformatics analysis revealed that in all the studied locations, the diazotroph communities comprised mainly of members of the diazotrophic cluster I and cluster III. The detected cluster III diazotrophs, which were composed entirely of sulfate-reducing bacteria, were more abundant in the less polluted locations. The metabolic capacities of these diazotrophs indicate the potential for bioremediation and resiliency of the ecosystem to anthropogenic impact. In heavily polluted locations, the diazotrophic community structures were markedly different and the diversity of species was significantly reduced when compared with those in a pristine location. This, together with the increased abundance of Marinobacterium, which is a bioindicator of pollution, suggests that anthropogenic activity has a negative impact on the genetic diversity of diazotrophs in the mangrove rhizosphere.

Exploring the Bacterial Microbiota of Colombian Fermented Maize Dough "Masa Agria" (Maiz Añejo).

PubMed

Chaves-Lopez, Clemencia; Serio, Annalisa; Delgado-Ospina, Johannes; Rossi, Chiara; Grande-Tovar, Carlos D; Paparella, Antonello

2016-01-01

Masa Agria is a naturally fermented maize dough produced in Colombia, very common in the traditional gastronomy. In this study we used culture-dependent and RNA-based pyrosequencing to investigate the bacterial community structure of Masa Agria samples produced in the south west of Colombia. The mean value of cell density was 7.6 log CFU/g of presumptive lactic acid bacteria, 5.4 log cfu/g for presumptive acetic bacteria and 5.6 og CFU/g for yeasts. The abundance of these microorganisms is also responsible for the low pH (3.1-3.7) registered. Although the 16S rRNA pyrosequencing revealed that the analyzed samples were different in bacteria richness and diversity, the genera Lactobacillus, Weissella, and Acetobacter were predominant. In particular, the most common species were Lactobacillus plantarum and Acetobacter fabarum, followed by L. fermentum, L. vaccinostercus, and Pediococcus argentinicus. Several microorganisms of environmental origin, such as Dechloromonas and most of all Sphingobium spp., revealed in each sample, were detected, and also bacteria related to maize, such as Phytoplasma. In conclusion, our results elucidated for the first time the structures of the bacterial communities of Masa Agria samples obtained from different producers, identifying the specific dominant species and revealing a complete picture of the bacterial consortium in this specific niche. The selective pressure of tropical environments may favor microbial biodiversity characterized by a useful technological potential.

Pyrosequencing of Plaque Microflora In Twin Children with Discordant Caries Phenotypes

PubMed Central

Zhang, Meng; Chen, Yongxing; Xie, Lingzhi; Li, Yuhong; Jiang, Han; Du, Minquan

2015-01-01

Despite recent successes in the control of dental caries, the mechanism of caries development remains unclear. To investigate the causes of dental decay, especially in early childhood caries, the supragingival microflora composition of 20 twins with discordant caries phenotypes were analyzed using high-throughput pyrosequencing. In addition, the parents completed a lifestyle questionnaire. A total of 228,789 sequencing reads revealed 10 phyla, 84 genera, and 155 species of microflora, the relative abundances of these strains varied dramatically among the children, Comparative analysis between groups revealed that Veillonella, Corynebacterium and Actinomyces were presumed to be caries-related genera, Fusobacterium, Kingella and Leptotrichia were presumed to be healthy-related genus, yet this six genera were not statistically significant (P>0.05). Moreover, a cluster analysis revealed that the microbial composition of samples in the same group was often dissimilar but that the microbial composition observed in twins was usually similar. Although the genetic and environmental factors that strongly influence the microbial composition of dental caries remains unknown, we speculate that genetic factors primarily influence the individual's susceptibility to dental caries and that environmental factors primarily regulate the microbial composition of the dental plaque and the progression to caries. By using improved twins models and increased sample sizes, our study can be extended to analyze the specific genetic and environmental factors that affect the development of caries. PMID:26524687

Biodiversity in Oscypek, a Traditional Polish Cheese, Determined by Culture-Dependent and -Independent Approaches

PubMed Central

Alegría, Ángel; Szczesny, Pawel; Mayo, Baltasar; Bardowski, Jacek

2012-01-01

Oscypek is a traditional Polish scalded-smoked cheese, with a protected-designation-of-origin (PDO) status, manufactured from raw sheep's milk without starter cultures in the Tatra Mountains region of Poland. This study was undertaken in order to gain insight into the microbiota that develops and evolves during the manufacture and ripening stages of Oscypek. To this end, we made use of both culturing and the culture-independent methods of PCR followed by denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing of 16S rRNA gene amplicons. The culture-dependent technique and PCR-DGGE fingerprinting detected the predominant microorganisms in traditional Oscypek, whereas the next-generation sequencing technique (454 pyrosequencing) revealed greater bacterial diversity. Besides members of the most abundant bacterial genera in dairy products, e.g., Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, and Enterococcus, identified by all three methods, other, subdominant bacteria belonging to the families Bifidobacteriaceae and Moraxellaceae (mostly Enhydrobacter), as well as various minor bacteria, were identified by pyrosequencing. The presence of bifidobacterial sequences in a cheese system is reported for the first time. In addition to bacteria, a great diversity of yeast species was demonstrated in Oscypek by the PCR-DGGE method. Culturing methods enabled the determination of a number of viable microorganisms from different microbial groups and their isolation for potential future applications in specific cheese starter cultures. PMID:22247135

Proteolytic bacterial dominance in a full-scale municipal solid waste anaerobic reactor assessed by 454 pyrosequencing technology.

PubMed

Cardinali-Rezende, Juliana; Rojas-Ojeda, Patricia; Nascimento, Andréa M A; Sanz, José L

2016-03-01

Biomethanization entails a good means to reduce the organic fraction (OF) derived from municipal solid wastes (MSW). The bacterial diversity of a full scale MSW anaerobic reactor located in Madrid (Spain) was investigated using high-throughput 454 pyrosequencing. Even though the proteolytic bacteria prevailed throughout all of the process, community shifts were observed from the start-up to the steady-state conditions, with an increasing biodiversity displayed over time. The Bacteroidetes and the Firmicutes were the majority phyla: 55.1 and 40.2% (start-up) and 18.7 and 78.7 (steady-state) of the total reads. The system's lack of evenness remains noteworthy as the sequences affiliated to the proteolytic non-saccharolytic Proteiniphylum, Gallicola and Fastidiosipila genera, together with the saccharolytic Saccharofermentans, were predominant on the system and this predominance appears to correlate with the presence of a high ammonium concentration. The 454 pyrosequencing revealed a great diversity of rare organisms which seemingly do not sustain any metabolic roles in the course of the OF-MSW degradation. However, this scarce and unique microbiota can confer great resilience to the system as a buffer against nutritional and environmental changing conditions, thus opening the door to increase the current knowledge about the bacterial community dynamics taking place during MSW treatment processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Humpback whale populations share a core skin bacterial community: towards a health index for marine mammals?

PubMed

Apprill, Amy; Robbins, Jooke; Eren, A Murat; Pack, Adam A; Reveillaud, Julie; Mattila, David; Moore, Michael; Niemeyer, Misty; Moore, Kathleen M T; Mincer, Tracy J

2014-01-01

Microbes are now well regarded for their important role in mammalian health. The microbiology of skin--a unique interface between the host and environment--is a major research focus in human health and skin disorders, but is less explored in other mammals. Here, we report on a cross-population study of the skin-associated bacterial community of humpback whales (Megaptera novaeangliae), and examine the potential for a core bacterial community and its variability with host (endogenous) or geographic/environmental (exogenous) specific factors. Skin biopsies or freshly sloughed skin from 56 individuals were sampled from populations in the North Atlantic, North Pacific and South Pacific oceans and bacteria were characterized using 454 pyrosequencing of SSU rRNA genes. Phylogenetic and statistical analyses revealed the ubiquity and abundance of bacteria belonging to the Flavobacteria genus Tenacibaculum and the Gammaproteobacteria genus Psychrobacter across the whale populations. Scanning electron microscopy of skin indicated that microbial cells colonize the skin surface. Despite the ubiquity of Tenacibaculum and Psychrobater spp., the relative composition of the skin-bacterial community differed significantly by geographic area as well as metabolic state of the animals (feeding versus starving during migration and breeding), suggesting that both exogenous and endogenous factors may play a role in influencing the skin-bacteria. Further, characteristics of the skin bacterial community from these free-swimming individuals were assembled and compared to two entangled and three dead individuals, revealing a decrease in the central or core bacterial community members (Tenacibaculum and Psychrobater spp.), as well as the emergence of potential pathogens in the latter cases. This is the first discovery of a cross-population, shared skin bacterial community. This research suggests that the skin bacteria may be connected to humpback health and immunity and could possibly serve as a useful index for health and skin disorder monitoring of threatened and endangered marine mammals.

Humpback Whale Populations Share a Core Skin Bacterial Community: Towards a Health Index for Marine Mammals?

PubMed Central

Apprill, Amy; Robbins, Jooke; Eren, A. Murat; Pack, Adam A.; Reveillaud, Julie; Mattila, David; Moore, Michael; Niemeyer, Misty; Moore, Kathleen M. T.; Mincer, Tracy J.

2014-01-01

Microbes are now well regarded for their important role in mammalian health. The microbiology of skin – a unique interface between the host and environment - is a major research focus in human health and skin disorders, but is less explored in other mammals. Here, we report on a cross-population study of the skin-associated bacterial community of humpback whales (Megaptera novaeangliae), and examine the potential for a core bacterial community and its variability with host (endogenous) or geographic/environmental (exogenous) specific factors. Skin biopsies or freshly sloughed skin from 56 individuals were sampled from populations in the North Atlantic, North Pacific and South Pacific oceans and bacteria were characterized using 454 pyrosequencing of SSU rRNA genes. Phylogenetic and statistical analyses revealed the ubiquity and abundance of bacteria belonging to the Flavobacteria genus Tenacibaculum and the Gammaproteobacteria genus Psychrobacter across the whale populations. Scanning electron microscopy of skin indicated that microbial cells colonize the skin surface. Despite the ubiquity of Tenacibaculum and Psychrobater spp., the relative composition of the skin-bacterial community differed significantly by geographic area as well as metabolic state of the animals (feeding versus starving during migration and breeding), suggesting that both exogenous and endogenous factors may play a role in influencing the skin-bacteria. Further, characteristics of the skin bacterial community from these free-swimming individuals were assembled and compared to two entangled and three dead individuals, revealing a decrease in the central or core bacterial community members (Tenacibaculum and Psychrobater spp.), as well as the emergence of potential pathogens in the latter cases. This is the first discovery of a cross-population, shared skin bacterial community. This research suggests that the skin bacteria may be connected to humpback health and immunity and could possibly serve as a useful index for health and skin disorder monitoring of threatened and endangered marine mammals. PMID:24671052

Metatranscriptomes reveal functional variation in diatom communities from the Antarctic Peninsula.

PubMed

Pearson, Gareth A; Lago-Leston, Asuncion; Cánovas, Fernando; Cox, Cymon J; Verret, Frederic; Lasternas, Sebastian; Duarte, Carlos M; Agusti, Susana; Serrão, Ester A

2015-10-01

Functional genomics of diatom-dominated communities from the Antarctic Peninsula was studied using comparative metatranscriptomics. Samples obtained from diatom-rich communities in the Bransfield Strait, the western Weddell Sea and sea ice in the Bellingshausen Sea/Wilkins Ice Shelf yielded more than 500K pyrosequencing reads that were combined to produce a global metatranscriptome assembly. Multi-gene phylogenies recovered three distinct communities, and diatom-assigned contigs further indicated little read-sharing between communities, validating an assembly-based annotation and analysis approach. Although functional analysis recovered a core of abundant shared annotations that were expressed across the three diatom communities, over 40% of annotations (but accounting for <10% of sequences) were community-specific. The two pelagic communities differed in their expression of N-metabolism and acquisition genes, which was almost absent in post-bloom conditions in the Weddell Sea community, while enrichment of transporters for ammonia and urea in Bransfield Strait diatoms suggests a physiological stance towards acquisition of reduced N-sources. The depletion of carbohydrate and energy metabolism pathways in sea ice relative to pelagic communities, together with increased light energy dissipation (via LHCSR proteins), photorespiration, and NO3(-) uptake and utilization all pointed to irradiance stress and/or inorganic carbon limitation within sea ice. Ice-binding proteins and cold-shock transcription factors were also enriched in sea ice diatoms. Surprisingly, the abundance of gene transcripts for the translational machinery tracked decreasing environmental temperature across only a 4 °C range, possibly reflecting constraints on translational efficiency and protein production in cold environments.

Wide Distribution of Closely Related, Antibiotic-Producing Arthrobacter Strains throughout the Arctic Ocean

PubMed Central

Wietz, Matthias; Månsson, Maria; Bowman, Jeff S.; Blom, Nikolaj; Ng, Yin

2012-01-01

We isolated 16 antibiotic-producing bacterial strains throughout the central Arctic Ocean, including seven Arthrobacter spp. with almost identical 16S rRNA gene sequences. These strains were numerically rare, as revealed using 454 pyrosequencing libraries. Arthrobacter spp. produced arthrobacilins A to C under different culture conditions, but other, unidentified compounds likely contributed to their antibiotic activity. PMID:22247128

Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

Treesearch

Daniel L. Lindner; Tor Carlsen; Henrik Nilsson; Marie Davey; Trond Schumacher; Havard. Kauserud

2013-01-01

The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular...

Monitoring of microbial communities in anaerobic digestion sludge for biogas optimisation.

PubMed

Lim, Jun Wei; Ge, Tianshu; Tong, Yen Wah

2018-01-01

This study characterised and compared the microbial communities of anaerobic digestion (AD) sludge using three different methods - (1) Clone library; (2) Pyrosequencing; and (3) Terminal restriction fragment length polymorphism (T-RFLP). Although high-throughput sequencing techniques are becoming increasingly popular and affordable, the reliance of such techniques for frequent monitoring of microbial communities may be a financial burden for some. Furthermore, the depth of microbial analysis revealed by high-throughput sequencing may not be required for monitoring purposes. This study aims to develop a rapid, reliable and economical approach for the monitoring of microbial communities in AD sludge. A combined approach where genetic information of sequences from clone library was used to assign phylogeny to T-RFs determined experimentally was developed in this study. In order to assess the effectiveness of the combined approach, microbial communities determined by the combined approach was compared to that characterised by pyrosequencing. Results showed that both pyrosequencing and clone library methods determined the dominant bacteria phyla to be Proteobacteria, Firmicutes, Bacteroidetes, and Thermotogae. Both methods also found that sludge A and B were predominantly dominated by acetogenic methanogens followed by hydrogenotrophic methanogens. The number of OTUs detected by T-RFLP was significantly lesser than that detected by the clone library. In this study, T-RFLP analysis identified majority of the dominant species of the archaeal consortia. However, many of the more highly diverse bacteria consortia were missed. Nevertheless, the combined approach developed in this study where clone sequences from the clone library were used to assign phylogeny to T-RFs determined experimentally managed to accurately predict the same dominant microbial groups for both sludge A and sludge B, as compared to the pyrosequencing results. Results showed that the combined approach of clone library and T-RFLP accurately predicted the dominant microbial groups and thus is a reliable and more economical way to monitor the evolution of microbial systems in AD sludge. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dental plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach

PubMed Central

Takeshita, Toru; Yasui, Masaki; Shibata, Yukie; Furuta, Michiko; Saeki, Yoji; Eshima, Nobuoki; Yamashita, Yoshihisa

2015-01-01

Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species including difficult-to-cultivate bacteria. We observed the assembly of a plaque bacterial community through 16S rRNA gene analysis. Plaque samples that accumulated on a hydroxyapatite disk for 1, 2, 3, 4, 5, and 7 days with saliva on day 0 were collected from 19 young adults using a removable resin splint. Quantitative PCR analysis showed that the total bacterial amount gradually increased and reached a plateau on day 4. Barcoded pyrosequencing analysis revealed that the microbial richness and diversity particularly increased between days 5 and 7. A principal coordinate analysis plot based on unweighted UniFrac showed the community assembly in a time-related manner, which became increasingly similar to the salivary microbiota. Facultative anaerobic bacteria such as Streptococcus, Neisseria, Abiotrophia, Gemella, and Rothia were predominant in the plaque bacterial community in the earlier days, whereas obligate anaerobes, such as Porphyromonas, Fusobacterium, Prevotella, and Capnocytophaga showed increased dominance on later days. UniFrac analysis also demonstrated that dental caries experience had a significant effect on the assembly process. Our results reveal the development pattern of the plaque bacterial community as well as the inter-individual differences associated with dental caries experience. PMID:25633431

Dental plaque development on a hydroxyapatite disk in young adults observed by using a barcoded pyrosequencing approach.

PubMed

Takeshita, Toru; Yasui, Masaki; Shibata, Yukie; Furuta, Michiko; Saeki, Yoji; Eshima, Nobuoki; Yamashita, Yoshihisa

2015-01-30

Dental plaque is a dynamic microbial biofilm ecosystem that comprises hundreds of species including difficult-to-cultivate bacteria. We observed the assembly of a plaque bacterial community through 16S rRNA gene analysis. Plaque samples that accumulated on a hydroxyapatite disk for 1, 2, 3, 4, 5, and 7 days with saliva on day 0 were collected from 19 young adults using a removable resin splint. Quantitative PCR analysis showed that the total bacterial amount gradually increased and reached a plateau on day 4. Barcoded pyrosequencing analysis revealed that the microbial richness and diversity particularly increased between days 5 and 7. A principal coordinate analysis plot based on unweighted UniFrac showed the community assembly in a time-related manner, which became increasingly similar to the salivary microbiota. Facultative anaerobic bacteria such as Streptococcus, Neisseria, Abiotrophia, Gemella, and Rothia were predominant in the plaque bacterial community in the earlier days, whereas obligate anaerobes, such as Porphyromonas, Fusobacterium, Prevotella, and Capnocytophaga showed increased dominance on later days. UniFrac analysis also demonstrated that dental caries experience had a significant effect on the assembly process. Our results reveal the development pattern of the plaque bacterial community as well as the inter-individual differences associated with dental caries experience.

Bacterial Pathogens and Community Composition in Advanced Sewage Treatment Systems Revealed by Metagenomics Analysis Based on High-Throughput Sequencing

PubMed Central

Lu, Xin; Zhang, Xu-Xiang; Wang, Zhu; Huang, Kailong; Wang, Yuan; Liang, Weigang; Tan, Yunfei; Liu, Bo; Tang, Junying

2015-01-01

This study used 454 pyrosequencing, Illumina high-throughput sequencing and metagenomic analysis to investigate bacterial pathogens and their potential virulence in a sewage treatment plant (STP) applying both conventional and advanced treatment processes. Pyrosequencing and Illumina sequencing consistently demonstrated that Arcobacter genus occupied over 43.42% of total abundance of potential pathogens in the STP. At species level, potential pathogens Arcobacter butzleri, Aeromonas hydrophila and Klebsiella pneumonia dominated in raw sewage, which was also confirmed by quantitative real time PCR. Illumina sequencing also revealed prevalence of various types of pathogenicity islands and virulence proteins in the STP. Most of the potential pathogens and virulence factors were eliminated in the STP, and the removal efficiency mainly depended on oxidation ditch. Compared with sand filtration, magnetic resin seemed to have higher removals in most of the potential pathogens and virulence factors. However, presence of the residual A. butzleri in the final effluent still deserves more concerns. The findings indicate that sewage acts as an important source of environmental pathogens, but STPs can effectively control their spread in the environment. Joint use of the high-throughput sequencing technologies is considered a reliable method for deep and comprehensive overview of environmental bacterial virulence. PMID:25938416

Characterization of Early Microbial Communities on Volcanic Deposits along a Vegetation Gradient on the Island of Miyake, Japan

PubMed Central

Guo, Yong; Fujimura, Reiko; Sato, Yoshinori; Suda, Wataru; Kim, Seok-won; Oshima, Kenshiro; Hattori, Masahira; Kamijo, Takashi; Narisawa, Kazuhiko; Ohta, Hiroyuki

2014-01-01

The 2000 eruption of Mount Oyama on the island of Miyake (Miyake-jima) created a unique opportunity to study the early ecosystem development on newly exposed terrestrial substrates. In this study, bacterial and fungal communities on 9- and 11-year-old volcanic deposits at poorly to fully vegetation-recovered sites in Miyake-jima, Japan, were characterized by conventional culture-based methods and pyrosequencing of 16S rRNA and 18S rRNA genes. Despite the differences in the vegetation cover, the upper volcanic deposit layer samples displayed low among-site variation for chemical properties (pH, total organic carbon, and total nitrogen) and microbial population densities (total direct count and culturable count). Statistical analyses of pyrosequencing data revealed that the microbial communities of volcanic deposit samples were phylogenetically diverse, in spite of very low-carbon environmental conditions, and their diversity was comparable to that in the lower soil layer (buried soil) samples. Comparing with the microbial communities in buried soil, the volcanic deposit communities were characterized by the presence of Betaproteobacteria and Gammaproteobacteria as the main bacterial class, Deinococcus- Thermus as the minor bacterial phyla, and Ascomycota as the major fungal phyla. Multivariate analysis revealed that several bacterial families and fungal classes correlated positively or negatively with plant species. PMID:24463576

Exploring the Bacterial Microbiota of Colombian Fermented Maize Dough “Masa Agria” (Maiz Añejo)

PubMed Central

Chaves-Lopez, Clemencia; Serio, Annalisa; Delgado-Ospina, Johannes; Rossi, Chiara; Grande-Tovar, Carlos D.; Paparella, Antonello

2016-01-01

Masa Agria is a naturally fermented maize dough produced in Colombia, very common in the traditional gastronomy. In this study we used culture-dependent and RNA-based pyrosequencing to investigate the bacterial community structure of Masa Agria samples produced in the south west of Colombia. The mean value of cell density was 7.6 log CFU/g of presumptive lactic acid bacteria, 5.4 log cfu/g for presumptive acetic bacteria and 5.6 og CFU/g for yeasts. The abundance of these microorganisms is also responsible for the low pH (3.1–3.7) registered. Although the 16S rRNA pyrosequencing revealed that the analyzed samples were different in bacteria richness and diversity, the genera Lactobacillus, Weissella, and Acetobacter were predominant. In particular, the most common species were Lactobacillus plantarum and Acetobacter fabarum, followed by L. fermentum, L. vaccinostercus, and Pediococcus argentinicus. Several microorganisms of environmental origin, such as Dechloromonas and most of all Sphingobium spp., revealed in each sample, were detected, and also bacteria related to maize, such as Phytoplasma. In conclusion, our results elucidated for the first time the structures of the bacterial communities of Masa Agria samples obtained from different producers, identifying the specific dominant species and revealing a complete picture of the bacterial consortium in this specific niche. The selective pressure of tropical environments may favor microbial biodiversity characterized by a useful technological potential. PMID:27524979

Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea

PubMed Central

Lee, On On; Wang, Yong; Yang, Jiangke; Lafi, Feras F; Al-Suwailem, Abdulaziz; Qian, Pei-Yuan

2011-01-01

Marine sponges are associated with a remarkable array of microorganisms. Using a tag pyrosequencing technology, this study was the first to investigate in depth the microbial communities associated with three Red Sea sponges, Hyrtios erectus, Stylissa carteri and Xestospongia testudinaria. We revealed highly diverse sponge-associated bacterial communities with up to 1000 microbial operational taxonomic units (OTUs) and richness estimates of up to 2000 species. Altogether, 26 bacterial phyla were detected from the Red Sea sponges, 11 of which were absent from the surrounding sea water and 4 were recorded in sponges for the first time. Up to 100 OTUs with richness estimates of up to 300 archaeal species were revealed from a single sponge species. This is by far the highest archaeal diversity ever recorded for sponges. A non-negligible proportion of unclassified reads was observed in sponges. Our results demonstrated that the sponge-associated microbial communities remained highly consistent in the same sponge species from different locations, although they varied at different degrees among different sponge species. A significant proportion of the tag sequences from the sponges could be assigned to one of the sponge-specific clusters previously defined. In addition, the sponge-associated microbial communities were consistently divergent from those present in the surrounding sea water. Our results suggest that the Red Sea sponges possess highly sponge-specific or even sponge-species-specific microbial communities that are resistant to environmental disturbance, and much of their microbial diversity remains to be explored. PMID:21085196

Molecular techniques revealed highly diverse microbial communities in natural marine biofilms on polystyrene dishes for invertebrate larval settlement.

PubMed

Lee, On On; Chung, Hong Chun; Yang, Jiangke; Wang, Yong; Dash, Swagatika; Wang, Hao; Qian, Pei-Yuan

2014-07-01

Biofilm microbial communities play an important role in the larval settlement response of marine invertebrates. However, the underlying mechanism has yet to be resolved, mainly because of the uncertainties in characterizing members in the communities using traditional 16S rRNA gene-based molecular methods and in identifying the chemical signals involved. In this study, pyrosequencing was used to characterize the bacterial communities in intertidal and subtidal marine biofilms developed during two seasons. We revealed highly diverse biofilm bacterial communities that varied with season and tidal level. Over 3,000 operational taxonomic units with estimates of up to 8,000 species were recovered in a biofilm sample, which is by far the highest number recorded in subtropical marine biofilms. Nineteen phyla were found, of which Cyanobacteria and Proteobacteria were the most dominant one in the intertidal and subtidal biofilms, respectively. Apart from these, Actinobacteria, Bacteroidetes, and Planctomycetes were the major groups recovered in both intertidal and subtidal biofilms, although their relative abundance varied among samples. Full-length 16S rRNA gene clone libraries were constructed for the four biofilm samples and showed similar bacterial compositions at the phylum level to those revealed by pyrosequencing. Laboratory assays confirmed that cyrids of the barnacle Balanus amphitrite preferred to settle on the intertidal rather than subtidal biofilms. This preference was independent of the biofilm bacterial density or biomass but was probably related to the biofilm community structure, particularly, the Proteobacterial and Cyanobacterial groups.

SNP-based real-time pyrosequencing as a sensitive and specific tool for identification and differentiation of Rickettsia species in Ixodes ricinus ticks.

PubMed

Janecek, Elisabeth; Streichan, Sabine; Strube, Christina

2012-10-18

Rickettsioses are caused by pathogenic species of the genus Rickettsia and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring Rickettsia species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German Ixodes ticks, which are R. helvetica, R. monacensis, R. massiliae, and R. felis. Tick material from a quantitative real-time PCR (qPCR) based study on Rickettsia-infections in I. ricinus allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing. A sequence stretch of rickettsial citrate synthase (gltA) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for Rickettsia species differentiation. Positive control plasmids inserting the respective target sequence of each Rickettsia species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen's PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 Ixodes tick samples already detected Rickettsia-positive in previous qPCR experiments. Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included Rickettsia species. The method was then applied to 263 Rickettsia-positive Ixodes ricinus samples, of which 153 (58.2%) could be identified for their species (151 R. helvetica and 2 R. monacensis) by previous custom Sanger sequencing. Real-time pyrosequencing identified all Sanger-determined ticks as well as 35 previously undifferentiated ticks resulting in a total number of 188 (71.5%) identified samples. Pyrosequencing sensitivity was found to be strongly dependent on gltA copy numbers in the reaction setup. Whereas less than 101 copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 101-102 copies, to 95.6% at >102-103 copies and reached 100% samples identified for their Rickettsia species if more than 103 copies were present in the template. The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of Rickettsia spp. present in I. ricinus diagnostic material and prevalence studies. Furthermore, the method proved to be faster, more cost-effective as well as more sensitive than custom Sanger sequencing with simultaneous high specificity.

Insights into fungal communities in composts revealed by 454-pyrosequencing: implications for human health and safety.

PubMed

De Gannes, Vidya; Eudoxie, Gaius; Hickey, William J

2013-01-01

Fungal community composition in composts of lignocellulosic wastes was assessed via 454-pyrosequencing of ITS1 libraries derived from the three major composting phases. Ascomycota represented most (93%) of the 27,987 fungal sequences. A total of 102 genera, 120 species, and 222 operational taxonomic units (OTUs; >97% similarity) were identified. Thirty genera predominated (ca. 94% of the sequences), and at the species level, sequences matching Chaetomium funicola and Fusarium oxysporum were the most abundant (26 and 12%, respectively). In all composts, fungal diversity in the mature phase exceeded that of the mesophilic phase, but there was no consistent pattern in diversity changes occurring in the thermophilic phase. Fifteen species of human pathogens were identified, eight of which have not been previously identified in composts. This study demonstrated that deep sequencing can elucidate fungal community diversity in composts, and that this information can have important implications for compost use and human health.

Insights into fungal communities in composts revealed by 454-pyrosequencing: implications for human health and safety

PubMed Central

De Gannes, Vidya; Eudoxie, Gaius; Hickey, William J.

2013-01-01

Fungal community composition in composts of lignocellulosic wastes was assessed via 454-pyrosequencing of ITS1 libraries derived from the three major composting phases. Ascomycota represented most (93%) of the 27,987 fungal sequences. A total of 102 genera, 120 species, and 222 operational taxonomic units (OTUs; >97% similarity) were identified. Thirty genera predominated (ca. 94% of the sequences), and at the species level, sequences matching Chaetomium funicola and Fusarium oxysporum were the most abundant (26 and 12%, respectively). In all composts, fungal diversity in the mature phase exceeded that of the mesophilic phase, but there was no consistent pattern in diversity changes occurring in the thermophilic phase. Fifteen species of human pathogens were identified, eight of which have not been previously identified in composts. This study demonstrated that deep sequencing can elucidate fungal community diversity in composts, and that this information can have important implications for compost use and human health. PMID:23785368

Effect of the soil type on the microbiome in the rhizosphere of field-grown lettuce.

PubMed

Schreiter, Susanne; Ding, Guo-Chun; Heuer, Holger; Neumann, Günter; Sandmann, Martin; Grosch, Rita; Kropf, Siegfried; Smalla, Kornelia

2014-01-01

The complex and enormous diversity of microorganisms associated with plant roots is important for plant health and growth and is shaped by numerous factors. This study aimed to unravel the effects of the soil type on bacterial communities in the rhizosphere of field-grown lettuce. We used an experimental plot system with three different soil types that were stored at the same site for 10 years under the same agricultural management to reveal differences directly linked to the soil type and not influenced by other factors such as climate or cropping history. Bulk soil and rhizosphere samples were collected 3 and 7 weeks after planting. The analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and pyrosequencing revealed soil type dependent differences in the bacterial community structure of the bulk soils and the corresponding rhizospheres. The rhizosphere effect differed depending on the soil type and the plant growth developmental stage. Despite the soil type dependent differences in the bacterial community composition several genera such as Sphingomonas, Rhizobium, Pseudomonas, and Variovorax were significantly increased in the rhizosphere of lettuce grown in all three soils. The number of rhizosphere responders was highest 3 weeks after planting. Interestingly, in the soil with the highest numbers of responders the highest shoot dry weights were observed. Heatmap analysis revealed that many dominant operational taxonomic units were shared among rhizosphere samples of lettuce grown in diluvial sand, alluvial loam, and loess loam and that only a subset was increased in relative abundance in the rhizosphere compared to the corresponding bulk soil. The findings of the study provide insights into the effect of soil types on the rhizosphere microbiome of lettuce.

Effect of the soil type on the microbiome in the rhizosphere of field-grown lettuce

PubMed Central

Schreiter, Susanne; Ding, Guo-Chun; Heuer, Holger; Neumann, Günter; Sandmann, Martin; Grosch, Rita; Kropf, Siegfried; Smalla, Kornelia

2014-01-01

The complex and enormous diversity of microorganisms associated with plant roots is important for plant health and growth and is shaped by numerous factors. This study aimed to unravel the effects of the soil type on bacterial communities in the rhizosphere of field-grown lettuce. We used an experimental plot system with three different soil types that were stored at the same site for 10 years under the same agricultural management to reveal differences directly linked to the soil type and not influenced by other factors such as climate or cropping history. Bulk soil and rhizosphere samples were collected 3 and 7 weeks after planting. The analysis of 16S rRNA gene fragments amplified from total community DNA by denaturing gradient gel electrophoresis and pyrosequencing revealed soil type dependent differences in the bacterial community structure of the bulk soils and the corresponding rhizospheres. The rhizosphere effect differed depending on the soil type and the plant growth developmental stage. Despite the soil type dependent differences in the bacterial community composition several genera such as Sphingomonas, Rhizobium, Pseudomonas, and Variovorax were significantly increased in the rhizosphere of lettuce grown in all three soils. The number of rhizosphere responders was highest 3 weeks after planting. Interestingly, in the soil with the highest numbers of responders the highest shoot dry weights were observed. Heatmap analysis revealed that many dominant operational taxonomic units were shared among rhizosphere samples of lettuce grown in diluvial sand, alluvial loam, and loess loam and that only a subset was increased in relative abundance in the rhizosphere compared to the corresponding bulk soil. The findings of the study provide insights into the effect of soil types on the rhizosphere microbiome of lettuce. PMID:24782839

A microbial spoilage profile of half shell Pacific oysters (Crassostrea gigas) and Sydney rock oysters (Saccostrea glomerata).

PubMed

Madigan, Thomas L; Bott, Nathan J; Torok, Valeria A; Percy, Nigel J; Carragher, John F; de Barros Lopes, Miguel A; Kiermeier, Andreas

2014-04-01

This study aimed to assess bacterial spoilage of half shell Pacific and Sydney rock oysters during storage using microbial culture and 16S rRNA pyrosequencing. Odour and pH of oyster meats were also investigated. Estimation of microbiological counts by microbial culture highlighted growth of psychrotrophic bacteria. During storage, odour scores (a score describing deterioration of fresh odours where a score of 1 is fresh and 4 is completely spoiled) increased from 1.0 to 3.0 for Pacific oysters and from 1.3 to 3.4 for Sydney rock oysters. pH results obtained for both species fluctuated during storage (range 6.28-6.73) with an overall increase at end of storage. Pyrosequencing revealed that the majority of bacteria at Day 0 represented taxa from amongst the Proteobacteria, Tenericutes and Spirochaetes that have not been cultured and systematically described. During storage, Proteobacteria became abundant with Pseudoalteromonas and Vibrio found to be dominant in both oyster species at Day 7. Analysis of the pyrosequencing data showed significant differences in bacterial profiles between oyster species and storage time (both P = 0.001). As oysters spoiled, bacterial profiles between oyster species became more similar indicating a common spoilage profile. Data presented here provides detailed insight into the changing bacterial profile of shucked oysters during storage and has identified two genera, Pseudoalteromonas and Vibrio, as being important in spoilage of shucked oysters. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

PubMed

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing

PubMed Central

Wang, Wei; Wang, Yejun; Zhang, Qing; Qi, Yan; Guo, Dianjing

2009-01-01

Background Glandular trichomes produce a wide variety of commercially important secondary metabolites in many plant species. The most prominent anti-malarial drug artemisinin, a sesquiterpene lactone, is produced in glandular trichomes of Artemisia annua. However, only limited genomic information is currently available in this non-model plant species. Results We present a global characterization of A. annua glandular trichome transcriptome using 454 pyrosequencing. Sequencing runs using two normalized cDNA collections from glandular trichomes yielded 406,044 expressed sequence tags (average length = 210 nucleotides), which assembled into 42,678 contigs and 147,699 singletons. Performing a second sequencing run only increased the number of genes identified by ~30%, indicating that massively parallel pyrosequencing provides deep coverage of the A. annua trichome transcriptome. By BLAST search against the NCBI non-redundant protein database, putative functions were assigned to over 28,573 unigenes, including previously undescribed enzymes likely involved in sesquiterpene biosynthesis. Comparison with ESTs derived from trichome collections of other plant species revealed expressed genes in common functional categories across different plant species. RT-PCR analysis confirmed the expression of selected unigenes and novel transcripts in A. annua glandular trichomes. Conclusion The presence of contigs corresponding to enzymes for terpenoids and flavonoids biosynthesis suggests important metabolic activity in A. annua glandular trichomes. Our comprehensive survey of genes expressed in glandular trichome will facilitate new gene discovery and shed light on the regulatory mechanism of artemisinin metabolism and trichome function in A. annua. PMID:19818120

Pyrosequencing for Microbial Identification and Characterization

PubMed Central

Cummings, Patrick J.; Ahmed, Ray; Durocher, Jeffrey A.; Jessen, Adam; Vardi, Tamar; Obom, Kristina M.

2013-01-01

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns. PMID:23995536

Pyrosequencing for microbial identification and characterization.

PubMed

Cummings, Patrick J; Ahmed, Ray; Durocher, Jeffrey A; Jessen, Adam; Vardi, Tamar; Obom, Kristina M

2013-08-22

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones ▿

PubMed Central

Bravo, Lulette Tricia C.; Tuohy, Marion J.; Ang, Concepcion; Destura, Raul V.; Mendoza, Myrna; Procop, Gary W.; Gordon, Steven M.; Hall, Geraldine S.; Shrestha, Nabin K.

2009-01-01

After isoniazid and rifampin (rifampicin), the next pivotal drug class in Mycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the rpoB, katG, and gyrA genes occur with frequencies of 97%, 50%, and 85% among M. tuberculosis isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine M. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 M. tuberculosis clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the rpoB, katG, and gyrA genes, and pyrosequencing was performed on the extracts. The M. tuberculosis H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting M. tuberculosis resistance to these three drugs. PMID:19846642

Fungal Diversity Is Not Determined by Mineral and Chemical Differences in Serpentine Substrates

PubMed Central

Daghino, Stefania; Murat, Claude; Sizzano, Elisa; Girlanda, Mariangela; Perotto, Silvia

2012-01-01

The physico-chemical properties of serpentine soils lead to strong selection of plant species. Whereas many studies have described the serpentine flora, little information is available on the fungal communities dwelling in these sites. Asbestos minerals, often associated with serpentine rocks, can be weathered by serpentine-isolated fungi, suggesting an adaptation to this substrate. In this study, we have investigated whether serpentine substrates characterized by the presence of rocks with distinct mineral composition could select for different fungal communities. Both fungal isolation and 454 pyrosequencing of amplicons obtained from serpentine samples following direct DNA extraction revealed some fungal taxa shared by the four ophiolitic substrates, but also highlighted several substrate-specific taxa. Bootstrap analysis of 454 OTU abundances indicated weak clustering of fungal assemblages from the different substrates, which did not match substrate classification based on exchangeable macronutrients and metals. Intra-substrate variability, as assessed by DGGE profiles, was similar across the four serpentine substrates, and comparable to inter-substrate variability. These findings indicate the absence of a correlation between the substrate (mineral composition and available cations) and the diversity of the fungal community. Comparison of culture-based and culture-independent methods supports the higher taxonomic precision of the former, as complementation of the better performance of the latter. PMID:23028507

Detection of polyoma virus in brain tissue of patients with progressive multifocal leukoencephalopathy by real-time PCR and pyrosequencing.

PubMed

Beck, Rose C; Kohn, Debra J; Tuohy, Marion J; Prayson, Richard A; Yen-Lieberman, Belinda; Procop, Gary W

2004-03-01

We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism

PubMed Central

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-01-01

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds. PMID:26585833

Position-specific automated processing of V3 env ultra-deep pyrosequencing data for predicting HIV-1 tropism.

PubMed

Jeanne, Nicolas; Saliou, Adrien; Carcenac, Romain; Lefebvre, Caroline; Dubois, Martine; Cazabat, Michelle; Nicot, Florence; Loiseau, Claire; Raymond, Stéphanie; Izopet, Jacques; Delobel, Pierre

2015-11-20

HIV-1 coreceptor usage must be accurately determined before starting CCR5 antagonist-based treatment as the presence of undetected minor CXCR4-using variants can cause subsequent virological failure. Ultra-deep pyrosequencing of HIV-1 V3 env allows to detect low levels of CXCR4-using variants that current genotypic approaches miss. However, the computation of the mass of sequence data and the need to identify true minor variants while excluding artifactual sequences generated during amplification and ultra-deep pyrosequencing is rate-limiting. Arbitrary fixed cut-offs below which minor variants are discarded are currently used but the errors generated during ultra-deep pyrosequencing are sequence-dependant rather than random. We have developed an automated processing of HIV-1 V3 env ultra-deep pyrosequencing data that uses biological filters to discard artifactual or non-functional V3 sequences followed by statistical filters to determine position-specific sensitivity thresholds, rather than arbitrary fixed cut-offs. It allows to retain authentic sequences with point mutations at V3 positions of interest and discard artifactual ones with accurate sensitivity thresholds.

Bacterial and diazotrophic diversities of endophytes in Dendrobium catenatum determined through barcoded pyrosequencing

PubMed Central

Li, Ou; Sun, Lihua; Guan, Chenglin; Kong, Dedong

2017-01-01

As an epiphyte orchid, Dendrobium catenatum relies on microorganisms for requisite nutrients. Metagenome pyrosequencing based on 16S rRNA and nifH genes was used to characterize the bacterial and diazotrophic communities associated with D. catenatum collected from 5 districts in China. Based on Meta-16S rRNA sequencing, 22 bacterial phyla and 699 genera were identified, distributed as 125 genera from 8 phyla and 319 genera from 10 phyla shared by all the planting bases and all the tissues, respectively. The predominant Proteobacteria varied from 71.81% (GZ) to 96.08% (YN), and Delftia (10.39–38.42%), Burkholderia (2.71–15.98%), Escherichia/Shigella (4.90–25.12%), Pseudomonas (2.68–30.72%) and Sphingomonas (1.83–2.05%) dominated in four planting bases. Pseudomonas (17.94–22.06%), Escherichia/Shigella (6.59–11.59%), Delftia (9.65–22.14%) and Burkholderia (3.12–11.05%) dominated in all the tissues. According to Meta-nifH sequencing, 4 phyla and 45 genera were identified, while 17 genera and 24 genera from 4 phyla were shared by all the planting bases and all the tissues, respectively. Burkholderia and Bradyrhizobium were the most popular in the planting bases, followed by Methylovirgula and Mesorhizobium. Mesorhizobium was the most popular in different tissues, followed by Beijerinckia, Xanthobacter, and Burkholderia. Among the genera, 39 were completely overlapped with the results based on the 16S rRNA gene. In conclusion, abundant bacteria and diazotrophs were identified in common in different tissues of D. catenatum from five planting bases, which might play a great role in the supply of nutrients such as nitrogen. The exact abundance of phylum and genus on the different tissues from different planting bases need deeper sequencing with more samples. PMID:28931073

Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians.

PubMed

Erwin, Patrick M; Pineda, Mari Carmen; Webster, Nicole; Turon, Xavier; López-Legentil, Susanna

2014-03-01

Ascidians are ecologically important components of marine ecosystems yet the ascidian microbiota remains largely unexplored beyond a few model species. We used 16S rRNA gene tag pyrosequencing to provide a comprehensive characterization of microbial symbionts in the tunic of 42 Great Barrier Reef ascidian samples representing 25 species. Results revealed high bacterial biodiversity (3 217 unique operational taxonomic units (OTU0.03) from 19 described and 14 candidate phyla) and the widespread occurrence of ammonia-oxidizing Thaumarchaeota in coral reef ascidians (24 of 25 host species). The ascidian microbiota was clearly differentiated from seawater microbial communities and included symbiont lineages shared with other invertebrate hosts as well as unique, ascidian-specific phylotypes. Several rare seawater microbes were markedly enriched (200-700 fold) in the ascidian tunic, suggesting that the rare biosphere of seawater may act as a conduit for horizontal symbiont transfer. However, most OTUs (71%) were rare and specific to single hosts and a significant correlation between host relatedness and symbiont community similarity was detected, indicating a high degree of host-specificity and potential role of vertical transmission in structuring these communities. We hypothesize that the complex ascidian microbiota revealed herein is maintained by the dynamic microenvironments within the ascidian tunic, offering optimal conditions for different metabolic pathways such as ample chemical substrate (ammonia-rich host waste) and physical habitat (high oxygen, low irradiance) for nitrification. Thus, ascidian hosts provide unique and fertile niches for diverse microorganisms and may represent an important and previously unrecognized habitat for nitrite/nitrate regeneration in coral reef ecosystems.

Effect of Increasing Total Solids Contents on Anaerobic Digestion of Food Waste under Mesophilic Conditions: Performance and Microbial Characteristics Analysis

PubMed Central

Jin, Jingwei; Dai, Xiaohu

2014-01-01

The total solids content of feedstocks affects the performances of anaerobic digestion and the change of total solids content will lead the change of microbial morphology in systems. In order to increase the efficiency of anaerobic digestion, it is necessary to understand the role of the total solids content on the behavior of the microbial communities involved in anaerobic digestion of organic matter from wet to dry technology. The performances of mesophilic anaerobic digestion of food waste with different total solids contents from 5% to 20% were compared and the microbial communities in reactors were investigated using 454 pyrosequencing technology. Three stable anaerobic digestion processes were achieved for food waste biodegradation and methane generation. Better performances mainly including volatile solids reduction and methane yield were obtained in the reactors with higher total solids content. Pyrosequencing results revealed significant shifts in bacterial community with increasing total solids contents. The proportion of phylum Chloroflexi decreased obviously with increasing total solids contents while other functional bacteria showed increasing trend. Methanosarcina absolutely dominated in archaeal communities in three reactors and the relative abundance of this group showed increasing trend with increasing total solids contents. These results revealed the effects of the total solids content on the performance parameters and the behavior of the microbial communities involved in the anaerobic digestion of food waste from wet to dry technologies. PMID:25051352

Effect of increasing total solids contents on anaerobic digestion of food waste under mesophilic conditions: performance and microbial characteristics analysis.

PubMed

Yi, Jing; Dong, Bin; Jin, Jingwei; Dai, Xiaohu

2014-01-01

The total solids content of feedstocks affects the performances of anaerobic digestion and the change of total solids content will lead the change of microbial morphology in systems. In order to increase the efficiency of anaerobic digestion, it is necessary to understand the role of the total solids content on the behavior of the microbial communities involved in anaerobic digestion of organic matter from wet to dry technology. The performances of mesophilic anaerobic digestion of food waste with different total solids contents from 5% to 20% were compared and the microbial communities in reactors were investigated using 454 pyrosequencing technology. Three stable anaerobic digestion processes were achieved for food waste biodegradation and methane generation. Better performances mainly including volatile solids reduction and methane yield were obtained in the reactors with higher total solids content. Pyrosequencing results revealed significant shifts in bacterial community with increasing total solids contents. The proportion of phylum Chloroflexi decreased obviously with increasing total solids contents while other functional bacteria showed increasing trend. Methanosarcina absolutely dominated in archaeal communities in three reactors and the relative abundance of this group showed increasing trend with increasing total solids contents. These results revealed the effects of the total solids content on the performance parameters and the behavior of the microbial communities involved in the anaerobic digestion of food waste from wet to dry technologies.

Time-Resolved DNA Stable Isotope Probing Links Desulfobacterales- and Coriobacteriaceae-Related Bacteria to Anaerobic Degradation of Benzene under Methanogenic Conditions

PubMed Central

Noguchi, Mana; Kurisu, Futoshi; Kasuga, Ikuro; Furumai, Hiroaki

2014-01-01

To identify the microorganisms involved in benzene degradation, DNA-stable isotope probing (SIP) with 13C-benzene was applied to a methanogenic benzene-degrading enrichment culture. Pyrosequencing of ribosomal RNA (rRNA) gene sequences revealed that the community structure was highly complex in spite of a 3-year incubation only with benzene. The culture degraded 98% of approximately 1 mM 13C-benzene and mineralized 72% of that within 63 d. The terminal restriction fragment length polymorphism (T-RFLP) profiles of the buoyant density fractions revealed the incorporation of 13C into two phylotypes after 64 d. These two phylotypes were determined to be Desulfobacterales- and Coriobacteriaceae-related bacteria by cloning and sequencing of the 16S rRNA gene in the 13C-labeled DNA abundant fraction. Comparative pyrosequencing analysis of the buoyant density fractions of 12C- and 13C-labeled samples indicated the incorporation of 13C into three bacterial and one archaeal OTUs related to Desulfobacterales, Coriobacteriales, Rhodocyclaceae, and Methanosarcinales. The first two OTUs included the bacteria detected by T-RFLP-cloning-sequencing analysis. Furthermore, time-resolved SIP analysis confirmed that the activity of all these microbes appeared at the earliest stage of degradation. In this methanogenic culture, Desulfobacterales- and Coriobacteriaceae-related bacteria were most likely to be the major benzene degraders. PMID:24909708

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing.

PubMed

Garofalo, Cristiana; Osimani, Andrea; Milanović, Vesna; Taccari, Manuela; Cardinali, Federica; Aquilanti, Lucia; Riolo, Paola; Ruschioni, Sara; Isidoro, Nunzio; Clementi, Francesca

2017-04-01

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of pyrosequencing to characterize the microbiota in the ileum of goats fed with increasing proportion of dietary grain.

PubMed

Mao, Shengyong; Huo, Wenjie; Zhu, Weiyun

2013-09-01

This study evaluated the effects of an increasing proportion of dietary grain on changes in bacterial populations in the goat ileum. Nine ruminally fistulated, castrated male goats were assigned to three diets in a completely randomized design. Goats were fed three different dietary treatments containing different proportions of corn grain (0, 25, and 50 %). The pH of the ileal contents and rumen fluid (P = 0.015) linearly decreased (P < 0.001), and the acetate, propionate, butyrate, and total volatile fatty acid in ileal contents increased (P < 0.05) with increases in dietary corn, and similar results were also observed in rumen fluid. The barcoded DNA pyrosequencing method was used to reveal 8 phyla, 70 genera, and 1,693 16S operational taxonomic units (OTUs). At the genus level, the proportions of Acetitomaculum, Enterococcus, Atopobium, unclassified Coriobacteriaceae, and unclassified Planctomycetaceae were linearly decreased (P < 0.05) with increases in corn grain. At the species level, high grain feeding linearly decreased the percentage of OTU8686 (unclassified Bacteria) (P = 0.004). To the best of our knowledge, this is the first study using barcoded DNA pyrosequencing method to survey the ileal microbiome of goats and the results suggest that increasing levels of dietary corn change the composition of the ileal bacterial community. These findings provide previously unknown information about the ileal microbiota of goats and a new understanding of the ileal microbial ecology, which may be useful in modulating the gut microbiome.

Pyrosequencing for detection of lamivudine-resistant hepatitis B virus.

PubMed

Lindström, Anna; Odeberg, Jacob; Albert, Jan

2004-10-01

Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

PubMed Central

Migheli, Francesca; Stoccoro, Andrea; Coppedè, Fabio; Wan Omar, Wan Adnan; Failli, Alessandra; Consolini, Rita; Seccia, Massimo; Spisni, Roberto; Miccoli, Paolo; Mathers, John C.; Migliore, Lucia

2013-01-01

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing. PMID:23326336

Isolation and metagenomic characterization of bacteria associated with calcium carbonate and struvite precipitation in a pure moving bed biofilm reactor-membrane bioreactor.

PubMed

Gonzalez-Martinez, A; Leyva-Díaz, J C; Rodriguez-Sanchez, A; Muñoz-Palazon, B; Rivadeneyra, A; Poyatos, J M; Rivadeneyra, M A; Martinez-Toledo, M V

2015-01-01

A bench-scale pure moving bed bioreactor-membrane bioreactor (MBBR-MBR) used for the treatment of urban wastewater was analyzed for the identification of bacterial strains with the potential capacity for calcium carbonate and struvite biomineral formation. Isolation of mineral-forming strains on calcium carbonate and struvite media revealed six major colonies with a carbonate or struvite precipitation capacity in the biofouling on the membrane surface and showed that heterotrophic bacteria with the ability to precipitate calcium carbonate and struvite constituted ~7.5% of the total platable bacteria. These belonged to the genera Lysinibacillus, Trichococcus, Comamomas and Bacillus. Pyrosequencing analysis of the microbial communities in the suspended cells and membrane biofouling showed a high degree of similarity in all the samples collected with respect to bacterial assemblage. The study of operational taxonomic units (OTUs) identified through pyrosequencing suggested that ~21% of the total bacterial community identified in the biofouling could potentially form calcium carbonate or struvite crystals in the pure MBBR-MBR system used for the treatment of urban wastewater.

Pyrosequencing reveals bacterial community differences in composting and vermicomposting on the stabilization of mixed sewage sludge and cattle dung.

PubMed

Lv, Baoyi; Xing, Meiyan; Yang, Jian; Zhang, Liangbo

2015-12-01

This study aimed to compare the microbial community structures and compositions in composting and vermicomposting processes. We applied 454 high-throughput pyrosequencing to analyze the 16S rRNA gene of bacteria obtained from bio-stabilization of sewage sludge and cattle dung. Results demonstrated that vermicomposting process presented higher operational taxonomic units and bacterial diversity than the composting. Analysis using weighted UniFrac indicated that composting exhibited higher effects on shaping microbial community structure than the vermicomposting. The succession of dominant bacteria was also detected during composting. Firmicutes was the dominant bacteria in the thermophilic phase of composting and shifted to Actinomycetes in the maturing stage. By contrast, Proteobacteria accounted for the highest proportions in the whole process of the vermicomposting. Furthermore, vermicomposting contained more uncultured and unidentified bacteria at the taxonomy level of genus than the composting. In summary, the bacterial community during composting significantly differed from that during vermicomposting. These two techniques played different roles in changing the diversity and composition of microbial communities.

Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts.

PubMed

Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W; Eyun, Seong-Il; Noriega, Daniel D; Siegfried, Blair

2016-01-01

The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest.

Pyrosequencing the Midgut Transcriptome of the Banana Weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) Reveals Multiple Protease-Like Transcripts

PubMed Central

Valencia, Arnubio; Wang, Haichuan; Soto, Alberto; Aristizabal, Manuel; Arboleda, Jorge W.; Eyun, Seong-il; Noriega, Daniel D.; Siegfried, Blair

2016-01-01

The banana weevil Cosmopolites sordidus is an important and serious insect pest in most banana and plantain-growing areas of the world. In spite of the economic importance of this insect pest very little genomic and transcriptomic information exists for this species. In the present study, we characterized the midgut transcriptome of C. sordidus using massive 454-pyrosequencing. We generated over 590,000 sequencing reads that assembled into 30,840 contigs with more than 400 bp, representing a significant expansion of existing sequences available for this insect pest. Among them, 16,427 contigs contained one or more GO terms. In addition, 15,263 contigs were assigned an EC number. In-depth transcriptome analysis identified genes potentially involved in insecticide resistance, peritrophic membrane biosynthesis, immunity-related function and defense against pathogens, and Bacillus thuringiensis toxins binding proteins as well as multiple enzymes involved with protein digestion. This transcriptome will provide a valuable resource for understanding larval physiology and for identifying novel target sites and management approaches for this important insect pest. PMID:26949943

High-throughput pyrosequencing used for the discovery of a novel cellulase from a thermophilic cellulose-degrading microbial consortium.

PubMed

Zhao, Chao; Chu, Yanan; Li, Yanhong; Yang, Chengfeng; Chen, Yuqing; Wang, Xumin; Liu, Bin

2017-01-01

To analyze the microbial diversity and gene content of a thermophilic cellulose-degrading consortium from hot springs in Xiamen, China using 454 pyrosequencing for discovering cellulolytic enzyme resources. A thermophilic cellulose-degrading consortium, XM70 that was isolated from a hot spring, used sugarcane bagasse as sole carbon and energy source. DNA sequencing of the XM70 sample resulted in 349,978 reads with an average read length of 380 bases, accounting for 133,896,867 bases of sequence information. The characterization of sequencing reads and assembled contigs revealed that most microbes were derived from four phyla: Geobacillus (Firmicutes), Thermus, Bacillus, and Anoxybacillus. Twenty-eight homologous genes belonging to 15 glycoside hydrolase families were detected, including several cellulase genes. A novel hot spring metagenome-derived thermophilic cellulase was expressed and characterized. The application value of thermostable sugarcane bagasse-degrading enzymes is shown for production of cellulosic biofuel. The practical power of using a short-read-based metagenomic approach for harvesting novel microbial genes is also demonstrated.

Pyrosequencing Analysis of Bench-Scale Nitrifying BiofiltersRemoving Trihalomethanes

EPA Science Inventory

The bacterial biofilm communities in four nitrifying biofilters degrading regulated drinking water trihalomethanes were characterized by 454 pyrosequencing. The three most abundant phylotypes based on total diversity were Nitrosomonas (70%), Nitrobacter (14%), and Chitinophagace...

Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing technology.

PubMed

Koontz, Deborah A; Huckins, Jacqueline J; Spencer, Antonina; Gallagher, Margaret L

2009-08-24

Identification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1-2 are derived from CYP2A7, and exons 3-9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology. A single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow. Pyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics. This Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.

Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

PubMed

Grasso, Chiara; Trevisan, Morena; Fiano, Valentina; Tarallo, Valentina; De Marco, Laura; Sacerdote, Carlotta; Richiardi, Lorenzo; Merletti, Franco; Gillio-Tos, Anna

2016-01-01

Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

Multiplex pyrosequencing method to determine CYP2C9*3, VKORC1*2, and CYP4F2*3 polymorphisms simultaneously: its application to a Korean population and comparisons with other ethnic groups.

PubMed

Kim, Kyoung-Ah; Song, Wan-Geun; Lee, Hae-Mi; Joo, Hyun-Jin; Park, Ji-Young

2014-11-01

Warfarin is an anticoagulant that is difficult to administer because of the wide variation in dose requirements to achieve a therapeutic effect. CYP2C9, VKROC1, and CYP4F2 play important roles in warfarin metabolism, and their genetic polymorphisms are related to the variability in dose determination. In this study we describe a new multiplex pyrosequencing method to identify CYP2C9*3 (rs1057910), VKORC1*2 (rs9923231), and CYP4F2*3 (rs2108661) simultaneously. A multiplex pyrosequencing method to simultaneously detect CYP2C9*3, VKORC1*2, and CYP4F2*3 alleles was designed. We assessed the allele frequencies of the polymorphisms in 250 Korean subjects using the multiplex pyrosequencing method. The results showed 100 % concordance between single and multiplex pyrosequencing methods, and the polymorphisms identified by pyrosequencing were also validated with the direct sequencing method. The allele frequencies of these polymorphisms in this population were as follows: 0.040 for CYP2C9*3, 0.918 for VKORC1*2, and 0.416 for CYP4F2*3. Although the allele frequencies of the CYP2C9*3 and VKROC1*2 were comparable to those in Japanese and Chinese populations, their frequencies in this Korean population differed from those in other ethnic groups; the CYP4F2*3 frequency was the highest among other ethnic populations including Chinese and Japanese populations. The pyrosequencing methods developed were rapid and reliable for detecting CYP2C9*3, VKORC1*2, and CYP4F2*3. Large ethnic differences in the frequency of these genetic polymorphisms were noted among ethnic groups. CYP4F2*3 exhibited its highest allele frequency among other ethnic populations compared to that in a Korean population.

Involvement of shared mucosal-associated microbiota in the duodenum and rectum in diarrhea-predominant irritable bowel syndrome.

PubMed

Li, Gangping; Yang, Min; Jin, Yu; Li, Ying; Qian, Wei; Xiong, Hanhua; Song, Jun; Hou, Xiaohua

2018-06-01

Most studies of diarrhea-predominant irritable bowel syndrome (IBS-D) focused on microbiota dysbiosis in a single segment of the intestine such as the colon. However, the intestine as a whole is involved in IBS-D and knowledge about the role of microbiota shared by the duodenum and rectum in IBS-D is limited. Here, we investigated the characteristics of mucosal microbiota shared by the duodenum and rectum in IBS-D patients. We collected duodenal and rectal mucosal samples from 33 adult IBS-D patients and 15 healthy control (HC) subjects. The 454 pyrosequencing method and multiple bioinformatics analyses were used to examine bacterial 16S rRNA. Clinical data including symptoms and Bristol Stool Form were analyzed. Mucosal microbiota in duodenal samples differed from rectal samples in HC, while less difference was shown in IBS-D. More numbers in terms of shared operational taxonomic units and genera found in IBS-D compared with HC. The frequency of genera in the duodenum and rectum of HC differed from that of IBS-D. We identified 24 genera shared in the duodenum and rectum, which both changed dramatically in IBS-D. Among these 24 genera, half had similar trends in frequency differences, and the other half had opposite trends. The frequency of Faecalibacterium and Hyphomicrobium were associated with clinical data of IBS-D patients. Shared mucosal-associated microbiota in the duodenum and rectum appear to contribute to the etiology and pathophysiology of whole intestine of IBS-D and to be potential therapeutic targets. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

PubMed

Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

2015-02-01

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Bacterial networks and co-occurrence relationships in the lettuce root microbiota.

PubMed

Cardinale, Massimiliano; Grube, Martin; Erlacher, Armin; Quehenberger, Julian; Berg, Gabriele

2015-01-01

Lettuce is one of the most common raw foods worldwide, but occasionally also involved in pathogen outbreaks. To understand the correlative structure of the bacterial community as a network, we studied root microbiota of eight ancient and modern Lactuca sativa cultivars and the wild ancestor Lactuca serriola by pyrosequencing of 16S rRNA gene amplicon libraries. The lettuce microbiota was dominated by Proteobacteria and Bacteriodetes, as well as abundant Chloroflexi and Actinobacteria. Cultivar specificity comprised 12.5% of the species. Diversity indices were not different between lettuce cultivar groups but higher than in L. serriola, suggesting that domestication lead to bacterial diversification in lettuce root system. Spearman correlations between operational taxonomic units (OTUs) showed that co-occurrence prevailed over co-exclusion, and complementary fluorescence in situ hybridization-confocal laser scanning microscopy (FISH-CLSM) analyses revealed that this pattern results from both potential interactions and habitat sharing. Predominant taxa, such as Pseudomonas, Flavobacterium and Sphingomonadaceae rather suggested interactions, even though these are not necessarily part of significant modules in the co-occurrence networks. Without any need for complex interactions, single organisms are able to invade into this microbial network and to colonize lettuce plants, a fact that can influence the susceptibility to pathogens. The approach to combine co-occurrence analysis and FISH-CLSM allows reliably reconstructing and interpreting microbial interaction networks. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

PubMed Central

Gulino, Lisa-Maree; Ouwerkerk, Diane; Kang, Alicia Y. H.; Maguire, Anita J.; Kienzle, Marco; Klieve, Athol V.

2013-01-01

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as ‘shared’ OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry. PMID:23626688

Highly heterogeneous bacterial communities associated with the South China Sea reef corals Porites lutea, Galaxea fascicularis and Acropora millepora.

PubMed

Li, Jie; Chen, Qi; Zhang, Si; Huang, Hui; Yang, Jian; Tian, Xin-Peng; Long, Li-Juan

2013-01-01

Coral harbor diverse and specific bacteria play significant roles in coral holobiont function. Bacteria associated with three of the common and phylogenetically divergent reef-building corals in the South China Sea, Porites lutea, Galaxea fascicularis and Acropora millepora, were investigated using 454 barcoded-pyrosequencing. Three colonies of each species were sampled, and 16S rRNA gene libraries were constructed individually. Analysis of pyrosequencing libraries showed that bacterial communities associated with the three coral species were more diverse than previous estimates based on corals from the Caribbean Sea, Indo-Pacific reefs and the Red Sea. Three candidate phyla, including BRC1, OD1 and SR1, were found for the first time in corals. Bacterial communities were separated into three groups: P. lutea and G. fascicular, A. millepora and seawater. P. lutea and G. fascicular displayed more similar bacterial communities, and bacterial communities associated with A. millepora differed from the other two coral species. The three coral species shared only 22 OTUs, which were distributed in Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, Chloroflexi, Actinobacteria, Acidobacteria and an unclassified bacterial group. The composition of bacterial communities within each colony of each coral species also showed variation. The relatively small common and large specific bacterial communities in these corals implies that bacterial associations may be structured by multiple factors at different scales and that corals may associate with microbes in terms of similar function, rather than identical species.

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage

PubMed Central

Ferrocino, Ilario; Greppi, Anna; La Storia, Antonietta; Rantsiou, Kalliopi; Ercolini, Danilo

2015-01-01

Beef burgers were stored at 4°C in a vacuum in nisin-activated antimicrobial packaging. Microbial ecology analyses were performed on samples collected between days 0 and 21 of storage to discover the population diversity. Two batches were analyzed using RNA-based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The active packaging retarded the growth of the total viable bacteria and lactic acid bacteria. Culture-independent analysis by pyrosequencing of RNA extracted directly from meat showed that Photobacterium phosphoreum, Lactococcus piscium, Lactobacillus sakei, and Leuconostoc carnosum were the major operational taxonomic units (OTUs) shared between control and treated samples. Beta diversity analysis of the 16S rRNA sequence data and RNA-DGGE showed a clear separation between two batches based on the microbiota. Control samples from batch B showed a significant high abundance of some taxa sensitive to nisin, such as Kocuria rhizophila, Staphylococcus xylosus, Leuconostoc carnosum, and Carnobacterium divergens, compared to control samples from batch A. However, only from batch B was it possible to find a significant difference between controls and treated samples during storage due to the active packaging. Predicted metagenomes confirmed differences between the two batches and indicated that the use of nisin-based antimicrobial packaging can determine a reduction in the abundance of specific metabolic pathways related to spoilage. The present study aimed to assess the viable bacterial communities in beef burgers stored in nisin-based antimicrobial packaging, and it highlights the efficacy of this strategy to prolong beef burger shelf life. PMID:26546424

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottesen, Elizabeth A.; Marin, Roman; Preston, Christina M.

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically undersampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at roommore » temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.« less

16S rRNA Gene Pyrosequencing Reveals Bacterial Dysbiosis in the Duodenum of Dogs with Idiopathic Inflammatory Bowel Disease

PubMed Central

Suchodolski, Jan S.; Dowd, Scot E.; Wilke, Vicky; Steiner, Jörg M.; Jergens, Albert E.

2012-01-01

Background Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Methodology/Principal Findings Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs. Conclusions/Significance In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation. PMID:22720094

16S rRNA gene pyrosequencing reveals bacterial dysbiosis in the duodenum of dogs with idiopathic inflammatory bowel disease.

PubMed

Suchodolski, Jan S; Dowd, Scot E; Wilke, Vicky; Steiner, Jörg M; Jergens, Albert E

2012-01-01

Canine idiopathic inflammatory bowel disease (IBD) is believed to be caused by a complex interaction of genetic, immunologic, and microbial factors. While mucosa-associated bacteria have been implicated in the pathogenesis of canine IBD, detailed studies investigating the enteric microbiota using deep sequencing techniques are lacking. The objective of this study was to evaluate mucosa-adherent microbiota in the duodenum of dogs with spontaneous idiopathic IBD using 16 S rRNA gene pyrosequencing. Biopsy samples of small intestinal mucosa were collected endoscopically from healthy dogs (n = 6) and dogs with moderate IBD (n = 7) or severe IBD (n = 7) as assessed by a clinical disease activity index. Total RNA was extracted from biopsy specimens and 454-pyrosequencing of the 16 S rRNA gene was performed on aliquots of cDNA from each dog. Intestinal inflammation was associated with significant differences in the composition of the intestinal microbiota when compared to healthy dogs. PCoA plots based on the unweighted UniFrac distance metric indicated clustering of samples between healthy dogs and dogs with IBD (ANOSIM, p<0.001). Proportions of Fusobacteria (p = 0.010), Bacteroidaceae (p = 0.015), Prevotellaceae (p = 0.022), and Clostridiales (p = 0.019) were significantly more abundant in healthy dogs. In contrast, specific bacterial genera within Proteobacteria, including Diaphorobacter (p = 0.044) and Acinetobacter (p = 0.040), were either more abundant or more frequently identified in IBD dogs. In conclusion, dogs with spontaneous IBD exhibit alterations in microbial groups, which bear resemblance to dysbiosis reported in humans with chronic intestinal inflammation. These bacterial groups may serve as useful targets for monitoring intestinal inflammation.

Pyrosequencing-Derived Bacterial, Archaeal, and Fungal Diversity of Spacecraft Hardware Destined for Mars

PubMed Central

Vaishampayan, Parag; Nilsson, Henrik R.; Torok, Tamas; Venkateswaran, Kasthuri

2012-01-01

Spacecraft hardware and assembly cleanroom surfaces (233 m2 in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m2) than colocated spacecraft hardware (187 OTU; 162 m2). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space. PMID:22729532

Allelic differences within and among sister spores of the arbuscular mycorrhizal fungus Glomus etunicatum suggest segregation at sporulation.

PubMed

Boon, Eva; Zimmerman, Erin; St-Arnaud, Marc; Hijri, Mohamed

2013-01-01

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF are made up of coenocytic networks of hyphae through which nuclei and organelles can freely migrate. In this study, we investigated the possibility of a genetic bottleneck and segregation of allelic variation at sporulation for a low-copy Polymerase1-like gene, PLS. Specifically, our objectives were (1) to estimate what allelic diversity is passed on to a single spore (2) to determine whether this diversity is less than the total amount of variation found in all spores (3) to investigate whether there is any differential segregation of allelic variation. We inoculated three tomato plants with a single spore of Glomus etunicatum each and after six months sampled between two and three daughter spores per tomato plant. Pyrosequencing PLS amplicons in eight spores revealed high levels of allelic diversity; between 43 and 152 alleles per spore. We corroborated the spore pyrosequencing results with Sanger- and pyrosequenced allele distributions from the original parent isolate. Both sequencing methods retrieved the most abundant alleles from the offspring spore allele distributions. Our results indicate that individual spores contain only a subset of the total allelic variation from the pooled spores and parent isolate. Patterns of allele diversity between spores suggest the possibility for segregation of PLS alleles among spores. We conclude that a genetic bottleneck could potentially occur during sporulation in AMF, with resulting differences in genetic variation among sister spores. We suggest that the effects of this bottleneck may be countered by anastomosis (hyphal fusion) between related hyphae.

Comparative Survey of Rumen Microbial Communities and Metabolites across One Caprine and Three Bovine Groups, Using Bar-Coded Pyrosequencing and 1H Nuclear Magnetic Resonance Spectroscopy

PubMed Central

Lee, Hyo Jung; Jung, Ji Young; Oh, Young Kyoon; Lee, Sang-Suk; Madsen, Eugene L.

2012-01-01

Pyrosequencing of 16S rRNA genes (targeting Bacteria and Archaea) and 1H nuclear magnetic resonance were applied to investigate the rumen microbiota and metabolites of Hanwoo steers in the growth stage (HGS), Hanwoo steers in the late fattening stage (HFS), Holstein-Friesian dairy cattle (HDC), and Korean native goats (KNG) in the late fattening stage. This was a two-part investigation. We began by comparing metabolites and microbiota of Hanwoo steers at two stages of husbandry. Statistical comparisons of metabolites and microbial communities showed no significant differences between HFS and HGS (differing by a dietary shift at 24 months and age [67 months versus 12 months]). We then augmented the study by extending the investigation to HDC and KNG. Overall, pyrosequencing of 16S rRNA genes showed that the rumens had highly diverse microbial communities containing many previously undescribed microorganisms. Bioinformatic analysis revealed that the bacterial sequences were predominantly affiliated with four phyla—Bacteroidetes, Firmicutes, Fibrobacteres, and Proteobacteria—in all ruminants. However, interestingly, the bacterial reads belonging to Fibrobacteres were present at a very low abundance (<0.1%) in KNG. Archaeal community analysis showed that almost all of these reads fell into a clade related to, but distinct from, known cultivated methanogens. Statistical analyses showed that the microbial communities and metabolites of KNG were clearly distinct from those of other ruminants. In addition, bacterial communities and metabolite profiles of HGS and HDC, fed similar diets, were distinctive. Our data indicate that bovine host breeds override diet as the key factor that determines bacterial community and metabolite profiles in the rumen. PMID:22706048

Allelic Differences within and among Sister Spores of the Arbuscular Mycorrhizal Fungus Glomus etunicatum Suggest Segregation at Sporulation

PubMed Central

St-Arnaud, Marc; Hijri, Mohamed

2013-01-01

Arbuscular mycorrhizal fungi (AMF) are root-inhabiting fungi that form mutualistic symbioses with their host plants. AMF are made up of coenocytic networks of hyphae through which nuclei and organelles can freely migrate. In this study, we investigated the possibility of a genetic bottleneck and segregation of allelic variation at sporulation for a low-copy Polymerase1-like gene, PLS. Specifically, our objectives were (1) to estimate what allelic diversity is passed on to a single spore (2) to determine whether this diversity is less than the total amount of variation found in all spores (3) to investigate whether there is any differential segregation of allelic variation. We inoculated three tomato plants with a single spore of Glomus etunicatum each and after six months sampled between two and three daughter spores per tomato plant. Pyrosequencing PLS amplicons in eight spores revealed high levels of allelic diversity; between 43 and 152 alleles per spore. We corroborated the spore pyrosequencing results with Sanger- and pyrosequenced allele distributions from the original parent isolate. Both sequencing methods retrieved the most abundant alleles from the offspring spore allele distributions. Our results indicate that individual spores contain only a subset of the total allelic variation from the pooled spores and parent isolate. Patterns of allele diversity between spores suggest the possibility for segregation of PLS alleles among spores. We conclude that a genetic bottleneck could potentially occur during sporulation in AMF, with resulting differences in genetic variation among sister spores. We suggest that the effects of this bottleneck may be countered by anastomosis (hyphal fusion) between related hyphae. PMID:24386173

Abundance and Diversity of Bacterial Nitrifiers and Denitrifiers and Their Functional Genes in Tannery Wastewater Treatment Plants Revealed by High-Throughput Sequencing

PubMed Central

Wang, Zhu; Zhang, Xu-Xiang; Lu, Xin; Liu, Bo; Li, Yan; Long, Chao; Li, Aimin

2014-01-01

Biological nitrification/denitrification is frequently used to remove nitrogen from tannery wastewater containing high concentrations of ammonia. However, information is limited about the bacterial nitrifiers and denitrifiers and their functional genes in tannery wastewater treatment plants (WWTPs) due to the low-throughput of the previously used methods. In this study, 454 pyrosequencing and Illumina high-throughput sequencing, combined with molecular methods, were used to comprehensively characterize structures and functions of nitrification and denitrification bacterial communities in aerobic and anaerobic sludge of two full-scale tannery WWTPs. Pyrosequencing of 16S rRNA genes showed that Proteobacteria and Synergistetes dominated in the aerobic and anaerobic sludge, respectively. Ammonia-oxidizing bacteria (AOB) amoA gene cloning revealed that Nitrosomonas europaea dominated the ammonia-oxidizing community in the WWTPs. Metagenomic analysis showed that the denitrifiers mainly included the genera of Thauera, Paracoccus, Hyphomicrobium, Comamonas and Azoarcus, which may greatly contribute to the nitrogen removal in the two WWTPs. It is interesting that AOB and ammonia-oxidizing archaea had low abundance although both WWTPs demonstrated high ammonium removal efficiency. Good correlation between the qPCR and metagenomic analysis is observed for the quantification of functional genes amoA, nirK, nirS and nosZ, indicating that the metagenomic approach may be a promising method used to comprehensively investigate the abundance of functional genes of nitrifiers and denitrifiers in the environment. PMID:25420093

Mineral Type and Solution Chemistry Affect the Structure and Composition of Actively Growing Bacterial Communities as Revealed by Bromodeoxyuridine Immunocapture and 16S rRNA Pyrosequencing.

PubMed

Kelly, L C; Colin, Y; Turpault, M-P; Uroz, S

2016-08-01

Understanding how minerals affect bacterial communities and their in situ activities in relation to environmental conditions are central issues in soil microbial ecology, as minerals represent essential reservoirs of inorganic nutrients for the biosphere. To determine the impact of mineral type and solution chemistry on soil bacterial communities, we compared the diversity, composition, and functional abilities of a soil bacterial community incubated in presence/absence of different mineral types (apatite, biotite, obsidian). Microcosms were prepared containing different liquid culture media devoid of particular essential nutrients, the nutrients provided only in the introduced minerals and therefore only available to the microbial community through mineral dissolution by biotic and/or abiotic processes. By combining functional screening of bacterial isolates and community analysis by bromodeoxyuridine DNA immunocapture and 16S rRNA gene pyrosequencing, we demonstrated that bacterial communities were mainly impacted by the solution chemistry at the taxonomic level and by the mineral type at the functional level. Metabolically active bacterial communities varied with solution chemistry and mineral type. Burkholderia were significantly enriched in the obsidian treatment compared to the biotite treatment and were the most effective isolates at solubilizing phosphorous or mobilizing iron, in all the treatments. A detailed analysis revealed that the 16S rRNA gene sequences of the OTUs or isolated strains assigned as Burkholderia in our study showed high homology with effective mineral-weathering bacteria previously recovered from the same experimental site.

Different continuous cropping spans significantly affect microbial community membership and structure in a vanilla-grown soil as revealed by deep pyrosequencing.

PubMed

Xiong, Wu; Zhao, Qingyun; Zhao, Jun; Xun, Weibing; Li, Rong; Zhang, Ruifu; Wu, Huasong; Shen, Qirong

2015-07-01

In the present study, soil bacterial and fungal communities across vanilla continuous cropping time-series fields were assessed through deep pyrosequencing of 16S ribosomal RNA (rRNA) genes and internal transcribed spacer (ITS) regions. The results demonstrated that the long-term monoculture of vanilla significantly altered soil microbial communities. Soil fungal diversity index increased with consecutive cropping years, whereas soil bacterial diversity was relatively stable. Bray-Curtis dissimilarity cluster and UniFrac-weighted principal coordinate analysis (PCoA) revealed that monoculture time was the major determinant for fungal community structure, but not for bacterial community structure. The relative abundances (RAs) of the Firmicutes, Actinobacteria, Bacteroidetes, and Basidiomycota phyla were depleted along the years of vanilla monoculture. Pearson correlations at the phyla level demonstrated that Actinobacteria, Armatimonadetes, Bacteroidetes, Verrucomicrobia, and Firmicutes had significant negative correlations with vanilla disease index (DI), while no significant correlation for fungal phyla was observed. In addition, the amount of the pathogen Fusarium oxysporum accumulated with increasing years and was significantly positively correlated with vanilla DI. By contrast, the abundance of beneficial bacteria, including Bradyrhizobium and Bacillus, significantly decreased over time. In sum, soil weakness and vanilla stem wilt disease after long-term continuous cropping can be attributed to the alteration of the soil microbial community membership and structure, i.e., the reduction of the beneficial microbes and the accumulation of the fungal pathogen.

Amplicon-Based Pyrosequencing Reveals High Diversity of Protistan Parasites in Ships' Ballast Water: Implications for Biogeography and Infectious Diseases.

PubMed

Pagenkopp Lohan, K M; Fleischer, R C; Carney, K J; Holzer, K K; Ruiz, G M

2016-04-01

Ships' ballast water (BW) commonly moves macroorganisms and microorganisms across the world's oceans and along coasts; however, the majority of these microbial transfers have gone undetected. We applied high-throughput sequencing methods to identify microbial eukaryotes, specifically emphasizing the protistan parasites, in ships' BW collected from vessels calling to the Chesapeake Bay (Virginia and Maryland, USA) from European and Eastern Canadian ports. We utilized tagged-amplicon 454 pyrosequencing with two general primer sets, amplifying either the V4 or V9 domain of the small subunit (SSU) of the ribosomal RNA (rRNA) gene complex, from total DNA extracted from water samples collected from the ballast tanks of bulk cargo vessels. We detected a diverse group of protistan taxa, with some known to contain important parasites in marine systems, including Apicomplexa (unidentified apicomplexans, unidentified gregarines, Cryptosporidium spp.), Dinophyta (Blastodinium spp., Euduboscquella sp., unidentified syndinids, Karlodinium spp., Syndinium spp.), Perkinsea (Parvilucifera sp.), Opisthokonta (Ichthyosporea sp., Pseudoperkinsidae, unidentified ichthyosporeans), and Stramenopiles (Labyrinthulomycetes). Further characterization of groups with parasitic taxa, consisting of phylogenetic analyses for four taxa (Cryptosporidium spp., Parvilucifera spp., Labyrinthulomycetes, and Ichthyosporea), revealed that sequences were obtained from both known and novel lineages. This study demonstrates that high-throughput sequencing is a viable and sensitive method for detecting parasitic protists when present and transported in the ballast water of ships. These data also underscore the potential importance of human-aided dispersal in the biogeography of these microbes and emerging diseases in the world's oceans.

Detection by real-time PCR and pyrosequencing of the cry1Ab and cry1Ac genes introduced in genetically modified (GM) constructs.

PubMed

Debode, Frederic; Janssen, Eric; Bragard, Claude; Berben, Gilbert

2017-08-01

The presence of genetically modified organisms (GMOs) in food and feed is mainly detected by the use of targets focusing on promoters and terminators. As some genes are frequently used in genetically modified (GM) construction, they also constitute excellent screening elements and their use is increasing. In this paper we propose a new target for the detection of cry1Ab and cry1Ac genes by real-time polymerase chain reaction (PCR) and pyrosequencing. The specificity, sensitivity and robustness of the real-time PCR method were tested following the recommendations of international guidelines and the method met the expected performance criteria. This paper also shows how the robustness testing was assessed. This new cry1Ab/Ac method can provide a positive signal with a larger number of GM events than do the other existing methods using double dye-probes. The method permits the analysis of results with less ambiguity than the SYBRGreen method recommended by the European Reference Laboratory (EURL) GM Food and Feed (GMFF). A pyrosequencing method was also developed to gain additional information thanks to the sequence of the amplicon. This method of sequencing-by-synthesis can determine the sequence between the primers used for PCR. Pyrosequencing showed that the sequences internal to the primers present differences following the GM events considered and three different sequences were observed. The sensitivity of the pyrosequencing was tested on reference flours with a low percentage GM content and different copy numbers. Improvements in the pyrosequencing protocol provided correct sequences with 50 copies of the target. Below this copy number, the quality of the sequence was more random.

Utilizing Pyrosequencing and Quantitative pCR to Characterize Fungal Populations among House Dust Samples

EPA Science Inventory

Molecular techniques are an alternative to culturing and counting methods in quantifying indoor fungal contamination. Pyrosequencing offers the possibility of identifying unexpected indoor fungi. In this study, 50 house dust samples were collected from homes in the Yakima Valley,...

Polycyclic Aromatic Hydrocarbon Degradation of Phytoplankton-Associated Arenibacter spp. and Description of Arenibacter algicola sp. nov., an Aromatic Hydrocarbon-Degrading Bacterium

PubMed Central

Rhodes, Glenn; Mishamandani, Sara; Berry, David; Whitman, William B.; Nichols, Peter D.; Semple, Kirk T.; Aitken, Michael D.

2014-01-01

Pyrosequencing of the bacterial community associated with a cosmopolitan marine diatom during enrichment with crude oil revealed several Arenibacter phylotypes, of which one (OTU-202) had become significantly enriched by the oil. Since members of the genus Arenibacter have not been previously shown to degrade hydrocarbons, we attempted to isolate a representative strain of this genus in order to directly investigate its hydrocarbon-degrading potential. Based on 16S rRNA sequencing, one isolate (designated strain TG409T) exhibited >99% sequence identity to three type strains of this genus. On the basis of phenotypic and genotypic characteristics, strain TG409T represents a novel species in the genus Arenibacter, for which the name Arenibacter algicola sp. nov. is proposed. We reveal for the first time that polycyclic aromatic hydrocarbon (PAH) degradation is a shared phenotype among members of this genus, indicating that it could be used as a taxonomic marker for this genus. Kinetic data for PAH mineralization rates showed that naphthalene was preferred to phenanthrene, and its mineralization was significantly enhanced in the presence of glass wool (a surrogate for diatom cell surfaces). During enrichment on hydrocarbons, strain TG409T emulsified n-tetradecane and crude oil, and cells were found to be preferentially attached to oil droplets, indicating an ability by the strain to express cell surface amphiphilic substances (biosurfactants or bioemulsifiers) as a possible strategy to increase the bioavailability of hydrocarbons. This work adds to our growing knowledge on the diversity of bacterial genera in the ocean contributing to the degradation of oil contaminants and of hydrocarbon-degrading bacteria found living in association with marine eukaryotic phytoplankton. PMID:24212584

Fish gut microbiota analysis differentiates physiology and behavior of invasive Asian carp and indigenous American fish

USGS Publications Warehouse

Ye, Lin; Amberg, Jon J.; Chapman, Duane C.; Gaikowski, Mark P.; Liu, Wen-Tso

2014-01-01

Gut microbiota of invasive Asian silver carp (SVCP) and indigenous planktivorous gizzard shad (GZSD) in Mississippi river basin were compared using 16S rRNA gene pyrosequencing. Analysis of more than 440 000 quality-filtered sequences obtained from the foregut and hindgut of GZSD and SVCP revealed high microbial diversity in these samples. GZSD hindgut (GZSD_H) samples (n=23) with >7000 operational taxonomy units (OTUs) exhibited the highest alpha-diversity indices followed by SVCP foregut (n=15), GZSD foregut (n=9) and SVCP hindgut (SVCP_H) (n=24). UniFrac distance-based non-metric multidimensional scaling (NMDS) analysis showed that the microbiota of GZSD_H and SVCP_H were clearly separated into two clusters: samples in the GZSD cluster were observed to vary by sampling location and samples in the SVCP cluster by sampling date. NMDS further revealed distinct microbial community between foregut to hindgut for individual GZSD and SVCP. Cyanobacteria, Proteobacteria, Actinobacteria and Bacteroidetes were detected as the predominant phyla regardless of fish or gut type. The high abundance of Cyanobacteria observed was possibly supported by their role as the fish’s major food source. Furthermore, unique and shared OTUs and OTUs in each gut type were identified, three OTUs from the order Bacteroidales, the genus Bacillariophyta and the genus Clostridium were found significantly more abundant in GZSD_H (14.9–22.8%) than in SVCP_H (0.13–4.1%) samples. These differences were presumably caused by the differences in the type of food sources including bacteria ingested, the gut morphology and digestion, and the physiological behavior between GZSD and SVCP.

Introduction of the hybcell-based compact sequencing technology and comparison to state-of-the-art methodologies for KRAS mutation detection.

PubMed

Zopf, Agnes; Raim, Roman; Danzer, Martin; Niklas, Norbert; Spilka, Rita; Pröll, Johannes; Gabriel, Christian; Nechansky, Andreas; Roucka, Markus

2015-03-01

The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.

Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing

PubMed Central

Lenchi, Nesrine; İnceoğlu, Özgül; Kebbouche-Gana, Salima; Gana, Mohamed Lamine; Llirós, Marc; Servais, Pierre; García-Armisen, Tamara

2013-01-01

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species. PMID:23805243

Mycoplasma ovipneumoniae - A Primary Cause of Severe Pneumonia Epizootics in the Norwegian Muskox (Ovibos moschatus) Population

PubMed Central

Handeland, Kjell; Tengs, Torstein; Kokotovic, Branko; Vikøren, Turid; Ayling, Roger D.; Bergsjø, Bjarne; Sigurðardóttir, Ólöf G.; Bretten, Tord

2014-01-01

The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004–2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection. PMID:25198695

Bacterial and fungal microbiota of spontaneously fermented Chinese products, Rubing milk cake and Yan-cai vegetable pickles.

PubMed

Liu, Xin; Kuda, Takashi; Takahashi, Hajime; Kimura, Bon

2018-06-01

The Rubing milk cake from Yunnan and the Yan-cai vegetable pickles from Guangdong are traditional spontaneously fermented foods in China. We evaluated the microbial properties of these products with the analysis of their bacterial and fungal microbiota using classical culture-dependent and culture-independent methods, including a 16S rDNA gene (V4) and an internal transcribed spacer (ITS) region pyrosequencing method with MiSeq system. The viable lactic acid bacteria (LAB) count was 8 and 6 log colony-forming units (CFU)/g in Rubing and Yan-cai samples, respectively. The yeast count was approximately 100-1000 times less than the LAB count in most samples, except one Yan-cai sample. In addition, the gram-negative rod count in half of the samples was similar to the LAB count. Pyrosequencing results revealed the high abundance (10%-20%) of gram-negative Pseudomonas spp. and Enterobacteriaceae in these samples. These results suggest that some of these traditional foods are undesirable as ready-to-eat (RTE) foods, even when these are typical lactic acid fermented foods. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative Analysis of Performance and Microbial Characteristics Between High-Solid and Low-Solid Anaerobic Digestion of Sewage Sludge Under Mesophilic Conditions.

PubMed

Lu, Qin; Yi, Jing; Yang, Dianhai

2016-01-01

High-solid anaerobic digestion of sewage sludge achieves highly efficient volatile solid reduction, and production of volatile fatty acid (VFA) and methane compared with conventional low-solid anaerobic digestion. In this study, the potential mechanisms of the better performance in high-solid anaerobic digestion of sewage sludge were investigated by using 454 high-throughput pyrosequencing and real-time PCR to analyze the microbial characteristics in sewage sludge fermentation reactors. The results obtained by 454 high-throughput pyrosequencing revealed that the phyla Chloroflexi, Bacteroidetes, and Firmicutes were the dominant functional microorganisms in high-solid and low-solid anaerobic systems. Meanwhile, the real-time PCR assays showed that high-solid anaerobic digestion significantly increased the number of total bacteria, which enhanced the hydrolysis and acidification of sewage sludge. Further study indicated that the number of total archaea (dominated by Methanosarcina) in a high-solid anaerobic fermentation reactor was also higher than that in a low-solid reactor, resulting in higher VFA consumption and methane production. Hence, the increased key bacteria and methanogenic archaea involved in sewage sludge hydrolysis, acidification, and methanogenesis resulted in the better performance of high-solid anaerobic sewage sludge fermentation.

Simultaneous detection of human mitochondrial DNA and nuclear-inserted mitochondrial-origin sequences (NumtS) using forensic mtDNA amplification strategies and pyrosequencing technology.

PubMed

Bintz, Brittania J; Dixon, Groves B; Wilson, Mark R

2014-07-01

Next-generation sequencing technologies enable the identification of minor mitochondrial DNA variants with higher sensitivity than Sanger methods, allowing for enhanced identification of minor variants. In this study, mixtures of human mtDNA control region amplicons were subjected to pyrosequencing to determine the detection threshold of the Roche GS Junior(®) instrument (Roche Applied Science, Indianapolis, IN). In addition to expected variants, a set of reproducible variants was consistently found in reads from one particular amplicon. A BLASTn search of the variant sequence revealed identity to a segment of a 611-bp nuclear insertion of the mitochondrial control region (NumtS) spanning the primer-binding sites of this amplicon (Nature 1995;378:489). Primers (Hum Genet 2012;131:757; Hum Biol 1996;68:847) flanking the insertion were used to confirm the presence or absence of the NumtS in buccal DNA extracts from twenty donors. These results further our understanding of human mtDNA variation and are expected to have a positive impact on the interpretation of mtDNA profiles using deep-sequencing methods in casework. © 2014 American Academy of Forensic Sciences.

Microbial diversity and evidence of novel homoacetogens in the gut of both geriatric and adult giant pandas (Ailuropoda melanoleuca).

PubMed

Tun, Hein Min; Mauroo, Nathalie France; Yuen, Chan San; Ho, John Chi Wang; Wong, Mabel Ting; Leung, Frederick Chi-Ching

2014-01-01

Recent studies have described the bacterial community residing in the guts of giant pandas, together with the presence of lignocellulolytic enzymes. However, a more comprehensive understanding of the intestinal microbial composition and its functional capacity in giant pandas remains a major goal. Here, we conducted a comparison of bacterial, fungal and homoacetogenic microbial communities from fecal samples taken from two geriatric and two adult captive giant pandas. 16S rDNA amplicon pyrosequencing revealed that Firmicutes and Proteobacteria are the most abundant microbiota in both geriatric and adult giant pandas. However, members of phylum Actinobacteria found in adult giant pandas were absent in their geriatric counterparts. Similarly, ITS1 amplicon pyrosequencing identified developmental changes in the most abundant fungal classes from Sordariomycetes in adult pandas to Saccharomycetes in geriatric pandas. Geriatric pandas exhibited significantly higher abundance of a potential probiotic fungus (Candida tropicalis) as compared to adult pandas, indicating their importance in the normal digestive physiology of aged pandas. Our study also reported the presence of a lignocellulolytic white-rot fungus, Perenniporia medulla-panis, and the evidence of novel homoacetogens residing in the guts of giant pandas.

Changes in the bacterial community of soybean rhizospheres during growth in the field.

PubMed

Sugiyama, Akifumi; Ueda, Yoshikatsu; Zushi, Takahiro; Takase, Hisabumi; Yazaki, Kazufumi

2014-01-01

Highly diverse communities of bacteria inhabiting soybean rhizospheres play pivotal roles in plant growth and crop production; however, little is known about the changes that occur in these communities during growth. We used both culture-dependent physiological profiling and culture independent DNA-based approaches to characterize the bacterial communities of the soybean rhizosphere during growth in the field. The physiological properties of the bacterial communities were analyzed by a community-level substrate utilization assay with BioLog Eco plates, and the composition of the communities was assessed by gene pyrosequencing. Higher metabolic capabilities were found in rhizosphere soil than in bulk soil during all stages of the BioLog assay. Pyrosequencing analysis revealed that differences between the bacterial communities of rhizosphere and bulk soils at the phylum level; i.e., Proteobacteria were increased, while Acidobacteria and Firmicutes were decreased in rhizosphere soil during growth. Analysis of operational taxonomic units showed that the bacterial communities of the rhizosphere changed significantly during growth, with a higher abundance of potential plant growth promoting rhizobacteria, including Bacillus, Bradyrhizobium, and Rhizobium, in a stage-specific manner. These findings demonstrated that rhizosphere bacterial communities were changed during soybean growth in the field.

Microbial Diversity and Evidence of Novel Homoacetogens in the Gut of Both Geriatric and Adult Giant Pandas (Ailuropoda melanoleuca)

PubMed Central

Tun, Hein Min; Mauroo, Nathalie France; Yuen, Chan San; Ho, John Chi Wang; Wong, Mabel Ting; Leung, Frederick Chi-Ching

2014-01-01

Recent studies have described the bacterial community residing in the guts of giant pandas, together with the presence of lignocellulolytic enzymes. However, a more comprehensive understanding of the intestinal microbial composition and its functional capacity in giant pandas remains a major goal. Here, we conducted a comparison of bacterial, fungal and homoacetogenic microbial communities from fecal samples taken from two geriatric and two adult captive giant pandas. 16S rDNA amplicon pyrosequencing revealed that Firmicutes and Proteobacteria are the most abundant microbiota in both geriatric and adult giant pandas. However, members of phylum Actinobacteria found in adult giant pandas were absent in their geriatric counterparts. Similarly, ITS1 amplicon pyrosequencing identified developmental changes in the most abundant fungal classes from Sordariomycetes in adult pandas to Saccharomycetes in geriatric pandas. Geriatric pandas exhibited significantly higher abundance of a potential probiotic fungus (Candida tropicalis) as compared to adult pandas, indicating their importance in the normal digestive physiology of aged pandas. Our study also reported the presence of a lignocellulolytic white-rot fungus, Perenniporia medulla-panis, and the evidence of novel homoacetogens residing in the guts of giant pandas. PMID:24475017

Bacterial communities in the gut and reproductive organs of Bactrocera minax (Diptera: Tephritidae) based on 454 pyrosequencing.

PubMed

Wang, Ailin; Yao, Zhichao; Zheng, Weiwei; Zhang, Hongyu

2014-01-01

The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs). A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%-95%). Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices) and community structure (PCA analysis) varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host's physiological demands.

Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine

USDA-ARS?s Scientific Manuscript database

Genomes from fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolates were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200...

Characterization of chlorinated and chloraminated drinking water microbial communities in a distribution system simulator using pyrosequencing data analysis

EPA Science Inventory

The molecular analysis of drinking water microbial communities has focused primarily on 16S rRNA gene sequence analysis. Since this approach provides limited information on function potential of microbial communities, analysis of whole-metagenome pyrosequencing data was used to...

Pyrosequencing vs. culture-dependent approaches to analyze lactic acid bacteria associated to chicha, a traditional maize-based fermented beverage from Northwestern Argentina.

PubMed

Elizaquível, Patricia; Pérez-Cataluña, Alba; Yépez, Alba; Aristimuño, Cecilia; Jiménez, Eugenia; Cocconcelli, Pier Sandro; Vignolo, Graciela; Aznar, Rosa

2015-04-02

The diversity of lactic acid bacteria (LAB) associated with chicha, a traditional maize-based fermented alcoholic beverage from Northwestern Argentina, was analyzed using culture-dependent and culture-independent approaches. Samples corresponding to 10 production steps were obtained from two local producers at Maimará (chicha M) and Tumbaya (chicha T). Whereas by culture-dependent approach a few number of species (Lactobacillus plantarum and Weissella viridescens in chicha M, and Enterococcus faecium and Leuconostoc mesenteroides in chicha T) were identified, a higher quantitative distribution of taxa was found in both beverages by pyrosequencing. The relative abundance of OTUs was higher in chicha M than in chicha T; six LAB genera were common for chicha M and T: Enterococcus, Lactococcus, Streptococcus, Weissella, Leuconostoc and Lactobacillus while Pediococcus only was detected in chicha M. Among the 46 identified LAB species, those of Lactobacillus were dominant in both chicha samples, exhibiting the highest diversity, whereas Enterococcus and Leuconostoc were recorded as the second dominant genera in chicha T and M, respectively. Identification at species level showed the predominance of Lb. plantarum, Lactobacillus rossiae, Leuconostoc lactis and W. viridescens in chicha M while Enterococcus hirae, E. faecium, Lc. mesenteroides and Weissella confusa predominated in chicha T samples. In parallel, when presumptive LAB isolates (chicha M: 146; chicha T: 246) recovered from the same samples were identified by ISR-PCR and RAPD-PCR profiles, species-specific PCR and 16S rRNA gene sequencing, most of them were assigned to the Leuconostoc genus (Lc. mesenteroides and Lc. lactis) in chicha M, Lactobacillus, Weissella and Enterococcus being also present. In contrast, chicha T exhibited the presence of Enterococcus and Leuconostoc, E. faecium being the most representative species. Massive sequencing approach was applied for the first time to study the diversity and evolution of microbial communities during chicha manufacture. Although differences in the LAB species profile between the two geographically different chicha productions were observed by culturing, a larger number for predominant LAB species as well as other minorities were revealed by pyrosequencing. The fine molecular inventory achieved by pyrosequencing provided more precise information on LAB population composition than culture-dependent analysis processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification of Marek's disease virus DNA for 454 pyrosequencing using micrococcal nuclease digestion and polyethylene glycol precipitation

USDA-ARS?s Scientific Manuscript database

Marek’s disease virus (MDV-1) is a cell-associated alphaherpesvirus that induces rapid-onset T-cell lymphomas in poultry. The genomes of 6 strains have been sequenced using both Sanger didoxy sequencing and 454 Life Science pyrosequencing. These genomes largely represent cell culture adapted strains...

Pyrosequencing analysis for characterization of bacterial diversity in a soil as affected by integrated livestock-cotton production systems

USDA-ARS?s Scientific Manuscript database

Impacts of integrated livestock-crop production systems compared to specialized systems on soil bacterial diversity have not been well documented. We used a bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) method to evaluate bacterial diversity of a clay loam soil (Fine, mixed, thermic To...

Analyses of the Stability and Core Taxonomic Memberships of the Human Microbiome

PubMed Central

Li, Kelvin; Bihan, Monika; Methé, Barbara A.

2013-01-01

Analyses of the taxonomic diversity associated with the human microbiome continue to be an area of great importance. The study of the nature and extent of the commonly shared taxa (“core”), versus those less prevalent, establishes a baseline for comparing healthy and diseased groups by quantifying the variation among people, across body habitats and over time. The National Institutes of Health (NIH) sponsored Human Microbiome Project (HMP) has provided an unprecedented opportunity to examine and better define what constitutes the taxonomic core within and across body habitats and individuals through pyrosequencing-based profiling of 16S rRNA gene sequences from oral, skin, distal gut (stool), and vaginal body habitats from over 200 healthy individuals. A two-parameter model is introduced to quantitatively identify the core taxonomic members of each body habitat’s microbiota across the healthy cohort. Using only cutoffs for taxonomic ubiquity and abundance, core taxonomic members were identified for each of the 18 body habitats and also for the 4 higher-level body regions. Although many microbes were shared at low abundance, they exhibited a relatively continuous spread in both their abundance and ubiquity, as opposed to a more discretized separation. The numbers of core taxa members in the body regions are comparatively small and stable, reflecting the relatively high, but conserved, interpersonal variability within the cohort. Core sizes increased across the body regions in the order of: vagina, skin, stool, and oral cavity. A number of “minor” oral taxonomic core were also identified by their majority presence across the cohort, but with relatively low and stable abundances. A method for quantifying the difference between two cohorts was introduced and applied to samples collected on a second visit, revealing that over time, the oral, skin, and stool body regions tended to be more transient in their taxonomic structure than the vaginal body region. PMID:23671663

Dynamics of bacterial communities before and after distribution in a full-scale drinking water network.

PubMed

El-Chakhtoura, Joline; Prest, Emmanuelle; Saikaly, Pascal; van Loosdrecht, Mark; Hammes, Frederik; Vrouwenvelder, Hans

2015-05-01

Understanding the biological stability of drinking water distribution systems is imperative in the framework of process control and risk management. The objective of this research was to examine the dynamics of the bacterial community during drinking water distribution at high temporal resolution. Water samples (156 in total) were collected over short time-scales (minutes/hours/days) from the outlet of a treatment plant and a location in its corresponding distribution network. The drinking water is treated by biofiltration and disinfectant residuals are absent during distribution. The community was analyzed by 16S rRNA gene pyrosequencing and flow cytometry as well as conventional, culture-based methods. Despite a random dramatic event (detected with pyrosequencing and flow cytometry but not with plate counts), the bacterial community profile at the two locations did not vary significantly over time. A diverse core microbiome was shared between the two locations (58-65% of the taxa and 86-91% of the sequences) and found to be dependent on the treatment strategy. The bacterial community structure changed during distribution, with greater richness detected in the network and phyla such as Acidobacteria and Gemmatimonadetes becoming abundant. The rare taxa displayed the highest dynamicity, causing the major change during water distribution. This change did not have hygienic implications and is contingent on the sensitivity of the applied methods. The concept of biological stability therefore needs to be revised. Biostability is generally desired in drinking water guidelines but may be difficult to achieve in large-scale complex distribution systems that are inherently dynamic. Copyright © 2015 Elsevier Ltd. All rights reserved.

Abiotic factors shape microbial diversity in Sonoran Desert soils.

PubMed

Andrew, David R; Fitak, Robert R; Munguia-Vega, Adrian; Racolta, Adriana; Martinson, Vincent G; Dontsova, Katerina

2012-11-01

High-throughput, culture-independent surveys of bacterial and archaeal communities in soil have illuminated the importance of both edaphic and biotic influences on microbial diversity, yet few studies compare the relative importance of these factors. Here, we employ multiplexed pyrosequencing of the 16S rRNA gene to examine soil- and cactus-associated rhizosphere microbial communities of the Sonoran Desert and the artificial desert biome of the Biosphere2 research facility. The results of our replicate sampling approach show that microbial communities are shaped primarily by soil characteristics associated with geographic locations, while rhizosphere associations are secondary factors. We found little difference between rhizosphere communities of the ecologically similar saguaro (Carnegiea gigantea) and cardón (Pachycereus pringlei) cacti. Both rhizosphere and soil communities were dominated by the disproportionately abundant Crenarchaeota class Thermoprotei, which comprised 18.7% of 183,320 total pyrosequencing reads from a comparatively small number (1,337 or 3.7%) of the 36,162 total operational taxonomic units (OTUs). OTUs common to both soil and rhizosphere samples comprised the bulk of raw sequence reads, suggesting that the shared community of soil and rhizosphere microbes constitute common and abundant taxa, particularly in the bacterial phyla Proteobacteria, Actinobacteria, Planctomycetes, Firmicutes, Bacteroidetes, Chloroflexi, and Acidobacteria. The vast majority of OTUs, however, were rare and unique to either soil or rhizosphere communities and differed among locations dozens of kilometers apart. Several soil properties, particularly soil pH and carbon content, were significantly correlated with community diversity measurements. Our results highlight the importance of culture-independent approaches in surveying microbial communities of extreme environments.

Abiotic Factors Shape Microbial Diversity in Sonoran Desert Soils

PubMed Central

Fitak, Robert R.; Munguia-Vega, Adrian; Racolta, Adriana; Martinson, Vincent G.; Dontsova, Katerina

2012-01-01

High-throughput, culture-independent surveys of bacterial and archaeal communities in soil have illuminated the importance of both edaphic and biotic influences on microbial diversity, yet few studies compare the relative importance of these factors. Here, we employ multiplexed pyrosequencing of the 16S rRNA gene to examine soil- and cactus-associated rhizosphere microbial communities of the Sonoran Desert and the artificial desert biome of the Biosphere2 research facility. The results of our replicate sampling approach show that microbial communities are shaped primarily by soil characteristics associated with geographic locations, while rhizosphere associations are secondary factors. We found little difference between rhizosphere communities of the ecologically similar saguaro (Carnegiea gigantea) and cardón (Pachycereus pringlei) cacti. Both rhizosphere and soil communities were dominated by the disproportionately abundant Crenarchaeota class Thermoprotei, which comprised 18.7% of 183,320 total pyrosequencing reads from a comparatively small number (1,337 or 3.7%) of the 36,162 total operational taxonomic units (OTUs). OTUs common to both soil and rhizosphere samples comprised the bulk of raw sequence reads, suggesting that the shared community of soil and rhizosphere microbes constitute common and abundant taxa, particularly in the bacterial phyla Proteobacteria, Actinobacteria, Planctomycetes, Firmicutes, Bacteroidetes, Chloroflexi, and Acidobacteria. The vast majority of OTUs, however, were rare and unique to either soil or rhizosphere communities and differed among locations dozens of kilometers apart. Several soil properties, particularly soil pH and carbon content, were significantly correlated with community diversity measurements. Our results highlight the importance of culture-independent approaches in surveying microbial communities of extreme environments. PMID:22885757

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage.

PubMed

Ferrocino, Ilario; Greppi, Anna; La Storia, Antonietta; Rantsiou, Kalliopi; Ercolini, Danilo; Cocolin, Luca

2016-01-15

Beef burgers were stored at 4°C in a vacuum in nisin-activated antimicrobial packaging. Microbial ecology analyses were performed on samples collected between days 0 and 21 of storage to discover the population diversity. Two batches were analyzed using RNA-based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The active packaging retarded the growth of the total viable bacteria and lactic acid bacteria. Culture-independent analysis by pyrosequencing of RNA extracted directly from meat showed that Photobacterium phosphoreum, Lactococcus piscium, Lactobacillus sakei, and Leuconostoc carnosum were the major operational taxonomic units (OTUs) shared between control and treated samples. Beta diversity analysis of the 16S rRNA sequence data and RNA-DGGE showed a clear separation between two batches based on the microbiota. Control samples from batch B showed a significant high abundance of some taxa sensitive to nisin, such as Kocuria rhizophila, Staphylococcus xylosus, Leuconostoc carnosum, and Carnobacterium divergens, compared to control samples from batch A. However, only from batch B was it possible to find a significant difference between controls and treated samples during storage due to the active packaging. Predicted metagenomes confirmed differences between the two batches and indicated that the use of nisin-based antimicrobial packaging can determine a reduction in the abundance of specific metabolic pathways related to spoilage. The present study aimed to assess the viable bacterial communities in beef burgers stored in nisin-based antimicrobial packaging, and it highlights the efficacy of this strategy to prolong beef burger shelf life. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Yeast diversity during the fermentation of Andean chicha: A comparison of high-throughput sequencing and culture-dependent approaches.

PubMed

Mendoza, Lucía M; Neef, Alexander; Vignolo, Graciela; Belloch, Carmela

2017-10-01

Diversity and dynamics of yeasts associated with the fermentation of Argentinian maize-based beverage chicha was investigated. Samples taken at different stages from two chicha productions were analyzed by culture-dependent and culture-independent methods. Five hundred and ninety six yeasts were isolated by classical microbiological methods and 16 species identified by RFLPs and sequencing of D1/D2 26S rRNA gene. Genetic typing of isolates from the dominant species, Saccharomyces cerevisiae, by PCR of delta elements revealed up to 42 different patterns. High-throughput sequencing (HTS) of D1/D2 26S rRNA gene amplicons from chicha samples detected more than one hundred yeast species and almost fifty filamentous fungi taxa. Analysis of the data revealed that yeasts dominated the fermentation, although, a significant percentage of filamentous fungi appeared in the first step of the process. Statistical analysis of results showed that very few taxa were represented by more than 1% of the reads per sample at any step of the process. S. cerevisiae represented more than 90% of the reads in the fermentative samples. Other yeast species dominated the pre-fermentative steps and abounded in fermented samples when S. cerevisiae was in percentages below 90%. Most yeasts species detected by pyrosequencing were not recovered by cultivation. In contrast, the cultivation-based methodology detected very few yeast taxa, and most of them corresponded with very few reads in the pyrosequencing analysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Microbial diversity and composition of the sediment in the drinking water reservoir Saidenbach (Saxonia, Germany).

PubMed

Röske, Kerstin; Sachse, René; Scheerer, Carola; Röske, Isolde

2012-02-01

Sediments contain a huge number and diversity of microorganisms that are important for the flux of material and are pivotal to all major biogeochemical cycles. Sediments of reservoirs are affected by a wide spectrum of allochthous and autochthonous influences providing versatile environments along the flow of water within the reservoir. Here we report on the microbial diversity in sediments of the mesotrophic drinking water reservoir Saidenbach, Germany, featuring a pronounced longitudinal gradient in sediment composition in the reservoir system. Three sampling sites were selected along the gradient, and the microbial communities in two sediment depths were characterized using catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and a bar-coded pyrosequencing approach. Multivariate statistic was used to reveal relationships between sequence diversity and the environmental conditions. The microbial communities were tremendously diverse with a Shannon index of diversity (H') ranging from 6.7 to 7.1. 18,986 sequences could be classified into 37 phyla including candidate divisions, but the full extent of genetic diversity was not captured. While CARD-FISH gave an overview about the community composition, more detailed information was gained by pyrosequencing. Bacteria were more abundant than Archaea. The dominating phylum in all samples was Proteobacteria, especially Betaproteobacteria and Deltaproteobacteria. Furthermore, sequences of Bacteroidetes, Verrucomicrobia, Acidobacteria, Chlorobi, Nitrospira, Spirochaetes, Gammaproteobacteria, Alphaproteobacteria, Chloroflexi, and Gemmatimonadetes were found. The site ammonium concentration, water content and organic matter content revealed to be strongest environmental predictors explaining the observed significant differences in the community composition between sampling sites. Copyright © 2011 Elsevier GmbH. All rights reserved.

Aberrant Promoter Methylation and Expression of UTF1 during Cervical Carcinogenesis

PubMed Central

Deplus, Rachel; Lampe, Xavier; Krusy, Nathalie; Calonne, Emilie; Delbecque, Katty; Kridelka, Frederic; Fuks, François; Ennaji, My Mustapha; Delvenne, Philippe

2012-01-01

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2′-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression. PMID:22880087

Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Xueling; Zhou, Tongqing; Zhu, Jiang

2013-03-04

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution ofmore » antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.« less

454 pyrosequencing project identifying expressed genes from the horn fly, Haematobia irritans

USDA-ARS?s Scientific Manuscript database

We used an EST approach to initiate a study of the genome of the horn fly, Haematobia irritans and have used 454 pyrosequencing techniques to sequence 73,512, 100,603, 71,550, and 85,769 expressed genes from the egg, first instar larvae, adult male, and adult female lifestages of the horn fly. cD...

Assessment of bacterial contamination of lipstick using pyrosequencing.

PubMed

Lee, So Y; Lee, Si Y

As soon as they are exposed to the environment, cosmetics become contaminated with microorganisms, and this contamination accumulates with increased use. In this study, we employed pyrosequencing to investigate the diversity of bacteria found on lipstick. Bacterial DNA was extracted from 20 lipstick samples and mixed in equal ratios for pyrosequencing analysis. As a result, 105 bacterial genera were detected, four of which ( Leifsonia , Methylobacterium , Streptococcus , and Haemophilus ) were predominant in 92% of the 19,863 total sequence reads. Potentially pathogenic genera such as Staphylococcus , Pseudomonas , Escherichia , Salmonella , Corynebacterium , Mycobacterium , and Neisseria accounted for 27.6% of the 105 genera. The most commonly identified oral bacteria belonged to the Streptococcus genus, although other oral genera such as Actinomyces , Fusobacterium , Porphyromonas , and Lactobacillus were also detected.

Characterization of the fecal microbiota using high-throughput sequencing reveals a stable microbial community during storage.

PubMed

Carroll, Ian M; Ringel-Kulka, Tamar; Siddle, Jennica P; Klaenhammer, Todd R; Ringel, Yehuda

2012-01-01

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at -80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or -80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or -80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Biochemical Association of Metabolic Profile and Microbiome in Chronic Pressure Ulcer Wounds

PubMed Central

Ammons, Mary Cloud B.; Morrissey, Kathryn; Tripet, Brian P.; Van Leuven, James T.; Han, Anne; Lazarus, Gerald S.; Zenilman, Jonathan M.; Stewart, Philip S.; James, Garth A.; Copié, Valérie

2015-01-01

Chronic, non-healing wounds contribute significantly to the suffering of patients with co-morbidities in the clinical population with mild to severely compromised immune systems. Normal wound healing proceeds through a well-described process. However, in chronic wounds this process seems to become dysregulated at the transition between resolution of inflammation and re-epithelialization. Bioburden in the form of colonizing bacteria is a major contributor to the delayed headlining in chronic wounds such as pressure ulcers. However how the microbiome influences the wound metabolic landscape is unknown. Here, we have used a Systems Biology approach to determine the biochemical associations between the taxonomic and metabolomic profiles of wounds colonized by bacteria. Pressure ulcer biopsies were harvested from primary chronic wounds and bisected into top and bottom sections prior to analysis of microbiome by pyrosequencing and analysis of metabolome using 1H nuclear magnetic resonance (NMR) spectroscopy. Bacterial taxonomy revealed that wounds were colonized predominantly by three main phyla, but differed significantly at the genus level. While taxonomic profiles demonstrated significant variability between wounds, metabolic profiles shared significant similarity based on the depth of the wound biopsy. Biochemical association between taxonomy and metabolic landscape indicated significant wound-to-wound similarity in metabolite enrichment sets and metabolic pathway impacts, especially with regard to amino acid metabolism. To our knowledge, this is the first demonstration of a statistically robust correlation between bacterial colonization and metabolic landscape within the chronic wound environment. PMID:25978400

The complete genome of klassevirus – a novel picornavirus in pediatric stool

PubMed Central

Greninger, Alexander L; Runckel, Charles; Chiu, Charles Y; Haggerty, Thomas; Parsonnet, Julie; Ganem, Donald; DeRisi, Joseph L

2009-01-01

Background Diarrhea kills 2 million children worldwide each year, yet an etiological agent is not found in approximately 30–50% of cases. Picornaviral genera such as enterovirus, kobuvirus, cosavirus, parechovirus, hepatovirus, teschovirus, and cardiovirus have all been found in human and animal diarrhea. Modern technologies, especially deep sequencing, allow rapid, high-throughput screening of clinical samples such as stool for new infectious agents associated with human disease. Results A pool of 141 pediatric gastroenteritis samples that were previously found to be negative for known diarrheal viruses was subjected to pyrosequencing. From a total of 937,935 sequence reads, a collection of 849 reads distantly related to Aichi virus were assembled and found to comprise 75% of a novel picornavirus genome. The complete genome was subsequently cloned and found to share 52.3% nucleotide pairwise identity and 38.9% amino acid identity to Aichi virus. The low level of sequence identity suggests a novel picornavirus genus which we have designated klassevirus. Blinded screening of 751 stool specimens from both symptomatic and asymptomatic individuals revealed a second positive case of klassevirus infection, which was subsequently found to be from the index case's 11-month old twin. Conclusion We report the discovery of human klassevirus 1, a member of a novel picornavirus genus, in stool from two infants from Northern California. Further characterization and epidemiological studies will be required to establish whether klasseviruses are significant causes of human infection. PMID:19538752

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

PubMed Central

Jeong, Ji Hun; Park, Soon Ho; Park, Mi Jung; Kim, Moon Jin; Kim, Kyung Hee; Park, Pil Whan; Seo, Yiel Hea; Lee, Jae Hoon; Park, Jinny; Hong, Junshik

2013-01-01

Background N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. Methods We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. Results The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. Conclusions There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses. PMID:23667841

454-Pyrosequencing survey of microbiota in adult Spotted Wing Drosophila (SWD) corroborates a core microbiome and additional symbiotic and entomopathogenic bacterial associates

USDA-ARS?s Scientific Manuscript database

Complete surveys of insect endosymbionts including species of economic importance have until recently been hampered by a lack of high-throughput genetic assays. We used 454-pyrosequencing of the 16S rRNA gene amplicon of adult spotted wing Drosophila (SWD) Drosophila suzukii (Matsumura) from souther...

Development of colonic microflora as assessed by pyrosequencing in dairy calves fed waste milk

USDA-ARS?s Scientific Manuscript database

The objective of the current study was to examine the effect of pasteurization of waste milk used to feed dairy calves on the bacterial diversity of their lower gut. Using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP), fecal samples from dairy calves aging from 1 week to 6 mon...

Microbial analysis in primary and persistent endodontic infections by using pyrosequencing.

PubMed

Hong, Bo-Young; Lee, Tae-Kwon; Lim, Sang-Min; Chang, Seok Woo; Park, Joonhong; Han, Seung Hyun; Zhu, Qiang; Safavi, Kamran E; Fouad, Ashraf F; Kum, Kee Yeon

2013-09-01

The aim of this study was to investigate the bacterial community profile of intracanal microbiota in primary and persistent endodontic infections associated with asymptomatic chronic apical periodontitis by using GS-FLX Titanium pyrosequencing. The null hypothesis was that there is no difference in diversity of overall bacterial community profiles between primary and persistent infections. Pyrosequencing analysis from 10 untreated and 8 root-filled samples was conducted. Analysis from 18 samples yielded total of 124,767 16S rRNA gene sequences (with a mean of 6932 reads per sample) that were taxonomically assigned into 803 operational taxonomic units (3% distinction), 148 genera, and 10 phyla including unclassified. Bacteroidetes was the most abundant phylum in both primary and persistent infections. There were no significant differences in bacterial diversity between the 2 infection groups (P > .05). The bacterial community profile that was based on dendrogram showed that bacterial population in both infections was not significantly different in their structure and composition (P > .05). The present pyrosequencing study demonstrates that persistent infections have as diverse bacterial community as primary infections. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.

PubMed

Leite, A M O; Mayo, B; Rachid, C T C C; Peixoto, R S; Silva, J T; Paschoalin, V M F; Delgado, S

2012-09-01

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem. Copyright © 2012 Elsevier Ltd. All rights reserved.

Clinical Neuropathology practice news 1-2014: Pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma

PubMed Central

Preusser, Matthias; Berghoff, Anna S.; Manzl, Claudia; Filipits, Martin; Weinhäusel, Andreas; Pulverer, Walter; Dieckmann, Karin; Widhalm, Georg; Wöhrer, Adelheid; Knosp, Engelbert; Marosi, Christine; Hainfellner, Johannes A.

2014-01-01

Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin-fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing-based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing. PMID:24359605

De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing

PubMed Central

2011-01-01

Background Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. Results From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. Conclusion The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition. PMID:21492485

De novo assembly and transcriptome analysis of five major tissues of Jatropha curcas L. using GS FLX titanium platform of 454 pyrosequencing.

PubMed

Natarajan, Purushothaman; Parani, Madasamy

2011-04-15

Jatropha curcas L. is an important non-edible oilseed crop with promising future in biodiesel production. However, factors like oil yield, oil composition, toxic compounds in oil cake, pests and diseases limit its commercial potential. Well established genetic engineering methods using cloned genes could be used to address these limitations. Earlier, 10,983 unigenes from Sanger sequencing of ESTs, and 3,484 unique assembled transcripts from 454 pyrosequencing of uncloned cDNAs were reported. In order to expedite the process of gene discovery, we have undertaken 454 pyrosequencing of normalized cDNAs prepared from roots, mature leaves, flowers, developing seeds, and embryos of J. curcas. From 383,918 raw reads, we obtained 381,957 quality-filtered and trimmed reads that are suitable for the assembly of transcript sequences. De novo contig assembly of these reads generated 17,457 assembled transcripts (contigs) and 54,002 singletons. Average length of the assembled transcripts was 916 bp. About 30% of the transcripts were longer than 1000 bases, and the size of the longest transcript was 7,173 bases. BLASTX analysis revealed that 2,589 of these transcripts are full-length. The assembled transcripts were validated by RT-PCR analysis of 28 transcripts. The results showed that the transcripts were correctly assembled and represent actively expressed genes. KEGG pathway mapping showed that 2,320 transcripts are related to major biochemical pathways including the oil biosynthesis pathway. Overall, the current study reports 14,327 new assembled transcripts which included 2589 full-length transcripts and 27 transcripts that are directly involved in oil biosynthesis. The large number of transcripts reported in the current study together with existing ESTs and transcript sequences will serve as an invaluable genetic resource for crop improvement in jatropha. Sequence information of those genes that are involved in oil biosynthesis could be used for metabolic engineering of jatropha to increase oil content, and to modify oil composition.

Next-generation sequencing sheds light on the natural history of hepatitis C infection in patients who fail treatment.

PubMed

Abdelrahman, Tamer; Hughes, Joseph; Main, Janice; McLauchlan, John; Thursz, Mark; Thomson, Emma

2015-01-01

High rates of sexually transmitted infection and reinfection with hepatitis C virus (HCV) have recently been reported in human immunodeficiency virus (HIV)-infected men who have sex with men and reinfection has also been described in monoinfected injecting drug users. The diagnosis of reinfection has traditionally been based on direct Sanger sequencing of samples pre- and posttreatment, but not on more sensitive deep sequencing techniques. We studied viral quasispecies dynamics in patients who failed standard of care therapy in a high-risk HIV-infected cohort of patients with early HCV infection to determine whether treatment failure was associated with reinfection or recrudescence of preexisting infection. Paired sequences (pre- and posttreatment) were analyzed. The HCV E2 hypervariable region-1 was amplified using nested reverse-transcription polymerase chain reaction (RT-PCR) with indexed genotype-specific primers and the same products were sequenced using both Sanger and 454 pyrosequencing approaches. Of 99 HIV-infected patients with acute HCV treated with 24-48 weeks of pegylated interferon alpha and ribavirin, 15 failed to achieve a sustained virological response (six relapsed, six had a null response, and three had a partial response). Using direct sequencing, 10/15 patients (66%) had evidence of a previously undetected strain posttreatment; in many studies, this is interpreted as reinfection. However, pyrosequencing revealed that 15/15 (100%) of patients had evidence of persisting infection; 6/15 (40%) patients had evidence of a previously undetected variant present in the posttreatment sample in addition to a variant that was detected at baseline. This could represent superinfection or a limitation of the sensitivity of pyrosequencing. In this high-risk group, the emergence of new viral strains following treatment failure is most commonly associated with emerging dominance of preexisting minority variants rather than reinfection. Superinfection may occur in this cohort but reinfection is overestimated by Sanger sequencing. © 2014 The Authors. Hepatology published by Wiley on behalf of the American Association for the Study of Liver Diseases.

Analysis of genetically modified organisms by pyrosequencing on a portable photodiode-based bioluminescence sequencer.

PubMed

Song, Qinxin; Wei, Guijiang; Zhou, Guohua

2014-07-01

A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Microbial Response to Soil Liming of Damaged Ecosystems Revealed by Pyrosequencing and Phospholipid Fatty Acid Analyses

PubMed Central

Narendrula-Kotha, Ramya; Nkongolo, Kabwe K.

2017-01-01

Aims To assess the effects of dolomitic limestone applications on soil microbial communities’ dynamics and bacterial and fungal biomass, relative abundance, and diversity in metal reclaimed regions. Methods and Results The study was conducted in reclaimed mining sites and metal uncontaminated areas. The limestone applications were performed over 35 years ago. Total microbial biomass was determined by Phospholipid fatty acids. Bacterial and fungal relative abundance and diversity were assessed using 454 pyrosequencing. There was a significant increase of total microbial biomass in limed sites (342 ng/g) compared to unlimed areas (149 ng/g). Chao1 estimates followed the same trend. But the total number of OTUs (Operational Taxonomic Units) in limed (463 OTUs) and unlimed (473 OTUs) soil samples for bacteria were similar. For fungi, OTUs were 96 and 81 for limed and unlimed soil samples, respectively. Likewise, Simpson and Shannon diversity indices revealed no significant differences between limed and unlimed sites. Bacterial and fungal groups specific to either limed or unlimed sites were identified. Five major bacterial phyla including Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, and Proteobacteria were found. The latter was the most prevalent phylum in all the samples with a relative abundance of 50%. Bradyrhizobiaceae family with 12 genera including the nitrogen fixing Bradirhizobium genus was more abundant in limed sites compared to unlimed areas. For fungi, Ascomycota was the most predominant phylum in unlimed soils (46%) while Basidiomycota phylum represented 86% of all fungi in the limed areas. Conclusion Detailed analysis of the data revealed that although soil liming increases significantly the amount of microbial biomass, the level of species diversity remain statistically unchanged even though the microbial compositions of the damaged and restored sites are different. Significance and Impact of the study Soil liming still have a significant beneficial effects on soil microbial abundance and composition > 35 years after dolomitic limestone applications. PMID:28052072

Effects of consecutive monoculture of Pseudostellaria heterophylla on soil fungal community as determined by pyrosequencing

NASA Astrophysics Data System (ADS)

Wu, Linkun; Chen, Jun; Wu, Hongmiao; Wang, Juanying; Wu, Yanhong; Lin, Sheng; Khan, Muhammad Umar; Zhang, Zhongyi; Lin, Wenxiong

2016-05-01

Under consecutive monoculture, the biomass and quality of Pseudostellaria heterophylla declines significantly. In this study, a three-year field experiment was conducted to identify typical growth inhibition effects caused by extended monoculturing of P. heterophylla. Deep pyrosequencing was used to examine changes in the structure and composition of soil fungal community along a three-year gradient of monoculture. The results revealed a distinct separation between the newly planted plot and the two-year, three-year monocultured plots. The Shannon and Simpson diversity indices were significantly higher in the two-year and three-year monoculture soils than in the newly planted soil. Consecutive monoculture of this plant led to a significant increase in relative abundance of Fusarium, Trichocladium and Myrothecium and Simplicillium, etc., but a significant decrease in the relative abundance of Penicillium. Quantitative PCR analysis confirmed a significant increase in Fusarium oxysporum, an agent known to cause wilt and rot disease of P. heterophylla. Furthermore, phenolic acid mixture at a ratio similar to that found in the rhizosphere could promote mycelial growth of pathogenic F. oxysporum. Overall, this study demonstrated that consecutive monoculture of P. heterophylla can alter the fungal community in the rhizosphere, including enrichment of host-specific pathogenic fungi at the expense of plant-beneficial fungi.

Diversity and distribution of lichen-associated fungi in the Ny-Ålesund Region (Svalbard, High Arctic) as revealed by 454 pyrosequencing

PubMed Central

Zhang, Tao; Wei, Xin-Li; Zhang, Yu-Qin; Liu, Hong-Yu; Yu, Li-Yan

2015-01-01

This study assessed the diversity and distribution of fungal communities associated with seven lichen species in the Ny-Ålesund Region (Svalbard, High Arctic) using Roche 454 pyrosequencing with fungal-specific primers targeting the internal transcribed spacer (ITS) region of the ribosomal rRNA gene. Lichen-associated fungal communities showed high diversity, with a total of 42,259 reads belonging to 370 operational taxonomic units (OTUs) being found. Of these OTUs, 294 belonged to Ascomycota, 54 to Basidiomycota, 2 to Zygomycota, and 20 to unknown fungi. Leotiomycetes, Dothideomycetes, and Eurotiomycetes were the major classes, whereas the dominant orders were Helotiales, Capnodiales, and Chaetothyriales. Interestingly, most fungal OTUs were closely related to fungi from various habitats (e.g., soil, rock, plant tissues) in the Arctic, Antarctic and alpine regions, which suggests that living in association with lichen thalli may be a transient stage of life cycle for these fungi and that long-distance dispersal may be important to the fungi in the Arctic. In addition, host-related factors shaped the lichen-associated fungal communities in this region. Taken together, these results suggest that lichens thalli act as reservoirs of diverse fungi from various niches, which may improve our understanding of fungal evolution and ecology in the Arctic. PMID:26463847

Vertical stratification of microbial communities in the Red Sea revealed by 16S rDNA pyrosequencing.

PubMed

Qian, Pei-Yuan; Wang, Yong; Lee, On On; Lau, Stanley C K; Yang, Jiangke; Lafi, Feras F; Al-Suwailem, Abdulaziz; Wong, Tim Y H

2011-03-01

The ecosystems of the Red Sea are among the least-explored microbial habitats in the marine environment. In this study, we investigated the microbial communities in the water column overlying the Atlantis II Deep and Discovery Deep in the Red Sea. Taxonomic classification of pyrosequencing reads of the 16S rRNA gene amplicons showed vertical stratification of microbial diversity from the surface water to 1500 m below the surface. Significant differences in both bacterial and archaeal diversity were observed in the upper (20 [corrected] and 50 m) and deeper layers (200 and 1500 m). There were no obvious differences in community structure at the same depth for the two sampling stations. The bacterial community in the upper layer was dominated by Cyanobacteria whereas the deeper layer harbored a large proportion of Proteobacteria. Among Archaea, Euryarchaeota, especially Halobacteriales, were dominant in the upper layer but diminished drastically in the deeper layer where Desulfurococcales belonging to Crenarchaeota became the dominant group. The results of our study indicate that the microbial communities sampled in this study are different from those identified in water column in other parts of the world. The depth-wise compositional variation in the microbial communities is attributable to their adaptations to the various environments in the Red Sea.

Bacterial and eukaryotic biodiversity patterns in terrestrial and aquatic habitats in the Sør Rondane Mountains, Dronning Maud Land, East Antarctica.

PubMed

Obbels, Dagmar; Verleyen, Elie; Mano, Marie-José; Namsaraev, Zorigto; Sweetlove, Maxime; Tytgat, Bjorn; Fernandez-Carazo, Rafael; De Wever, Aaike; D'hondt, Sofie; Ertz, Damien; Elster, Josef; Sabbe, Koen; Willems, Anne; Wilmotte, Annick; Vyverman, Wim

2016-06-01

The bacterial and microeukaryotic biodiversity were studied using pyrosequencing analysis on a 454 GS FLX+ platform of partial SSU rRNA genes in terrestrial and aquatic habitats of the Sør Rondane Mountains, including soils, on mosses, endolithic communities, cryoconite holes and supraglacial and subglacial meltwater lenses. This inventory was complemented with Denaturing Gradient Gel Electrophoresis targeting Chlorophyta and Cyanobacteria. OTUs belonging to the Rotifera, Chlorophyta, Tardigrada, Ciliophora, Cercozoa, Fungi, Bryophyta, Bacillariophyta, Collembola and Nematoda were present with a relative abundance of at least 0.1% in the eukaryotic communities. Cyanobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, FBP and Actinobacteria were the most abundant bacterial phyla. Multivariate analyses of the pyrosequencing data revealed a general lack of differentiation of both eukaryotes and prokaryotes according to habitat type. However, the bacterial community structure in the aquatic habitats was dominated by the filamentous cyanobacteria Leptolyngbya and appeared to be significantly different compared with those in dry soils, on mosses, and in endolithic habitats. A striking feature in all datasets was the detection of a relatively large amount of sequences new to science, which underscores the need for additional biodiversity assessments in Antarctic inland locations. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Microbiome analysis and confocal microscopy of used kitchen sponges reveal massive colonization by Acinetobacter, Moraxella and Chryseobacterium species.

PubMed

Cardinale, Massimiliano; Kaiser, Dominik; Lueders, Tillmann; Schnell, Sylvia; Egert, Markus

2017-07-19

The built environment (BE) and in particular kitchen environments harbor a remarkable microbial diversity, including pathogens. We analyzed the bacterial microbiome of used kitchen sponges by 454-pyrosequencing of 16S rRNA genes and fluorescence in situ hybridization coupled with confocal laser scanning microscopy (FISH-CLSM). Pyrosequencing showed a relative dominance of Gammaproteobacteria within the sponge microbiota. Five of the ten most abundant OTUs were closely related to risk group 2 (RG2) species, previously detected in the BE and kitchen microbiome. Regular cleaning of sponges, indicated by their users, significantly affected the microbiome structure. Two of the ten dominant OTUs, closely related to the RG2-species Chryseobacterium hominis and Moraxella osloensis, showed significantly greater proportions in regularly sanitized sponges, thereby questioning such sanitation methods in a long term perspective. FISH-CLSM showed an ubiquitous distribution of bacteria within the sponge tissue, concentrating in internal cavities and on sponge surfaces, where biofilm-like structures occurred. Image analysis showed local densities of up to 5.4 * 10 10 cells per cm 3 , and confirmed the dominance of Gammaproteobacteria. Our study stresses and visualizes the role of kitchen sponges as microbiological hot spots in the BE, with the capability to collect and spread bacteria with a probable pathogenic potential.

Enteral tube feeding alters the oral indigenous microbiota in elderly adults.

PubMed

Takeshita, Toru; Yasui, Masaki; Tomioka, Mikiko; Nakano, Yoshio; Shimazaki, Yoshihiro; Yamashita, Yoshihisa

2011-10-01

Enteral tube feeding is widely used to maintain nutrition for elderly adults with eating difficulties, but its long-term use alters the environment of the oral ecosystem. This study characterized the tongue microbiota of tube-fed elderly adults by analyzing the 16S rRNA gene. The terminal restriction fragment length polymorphism (T-RFLP) profiles of 44 tube-fed subjects were compared with those of 54 subjects fed orally (average age, 86.4 ± 6.9 years). Bar-coded pyrosequencing data were also obtained for a subset of the subjects from each group (15 tube-fed subjects and 16 subjects fed orally). The T-RFLP profiles demonstrated that the microbiota of the tube-fed subjects was distinct from that of the subjects fed orally (permutational multivariate analysis of variance [perMANOVA], P < 0.001). The pyrosequencing data revealed that 22 bacterial genera, including Corynebacterium, Peptostreptococcus, and Fusobacterium, were significantly more predominant in tube-fed subjects, whereas the dominant genera in the subjects fed orally, such as Streptococcus and Veillonella, were present in much lower proportions. Opportunistic pathogens rarely detected in the normal oral microbiota, such as Corynebacterium striatum and Streptococcus agalactiae, were often found in high proportions in tube-fed subjects. The oral indigenous microbiota is disrupted by the use of enteral feeding, allowing health-threatening bacteria to thrive.

Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production.

PubMed

Zheng, Xiong; Su, Yinglong; Li, Xiang; Xiao, Naidong; Wang, Dongbo; Chen, Yinguang

2013-05-07

Short-chain fatty acids (SCFAs) have been regarded as the excellent carbon source of wastewater biological nutrient removal, and sludge alkaline (pH 10) fermentation has been reported to achieve highly efficient SCFAs production. In this study, the underlying mechanisms for the improved SCFAs production at pH 10 were investigated by using 454 pyrosequencing and fluorescent in situ hybridization (FISH) to analyze the microbial community structures in sludge fermentation reactors. It was found that sludge fermentation at pH 10 increased the abundances of Pseudomonas sp. and Alcaligenes sp., which were able to excrete extracellular proteases and depolymerases, and thus enhanced the hydrolysis of insoluble sludge protein and polyhydroxyalkanoates (PHA). Meanwhile, the abundance of acid-producing bacteria (such as Clostridium sp.) in the reactor of pH 10 was also higher than that of uncontrolled pH, which benefited the acidification of soluble organic substrates. Further study indicated that sludge fermentation at pH 10 significantly decreased the number of methanogenic archaea, resulting in lower SCFAs consumption and lower methane production. Therefore, anaerobic sludge fermentation under alkaline conditions increased the abundances of bacteria involved in sludge hydrolysis and acidification, and decreased the abundance of methanogenic archaea, which favored the competition of bacteria over methanogens and resulted in the efficient production of SCFAs.

Gut content microbiota of introduced bigheaded carps (Hypophthalmichthys spp.) inhabiting the largest shallow lake in Central Europe.

PubMed

Borsodi, Andrea K; Szabó, Attila; Krett, Gergely; Felföldi, Tamás; Specziár, András; Boros, Gergely

2017-01-01

Studying the microbiota in the alimentary tract of bigheaded carps (Hypophthalmichthys spp.) gained special interest recently, as these types of investigations on non-native fish species may lead to a better understanding of their ecological role and feeding habits in an invaded habitat. For microbiological examinations, bigheaded carp gut contents and water column samples from Lake Balaton (Hungary) were collected from spring to autumn in 2013. Denaturing Gradient Gel Electrophoresis (DGGE) and pyrosequencing of the 16S rRNA gene were performed to reveal the composition. According to the DGGE patterns, bacterial communities of water samples separated clearly from that of the intestines. Moreover, the bacterial communities in the foreguts and hindguts were also strikingly dissimilar. Based on pyrosequencing, both foregut and hindgut samples were predominated by the fermentative genus Cetobacterium (Fusobacteria). The presence of some phytoplankton taxa and the high relative abundance of cellulose-degrading bacteria in the guts suggest that intestinal microbes may have an important role in digesting algae and making them utilizable for bigheaded carps that lack cellulase enzyme. In turn, the complete absence of typical heterotrophic freshwater bacteria in all studied sections of the intestines indicated that bacterioplankton probably has a negligible role in the nutrition of bigheaded carps. Copyright © 2016 Elsevier GmbH. All rights reserved.

Molecular Analysis of Bacterial Communities in Biofilms of a Drinking Water Clearwell

PubMed Central

Zhang, Minglu; Liu, Wenjun; Nie, Xuebiao; Li, Cuiping; Gu, Junnong; Zhang, Can

2012-01-01

Microbial community structures in biofilms of a clearwell in a drinking water supply system in Beijing, China were examined by clone library, terminal restriction fragment length polymorphism (T-RFLP) and 454 pyrosequencing of the amplified 16S rRNA gene. Six biofilm samples (designated R1–R6) collected from six locations (upper and lower sites of the inlet, middle and outlet) of the clearwell revealed similar bacterial patterns by T-RFLP analysis. With respect to the dominant groups, the phylotypes detected by clone library and T-RFLP generally matched each other. A total of 9,543 reads were obtained from samples located at the lower inlet and the lower outlet sites by pyrosequencing. The bacterial diversity of the two samples was compared at phylum and genus levels. Alphaproteobacteria dominated the communities in both samples and the genus of Sphingomonas constituted 75.1%–99.6% of this phylum. A high level of Sphingomonas sp. was first observed in the drinking water biofilms with 0.6–1.0 mg L−1 of chlorine residual. Disinfectant-resistant microorganisms deserve special attention in drinking water management. This study provides novel insights into the microbial populations in drinking water systems and highlights the important role of Sphingomonas species in biofilm formation. PMID:23059725

Pyrosequencing Analysis of Subgingival Microbiota in Distinct Periodontal Conditions.

PubMed

Park, O-J; Yi, H; Jeon, J H; Kang, S-S; Koo, K-T; Kum, K-Y; Chun, J; Yun, C-H; Han, S H

2015-07-01

Subgingival microorganisms are potentially associated with periodontal diseases. However, changes in the subgingival microbiota during the progress of periodontal diseases are poorly understood. In this study, we analyzed bacterial communities in the subgingival paper point samples from 32 Korean individuals with no sign of disease, gingivitis, or periodontitis using 454 FLX Titanium pyrosequencing. A total of 256,113 reads representing 26 phyla, 433 genera, and 1,016 species were detected. Bacteroidetes, Fusobacteria, Synergistetes, and Spirochaetes were the abundant phyla in periodontitis subjects, whereas Firmicutes and Proteobacteria were identified as the dominant phyla in the gingivitis and healthy subjects, respectively. Although high levels of Porphyromonas, Fusobacterium, Fretibacterium, Rothia, Filifactor, and Treponema genera were observed in the periodontitis subjects, Streptococcus, Capnocytophaga, Leptotrichia, and Haemophilus genera were found at high frequency in the gingivitis subjects. Species including Porphyromonas gingivalis, Fusobacterium nucleatum, and Fretibacterium fastidiosum were significantly increased in periodontitis subjects. On the other hand, Streptococcus pseudopneumoniae, Haemophilus parainfluenzae, and Leptotrichia hongkongensis were preferentially observed in the gingivitis subjects. Intriguingly, the halophile Halomonas hamiltonii was revealed as a predominant species in the healthy subjects. Based on Fast UniFrac analysis, distinctive bacterial clusters were classified for the healthy, gingivitis, and periodontitis state. The current findings might be useful for understanding the pathogenesis, diagnosis, and treatment of periodontal diseases. © International & American Associations for Dental Research 2015.

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

PubMed

Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

2016-01-01

Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested-MSP, pyrosequencing, and MS-HRM varied, the prognostic effect seemed similar (HR 1.74, 95 % CI 0.97-3.15; HR 1.85, 95 % CI 0.93-3.86; HR 1.83, 95 % CI 0.92-3.65, respectively). Our results show that upon optimizing and aligning four RET methylation assays with regard to primer location and sensitivity, differences in methylation frequencies and clinical sensitivities are observed; however, the effect on the marker's prognostic outcome is minimal.

Draft genome sequence of Thermoanaerobacterium sp. strain PSU-2 isolated from thermophilic hydrogen producing reactor.

PubMed

O-Thong, Sompong; Khongkliang, Peerawat; Mamimin, Chonticha; Singkhala, Apinya; Prasertsan, Poonsuk; Birkeland, Nils-Kåre

2017-06-01

Thermoanaerobacterium sp. strain PSU-2 was isolated from thermophilic hydrogen producing reactor and subjected to draft genome sequencing on 454 pyrosequencing and annotated on RAST. The draft genome sequence of strain PSU-2 contains 2,552,497 bases with an estimated G + C content of 35.2%, 2555 CDS, 8 rRNAs and 57 tRNAs. The strain had a number of genes responsible for carbohydrates metabolic, amino acids and derivatives, and protein metabolism of 17.7%, 14.39% and 9.81%, respectively. Strain PSU-2 also had gene responsible for hydrogen biosynthesis as well as the genes related to Ni-Fe hydrogenase. Comparative genomic analysis indicates strain PSU-2 shares about 94% genome sequence similarity with Thermoanaerobacterium xylanolyticum LX-11. The nucleotide sequence of this draft genome was deposited into DDBJ/ENA/GenBank under the accession MSQD00000000.

Using expected sequence features to improve basecalling accuracy of amplicon pyrosequencing data.

PubMed

Rask, Thomas S; Petersen, Bent; Chen, Donald S; Day, Karen P; Pedersen, Anders Gorm

2016-04-22

Amplicon pyrosequencing targets a known genetic region and thus inherently produces reads highly anticipated to have certain features, such as conserved nucleotide sequence, and in the case of protein coding DNA, an open reading frame. Pyrosequencing errors, consisting mainly of nucleotide insertions and deletions, are on the other hand likely to disrupt open reading frames. Such an inverse relationship between errors and expectation based on prior knowledge can be used advantageously to guide the process known as basecalling, i.e. the inference of nucleotide sequence from raw sequencing data. The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data. This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria virulence gene family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. This novel method can be used to increase accuracy of existing and future amplicon sequencing data, particularly where extensive prior knowledge is available about the obtained sequences, for example in analysis of the immunoglobulin VDJ region where Multipass can be combined with a model for the known recombining germline genes. Multipass is available for Roche 454 data at http://www.cbs.dtu.dk/services/MultiPass-1.0 , and the concept can potentially be implemented for other sequencing technologies as well.

Diversity, Biogeography, and Biodegradation Potential of Actinobacteria in the Deep-Sea Sediments along the Southwest Indian Ridge

PubMed Central

Chen, Ping; Zhang, Limin; Guo, Xiaoxuan; Dai, Xin; Liu, Li; Xi, Lijun; Wang, Jian; Song, Lei; Wang, Yuezhu; Zhu, Yaxin; Huang, Li; Huang, Ying

2016-01-01

The phylum Actinobacteria has been reported to be common or even abundant in deep marine sediments, however, knowledge about the diversity, distribution, and function of actinobacteria is limited. In this study, actinobacterial diversity in the deep sea along the Southwest Indian Ridge (SWIR) was investigated using both 16S rRNA gene pyrosequencing and culture-based methods. The samples were collected at depths of 1662–4000 m below water surface. Actinobacterial sequences represented 1.2–9.1% of all microbial 16S rRNA gene amplicon sequences in each sample. A total of 5 actinobacterial classes, 17 orders, 28 families, and 52 genera were detected by pyrosequencing, dominated by the classes Acidimicrobiia and Actinobacteria. Differences in actinobacterial community compositions were found among the samples. The community structure showed significant correlations to geochemical factors, notably pH, calcium, total organic carbon, total phosphorus, and total nitrogen, rather than to spatial distance at the scale of the investigation. In addition, 176 strains of the Actinobacteria class, belonging to 9 known orders, 18 families, and 29 genera, were isolated. Among these cultivated taxa, 8 orders, 13 families, and 15 genera were also recovered by pyrosequencing. At a 97% 16S rRNA gene sequence similarity, the pyrosequencing data encompassed 77.3% of the isolates but the isolates represented only 10.3% of the actinobacterial reads. Phylogenetic analysis of all the representative actinobacterial sequences and isolates indicated that at least four new orders within the phylum Actinobacteria were detected by pyrosequencing. More than half of the isolates spanning 23 genera and all samples demonstrated activity in the degradation of refractory organics, including polycyclic aromatic hydrocarbons and polysaccharides, suggesting their potential ecological functions and biotechnological applications for carbon recycling. PMID:27621725

Changes in N-Transforming Archaea and Bacteria in Soil during the Establishment of Bioenergy Crops

PubMed Central

Mao, Yuejian; Yannarell, Anthony C.; Mackie, Roderick I.

2011-01-01

Widespread adaptation of biomass production for bioenergy may influence important biogeochemical functions in the landscape, which are mainly carried out by soil microbes. Here we explore the impact of four potential bioenergy feedstock crops (maize, switchgrass, Miscanthus X giganteus, and mixed tallgrass prairie) on nitrogen cycling microorganisms in the soil by monitoring the changes in the quantity (real-time PCR) and diversity (barcoded pyrosequencing) of key functional genes (nifH, bacterial/archaeal amoA and nosZ) and 16S rRNA genes over two years after bioenergy crop establishment. The quantities of these N-cycling genes were relatively stable in all four crops, except maize (the only fertilized crop), in which the population size of AOB doubled in less than 3 months. The nitrification rate was significantly correlated with the quantity of ammonia-oxidizing archaea (AOA) not bacteria (AOB), indicating that archaea were the major ammonia oxidizers. Deep sequencing revealed high diversity of nifH, archaeal amoA, bacterial amoA, nosZ and 16S rRNA genes, with 229, 309, 330, 331 and 8989 OTUs observed, respectively. Rarefaction analysis revealed the diversity of archaeal amoA in maize markedly decreased in the second year. Ordination analysis of T-RFLP and pyrosequencing results showed that the N-transforming microbial community structures in the soil under these crops gradually differentiated. Thus far, our two-year study has shown that specific N-transforming microbial communities develop in the soil in response to planting different bioenergy crops, and each functional group responded in a different way. Our results also suggest that cultivation of maize with N-fertilization increases the abundance of AOB and denitrifiers, reduces the diversity of AOA, and results in significant changes in the structure of denitrification community. PMID:21935454

Deep 16S rRNA Pyrosequencing Reveals a Bacterial Community Associated with Banana Fusarium Wilt Disease Suppression Induced by Bio-Organic Fertilizer Application

PubMed Central

Ruan, Yunze; Xue, Chao; Zhang, Jian; Li, Rong; Shen, Qirong

2014-01-01

Our previous work demonstrated that application of a bio-organic fertilizer (BIO) to a banana mono-culture orchard with serious Fusarium wilt disease effectively decreased the number of soil Fusarium sp. and controlled the soil-borne disease. Because bacteria are an abundant and diverse group of soil organisms that responds to soil health, deep 16 S rRNA pyrosequencing was employed to characterize the composition of the bacterial community to investigate how it responded to BIO or the application of other common composts and to explore the potential correlation between bacterial community, BIO application and Fusarium wilt disease suppression. After basal quality control, 137,646 sequences and 9,388 operational taxonomic units (OTUs) were obtained from the 15 soil samples. Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria were the most frequent phyla and comprised up to 75.3% of the total sequences. Compared to the other soil samples, BIO-treated soil revealed higher abundances of Gemmatimonadetes and Acidobacteria, while Bacteroidetes were found in lower abundance. Meanwhile, on genus level, higher abundances compared to other treatments were observed for Gemmatimonas and Gp4. Correlation and redundancy analysis showed that the abundance of Gemmatimonas and Sphingomonas and the soil total nitrogen and ammonium nitrogen content were higher after BIO application, and they were all positively correlated with disease suppression. Cumulatively, the reduced Fusarium wilt disease incidence that was seen after BIO was applied for 1-year might be attributed to the general suppression based on a shift within the bacteria soil community, including specific enrichment of Gemmatimonas and Sphingomonas. PMID:24871319

Stratified Microbial Structure and Activity in Sulfide- and Methane-Producing Anaerobic Sewer Biofilms

PubMed Central

Sun, Jing; Hu, Shihu; Sharma, Keshab Raj; Ni, Bing-Jie

2014-01-01

Simultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 μm, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescence in situ hybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 μm and 700 μm, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera, Desulfobulbus, Desulfomicrobium, Desulfovibrio, Desulfatiferula, and Desulforegula, while about 90% of the MA population belonged to the genus Methanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates. PMID:25192994

Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

PubMed

Kröber, Magdalena; Bekel, Thomas; Diaz, Naryttza N; Goesmann, Alexander; Jaenicke, Sebastian; Krause, Lutz; Miller, Dimitri; Runte, Kai J; Viehöver, Prisca; Pühler, Alfred; Schlüter, Andreas

2009-06-01

The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

Culture-Independent Analysis of Bacterial Fuel Contamination Provides Insight into the Level of Concordance with the Standard Industry Practice of Aerobic Cultivation ▿ †

PubMed Central

White, Judith; Gilbert, Jack; Hill, Graham; Hill, Edward; Huse, Susan M.; Weightman, Andrew J.; Mahenthiralingam, Eshwar

2011-01-01

Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of Pseudomonas (21%), Burkholderia (7%), and Bacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed Proteobacteria and Firmicutes to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samples (indicated by “JW”) was performed, Betaproteobacteria (42.8%) and Gammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera were Marinobacter (15.4%; JW57), Achromobacter (41.6%; JW63), Burkholderia (80.7%; JW76), and Halomonas (66.2%; JW78), all of which were also observed by DGGE. However, the Clostridia (38.5%) and Deltaproteobacteria (11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a Pseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a Mangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a Burkholderia vietnamiensis strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as Pseudomonas. PMID:21602386

Culture-independent analysis of bacterial fuel contamination provides insight into the level of concordance with the standard industry practice of aerobis cultivation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, J.; Gilbert, J. A.; Hill, G.

2011-07-01

Bacterial diversity in contaminated fuels has not been systematically investigated using cultivation-independent methods. The fuel industry relies on phenotypic cultivation-based contaminant identification, which may lack accuracy and neglect difficult-to-culture taxa. By the use of industry practice aerobic cultivation, 16S rRNA gene sequencing, and strain genotyping, a collection of 152 unique contaminant isolates from 54 fuel samples was assembled, and a dominance of Pseudomonas (21%), Burkholderia (7%), and Bacillus (7%) was demonstrated. Denaturing gradient gel electrophoresis (DGGE) of 15 samples revealed Proteobacteria and Firmicutes to be the most abundant phyla. When 16S rRNA V6 gene pyrosequencing of four selected fuel samplesmore » (indicated by 'JW') was performed, Betaproteobacteria (42.8%) and Gammaproteobacteria (30.6%) formed the largest proportion of reads; the most abundant genera were Marinobacter (15.4%; JW57), Achromobacter (41.6%; JW63), Burkholderia (80.7%; JW76), and Halomonas (66.2%; JW78), all of which were also observed by DGGE. However, the Clostridia (38.5%) and Deltaproteobacteria (11.1%) identified by pyrosequencing in sample JW57 were not observed by DGGE or aerobic culture. Genotyping revealed three instances where identical strains were found: (i) a Pseudomonas sp. strain recovered from 2 different diesel fuel tanks at a single industrial site; (ii) a Mangroveibacter sp. strain isolated from 3 biodiesel tanks at a single refinery site; and (iii) a Burkholderia vietnamiensis strain present in two unrelated automotive diesel samples. Overall, aerobic cultivation of fuel contaminants recovered isolates broadly representative of the phyla and classes present but lacked accuracy by overrepresenting members of certain groups such as Pseudomonas.« less

Next-generation pyrosequencing analysis of microbial biofilm communities on granular activated carbon in treatment of oil sands process-affected water.

PubMed

Islam, M Shahinoor; Zhang, Yanyan; McPhedran, Kerry N; Liu, Yang; Gamal El-Din, Mohamed

2015-06-15

The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>10(9) gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O3/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Deep sequencing of hepatitis C virus hypervariable region 1 reveals no correlation between genetic heterogeneity and antiviral treatment outcome

PubMed Central

2014-01-01

Background Hypervariable region 1 (HVR1) contained within envelope protein 2 (E2) gene is the most variable part of HCV genome and its translation product is a major target for the host immune response. Variability within HVR1 may facilitate evasion of the immune response and could affect treatment outcome. The aim of the study was to analyze the impact of HVR1 heterogeneity employing sensitive ultra-deep sequencing, on the outcome of PEG-IFN-α (pegylated interferon α) and ribavirin treatment. Methods HVR1 sequences were amplified from pretreatment serum samples of 25 patients infected with genotype 1b HCV (12 responders and 13 non-responders) and were subjected to pyrosequencing (GS Junior, 454/Roche). Reads were corrected for sequencing error using ShoRAH software, while population reconstruction was done using three different minimal variant frequency cut-offs of 1%, 2% and 5%. Statistical analysis was done using Mann–Whitney and Fisher’s exact tests. Results Complexity, Shannon entropy, nucleotide diversity per site, genetic distance and the number of genetic substitutions were not significantly different between responders and non-responders, when analyzing viral populations at any of the three frequencies (≥1%, ≥2% and ≥5%). When clonal sample was used to determine pyrosequencing error, 4% of reads were found to be incorrect and the most abundant variant was present at a frequency of 1.48%. Use of ShoRAH reduced the sequencing error to 1%, with the most abundant erroneous variant present at frequency of 0.5%. Conclusions While deep sequencing revealed complex genetic heterogeneity of HVR1 in chronic hepatitis C patients, there was no correlation between treatment outcome and any of the analyzed quasispecies parameters. PMID:25016390

Next-Generation Pyrosequencing Analysis of Microbial Biofilm Communities on Granular Activated Carbon in Treatment of Oil Sands Process-Affected Water

PubMed Central

Islam, M. Shahinoor; Zhang, Yanyan; McPhedran, Kerry N.

2015-01-01

The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>109 gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O3/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds. PMID:25841014

High-resolution SAR11 ecotype dynamics at the Bermuda Atlantic Time-series Study site by phylogenetic placement of pyrosequences

PubMed Central

Vergin, Kevin L; Beszteri, Bánk; Monier, Adam; Cameron Thrash, J; Temperton, Ben; Treusch, Alexander H; Kilpert, Fabian; Worden, Alexandra Z; Giovannoni, Stephen J

2013-01-01

Advances in next-generation sequencing technologies are providing longer nucleotide sequence reads that contain more information about phylogenetic relationships. We sought to use this information to understand the evolution and ecology of bacterioplankton at our long-term study site in the Western Sargasso Sea. A bioinformatics pipeline called PhyloAssigner was developed to align pyrosequencing reads to a reference multiple sequence alignment of 16S ribosomal RNA (rRNA) genes and assign them phylogenetic positions in a reference tree using a maximum likelihood algorithm. Here, we used this pipeline to investigate the ecologically important SAR11 clade of Alphaproteobacteria. A combined set of 2.7 million pyrosequencing reads from the 16S rRNA V1–V2 regions, representing 9 years at the Bermuda Atlantic Time-series Study (BATS) site, was quality checked and parsed into a comprehensive bacterial tree, yielding 929 036 Alphaproteobacteria reads. Phylogenetic structure within the SAR11 clade was linked to seasonally recurring spatiotemporal patterns. This analysis resolved four new SAR11 ecotypes in addition to five others that had been described previously at BATS. The data support a conclusion reached previously that the SAR11 clade diversified by subdivision of niche space in the ocean water column, but the new data reveal a more complex pattern in which deep branches of the clade diversified repeatedly across depth strata and seasonal regimes. The new data also revealed the presence of an unrecognized clade of Alphaproteobacteria, here named SMA-1 (Sargasso Mesopelagic Alphaproteobacteria, group 1), in the upper mesopelagic zone. The high-resolution phylogenetic analyses performed herein highlight significant, previously unknown, patterns of evolutionary diversification, within perhaps the most widely distributed heterotrophic marine bacterial clade, and strongly links to ecosystem regimes. PMID:23466704

High-resolution SAR11 ecotype dynamics at the Bermuda Atlantic Time-series Study site by phylogenetic placement of pyrosequences.

PubMed

Vergin, Kevin L; Beszteri, Bánk; Monier, Adam; Thrash, J Cameron; Temperton, Ben; Treusch, Alexander H; Kilpert, Fabian; Worden, Alexandra Z; Giovannoni, Stephen J

2013-07-01

Advances in next-generation sequencing technologies are providing longer nucleotide sequence reads that contain more information about phylogenetic relationships. We sought to use this information to understand the evolution and ecology of bacterioplankton at our long-term study site in the Western Sargasso Sea. A bioinformatics pipeline called PhyloAssigner was developed to align pyrosequencing reads to a reference multiple sequence alignment of 16S ribosomal RNA (rRNA) genes and assign them phylogenetic positions in a reference tree using a maximum likelihood algorithm. Here, we used this pipeline to investigate the ecologically important SAR11 clade of Alphaproteobacteria. A combined set of 2.7 million pyrosequencing reads from the 16S rRNA V1-V2 regions, representing 9 years at the Bermuda Atlantic Time-series Study (BATS) site, was quality checked and parsed into a comprehensive bacterial tree, yielding 929 036 Alphaproteobacteria reads. Phylogenetic structure within the SAR11 clade was linked to seasonally recurring spatiotemporal patterns. This analysis resolved four new SAR11 ecotypes in addition to five others that had been described previously at BATS. The data support a conclusion reached previously that the SAR11 clade diversified by subdivision of niche space in the ocean water column, but the new data reveal a more complex pattern in which deep branches of the clade diversified repeatedly across depth strata and seasonal regimes. The new data also revealed the presence of an unrecognized clade of Alphaproteobacteria, here named SMA-1 (Sargasso Mesopelagic Alphaproteobacteria, group 1), in the upper mesopelagic zone. The high-resolution phylogenetic analyses performed herein highlight significant, previously unknown, patterns of evolutionary diversification, within perhaps the most widely distributed heterotrophic marine bacterial clade, and strongly links to ecosystem regimes.

Stratified microbial structure and activity in sulfide- and methane-producing anaerobic sewer biofilms.

PubMed

Sun, Jing; Hu, Shihu; Sharma, Keshab Raj; Ni, Bing-Jie; Yuan, Zhiguo

2014-11-01

Simultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 μm, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescence in situ hybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 μm and 700 μm, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera, Desulfobulbus, Desulfomicrobium, Desulfovibrio, Desulfatiferula, and Desulforegula, while about 90% of the MA population belonged to the genus Methanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Descriptive Biomarkers for Assessing Breast Cancer Risk

DTIC Science & Technology

2010-10-01

and we are making significant progress on Tasks 6 and 7. We completed methylation analyses of three genes (RASSF1, SFRP1 and GSTP1 ) on all samples...promoter hypermethylation; RASSF1, GSTP1 , SFRP1 12 karcaro@nre.umass.edu Arcaro, Kathleen F Annual Report...methylation analysis by pyrosequencing. PCR amplification and pyrosequencing has been completed for three genes, RASSF1, SFRP1 and GSTP1 and have

Pyrosequencing Analysis of Norovirus Genogroup II Distribution in Sewage and Oysters: First Detection of GII.17 Kawasaki 2014 in Oysters.

PubMed

Pu, Jian; Kazama, Shinobu; Miura, Takayuki; Azraini, Nabila Dhyan; Konta, Yoshimitsu; Ito, Hiroaki; Ueki, You; Cahyaningrum, Ermaya Eka; Omura, Tatsuo; Watanabe, Toru

2016-12-01

Norovirus GII.3, GII.4, and GII.17 were detected using pyrosequencing in sewage and oysters in January and February 2015, in Japan. The strains in sewage and oyster samples were genetically identical or similar, predominant strains belonging to GII.17 Kawasaki 2014 lineage. This is the first report of GII.17 Kawasaki 2014 in oysters.

Combined Real-Time PCR and Pyrosequencing Strategy for Objective, Sensitive, Specific, and High-Throughput Identification of Reduced Susceptibility to Penicillins in Neisseria meningitidis▿

PubMed Central

Thulin, Sara; Olcén, Per; Fredlund, Hans; Unemo, Magnus

2008-01-01

A segment of penA in Neisseria meningitidis strains (n = 127), including two nucleotide sites closely associated to reduced susceptibility to penicillins, was amplified and pyrosequenced. All results were in concordance with Sanger sequencing, and a high correlation between alterations in the two Peni-specific sites and reduced susceptibility to penicillins was identified. PMID:18070955

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

PubMed

Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

2002-01-01

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Identification and genotyping of molluscum contagiosum virus from genital swab samples by real-time PCR and Pyrosequencing.

PubMed

Trama, Jason P; Adelson, Martin E; Mordechai, Eli

2007-12-01

Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. To develop a rapid method for identifying patients infected with MCV via swab sampling. Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.

The impact of rearing environment on the development of gut microbiota in tilapia larvae

PubMed Central

Giatsis, Christos; Sipkema, Detmer; Smidt, Hauke; Heilig, Hans; Benvenuti, Giulia; Verreth, Johan; Verdegem, Marc

2015-01-01

This study explores the effect of rearing environment on water bacterial communities (BC) and the association with those present in the gut of Nile tilapia larvae (Oreochromis niloticus, Linnaeus) grown in either recirculating or active suspension systems. 454 pyrosequencing of PCR-amplified 16S rRNA gene fragments was applied to characterize the composition of water, feed and gut bacteria communities. Observed changes in water BC over time and differences in water BCs between systems were highly correlated with corresponding water physico-chemical properties. Differences in gut bacterial communities during larval development were correlated with differences in water communities between systems. The correlation of feed BC with those in the gut was minor compared to that between gut and water, reflected by the fact that 4 to 43 times more OTUs were shared between water and gut than between gut and feed BC. Shared OTUs between water and gut suggest a successful transfer of microorganisms from water into the gut, and give insight about the niche and ecological adaptability of water microorganisms inside the gut. These findings suggest that steering of gut microbial communities could be possible through water microbial management derived by the design and functionality of the rearing system. PMID:26658351

Major histocompatibility complex class II compatibility, but not class I, predicts mate choice in a bird with highly developed olfaction.

PubMed

Strandh, Maria; Westerdahl, Helena; Pontarp, Mikael; Canbäck, Björn; Dubois, Marie-Pierre; Miquel, Christian; Taberlet, Pierre; Bonadonna, Francesco

2012-11-07

Mate choice for major histocompatibility complex (MHC) compatibility has been found in several taxa, although rarely in birds. MHC is a crucial component in adaptive immunity and by choosing an MHC-dissimilar partner, heterozygosity and potentially broad pathogen resistance is maximized in the offspring. The MHC genotype influences odour cues and preferences in mammals and fish and hence olfactory-based mate choice can occur. We tested whether blue petrels, Halobaena caerulea, choose partners based on MHC compatibility. This bird is long-lived, monogamous and can discriminate between individual odours using olfaction, which makes it exceptionally well suited for this analysis. We screened MHC class I and II B alleles in blue petrels using 454-pyrosequencing and quantified the phylogenetic, functional and allele-sharing similarity between individuals. Partners were functionally more dissimilar at the MHC class II B loci than expected from random mating (p = 0.033), whereas there was no such difference at the MHC class I loci. Phylogenetic and non-sequence-based MHC allele-sharing measures detected no MHC dissimilarity between partners for either MHC class I or II B. Our study provides evidence of mate choice for MHC compatibility in a bird with a high dependency on odour cues, suggesting that MHC odour-mediated mate choice occurs in birds.

Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments.

PubMed

Korsak, N; Taminiau, B; Leclercq, M; Nezer, C; Crevecoeur, S; Ferauche, C; Detry, E; Delcenserie, V; Daube, G

2015-06-01

Milk kefir is produced by fermenting milk in the presence of kefir grains. This beverage has several benefits for human health. The aim of this experiment was to analyze 5 kefir grains (and their products) using a targeted metagenetic approach. Of the 5 kefir grains analyzed, 1 was purchased in a supermarket, 2 were provided by the Ministry of Agriculture (Namur, Belgium), and 2 were provided by individuals. The metagenetic approach targeted the V1-V3 fragment of the 16S ribosomal (r)DNA for the grains and the resulting beverages at 2 levels of grain incorporation (5 and 10%) to identify the bacterial species population. In contrast, the 26S rDNA pyrosequencing was performed only on kefir grains with the aim of assessing the yeast populations. In parallel, pH measurements were performed on the kefir obtained from the kefir grains using 2 incorporation rates. Regarding the bacterial population, 16S pyrosequencing revealed the presence of 20 main bacterial species, with a dominance of the following: Lactobacillus kefiranofaciens, Lactococcus lactis ssp. cremoris, Gluconobacter frateurii, Lactobacillus kefiri, Acetobacter orientalis, and Acetobacter lovaniensis. An important difference was noticed between the kefir samples: kefir grain purchased from a supermarket (sample E) harbored a much higher proportion of several operational taxonomic units of Lactococcus lactis and Leuconostoc mesenteroides. This sample of grain was macroscopically different from the others in terms of size, apparent cohesion of the grains, structure, and texture, probably associated with a lower level of Lactobacillus kefiranofaciens. The kefir (at an incorporation rate of 5%) produced from this sample of grain was characterized by a lower pH value (4.5) than the others. The other 4 samples of kefir (5%) had pH values above 5. Comparing the kefir grain and the kefir, an increase in the population of Gluconobacter in grain sample B was observed. This was also the case for Acetobacter orientalis in sample D. In relation to 26S pyrosequencing, our study revealed the presence of 3 main yeast species: Naumovozyma spp., Kluyveromyces marxianus, and Kazachastania khefir. For Naumovozyma, further studies are needed to assess the isolation of new species. In conclusion, this study has proved that it is possible to establish the patterns of bacterial and yeast composition of kefir and kefir grain. This was only achieved with the use of high-throughput sequencing techniques. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Bacterial communities in the phylloplane of Prunus species.

PubMed

Jo, Yeonhwa; Cho, Jin Kyong; Choi, Hoseong; Chu, Hyosub; Lian, Sen; Cho, Won Kyong

2015-04-01

Bacterial populations in the phylloplane of four different Prunus species were investigated by 16 S rRNA pyrosequencing. Bioinformatic analysis identified an average of 510 operational taxonomic units belonging to 159 genera in 76 families. The two genera, Sphingomonas and Methylobacterium, were dominant in the phylloplane of four Prunus species. Twenty three genera were commonly identified in the four Prunus species, indicating a high level of bacterial diversity dependent on the plant species. Our study based on 16 S rRNA sequencing reveals the complexity of bacterial diversity in the phylloplane of Prunus species in detail. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two novel Alphaflexiviridae members revealed by deep sequencing of the Vanilla (Orchidaceae) virome.

PubMed

Grisoni, Michel; Marais, Armelle; Filloux, Denis; Saison, Anne; Faure, Chantal; Julian, Charlotte; Theil, Sébastien; Contreras, Sandy; Teycheney, Pierre-Yves; Roumagnac, Philippe; Candresse, Thierry

2017-12-01

The genomes of two novel viruses were assembled from 454 pyrosequencing data obtained from vanilla leaves from La Réunion. Based on genome organization and homologies, one agent was unambiguously classified as a member of the genus Potexvirus and named vanilla virus X (VVX). The second one, vanilla latent virus (VLV), is phylogenetically close to three unclassified members of the family Alphaflexiviridae with similarity to allexiviruses, and despite the presence of an additional 8-kDa open reading frame, we propose to include VLV as a new member of the genus Allexivirus. Both VVX and VLV were mechanically transmitted to vanilla plants, resulting in asymptomatic infections.

Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing

DTIC Science & Technology

2010-08-25

or intentional genetic modifications that circumvent the targets of the detection assays or in the case of a biological attack using an antibiotic ...genetic changes conferring antibiotic resistance can be deciphered rapidly and accurately using WGS. We demonstrate the utility of Roche 454...Rapid Identification of Genetic Modifications in Bacillus anthracis Using Whole Genome Draft Sequences Generated by 454 Pyrosequencing Peter E. Chen1

Microbial community structure in the gut of the New Zealand insect Auckland tree weta (Hemideina thoracica).

PubMed

Waite, David W; Dsouza, Melissa; Biswas, Kristi; Ward, Darren F; Deines, Peter; Taylor, Michael W

2015-05-01

The endemic New Zealand weta is an enigmatic insect. Although the insect is well known by its distinctive name, considerable size, and morphology, many basic aspects of weta biology remain unknown. Here, we employed cultivation-independent enumeration techniques and rRNA gene sequencing to investigate the gut microbiota of the Auckland tree weta (Hemideina thoracica). Fluorescence in situ hybridisation performed on different sections of the gut revealed a bacterial community of fluctuating density, while rRNA gene-targeted amplicon pyrosequencing revealed the presence of a microbial community containing high bacterial diversity, but an apparent absence of archaea. Bacteria were further studied using full-length 16S rRNA gene sequences, with statistical testing of bacterial community membership against publicly available termite- and cockroach-derived sequences, revealing that the weta gut microbiota is similar to that of cockroaches. These data represent the first analysis of the weta microbiota and provide initial insights into the potential function of these microorganisms.

Genome-wide placental DNA methylation analysis of severely growth-discordant monochorionic twins reveals novel epigenetic targets for intrauterine growth restriction.

PubMed

Roifman, Maian; Choufani, Sanaa; Turinsky, Andrei L; Drewlo, Sascha; Keating, Sarah; Brudno, Michael; Kingdom, John; Weksberg, Rosanna

2016-01-01

Intrauterine growth restriction (IUGR), which refers to reduced fetal growth in the context of placental insufficiency, is etiologically heterogeneous. IUGR is associated not only with perinatal morbidity and mortality but also with adult-onset disorders, such as cardiovascular disease and diabetes, posing a major health burden. Placental epigenetic dysregulation has been proposed as one mechanism that causes IUGR; however, the spectrum of epigenetic pathophysiological mechanisms leading to IUGR remains to be elucidated. Monozygotic monochorionic twins are particularly affected by IUGR, in the setting of severe discordant growth. Because monozygotic twins have the same genotype at conception and a shared maternal environment, they provide an ideal model system for studying epigenetic dysregulation of the placenta. We compared genome-wide placental DNA methylation patterns of severely growth-discordant twins to identify novel candidate genes for IUGR. Snap-frozen placental samples for eight severely growth-discordant monozygotic monochorionic twin pairs were obtained at delivery from each twin. A high-resolution DNA methylation array platform was used to identify methylation differences between IUGR and normal twins. Our analysis revealed differentially methylated regions in the promoters of eight genes: DECR1, ZNF300, DNAJA4, CCL28, LEPR, HSPA1A/L, GSTO1, and GNE. The largest methylation differences between the two groups were in the promoters of DECR1 and ZNF300. The significance of these group differences was independently validated by bisulfite pyrosequencing, implicating aberrations in fatty acid beta oxidation and transcriptional regulation, respectively. Further analysis of the array data identified methylation changes most prominently affecting the Wnt and cadherin pathways in the IUGR cohort. Our results suggest that IUGR in monozygotic twins is associated with impairments in lipid metabolism and transcriptional regulation as well as cadherin and Wnt signaling. We show that monozygotic monochorionic twins discordant for growth provide a useful model to study one type of the epigenetic placental dysregulation that drives IUGR.

Actinobacterial Diversity in Volcanic Caves and Associated Geomicrobiological Interactions

PubMed Central

Riquelme, Cristina; Marshall Hathaway, Jennifer J.; Enes Dapkevicius, Maria de L. N.; Miller, Ana Z.; Kooser, Ara; Northup, Diana E.; Jurado, Valme; Fernandez, Octavio; Saiz-Jimenez, Cesareo; Cheeptham, Naowarat

2015-01-01

Volcanic caves are filled with colorful microbial mats on the walls and ceilings. These volcanic caves are found worldwide, and studies are finding vast bacteria diversity within these caves. One group of bacteria that can be abundant in volcanic caves, as well as other caves, is Actinobacteria. As Actinobacteria are valued for their ability to produce a variety of secondary metabolites, rare and novel Actinobacteria are being sought in underexplored environments. The abundance of novel Actinobacteria in volcanic caves makes this environment an excellent location to study these bacteria. Scanning electron microscopy (SEM) from several volcanic caves worldwide revealed diversity in the morphologies present. Spores, coccoid, and filamentous cells, many with hair-like or knobby extensions, were some of the microbial structures observed within the microbial mat samples. In addition, the SEM study pointed out that these features figure prominently in both constructive and destructive mineral processes. To further investigate this diversity, we conducted both Sanger sequencing and 454 pyrosequencing of the Actinobacteria in volcanic caves from four locations, two islands in the Azores, Portugal, and Hawai'i and New Mexico, USA. This comparison represents one of the largest sequencing efforts of Actinobacteria in volcanic caves to date. The diversity was shown to be dominated by Actinomycetales, but also included several newly described orders, such as Euzebyales, and Gaiellales. Sixty-two percent of the clones from the four locations shared less than 97% similarity to known sequences, and nearly 71% of the clones were singletons, supporting the commonly held belief that volcanic caves are an untapped resource for novel and rare Actinobacteria. The amplicon libraries depicted a wider view of the microbial diversity in Azorean volcanic caves revealing three additional orders, Rubrobacterales, Solirubrobacterales, and Coriobacteriales. Studies of microbial ecology in volcanic caves are still very limited. To rectify this deficiency, the results from our study help fill in the gaps in our knowledge of actinobacterial diversity and their potential roles in the volcanic cave ecosystems. PMID:26696966

Stability and phylogenetic correlation in gut microbiota: lessons from ants and apes.

PubMed

Sanders, Jon G; Powell, Scott; Kronauer, Daniel J C; Vasconcelos, Heraldo L; Frederickson, Megan E; Pierce, Naomi E

2014-03-01

Correlation between gut microbiota and host phylogeny could reflect codiversification over shared evolutionary history or a selective environment that is more similar in related hosts. These alternatives imply substantial differences in the relationship between host and symbiont, but can they be distinguished based on patterns in the community data themselves? We explored patterns of phylogenetic correlation in the distribution of gut bacteria among species of turtle ants (genus Cephalotes), which host a dense gut microbial community. We used 16S rRNA pyrosequencing from 25 Cephalotes species to show that their gut community is remarkably stable, from the colony to the genus level. Despite this overall similarity, the existing differences among species' microbiota significantly correlated with host phylogeny. We introduced a novel analytical technique to test whether these phylogenetic correlations are derived from recent bacterial evolution, as would be expected in the case of codiversification, or from broader shifts more likely to reflect environmental filters imposed by factors such as diet or habitat. We also tested this technique on a published data set of ape microbiota, confirming earlier results while revealing previously undescribed patterns of phylogenetic correlation. Our results indicated a high degree of partner fidelity in the Cephalotes microbiota, suggesting that vertical transmission of the entire community could play an important role in the evolution and maintenance of the association. As additional comparative microbiota data become available, the techniques presented here can be used to explore trends in the evolution of host-associated microbial communities. © 2014 John Wiley & Sons Ltd.

Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

PubMed

Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc

2015-06-25

Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex analysis. Using this combined multiplex approach also substantially reduced the production of waste material. These genotyping assays appear therefore to be biologically, economically and ecologically highly relevant, being worth to be integrated in genetic-based predictive strategies for better selecting patients at risk for prostate cancer. In addition, the same approach could now equally be transposed to other clinical/research applications relying on the computation of MGRS based on multi-SNP genotyping.

The Effect of Long-Term Continuous Cropping of Black Pepper on Soil Bacterial Communities as Determined by 454 Pyrosequencing.

PubMed

Xiong, Wu; Li, Zhigang; Liu, Hongjun; Xue, Chao; Zhang, Ruifu; Wu, Huasong; Li, Rong; Shen, Qirong

2015-01-01

In the present study, 3 replanted black pepper orchards with continuously cropping histories for 10, 21, and 55 years in tropical China, were selected for investigating the effect of monoculture on soil physiochemical properties, enzyme activities, bacterial abundance, and bacterial community structures. Results showed long-term continuous cropping led to a significant decline in soil pH, organic matter contents, enzymatic activities, and resulted in a decrease in soil bacterial abundance. 454 pyrosequencing analysis of 16S rRNA genes revealed that the Acidobacteria and Proteobacteria were the main phyla in the replanted black pepper orchard soils, comprising up to 73.82% of the total sequences; the relative abundances of Bacteroidetes and Firmicutes phyla decreased with long-term continuous cropping; and at genus level, the Pseudomonas abundance significantly depleted after 21 years continuous cropping. In addition, bacterial diversity significantly decreased after 55 years black pepper continuous cropping; obvious variations for community structures across the 3 time-scale replanted black pepper orchards were observed, suggesting monoculture duration was the major determinant for bacterial community structure. Overall, continuous cropping during black pepper cultivation led to a significant decline in soil pH, organic matter contents, enzymatic activities, resulted a decrease in soil bacterial abundance, and altered soil microbial community membership and structure, which in turn resulted in black pepper poor growth in the continuous cropping system.

Bacterial community variations in an alfalfa-rice rotation system revealed by 16S rRNA gene 454-pyrosequencing.

PubMed

Lopes, Ana R; Manaia, Célia M; Nunes, Olga C

2014-03-01

Crop rotation is a practice harmonized with the sustainable rice production. Nevertheless, the implications of this empirical practice are not well characterized, mainly in relation to the bacterial community composition and structure. In this study, the bacterial communities of two adjacent paddy fields in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot were characterized before rice seeding and after harvesting, using 454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria, Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all the samples, there were variations in relative abundance of these groups. Samples from the 3rd and 4th years of the crop rotation differed on the higher abundance of groups of presumable aerobic bacteria and of presumable anaerobic and acidobacterial groups, respectively. Members of the phylum Nitrospira were more abundant after rice harvest than in the previously sampled period. Rice cropping was positively correlated with the abundance of members of the orders Acidobacteriales and 'Solibacterales' and negatively with lineages such as Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations occurring in the microbial communities in soils under sustainable rice production, based on real-world data. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

SeqTrim: a high-throughput pipeline for pre-processing any type of sequence read

PubMed Central

2010-01-01

Background High-throughput automated sequencing has enabled an exponential growth rate of sequencing data. This requires increasing sequence quality and reliability in order to avoid database contamination with artefactual sequences. The arrival of pyrosequencing enhances this problem and necessitates customisable pre-processing algorithms. Results SeqTrim has been implemented both as a Web and as a standalone command line application. Already-published and newly-designed algorithms have been included to identify sequence inserts, to remove low quality, vector, adaptor, low complexity and contaminant sequences, and to detect chimeric reads. The availability of several input and output formats allows its inclusion in sequence processing workflows. Due to its specific algorithms, SeqTrim outperforms other pre-processors implemented as Web services or standalone applications. It performs equally well with sequences from EST libraries, SSH libraries, genomic DNA libraries and pyrosequencing reads and does not lead to over-trimming. Conclusions SeqTrim is an efficient pipeline designed for pre-processing of any type of sequence read, including next-generation sequencing. It is easily configurable and provides a friendly interface that allows users to know what happened with sequences at every pre-processing stage, and to verify pre-processing of an individual sequence if desired. The recommended pipeline reveals more information about each sequence than previously described pre-processors and can discard more sequencing or experimental artefacts. PMID:20089148

Enteral Tube Feeding Alters the Oral Indigenous Microbiota in Elderly Adults ▿ †

PubMed Central

Takeshita, Toru; Yasui, Masaki; Tomioka, Mikiko; Nakano, Yoshio; Shimazaki, Yoshihiro; Yamashita, Yoshihisa

2011-01-01

Enteral tube feeding is widely used to maintain nutrition for elderly adults with eating difficulties, but its long-term use alters the environment of the oral ecosystem. This study characterized the tongue microbiota of tube-fed elderly adults by analyzing the 16S rRNA gene. The terminal restriction fragment length polymorphism (T-RFLP) profiles of 44 tube-fed subjects were compared with those of 54 subjects fed orally (average age, 86.4 ± 6.9 years). Bar-coded pyrosequencing data were also obtained for a subset of the subjects from each group (15 tube-fed subjects and 16 subjects fed orally). The T-RFLP profiles demonstrated that the microbiota of the tube-fed subjects was distinct from that of the subjects fed orally (permutational multivariate analysis of variance [perMANOVA], P < 0.001). The pyrosequencing data revealed that 22 bacterial genera, including Corynebacterium, Peptostreptococcus, and Fusobacterium, were significantly more predominant in tube-fed subjects, whereas the dominant genera in the subjects fed orally, such as Streptococcus and Veillonella, were present in much lower proportions. Opportunistic pathogens rarely detected in the normal oral microbiota, such as Corynebacterium striatum and Streptococcus agalactiae, were often found in high proportions in tube-fed subjects. The oral indigenous microbiota is disrupted by the use of enteral feeding, allowing health-threatening bacteria to thrive. PMID:21821752

The Effect of Long-Term Continuous Cropping of Black Pepper on Soil Bacterial Communities as Determined by 454 Pyrosequencing

PubMed Central

Xiong, Wu; Li, Zhigang; Liu, Hongjun; Xue, Chao; Zhang, Ruifu; Wu, Huasong; Li, Rong; Shen, Qirong

2015-01-01

In the present study, 3 replanted black pepper orchards with continuously cropping histories for 10, 21, and 55 years in tropical China, were selected for investigating the effect of monoculture on soil physiochemical properties, enzyme activities, bacterial abundance, and bacterial community structures. Results showed long-term continuous cropping led to a significant decline in soil pH, organic matter contents, enzymatic activities, and resulted in a decrease in soil bacterial abundance. 454 pyrosequencing analysis of 16S rRNA genes revealed that the Acidobacteria and Proteobacteria were the main phyla in the replanted black pepper orchard soils, comprising up to 73.82% of the total sequences; the relative abundances of Bacteroidetes and Firmicutes phyla decreased with long-term continuous cropping; and at genus level, the Pseudomonas abundance significantly depleted after 21 years continuous cropping. In addition, bacterial diversity significantly decreased after 55 years black pepper continuous cropping; obvious variations for community structures across the 3 time-scale replanted black pepper orchards were observed, suggesting monoculture duration was the major determinant for bacterial community structure. Overall, continuous cropping during black pepper cultivation led to a significant decline in soil pH, organic matter contents, enzymatic activities, resulted a decrease in soil bacterial abundance, and altered soil microbial community membership and structure, which in turn resulted in black pepper poor growth in the continuous cropping system. PMID:26317364

Comparative microbial diversity analyses of modern marine thrombolitic mats by barcoded pyrosequencing.

PubMed

Mobberley, Jennifer M; Ortega, Maya C; Foster, Jamie S

2012-01-01

Thrombolites are unlaminated carbonate structures that form as a result of the metabolic interactions of complex microbial mat communities. Thrombolites have a long geological history; however, little is known regarding the microbes associated with modern structures. In this study, we use a barcoded 16S rRNA gene-pyrosequencing approach coupled with morphological analysis to assess the bacterial, cyanobacterial and archaeal diversity associated with actively forming thrombolites found in Highborne Cay, Bahamas. Analyses revealed four distinct microbial mat communities referred to as black, beige, pink and button mats on the surfaces of the thrombolites. At a coarse phylogenetic resolution, the domain bacterial sequence libraries from the four mats were similar, with Proteobacteria and Cyanobacteria being the most abundant. At the finer resolution of the rRNA gene sequences, significant differences in community structure were observed, with dramatically different cyanobacterial communities. Of the four mat types, the button mats contained the highest diversity of Cyanobacteria, and were dominated by two sequence clusters with high similarity to the genus Dichothrix, an organism associated with the deposition of carbonate. Archaeal diversity was low, but varied in all mat types, and the archaeal community was predominately composed of members of the Thaumarchaeota and Euryarchaeota. The morphological and genetic data support the hypothesis that the four mat types are distinctive thrombolitic mat communities. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

An Insight Into the Microbiome of the Amblyomma maculatum (Acari: Ixodidae)

PubMed Central

BUDACHETRI, KHEMRAJ; BROWNING, REBECCA E.; ADAMSON, STEVEN W.; DOWD, SCOT E.; CHAO, CHIEN-CHUNG; CHING, WEI-MEI; KARIM, SHAHID

2014-01-01

The aim of this study was to survey the bacterial diversity of Amblyomma maculatum Koch, 1844, and characterize its infection with Rickettsia parkeri. Pyrosequencing of the bacterial 16S rRNA was used to determine the total bacterial population in A. maculatum. Pyrosequencing analysis identified Rickettsia in A. maculatum midguts, salivary glands, and saliva, which indicates successful trafficking in the arthropod vector. The identity of Rickettsia spp. was determined based on sequencing the rickettsial outer membrane protein A (rompA) gene. The sequence homology search revealed the presence of R. parkeri, Rickettsia amblyommii, and Rickettsia endosymbiont of A. maculatum in midgut tissues, whereas the only rickettsia detected in salivary glands was R. parkeri, suggesting it is unique in its ability to migrate from midgut to salivary glands, and colonize this tissue before dissemination to the host. Owing to its importance as an emerging infectious disease, the R. parkeri pathogen burden was quantified by a rompB-based quantitative polymerase chain reaction (qPCR) assay and the diagnostic effectiveness of using R. parkeri polyclonal antibodies in tick tissues was tested. Together, these data indicate that field-collected A. maculatum had a R. parkeri infection rate of 12–32%. This study provides an insight into the A. maculatum microbiome and confirms the presence of R. parkeri, which will serve as the basis for future tick and microbiome interaction studies. PMID:24605461

Protist communities in a marine oxygen minimum zone off Costa Rica by 454 pyrosequencing

NASA Astrophysics Data System (ADS)

Jing, H.; Rocke, E.; Kong, L.; Xia, X.; Liu, H.; Landry, M. R.

2015-08-01

Marine planktonic protists, including microalgae and protistan grazers, are an important contributor to global primary production and carbon and mineral cycles, however, little is known about their population shifts along the oxic-anoxic gradient in the water column. We used 454 pyrosequencing of the 18S rRNA gene and gene transcripts to study the community composition of whole and active protists throughout a water column in the Costa Rica Dome, where a stable oxygen minimum zone (OMZ) exists at a depth of 400~700 m. A clear shift of protist composition from photosynthetic Dinoflagellates in the surface to potential parasitic Dinoflagellates and Ciliates in the deeper water was revealed along the vertical profile at both rRNA and rDNA levels. Those protist groups recovered only at the rDNA level represent either lysed aggregates sinking from the upper waters or potential hosts for parasitic groups. UPGMA clustering demonstrated that total and active protists in the anoxic core of OMZ (550 m) were distinct from those in other water depths. The reduced community diversity and presence of a parasitic/symbiotic trophic lifestyle in the OMZ, especially the anoxic core, suggests that OMZs can exert a selective pressure on protist communities. Such changes in community structure and a shift in trophic lifestyle could result in a modulation of the microbial loop and associated biogeochemical cycling.

Detection of novel NF1 mutations and rapid mutation prescreening with Pyrosequencing.

PubMed

Brinckmann, Anja; Mischung, Claudia; Bässmann, Ingelore; Kühnisch, Jirko; Schuelke, Markus; Tinschert, Sigrid; Nürnberg, Peter

2007-12-01

Neurofibromatosis type 1 (NF1) is caused by mutations in the neurofibromin (NF1) gene. Mutation analysis of NF1 is complicated by its large size, the lack of mutation hotspots, pseudogenes and frequent de novo mutations. Additionally, the search for NF1 mutations on the mRNA level is often hampered by nonsense-mediated mRNA decay (NMD) of the mutant allele. In this study we searched for mutations in a cohort of 38 patients and investigated the relationship between mutation type and allele-specific transcription from the wild-type versus mutant alleles. Quantification of relative mRNA transcript numbers was done by Pyrosequencing, a novel real-time sequencing method whose signals can be quantified very accurately. We identified 21 novel mutations comprising various mutation types. Pyrosequencing detected a definite relationship between allelic NF1 transcript imbalance due to NMD and mutation type in 24 of 29 patients who all carried frame-shift or nonsense mutations. NMD was absent in 5 patients with missense and silent mutations, as well as in 4 patients with splice-site mutations that did not disrupt the reading frame. Pyrosequencing was capable of detecting NMD even when the effects were only moderate. Diagnostic laboratories could thus exploit this effect for rapid prescreening for NF1 mutations as more than 60% of the mutations in this gene disrupt the reading frame and are prone to NMD.

Validation of the VE1 Immunostain for the BRAF V600E Mutation in Melanoma

PubMed Central

Pearlstein, Michelle V.; Zedek, Daniel C.; Ollila, David W.; Treece, Amanda; Gulley, Margaret L.; Groben, Pamela A.; Thomas, Nancy E.

2014-01-01

BACKGROUND BRAF mutation status, and therefore eligibility for BRAF inhibitors, is currently determined by sequencing methods. We assessed the validity of VE1, a monoclonal antibody against the BRAF V600E mutant protein, in the detection of mutant BRAF V600E melanomas as classified by DNA pyrosequencing. METHODS The cases were 76 metastatic melanoma patients with only one known primary melanoma who had had BRAF codon 600 pyrosequencing of either their primary (n=19), metastatic (n=57) melanoma, or both (n=17). All melanomas (n=93) were immunostained with the BRAF VE1 antibody using a red detection system. The staining intensity of these specimens was scored from 0 – 3+ by a dermatopathologist. Scores of 0 and 1+ were considered as negative staining while scores of 2+ and 3+ were considered positive. RESULTS The VE1 antibody demonstrated a sensitivity of 85% and a specificity of 100% as compared to DNA pyrosequencing results. There was 100% concordance between VE1 immunostaining of primary and metastatic melanomas from the same patient. V600K, V600Q, and V600R BRAF melanomas did not positively stain with VE1. CONCLUSIONS This hospital-based study finds high sensitivity and specificity for the BRAF VE1 immunostain in comparison to pyrosequencing in detection of BRAF V600E in melanomas. PMID:24917033

An Appropriate Cutoff Value for Determining the Colonization of Helicobacter pylori by the Pyrosequencing Method: Comparison with Conventional Methods.

PubMed

Kim, Jaeyeon; Kim, Nayoung; Jo, Hyun Jin; Park, Ji Hyun; Nam, Ryoung Hee; Seok, Yeong-Jae; Kim, Yeon-Ran; Kim, Joo Sung; Kim, Jung Mogg; Kim, Jung Min; Lee, Dong Ho; Jung, Hyun Chae

2015-10-01

Sequencing of 16S ribosomal RNA (rRNA) gene has improved the characterization of microbial communities. It enabled the detection of low abundance gastric Helicobacter pylori sequences even in subjects that were found to be H. pylori negative with conventional methods. The objective of this study was to obtain a cutoff value for H. pylori colonization in gastric mucosa samples by pyrosequencing method. Gastric mucosal biopsies were taken from 63 subjects whose H. pylori status was determined by a combination of serology, rapid urease test, culture, and histology. Microbial DNA from mucosal samples was amplified by PCR using universal bacterial primers. 16S rDNA amplicons were pyrosequenced. ROC curve analysis was performed to determine the cutoff value for H. pylori colonization by pyrosequencing. In addition, temporal changes in the stomach microbiota were observed in eight initially H. pylori-positive and eight H. pylori-negative subjects at a single time point 1-8 years later. Of the 63 subjects, the presence of H. pylori sequences was detected in all (28/28) conventionally H. pylori-positive samples and in 60% (21/35) of H. pylori-negative samples. The average percent of H. pylori reads in each sample was 0.67 ± 1.09% in the H. pylori-negative group. Cutoff value for clinically positive H. pylori status was approximately 1.22% based on ROC curve analysis (AUC = 0.957; p < .001). Helicobacter pylori was successfully eradicated in five of seven treated H. pylori-positive subjects (71.4%), and the percentage of H. pylori reads in these five subjects dropped from 1.3-95.18% to 0-0.16% after eradication. These results suggest that the cutoff value of H. pylori sequence percentage for H. pylori colonization by pyrosequencing could be set at approximately 1%. It might be helpful to analyze gastric microbiota related to H. pylori sequence status. © 2015 John Wiley & Sons Ltd.

454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

PubMed Central

Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D’Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

2013-01-01

Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues. PMID:23950653

Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae.

PubMed

Castro-Carrera, T; Toral, P G; Frutos, P; McEwan, N R; Hervás, G; Abecia, L; Pinloche, E; Girdwood, S E; Belenguer, A

2014-03-01

Developing novel strategies to increase the content of bioactive unsaturated fatty acids (FA) in ruminant-derived products requires a deeper understanding of rumen biohydrogenation and bacteria involved in this process. Although high-throughput pyrosequencing may allow for a great coverage of bacterial diversity, it has hardly been used to investigate the microbiology of ruminal FA metabolism. In this experiment, 454 pyrosequencing and a molecular fingerprinting technique (terminal restriction fragment length polymorphism; T-RFLP) were used concurrently to assess the effect of diet supplementation with marine algae (MA) on the rumen bacterial community of dairy sheep. Eleven lactating ewes were divided in 2 lots and offered a total mixed ration based on alfalfa hay and concentrate (40:60), supplemented with 0 (control) or 8 (MA) g of MA/kg of dry matter. After 54 d on treatments, animals were slaughtered and samples of rumen content and fluid were collected separately for microbial analysis. Pyrosequencing yielded a greater coverage of bacterial diversity than T-RFLP and allowed the identification of low abundant populations. Conversely, both molecular approaches pointed to similar conclusions and showed that relevant changes due to MA addition were observed within the major ruminal phyla, namely Bacteroidetes, Firmicutes, and Proteobacteria. Decreases in the abundance of unclassified Bacteroidales, Porphyromonadaceae, and Ruminococcaceae and increases in as-yet uncultured species of the family Succinivibrionaceae, might be related to a potential role of these groups in different pathways of rumen FA metabolism. Diet supplementation with MA, however, had no effect on the relative abundance of Butyrivibrio and Pseudobutyrivibrio genera. In addition, results from both 454 pyrosequencing and T-RFLP indicate that the effect of MA was rather consistent in rumen content or fluid samples, despite inherent differences between these fractions in their bacterial composition. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

A Bioluminometric Method of DNA Sequencing

NASA Technical Reports Server (NTRS)

Ronaghi, Mostafa; Pourmand, Nader; Stolc, Viktor; Arnold, Jim (Technical Monitor)

2001-01-01

Pyrosequencing is a bioluminometric single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis. In this sequencing-by-synthesis method, a cascade of enzymatic reactions yields detectable light, which is proportional to incorporated nucleotides. Pyrosequencing has the advantages of accuracy, flexibility and parallel processing. It can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides and gel-electrophoresis. In this chapter, the use of this technique for different applications is discussed.

Picoliter DNA Sequencing Chemistry on an Electrowetting-based Digital Microfluidic Platform

PubMed Central

Ferguson Welch, Erin R.; Lin, Yan-You; Madison, Andrew; Fair, R.B.

2011-01-01

The results of investigations into performing DNA sequencing chemistry on a picoliter-scale electrowetting digital microfluidic platform are reported. Pyrosequencing utilizes pyrophosphate produced during nucleotide base addition to initiate a process ending with detection through a chemiluminescence reaction using firefly luciferase. The intensity of light produced during the reaction can be quantified to determine the number of bases added to the DNA strand. The logic-based control and discrete fluid droplets of a digital microfluidic device lend themselves well to the pyrosequencing process. Bead-bound DNA is magnetically held in a single location, and wash or reagent droplets added or split from it to circumvent product dilution. Here we discuss the dispensing, control, and magnetic manipulation of the paramagnetic beads used to hold target DNA. We also demonstrate and characterize the picoliter-scale reaction of luciferase with adenosine triphosphate to represent the detection steps of pyrosequencing and all necessary alterations for working on this scale. PMID:21298802

Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

PubMed Central

Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

2014-01-01

Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

Pyrosequencing analysis of the gyrB gene to differentiate bacteria responsible for diarrheal diseases.

PubMed

Hou, X-L; Cao, Q-Y; Jia, H-Y; Chen, Z

2008-07-01

Pathogens causing acute diarrhea include a large variety of species from Enterobacteriaceae and Vibrionaceae. A method based on pyrosequencing was used here to differentiate bacteria commonly associated with diarrhea in China; the method is targeted to a partial amplicon of the gyrB gene, which encodes the B subunit of DNA gyrase. Twenty-eight specific polymorphic positions were identified from sequence alignment of a large sequence dataset and targeted using 17 sequencing primers. Of 95 isolates tested, belonging to 13 species within 7 genera, most could be identified to the species level; O157 type could be differentiated from other E. coli types; Salmonella enterica subsp. enterica could be identified at the serotype level; the genus Shigella, except for S. boydii and S. dysenteriae, could also be identified. All these isolates were also subjected to conventional sequencing of a relatively long ( approximately1.2 kb) region of gyrB DNA; these results confirmed those with pyrosequencing. Twenty-two fecal samples were surveyed, the results of which were concordant with culture-based bacterial identification, and the pathogen detection limit with simulated stool specimens was 10(4) CFU/ml. DNA from different pathogens was also mixed to simulate a case of multibacterial infection, and the generated signals correlated well with the mix ratio. In summary, the gyrB-based pyrosequencing approach proved to have significant reliability and discriminatory power for enteropathogenic bacterial identification and provided a fast and effective method for clinical diagnosis.

Differences in Bacterial Diversity and Communities Between Glacial Snow and Glacial Soil on the Chongce Ice Cap, West Kunlun Mountains.

PubMed

Yang, Guang Li; Hou, Shu Gui; Le Baoge, Ri; Li, Zhi Guo; Xu, Hao; Liu, Ya Ping; Du, Wen Tao; Liu, Yong Qin

2016-11-04

A detailed understanding of microbial ecology in different supraglacial habitats is important due to the unprecedented speed of glacier retreat. Differences in bacterial diversity and community structure between glacial snow and glacial soil on the Chongce Ice Cap were assessed using 454 pyrosequencing. Based on rarefaction curves, Chao1, ACE, and Shannon indices, we found that bacterial diversity in glacial snow was lower than that in glacial soil. Principal coordinate analysis (PCoA) and heatmap analysis indicated that there were major differences in bacterial communities between glacial snow and glacial soil. Most bacteria were different between the two habitats; however, there were some common bacteria shared between glacial snow and glacial soil. Some rare or functional bacterial resources were also present in the Chongce Ice Cap. These findings provide a preliminary understanding of the shifts in bacterial diversity and communities from glacial snow to glacial soil after the melting and inflow of glacial snow into glacial soil.

Next Generation Sequencing Plus (NGS+) with Y-chromosomal Markers for Forensic Pedigree Searches.

PubMed

Qian, Xiaoqin; Hou, Jiayi; Wang, Zheng; Ye, Yi; Lang, Min; Gao, Tianzhen; Liu, Jing; Hou, Yiping

2017-09-12

There is high demand for forensic pedigree searches with Y-chromosome short tandem repeat (Y-STR) profiling in large-scale crime investigations. However, when two Y-STR haplotypes have a few mismatched loci, it is difficult to determine if they are from the same male lineage because of the high mutation rate of Y-STRs. Here we design a new strategy to handle cases in which none of pedigree samples shares identical Y-STR haplotype. We combine next generation sequencing (NGS), capillary electrophoresis and pyrosequencing under the term 'NGS+' for typing Y-STRs and Y-chromosomal single nucleotide polymorphisms (Y-SNPs). The high-resolution Y-SNP haplogroup and Y-STR haplotype can be obtained with NGS+. We further developed a new data-driven decision rule, FSindex, for estimating the likelihood for each retrieved pedigree. Our approach enables positive identification of pedigree from mismatched Y-STR haplotypes. It is envisaged that NGS+ will revolutionize forensic pedigree searches, especially when the person of interest was not recorded in forensic DNA database.

Major histocompatibility complex class II compatibility, but not class I, predicts mate choice in a bird with highly developed olfaction

PubMed Central

Strandh, Maria; Westerdahl, Helena; Pontarp, Mikael; Canbäck, Björn; Dubois, Marie-Pierre; Miquel, Christian; Taberlet, Pierre; Bonadonna, Francesco

2012-01-01

Mate choice for major histocompatibility complex (MHC) compatibility has been found in several taxa, although rarely in birds. MHC is a crucial component in adaptive immunity and by choosing an MHC-dissimilar partner, heterozygosity and potentially broad pathogen resistance is maximized in the offspring. The MHC genotype influences odour cues and preferences in mammals and fish and hence olfactory-based mate choice can occur. We tested whether blue petrels, Halobaena caerulea, choose partners based on MHC compatibility. This bird is long-lived, monogamous and can discriminate between individual odours using olfaction, which makes it exceptionally well suited for this analysis. We screened MHC class I and II B alleles in blue petrels using 454-pyrosequencing and quantified the phylogenetic, functional and allele-sharing similarity between individuals. Partners were functionally more dissimilar at the MHC class II B loci than expected from random mating (p = 0.033), whereas there was no such difference at the MHC class I loci. Phylogenetic and non-sequence-based MHC allele-sharing measures detected no MHC dissimilarity between partners for either MHC class I or II B. Our study provides evidence of mate choice for MHC compatibility in a bird with a high dependency on odour cues, suggesting that MHC odour-mediated mate choice occurs in birds. PMID:22951737

Characterization of microsatellite loci from two-spotted octopus Octopus bimaculatus Verrill 1883 from pyrosequencing reads

USGS Publications Warehouse

Domínguez-Contreras, J. F.; Munguía-Vega, A.; Ceballos-Vázquez, B. P.; Arellano-Martínez, M.; Culver, Melanie

2014-01-01

We characterized 22 novel microsatellite loci in the two-spotted octopus Octopus bimaculatus using 454 pyrosequencing reads. All loci were polymorphic and will be used in studies of marine connectivity aimed at increasing sustainability of the resource. The mean number alleles per locus was 13.09 (range 7–19) and observed heterozygosities ranged from 0.50 to 1.00. Four loci pairs were linked and three deviated from Hardy–Weinberg equilibrium. Eighteen and 12 loci were polymorphic in Octopus bimaculoides and Octopus hubbsorum, respectively.

Bacterial communities associated with four ctenophore genera from the German Bight (North Sea).

PubMed

Hao, Wenjin; Gerdts, Gunnar; Peplies, Jörg; Wichels, Antje

2015-01-01

Intense research has been conducted on jellyfish and ctenophores in recent years. They are increasingly recognized as key elements in the marine ecosystem that serve as critical indicators and drivers of ecosystem performance and change. However, the bacterial community associated with ctenophores is still poorly investigated. Based on automated ribosomal intergenic spacer analysis (ARISA) and 16S ribosomal RNA gene amplicon pyrosequencing, we investigated bacterial communities associated with the frequently occurring ctenophore species Mnemiopsis leidyi, Beroe sp., Bolinopsis infundibulum and Pleurobrachia pileus at Helgoland Roads in the German Bight (North Sea). We observed significant differences between the associated bacterial communities of the different ctenophore species based on ARISA patterns. With respect to bacterial taxa, all ctenophore species were dominated by Proteobacteria as revealed by pyrosequencing. Mnemiopsis leidyi and P. pileus mainly harboured Gammaproteobacteria, with Marinomonas as the dominant phylotype of M. leidyi. By contrast, Pseudoalteromonas and Psychrobacter were the most abundant Gammaproteobacteria in P. pileus. Beroe sp. was mainly dominated by Alphaproteobacteria, particularly by the genus Thalassospira. For B. infundibulum, the bacterial community was composed of Alphaproteobacteria and Gammaproteobacteria in equal parts, which consisted of the genera Thalassospira and Marinomonas. In addition, the bacterial communities associated with M. leidyi display a clear variation over time that needs further investigation. Our results indicate that the bacterial communities associated with ctenophores are highly species- specific. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Influence of Hand Rearing and Bird Age on the Fecal Microbiota of the Critically Endangered Kakapo

PubMed Central

Waite, David W.; Eason, Daryl K.

2014-01-01

The critically endangered New Zealand parrot, the kakapo, is subject to an intensive management regime aiming to maintain bird health and boost population size. Newly hatched kakapo chicks are subjected to human intervention and are frequently placed in captivity throughout their formative months. Hand rearing greatly reduces mortality among juveniles, but the potential long-term impact on the kakapo gut microbiota is uncertain. To track development of the kakapo gut microbiota, fecal samples from healthy, prefledged juvenile kakapos, as well as from unrelated adults, were analyzed by using 16S rRNA gene amplicon pyrosequencing. Following the original sampling, juvenile kakapos underwent a period of captivity, so further sampling during and after captivity aimed to elucidate the impact of captivity on the juvenile gut microbiota. Variation in the fecal microbiota over a year was also investigated, with resampling of the original juvenile population. Amplicon pyrosequencing revealed a juvenile fecal microbiota enriched with particular lactic acid bacteria compared to the microbiota of adults, although the overall community structure did not differ significantly among kakapos of different ages. The abundance of key operational taxonomic units (OTUs) was correlated with antibiotic treatment and captivity, although the importance of these factors could not be proven unequivocally within the bounds of this study. Finally, the microbial community structure of juvenile and adult kakapos changed over time, reinforcing the need for continual monitoring of the microbiota as part of regular health screening. PMID:24837385

Pyrosequencing the Canine Faecal Microbiota: Breadth and Depth of Biodiversity

PubMed Central

Hand, Daniel; Wallis, Corrin; Colyer, Alison; Penn, Charles W.

2013-01-01

Mammalian intestinal microbiota remain poorly understood despite decades of interest and investigation by culture-based and other long-established methodologies. Using high-throughput sequencing technology we now report a detailed analysis of canine faecal microbiota. The study group of animals comprised eleven healthy adult miniature Schnauzer dogs of mixed sex and age, some closely related and all housed in kennel and pen accommodation on the same premises with similar feeding and exercise regimes. DNA was extracted from faecal specimens and subjected to PCR amplification of 16S rDNA, followed by sequencing of the 5′ region that included variable regions V1 and V2. Barcoded amplicons were sequenced by Roche-454 FLX high-throughput pyrosequencing. Sequences were assigned to taxa using the Ribosomal Database Project Bayesian classifier and revealed dominance of Fusobacterium and Bacteroidetes phyla. Differences between animals in the proportions of different taxa, among 10,000 reads per animal, were clear and not supportive of the concept of a “core microbiota”. Despite this variability in prominent genera, littermates were shown to have a more similar faecal microbial composition than unrelated dogs. Diversity of the microbiota was also assessed by assignment of sequence reads into operational taxonomic units (OTUs) at the level of 97% sequence identity. The OTU data were then subjected to rarefaction analysis and determination of Chao1 richness estimates. The data indicated that faecal microbiota comprised possibly as many as 500 to 1500 OTUs. PMID:23382835

454 pyrosequencing analysis of bacterial diversity revealed by a comparative study of soils from mining subsidence and reclamation areas.

PubMed

Li, Yuanyuan; Chen, Longqian; Wen, Hongyu; Zhou, Tianjian; Zhang, Ting; Gao, Xiali

2014-03-28

Significant alteration in the microbial community can occur across reclamation areas suffering subsidence from mining. A reclamation site undergoing fertilization practices and an adjacent coal-excavated subsidence site (sites A and B, respectively) were examined to characterize the bacterial diversity using 454 high-throughput 16S rDNA sequencing. The dominant taxonomic groups in both the sites were Proteobacteria, Acidobacteria, Bacteroidetes, Betaproteobacteria, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Chloroflexi, and Firmicutes. However, the bacterial communities' abundance, diversity, and composition differed significantly between the sites. Site A presented higher bacterial diversity and more complex community structures than site B. The majority of sequences related to Proteobacteria, Gemmatimonadetes, Chloroflexi, Nitrospirae, Firmicutes, Betaproteobacteria, Deltaproteobacteria, and Anaerolineae were from site A; whereas those related to Actinobacteria, Planctomycetes, Bacteroidetes, Verrucomicrobia, Gammaproteobacteria, Nitriliruptoria, Alphaproteobacteria, and Phycisphaerae originated from site B. The distribution of some bacterial groups and subgroups in the two sites correlated with soil properties and vegetation due to reclamation practice. Site A exhibited enriched bacterial community, soil organic matter (SOM), and total nitrogen (TN), suggesting the presence of relatively diverse microorganisms. SOM and TN were important factors shaping the underlying microbial communities. Furthermore, the specific plant functional group (legumes) was also an important factor influencing soil microbial community composition. Thus, the effectiveness of 454 pyrosequencing in analyzing soil bacterial diversity was validated and an association between land ecological system restoration, mostly mediated by microbial communities, and an improvement in soil properties in coalmining reclamation areas was suggested.

Impact of aerobic acclimation on the nitrification performance and microbial community of landfill leachate sludge.

PubMed

Hira, Daisuke; Aiko, Nobuyuki; Yabuki, Yoshinori; Fujii, Takao

2018-03-01

Nitrogenous pollution of water is regarded as a global environmental problem, and nitrogen removal has become an important issue in wastewater treatment processes. Landfill leachate is a typical large source of nitrogenous wastewater. Although the characteristics of leachate vary according to the age of the landfill, leachates of mature landfill have high concentrations of nitrogenous compounds. Most nitrogen in these leachates is in the form of ammonium nitrogen. In this study, we investigated the bacterial community of sludge from a landfill leachate lagoon by pyrosequencing of the bacterial 16S rRNA gene. The sludge was acclimated in a laboratory-scale reactor with aeration using a mechanical stirrer to promote nitrification. On 149 days, nitrification was achieved and then the bacterial community was also analyzed. The bacterial community was also analyzed after nitrification was achieved. Pyrosequencing analyses revealed that the abundances of ammonia-oxidizing and nitrite-oxidizing bacteria were increased by acclimation and their total proportions increased to >15% of total biomass. Changes in the sulfate-reducing and sulfur-oxidizing bacteria were also observed during the acclimation process. The aerobic acclimation process enriched a nitrifying microbial community from the landfill leachate sludge. These results suggested that the aerobic acclimation is a processing method for the nitrification ammonium oxidizing throw the enrichment of nitrifiers. Improvement of this acclimation method would allow nitrogen removal from leachate by nitrification and sulfur denitrification. Copyright © 2017 Elsevier Ltd. All rights reserved.

Unique Vaginal Microbiota That Includes an Unknown Mycoplasma-Like Organism Is Associated With Trichomonas vaginalis Infection

PubMed Central

Martin, David H.; Zozaya, Marcela; Lillis, Rebecca A.; Myers, Leann; Nsuami, M. Jacques; Ferris, Michael J.

2013-01-01

Background. The prevalence of Trichomonas vaginalis infection is highest in women with intermediate Nugent scores. We hypothesized that the vaginal microbiota in T. vaginalis–infected women differs from that in T. vaginalis–uninfected women. Methods. Vaginal samples from 30 T. vaginalis–infected women were matched by Nugent score to those from 30 T. vaginalis–uninfected women. Equal numbers of women with Nugent scores categorized as normal, intermediate, and bacterial vaginosis were included. The vaginal microbiota was assessed using 454 pyrosequencing analysis of polymerase chain reaction–amplified 16S ribosomal RNA gene sequences. The 16S ribosomal RNA gene sequence of an unknown organism was obtained by universal bacterial polymerase chain reaction amplification, cloning, and sequencing. Results. Principal coordinates analysis of the pyrosequencing data showed divergence of the vaginal microbiota in T. vaginalis–infected and T. vaginalis–uninfected patients among women with normal and those with intermediate Nugent scores but not among women with bacterial vaginosis. Cluster analysis revealed 2 unique groups of T. vaginalis–infected women. One had high abundance of Mycoplasma hominis and other had high abundance of an unknown Mycoplasma species. Women in the former group had clinical evidence of enhanced vaginal inflammation. Conclusions. T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response. PMID:23482642

Analysis of the melon (Cucumis melo) small RNAome by high-throughput pyrosequencing

PubMed Central

2011-01-01

Background Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. Results We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. Conclusion We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions. PMID:21812964

Pyrosequencing reveals benthic bacteria changes responsing to heavy deposition of Microcystis scum in lab — searching bacteria for bloom control

NASA Astrophysics Data System (ADS)

Tang, Yali; Cheng, Dongmei; Guan, Baohua; Zhang, Xiufeng; Liu, Zhengwen; Liu, Zejun

2017-05-01

Bacteria capable of degrading cyanobacteria Microcystis are crucial for determining the ecological consequences of Microcystis blooms in freshwater lakes. Scum derived from Microcystis blooms tends to accumulate in bays of large lakes and then sink to the sediments where it is finally consumed by benthic bacteria. Understanding the response of benthic bacterial communities to massive Microcystis deposition events may help identify the bacteria best suited to Microcystis hydrolyzation and even bloom control. For that purpose, an experimental system was set up in which intact sediment cores were incubated in the laboratory with normal and heavy deposits of Microcystis detritus. Pyrosequencing was performed in order to describe a phylogenetic inventory of bacterial communities in samples taken at 0-1, 1-2 and 2-3 cm depths in incubated sediments and in original untreated sediment. A hierarchical cluster tree was constructed expose differences between sediments. Similarity percentage calculations were also performed to identify the bacterial species contributing to variation. The results of this study suggest that: (1) deposition of Microcystis scums exerts a strong effect on the bacterial community composition in the surface (0-1 cm) and sub-surface (1-2 cm) sediment layers; (2) bacterial community responses to Microcystis detritus deposition vary across vertical gradients. A list of bacteria with potential roles in Microcystis degradation was compiled. These findings may inform the development of future measures for Microcystis bloom control in lakes.

Naphthalene biodegradation in temperate and arctic marine microcosms.

PubMed

Bagi, Andrea; Pampanin, Daniela M; Lanzén, Anders; Bilstad, Torleiv; Kommedal, Roald

2014-02-01

Naphthalene, the smallest polycyclic aromatic hydrocarbon (PAH), is found in abundance in crude oil, its major source in marine environments. PAH removal occurs via biodegradation, a key process determining their fate in the sea. Adequate estimation of PAH biodegradation rates is essential for environmental risk assessment and response planning using numerical models such as the oil spill contingency and response (OSCAR) model. Using naphthalene as a model compound, biodegradation rate, temperature response and bacterial community composition of seawaters from two climatically different areas (North Sea and Arctic Ocean) were studied and compared. Naphthalene degradation was followed by measuring oxygen consumption in closed bottles using the OxiTop(®) system. Microbial communities of untreated and naphthalene exposed samples were analysed by polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. Three times higher naphthalene degradation rate coefficients were observed in arctic seawater samples compared to temperate, at all incubation temperatures. Rate coefficients at in situ temperatures were however, similar (0.048 day(-1) for temperate and 0.068 day(-1) for arctic). Naphthalene biodegradation rates decreased with similar Q10 ratios (3.3 and 3.5) in both seawaters. Using the temperature compensation method implemented in the OSCAR model, Q10 = 2, biodegradation in arctic seawater was underestimated when calculated from the measured temperate k1 value, showing that temperature difference alone could not predict biodegradation rates adequately. Temperate and arctic untreated seawater communities were different as revealed by pyrosequencing. Geographic origin of seawater affected the community composition of exposed samples.

Unique vaginal microbiota that includes an unknown Mycoplasma-like organism is associated with Trichomonas vaginalis infection.

PubMed

Martin, David H; Zozaya, Marcela; Lillis, Rebecca A; Myers, Leann; Nsuami, M Jacques; Ferris, Michael J

2013-06-15

The prevalence of Trichomonas vaginalis infection is highest in women with intermediate Nugent scores. We hypothesized that the vaginal microbiota in T. vaginalis-infected women differs from that in T. vaginalis-uninfected women. Vaginal samples from 30 T. vaginalis-infected women were matched by Nugent score to those from 30 T. vaginalis-uninfected women. Equal numbers of women with Nugent scores categorized as normal, intermediate, and bacterial vaginosis were included. The vaginal microbiota was assessed using 454 pyrosequencing analysis of polymerase chain reaction-amplified 16S ribosomal RNA gene sequences. The 16S ribosomal RNA gene sequence of an unknown organism was obtained by universal bacterial polymerase chain reaction amplification, cloning, and sequencing. Principal coordinates analysis of the pyrosequencing data showed divergence of the vaginal microbiota in T. vaginalis-infected and T. vaginalis-uninfected patients among women with normal and those with intermediate Nugent scores but not among women with bacterial vaginosis. Cluster analysis revealed 2 unique groups of T. vaginalis-infected women. One had high abundance of Mycoplasma hominis and other had high abundance of an unknown Mycoplasma species. Women in the former group had clinical evidence of enhanced vaginal inflammation. T. vaginalis may alter the vaginal microbiota in a manner that is favorable to its survival and/or transmissibility. An unknown Mycoplasma species plays a role in some of these transformations. In other cases, these changes may result in a heightened host inflammatory response.

Comparative Analysis of Korean Human Gut Microbiota by Barcoded Pyrosequencing

PubMed Central

Nam, Young-Do; Jung, Mi-Ja; Roh, Seong Woon; Kim, Min-Soo; Bae, Jin-Woo

2011-01-01

Human gut microbiota plays important roles in harvesting energy from the diet, stimulating the proliferation of the intestinal epithelium, developing the immune system, and regulating fat storage in the host. Characterization of gut microbiota, however, has been limited to western people and is not sufficiently extensive to fully describe microbial communities. In this study, we investigated the overall composition of the gut microbiota and its host specificity and temporal stability in 20 Koreans using 454-pyrosequencing with barcoded primers targeting the V1 to V3 region of the bacterial 16S rRNA gene. A total of 303,402 high quality reads covered each sample and 8,427 reads were analyzed on average. The results were compared with those of individuals from the USA, China and Japan. In general, microbial communities were dominated by five previously identified phyla: Actinobacteria, Firmicutes, Bacteroidetes, Fusobacteria, and Proteobacteria. UPGMA cluster analysis showed that the species composition of gut microbiota was host-specific and stable over the duration of the test period, but the relative abundance of each member fluctuated. 43 core Korean gut microbiota were identified by comparison of sequences from each individual, of which 15 species level phylotypes were related to previously-reported butyrate-producing bacteria. UniFrac analysis revealed that human gut microbiota differed between countries: Korea, USA, Japan and China, but tended to vary less between individual Koreans, suggesting that gut microbial composition is related to internal and external characteristics of each country member such as host genetics and diet styles. PMID:21829445

The predominance of Lactobacillus sanfranciscensis in French organic sourdoughs and its impact on related bread characteristics.

PubMed

Lhomme, Emilie; Orain, Servane; Courcoux, Philippe; Onno, Bernard; Dousset, Xavier

2015-11-20

Fourteen bakeries located in different regions of France were selected. These bakers use natural sourdough and organic ingredients. Consequently, different organic sourdoughs used for the manufacture of French bread were studied by the enumeration of lactic acid bacteria (LAB) and 16S rRNA sequencing of the isolates. In addition, after DNA extraction the bacterial diversity was assessed by pyrosequencing of the 16S rDNA V1-V3 region. Although LAB counts showed significant variations (7.6-9.5log10CFU/g) depending on the sourdough studied, their identification through a polyphasic approach revealed a large predominance of Lactobacillus sanfranciscensis in all samples. In ten sourdoughs, both culture and independent methods identified L. sanfranciscensis as the dominant LAB species identified. In the remaining sourdoughs, culture methods identified 30-80% of the LAB as L. sanfranciscensis whereas more than 95% of the reads obtained by pyrosequencing belonged to L. sanfranciscensis. Other sub-dominant species, such as Lactobacillus curvatus, Lactobacillus hammesii, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus sakei, were also identified. Quantification of L. sanfranciscensis by real-time PCR confirmed the predominance of this species ranging from 8.24 to 10.38log10CFU/g. Regarding the acidification characteristics, sourdough and related bread physico-chemical characteristics varied, questioning the involvement of sub-dominant species or L. sanfranciscensis intra-species diversity and/or the role of the baker's practices. Copyright © 2015 Elsevier B.V. All rights reserved.

Androgen receptor mutations are associated with altered epigenomic programming as evidenced by HOXA5 methylation.

PubMed

Bens, S; Ammerpohl, O; Martin-Subero, J I; Appari, M; Richter, J; Hiort, O; Werner, R; Riepe, F G; Siebert, R; Holterhus, P-M

2011-01-01

Male external genital differentiation is accompanied by implementation of a long-term, male-specific gene expression pattern indicating androgen programming in cultured genital fibroblasts. We hypothesized the existence of an epigenetic background contributing to this phenomenon. DNA methylation levels in 2 normal scrotal fibroblast strains from 46,XY males compared to 2 labia majora fibroblast strains from 46,XY females with complete androgen insensitivity syndrome (AIS) due to androgen receptor (AR) mutations were analyzed by Illumina GoldenGate methylation arrays®. Results were validated with pyrosequencing in labia majora fibroblast strains from fifteen 46,XY patients and compared to nine normal male scrotal fibroblast strains. HOXA5 showed a significantly higher methylation level in complete AIS. This finding was confirmed by bisulfite pyrosequencing of 14 CpG positions within the HOXA5 promoter in the same strains. Extension of the 2 groups revealed a constant low HOXA5 methylation pattern in the controls in contrast to a highly variable methylation pattern in the AIS patients. HOXA5 represents a candidate gene of androgen-mediated promoter methylation. The constantly low HOXA5 DNA methylation level of normal male scrotal fibroblast strains and the frequently high methylation levels in labia majora fibroblast strains in AIS indicate for the first time that androgen programming in sexual differentiation is not restricted to global gene transcription but also occurs at the epigenetic level. 2011 S. Karger AG, Basel.

Endophyte Microbiome Diversity in Micropropagated Atriplex canescens and Atriplex torreyi var griffithsii

PubMed Central

Lucero, Mary E.; Unc, Adrian; Cooke, Peter; Dowd, Scot; Sun, Shulei

2011-01-01

Microbial diversity associated with micropropagated Atriplex species was assessed using microscopy, isolate culturing, and sequencing. Light, electron, and confocal microscopy revealed microbial cells in aseptically regenerated leaves and roots. Clone libraries and tag-encoded FLX amplicon pyrosequencing (TEFAP) analysis amplified sequences from callus homologous to diverse fungal and bacterial taxa. Culturing isolated some seed borne endophyte taxa which could be readily propagated apart from the host. Microbial cells were observed within biofilm-like residues associated with plant cell surfaces and intercellular spaces. Various universal primers amplified both plant and microbial sequences, with different primers revealing different patterns of fungal diversity. Bacterial and fungal TEFAP followed by alignment with sequences from curated databases revealed 7 bacterial and 17 ascomycete taxa in A. canescens, and 5 bacterial taxa in A. torreyi. Additional diversity was observed among isolates and clone libraries. Micropropagated Atriplex retains a complex, intimately associated microbiome which includes diverse strains well poised to interact in manners that influence host physiology. Microbiome analysis was facilitated by high throughput sequencing methods, but primer biases continue to limit recovery of diverse sequences from even moderately complex communities. PMID:21437280

Novel microbial diversity retrieved by autonomous robotic exploration of the world's deepest vertical phreatic sinkhole.

PubMed

Sahl, Jason W; Fairfield, Nathaniel; Harris, J Kirk; Wettergreen, David; Stone, William C; Spear, John R

2010-03-01

The deep phreatic thermal explorer (DEPTHX) is an autonomous underwater vehicle designed to navigate an unexplored environment, generate high-resolution three-dimensional (3-D) maps, collect biological samples based on an autonomous sampling decision, and return to its origin. In the spring of 2007, DEPTHX was deployed in Zacatón, a deep (approximately 318 m), limestone, phreatic sinkhole (cenote) in northeastern Mexico. As DEPTHX descended, it generated a 3-D map based on the processing of range data from 54 onboard sonars. The vehicle collected water column samples and wall biomat samples throughout the depth profile of the cenote. Post-expedition sample analysis via comparative analysis of 16S rRNA gene sequences revealed a wealth of microbial diversity. Traditional Sanger gene sequencing combined with a barcoded-amplicon pyrosequencing approach revealed novel, phylum-level lineages from the domains Bacteria and Archaea; in addition, several novel subphylum lineages were also identified. Overall, DEPTHX successfully navigated and mapped Zacatón, and collected biological samples based on an autonomous decision, which revealed novel microbial diversity in a previously unexplored environment.

Microbial Ecology Dynamics Reveal a Succession in the Core Microbiota Involved in the Ripening of Pasta Filata Caciocavallo Pugliese Cheese

PubMed Central

De Pasquale, Ilaria; Buchin, Solange; De Angelis, Maria; Gobbetti, Marco

2014-01-01

Pyrosequencing of the 16S rRNA targeting RNA, community-level physiological profiles made with Biolog EcoPlates, proteolysis, and volatile component (VOC) analyses were mainly used to characterize the manufacture and ripening of the pasta filata cheese Caciocavallo Pugliese. Plate counts revealed that cheese manufacture affected the microbial ecology. The results agreed with those from culture-independent approaches. As shown by urea-PAGE, reverse-phase high pressure liquid chromatography (RP-HPLC), and free-amino-acid (FAA) analyses, the extent of secondary proteolysis mainly increased after 30 to 45 days of ripening. VOCs and volatile free fatty acids (VFFA) were identified by a purge-and-trap method (PT) and solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS), respectively. Except for aldehydes, the levels of most of VOCs and VFFA mainly increased from 30 to 45 days onwards. As shown through pyrosequencing analysis, raw cows' milk was contaminated by Firmicutes (53%), Proteobacteria (39%), Bacteroidetes (7.8%), Actinobacteria (0.06%), and Fusobacteria (0.03%), with heterogeneity at the genus level. The primary starter Streptococcus thermophilus dominated the curd population. Other genera occurred at low incidence or sporadically. The microbial dynamics reflected on the overall physiological diversity. At 30 days, a microbial succession was clearly highlighted. The relative abundance of Streptococcus sp. and especially St. thermophilus decreased, while that of Lactobacillus casei, Lactobacillus sp., and especially Lactobacillus paracasei increased consistently. Despite the lower relative abundance compared to St. thermophilus, mesophilic lactobacilli were the only organisms positively correlated with the concentration of FAAs, area of hydrophilic peptide peaks, and several VOCs (e.g., alcohols, ketones, esters and all furans). This study showed that a core microbiota was naturally selected during middle ripening, which seemed to be the main factor responsible for cheese ripening. PMID:25085486

Pyrosequencing reveals bacteria carried in different wind-eroded sediments.

PubMed

Gardner, Terrence; Acosta-Martinez, Veronica; Calderón, Francisco J; Zobeck, Ted M; Baddock, Matthew; Van Pelt, R Scott; Senwo, Zachary; Dowd, Scot; Cox, Stephen

2012-01-01

Little is known about the microbial communities carried in wind-eroded sediments from various soil types and land management systems. The novel technique of pyrosequencing promises to expand our understanding of the microbial diversity of soils and eroded sediments because it can sequence 10 to 100 times more DNA fragments than previous techniques, providing enhanced exploration into what microbes are being lost from soil due to wind erosion. Our study evaluated the bacterial diversity of two types of wind-eroded sediments collected from three different organic-rich soils in Michigan using a portable field wind tunnel. The wind-eroded sediments evaluated were a coarse sized fraction with 66% of particles >106 μm (coarse eroded sediment) and a finer eroded sediment with 72% of particles <106 μm. Our findings suggested that (i) bacteria carried in the coarser sediment and fine dust were effective fingerprints of the source soil, although their distribution may vary depending on the soil characteristics because certain bacteria may be more protected in soil surfaces than others; (ii) coarser wind-eroded sediment showed higher bacterial diversity than fine dust in two of the three soils evaluated; and (iii) certain bacteria were more predominant in fine dust (, , and ) than coarse sediment ( and ), revealing different locations and niches of bacteria in soil, which, depending on wind erosion processes, can have important implications on the soil sustainability and functioning. Infrared spectroscopy showed that wind erosion preferentially removes particular kinds of C from the soil that are lost via fine dust. Our study shows that eroded sediments remove the active labile organic soil particulates containing key microorganisms involved in soil biogeochemical processes, which can have a negative impact on the quality and functioning of the source soil. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Bacterial Community Profiling of H2/CO2 or Formate-Utilizing Acetogens Enriched from Diverse Ecosystems

NASA Astrophysics Data System (ADS)

Han, R.; Zhang, L.; Fu, B.; Liu, H.

2014-12-01

Synthetic gases are usually generated from either cellulosic agricultural waste combustion or industrial release and could be subsequently transformed into acetate, ethanol, and/or butyrate by homoacetogenic bacteria, which commonly possess reductive acetyl-CoA synthesis pathway. Homoacetogen-based syngas fermentation technology provides an alternative solution to link greenhouse gas emission control and cellulosic solid waste treatment with biofuels production. The objective of our current project is to hunt for homoacetogens with capabilities of highly efficiently converting syngases to chemical solvents. In this study, we evaluated homoacetogens population dynamics during enrichments and pinpointed dominant homoacetogens representing diverse ecosystems enriched by different substrates. We enriched homoacetogens from four different samples including waste activate sludge, freshwater sediment, anaerobic methanogenic sludge, and cow manure using H2/CO2 (4:1) or formate as substrate for homoacetogen enrichment. Along with the formyltetrahydrofolate synthetase (FTHFS) gene (fhs gene)-specific real time qPCR assay and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, 16S rRNA based 454 high-throughput pyrosequencing was applied to reveal the population dynamic and community structure during enrichment from different origins. Enrichment of homoacetogenic populations coincided with accumulations of short chain fatty acids such as acetate and butyrate. 454 high-throughput pyrosequencing revealed Firmicutes and Spirochaetes populations became dominant while the overall microbial diversity decreased after enrichment. The most abundant sequences among the four origins belonged to the following phyla: Firmicutes, Spirochaetes, Proteobacteria, and Bacteroidetes, accounting for 62.1%-99.1% of the total reads. The major putative homoacetogenic species enriched on H2/CO2 or formate belonged to Clostridium spp., Acetobacterium spp., Acetoanaerobium spp., Eubacterium spp., Sporomusa spp. This comprehensive molecular ecology study on homoacetogen enrichments provides molecular evidences for shaping homoacetogenic populations and targeting novel homoacetogenic species enriched from diverse ecosystems.

Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice.

PubMed

De Minicis, Samuele; Rychlicki, Chiara; Agostinelli, Laura; Saccomanno, Stefania; Candelaresi, Cinzia; Trozzi, Luciano; Mingarelli, Eleonora; Facinelli, Bruna; Magi, Gloria; Palmieri, Claudio; Marzioni, Marco; Benedetti, Antonio; Svegliati-Baroni, Gianluca

2014-05-01

Nonalcoholic fatty liver disease (NAFLD) may lead to hepatic fibrosis. Dietary habits affect gut microbiota composition, whereas endotoxins produced by Gram-negative bacteria stimulate hepatic fibrogenesis. However, the mechanisms of action and the potential effect of microbiota in the liver are still unknown. Thus, we sought to analyze whether microbiota may interfere with liver fibrogenesis. Mice fed control (CTRL) or high-fat diet (HFD) were subjected to either bile duct ligation (BDL) or CCl4 treatment. Previously gut-sterilized mice were subjected to microbiota transplantation by oral gavage of cecum content obtained from donor CTRL- or HFD-treated mice. Fibrosis, intestinal permeability, bacterial translocation, and serum endotoxemia were measured. Inflammasome components were evaluated in gut and liver. Microbiota composition (dysbiosis) was evaluated by Pyrosequencing. Fibrosis degree was increased in HFD+BDL versus CTRL+BDL mice, whereas no differences were observed between CTRL+CCl4 and HFD+CCl4 mice. Culture of mesenteric lymph nodes showed higher density of infection in HFD+BDL mice versus CTRL+BDL mice, suggesting higher bacterial translocation rate. Pyrosequencing revealed an increase in percentage of Gram-negative versus Gram-postive bacteria, a reduced ratio between Bacteroidetes and Firmicutes, as well as a dramatic increase of Gram-negative Proteobacteria in HFD+BDL versus CTRL+BDL mice. Inflammasome expression was increased in liver of fibrotic mice, but significantly reduced in gut. Furthermore, microbiota transplantation revealed more liver damage in chimeric mice fed CTRL diet, but receiving the microbiota of HFD-treated mice; liver damage was further enhanced by transplantation of selected Gram-negative bacteria obtained from cecum content of HFD+BDL-treated mice. Dietary habits, by increasing the percentage of intestinal Gram-negative endotoxin producers, may accelerate liver fibrogenesis, introducing dysbiosis as a cofactor contributing to chronic liver injury in NAFLD. © 2014 by the American Association for the Study of Liver Diseases.

Microbiota of Cow’s Milk; Distinguishing Healthy, Sub-Clinically and Clinically Diseased Quarters

PubMed Central

Oikonomou, Georgios; Bicalho, Marcela Lucas; Meira, Enoch; Rossi, Rodolfo Elke; Foditsch, Carla; Machado, Vinicius Silva; Teixeira, Andre Gustavo Vieira; Santisteban, Carlos; Schukken, Ynte Hein; Bicalho, Rodrigo Carvalho

2014-01-01

The objective of this study was to use pyrosequencing of the 16S rRNA genes to describe the microbial diversity of bovine milk samples derived from clinically unaffected quarters across a range of somatic cell counts (SCC) values or from clinical mastitis, culture negative quarters. The obtained microbiota profiles were used to distinguish healthy, subclinically and clinically affected quarters. Two dairy farms were used for the collection of milk samples. A total of 177 samples were used. Fifty samples derived from healthy, culture negative quarters with a SCC of less than 20,000 cells/ml (group 1); 34 samples derived from healthy, culture negative quarters, with a SCC ranging from 21,000 to 50,000 cells/ml (group 2); 26 samples derived from healthy, culture negative quarters with a SCC greater than 50,000 cells/ml (group 3); 34 samples derived from healthy, culture positive quarters, with a SCC greater than 400,000 (group 4, subclinical); and 33 samples derived from clinical mastitis, culture negative quarters (group 5, clinical). Bacterial DNA was isolated from these samples and the 16S rRNA genes were individually amplified and pyrosequenced. All samples analyzed revealed great microbial diversity. Four bacterial genera were present in every sample obtained from healthy quarters (Faecalibacterium spp., unclassified Lachnospiraceae, Propionibacterium spp. and Aeribacillus spp.). Discriminant analysis models showed that samples derived from healthy quarters were easily discriminated based on their microbiota profiles from samples derived from clinical mastitis, culture negative quarters; that was also the case for samples obtained from different farms. Staphylococcus spp. and Streptococcus spp. were among the most prevalent genera in all groups while a general multivariable linear model revealed that Sphingobacterium and Streptococcus prevalences were associated with increased 10 log SCC. Conversely, Nocardiodes and Paenibacillus were negatively correlated, and a higher percentage of the genera was associated with a lower 10 log SCC. PMID:24465777

Competitive interactions between methane- and ammonia-oxidizing bacteria modulate carbon and nitrogen cycling in paddy soil

NASA Astrophysics Data System (ADS)

Zheng, Y.; Huang, R.; Wang, B. Z.; Bodelier, P. L. E.; Jia, Z. J.

2014-06-01

Pure culture studies have demonstrated that methanotrophs and ammonia oxidizers can both carry out the oxidation of methane and ammonia. However, the expected interactions resulting from these similarities are poorly understood, especially in complex, natural environments. Using DNA-based stable isotope probing and pyrosequencing of 16S rRNA and functional genes, we report on biogeochemical and molecular evidence for growth stimulation of methanotrophic communities by ammonium fertilization, and that methane modulates nitrogen cycling by competitive inhibition of nitrifying communities in a rice paddy soil. Pairwise comparison between microcosms amended with CH4, CH4+Urea, and Urea indicated that urea fertilization stimulated methane oxidation activity 6-fold during a 19-day incubation period, while ammonia oxidation activity was significantly suppressed in the presence of CH4. Pyrosequencing of the total 16S rRNA genes revealed that urea amendment resulted in rapid growth of Methylosarcina-like MOB, and nitrifying communities appeared to be partially inhibited by methane. High-throughput sequencing of the 13C-labeled DNA further revealed that methane amendment resulted in clear growth of Methylosarcina-related MOB while methane plus urea led to an equal increase in Methylosarcina and Methylobacter-related type Ia MOB, indicating the differential growth requirements of representatives of these genera. An increase in 13C assimilation by microorganisms related to methanol oxidizers clearly indicated carbon transfer from methane oxidation to other soil microbes, which was enhanced by urea addition. The active growth of type Ia methanotrops was significantly stimulated by urea amendment, and the pronounced growth of methanol-oxidizing bacteria occurred in CH4-treated microcosms only upon urea amendment. Methane addition partially inhibited the growth of Nitrosospira and Nitrosomonas in urea-amended microcosms, as well as growth of nitrite-oxidizing bacteria. These results suggest that type I methanotrophs can outcompete type II methane oxidizers in nitrogen-rich environments, rendering the interactions among methane and ammonia oxidizers more complicated than previously appreciated.

In-depth diversity analysis of the bacterial community resident in the camel rumen.

PubMed

Gharechahi, Javad; Zahiri, Hossein Shahbani; Noghabi, Kambiz Akbari; Salekdeh, Ghasem Hosseini

2015-02-01

The rumen compartment of the ruminant digestive tract is an enlarged fermentation chamber which houses a diverse collection of symbiotic microorganisms that provide the host animal with a remarkable ability to digest plant lignocellulosic materials. Characterization of the ruminal microbial community provides opportunities to improve animal food digestion efficiency, mitigate methane emission, and develop efficient fermentation systems to convert plant biomasses into biofuels. In this study, 16S rRNA gene amplicon pyrosequencing was applied in order to explore the structure of the bacterial community inhabiting the camel rumen. Using 76,333 quality-checked, chimera- and singleton-filtered reads, 4954 operational taxonomic units (OTUs) were identified at a 97% species level sequence identity. At the phylum level, more than 96% of the reads were affiliated to OTUs belonging to Bacteroidetes (51%), Firmicutes (31%), Proteobacteria (4.8%), Spirochaetes (3.5%), Fibrobacteres (3.1%), Verrucomicrobia (2.7%), and Tenericutes (0.95%). A total of 15% of the OTUs (746) that contained representative sequences from all major taxa were shared by all animals and they were considered as candidate members of the core camel rumen microbiome. Analysis of microbial composition through the solid and liquid fractions of rumen digesta revealed differential enrichment of members of Fibrobacter, Clostridium, Ruminococcus, and Treponema in the solid fraction, as well as members of Prevotella, Verrucomicrobia, Cyanobacteria, and Succinivibrio in the liquid fraction. The results clearly showed that the camel rumen microbiome was structurally similar but compositionally distinct from that of other ruminants, such as the cow. The unique characteristic of the camel rumen microbiome that differentiated it from those of other ruminants was the significant enrichment for cellulolytic bacteria. Copyright © 2014. Published by Elsevier GmbH.

Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre.

PubMed

Poretsky, Rachel S; Hewson, Ian; Sun, Shulei; Allen, Andrew E; Zehr, Jonathan P; Moran, Mary Ann

2009-06-01

Metatranscriptomic analyses of microbial assemblages (< 5 microm) from surface water at the Hawaiian Ocean Time-Series (HOT) revealed community-wide metabolic activities and day/night patterns of differential gene expression. Pyrosequencing produced 75 558 putative mRNA reads from a day transcriptome and 75 946 from a night transcriptome. Taxonomic binning of annotated mRNAs indicated that Cyanobacteria contributed a greater percentage of the transcripts (54% of annotated sequences) than expected based on abundance (35% of cell counts and 21% 16S rRNA of libraries), and may represent the most actively transcribing cells in this surface ocean community in both the day and night. Major heterotrophic taxa contributing to the community transcriptome included alpha-Proteobacteria (19% of annotated sequences, most of which were SAR11-related) and gamma-Proteobacteria (4%). The composition of transcript pools was consistent with models of prokaryotic gene expression, including operon-based transcription patterns and an abundance of genes predicted to be highly expressed. Metabolic activities that are shared by many microbial taxa (e.g. glycolysis, citric acid cycle, amino acid biosynthesis and transcription and translation machinery) were well represented among the community transcripts. There was an overabundance of transcripts for photosynthesis, C1 metabolism and oxidative phosphorylation in the day compared with night, and evidence that energy acquisition is coordinated with solar radiation levels for both autotrophic and heterotrophic microbes. In contrast, housekeeping activities such as amino acid biosynthesis, membrane synthesis and repair, and vitamin biosynthesis were overrepresented in the night transcriptome. Direct sequencing of these environmental transcripts has provided detailed information on metabolic and biogeochemical responses of a microbial community to solar forcing.

Comparative fecal metagenomics unveils unique functional capacity of the swine gut

PubMed Central

2011-01-01

Background Uncovering the taxonomic composition and functional capacity within the swine gut microbial consortia is of great importance to animal physiology and health as well as to food and water safety due to the presence of human pathogens in pig feces. Nonetheless, limited information on the functional diversity of the swine gut microbiome is available. Results Analysis of 637, 722 pyrosequencing reads (130 megabases) generated from Yorkshire pig fecal DNA extracts was performed to help better understand the microbial diversity and largely unknown functional capacity of the swine gut microbiome. Swine fecal metagenomic sequences were annotated using both MG-RAST and JGI IMG/M-ER pipelines. Taxonomic analysis of metagenomic reads indicated that swine fecal microbiomes were dominated by Firmicutes and Bacteroidetes phyla. At a finer phylogenetic resolution, Prevotella spp. dominated the swine fecal metagenome, while some genes associated with Treponema and Anareovibrio species were found to be exclusively within the pig fecal metagenomic sequences analyzed. Functional analysis revealed that carbohydrate metabolism was the most abundant SEED subsystem, representing 13% of the swine metagenome. Genes associated with stress, virulence, cell wall and cell capsule were also abundant. Virulence factors associated with antibiotic resistance genes with highest sequence homology to genes in Bacteroidetes, Clostridia, and Methanosarcina were numerous within the gene families unique to the swine fecal metagenomes. Other abundant proteins unique to the distal swine gut shared high sequence homology to putative carbohydrate membrane transporters. Conclusions The results from this metagenomic survey demonstrated the presence of genes associated with resistance to antibiotics and carbohydrate metabolism suggesting that the swine gut microbiome may be shaped by husbandry practices. PMID:21575148

Patterns of Symbiodinium (Dinophyceae) diversity and assemblages among diverse hosts and the coral reef environment of Lizard Island, Australia.

PubMed

Ziegler, Maren; Stone, Elizabeth; Colman, Daniel; Takacs-Vesbach, Cristina; Shepherd, Ursula

2018-04-26

Large-scale environmental disturbances may impact both partners in coral host-Symbiodinium systems. Elucidation of the assembly patterns in such complex and interdependent communities may enable better prediction of environmental impacts across coral reef ecosystems. In this study, we investigated how the community composition and diversity of dinoflagellate symbionts in the genus Symbiodinium were distributed among 12 host species from six taxonomic orders (Actinaria, Alcyonacea, Miliolida, Porifera, Rhizostoma, Scleractinia) and in the reef water and sediments at Lizard Island, Great Barrier Reef before the 3 rd Global Coral Bleaching Event. 454 pyrosequencing of the ITS2 region of Symbiodinium yielded 83 Operational Taxonomic Units (OTUs) at a 97% similarity cut-off. Approximately half of the Symbiodinium OTUs from reef water or sediments were also present in symbio. OTUs belonged to six clades (A-D, F-G), but community structure was uneven. The two most abundant OTUs (100% matches to types C1 and A3) comprised 91% of reads and OTU C1 was shared by all species. However, sequence-based analysis of these dominant OTUs revealed host species-specificity, suggesting that genetic similarity cut-offs of Symbiodinium ITS2 data sets need careful evaluation. Of the less abundant OTUs, roughly half occurred at only one site or in one species and the background Symbiodinium communities were distinct between individual samples. We conclude that sampling multiple host taxa with differing life history traits will be critical to fully understand the symbiont diversity of a given system and to predict coral ecosystem responses to environmental change and disturbance considering the differential stress response of the taxa within. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.

PubMed

Owa, Chie; Poulin, Matthew; Yan, Liying; Shioda, Toshi

2018-01-01

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.

Lyssavirus Detection and Typing Using Pyrosequencing▿#‖

PubMed Central

De Benedictis, Paola; De Battisti, Cristian; Dacheux, Laurent; Marciano, Sabrina; Ormelli, Silvia; Salomoni, Angela; Caenazzo, Silvia Tiozzo; Lepelletier, Anthony; Bourhy, Hervé; Capua, Ilaria; Cattoli, Giovanni

2011-01-01

Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3′ terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin. PMID:21389152

Application of Pyrosequencing® in Food Biodefense.

PubMed

Amoako, Kingsley Kwaku

2015-01-01

The perpetration of a bioterrorism attack poses a significant risk for public health with potential socioeconomic consequences. It is imperative that we possess reliable assays for the rapid and accurate identification of biothreat agents to make rapid risk-informed decisions on emergency response. The development of advanced methodologies for the detection of biothreat agents has been evolving rapidly since the release of the anthrax spores in the mail in 2001, and recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence-based approaches such as Pyrosequencing(®), which has the capability to determine short DNA stretches in real time using biotinylated PCR amplicons, have potential biodefense applications. Using markers from the virulence plasmids and chromosomal regions, my laboratory has demonstrated the power of this technology in the rapid, specific, and sensitive detection of B. anthracis spores and Yersinia pestis in food. These are the first applications for the detection of the two organisms in food. Furthermore, my lab has developed a rapid assay to characterize the antimicrobial resistance (AMR) gene profiles for Y. pestis using Pyrosequencing. Pyrosequencing is completed in about 60 min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence, thus enabling rapid risk-informed decisions to be made. A typical run yields 40-84 bp reads with 94-100 % identity to the expected sequence. It also provides a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The method described is proposed as a novel detection system for potential application in food biodefense.

Field Monitoring of Avian Influenza Viruses: Whole-Genome Sequencing and Tracking of Neuraminidase Evolution Using 454 Pyrosequencing

PubMed Central

Croville, Guillaume; Soubies, Sébastien Mathieu; Barbieri, Johanna; Klopp, Christophe; Mariette, Jérôme; Bouchez, Olivier; Camus-Bouclainville, Christelle

2012-01-01

Adaptation of avian influenza viruses (AIVs) from waterfowl to domestic poultry with a deletion in the neuraminidase (NA) stalk has already been reported. The way the virus undergoes this evolution, however, is thus far unclear. We address this question using pyrosequencing of duck and turkey low-pathogenicity AIVs. Ducks and turkeys were sampled at the very beginning of an H6N1 outbreak, and turkeys were swabbed again 8 days later. NA stalk deletions were evidenced in turkeys by Sanger sequencing. To further investigate viral evolution, 454 pyrosequencing was performed: for each set of samples, up to 41,500 reads of ca. 400 bp were generated and aligned. Genetic polymorphisms between duck and turkey viruses were tracked on the whole genome. NA deletion was detected in less than 2% of reads in duck feces but in 100% of reads in turkey tracheal specimens collected at the same time. Further variations in length were observed in NA from turkeys 8 days later. Similarly, minority mutants emerged on the hemagglutinin (HA) gene, with substitutions mostly in the receptor binding site on the globular head. These critical changes suggest a strong evolutionary pressure in turkeys. The increasing performances of next-generation sequencing technologies should enable us to monitor the genomic diversity of avian influenza viruses and early emergence of potentially pathogenic variants within bird flocks. The present study, based on 454 pyrosequencing, suggests that NA deletion, an example of AIV adaptation from waterfowl to domestic poultry, occurs by selection rather than de novo emergence of viral mutants. PMID:22718944

Metatranscriptomics and Pyrosequencing Facilitate Discovery of Potential Viral Natural Enemies of the Invasive Caribbean Crazy Ant, Nylanderia pubens

PubMed Central

Valles, Steven M.; Oi, David H.; Yu, Fahong; Tan, Xin-Xing; Buss, Eileen A.

2012-01-01

Background Nylanderia pubens (Forel) is an invasive ant species that in recent years has developed into a serious nuisance problem in the Caribbean and United States. A rapidly expanding range, explosive localized population growth, and control difficulties have elevated this ant to pest status. Professional entomologists and the pest control industry in the United States are urgently trying to understand its biology and develop effective control methods. Currently, no known biological-based control agents are available for use in controlling N. pubens. Methodology and Principal Findings Metagenomics and pyrosequencing techniques were employed to examine the transcriptome of field-collected N. pubens colonies in an effort to identify virus infections with potential to serve as control agents against this pest ant. Pyrosequencing (454-platform) of a non-normalized N. pubens expression library generated 1,306,177 raw sequence reads comprising 450 Mbp. Assembly resulted in generation of 59,017 non-redundant sequences, including 27,348 contigs and 31,669 singlets. BLAST analysis of these non-redundant sequences identified 51 of potential viral origin. Additional analyses winnowed this list of potential viruses to three that appear to replicate in N. pubens. Conclusions Pyrosequencing the transcriptome of field-collected samples of N. pubens has identified at least three sequences that are likely of viral origin and, in which, N. pubens serves as host. In addition, the N. pubens transcriptome provides a genetic resource for the scientific community which is especially important at this early stage of developing a knowledgebase for this new pest. PMID:22384082

CYP3A4 and CYP3A5 genotyping by Pyrosequencing

PubMed Central

Garsa, Adam A; McLeod, Howard L; Marsh, Sharon

2005-01-01

Background Human cytochrome P450 3A enzymes, particularly CYP3A4 and CYP3A5, play an important role in drug metabolism. CYP3A expression exhibits substantial interindividual variation, much of which may result from genetic variation. This study describes Pyrosequencing assays for key SNPs in CYP3A4 (CYP3A4*1B, CYP3A4*2, and CYP3A4*3) and CYP3A5 (CYP3A5*3C and CYP3A5*6). Methods Genotyping of 95 healthy European and 95 healthy African volunteers was performed using Pyrosequencing. Linkage disequilibrium, haplotype inference, Hardy-Weinberg equilibrium, and tag SNPs were also determined for these samples. Results CYP3A4*1B allele frequencies were 4% in Europeans and 82% in Africans. The CYP3A4*2 allele was found in neither population sample. CYP3A4*3 had an allele frequency of 2% in Europeans and 0% in Africans. The frequency of CYP3A5*3C was 94% in Europeans and 12% in Africans. No CYP3A5*6 variants were found in the European samples, but this allele had a frequency of 16% in the African samples. Allele frequencies and haplotypes show interethnic variation, highlighting the need to analyze clinically relevant SNPs and haplotypes in a variety of ethnic groups. Conclusion Pyrosequencing is a versatile technique that could improve the efficiency of SNP analysis for pharmacogenomic research with the ultimate goal of pre-screening patients for individual therapy selection. PMID:15882469

Comparative analysis of the intestinal bacterial communities in different species of carp by pyrosequencing.

PubMed

Li, Tongtong; Long, Meng; Gatesoupe, François-Joël; Zhang, Qianqian; Li, Aihua; Gong, Xiaoning

2015-01-01

Gut microbiota is increasingly regarded as an integral component of the host, due to important roles in the modulation of the immune system, the proliferation of the intestinal epithelium and the regulation of the dietary energy intake. Understanding the factors that influence the composition of these microbial communities is essential to health management, and the application to aquatic animals still requires basic investigation. In this study, we compared the bacterial communities harboured in the intestines and in the rearing water of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius cuvieri), and bighead carp (Hypophthalmichthys nobilis), by using 454-pyrosequencing with barcoded primers targeting the V4 to V5 regions of the bacterial 16S rRNA gene. The specimens of the three species were cohabiting in the same pond. Between 6,218 and 10,220 effective sequences were read from each sample, resulting in a total of 110,398 sequences for 13 samples from gut microbiota and pond water. In general, the microbial communities of the three carps were dominated by Fusobacteria, Firmicutes, Proteobacteria and Bacteroidetes, but the abundance of each phylum was significantly different between species. At the genus level, the overwhelming group was Cetobacterium (97.29 ± 0.46 %) in crucian carp, while its abundance averaged c. 40 and 60 % of the sequences read in the other two species. There was higher microbial diversity in the gut of filter-feeding bighead carp than the gut of the two other species, with grazing feeding habits. The composition of intestine microbiota of grass carp and crucian carp shared higher similarity when compared with bighead carp. The principal coordinates analysis (PCoA) with the weighted UniFrac distance and the heatmap analysis suggested that gut microbiota was not a simple reflection of the microbial community in the local habitat but resulted from species-specific selective pressures, possibly dependent on behavioural, immune and metabolic characteristics.

454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature

PubMed Central

Rodriguez-Sanchez, Alejandro; Rodelas, Belén; Abbas, Ben A.; Martinez-Toledo, Maria Victoria; van Loosdrecht, Mark C. M.; Osorio, F.; Gonzalez-Lopez, Jesus

2015-01-01

Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44–49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed. PMID:26421306

454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature.

PubMed

Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Rodelas, Belén; Abbas, Ben A; Martinez-Toledo, Maria Victoria; van Loosdrecht, Mark C M; Osorio, F; Gonzalez-Lopez, Jesus

2015-01-01

Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44-49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.

Detection of Drug-Resistant Mycobacterium tuberculosis.

PubMed

Engström, Anna; Juréen, Pontus

2015-01-01

Tuberculosis (TB) remains a global health problem. The increasing prevalence of drug-resistant Mycobacterium tuberculosis, the causative agent of TB, demands new measures to combat the situation. Rapid and accurate diagnosis of the pathogen and its drug susceptibility pattern is essential for timely initiation of optimal treatment, and, ultimately, control of the disease. We have developed a molecular method for detection of first- and second-line drug resistance in M. tuberculosis by Pyrosequencing(®). The method consists of seven Pyrosequencing assays for the detection of mutations in the genes or promoter regions, which are most commonly responsible for resistance to the drugs rifampicin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and fluoroquinolones. The method was validated on clinical isolates and it was shown that the sensitivity and specificity of the method were comparable to those of Sanger sequencing. In the protocol in this chapter we describe the steps necessary for setting up and performing Pyrosequencing for M. tuberculosis. The first part of the protocol describes the assay development and the second part of the protocol describes utilization of the method.

Antimicrobial peptide expression in a wild tobacco plant reveals the limits of host-microbe-manipulations in the field

PubMed Central

Karimi Dorcheh, Elham; Li, Ran; Rameshkumar, Natarajan; Baldwin, Ian T

2018-01-01

Plant-microbe associations are thought to be beneficial for plant growth and resistance against biotic or abiotic stresses, but for natural ecosystems, the ecological analysis of microbiome function remains in its infancy. We used transformed wild tobacco plants (Nicotiana attenuata) which constitutively express an antimicrobial peptide (Mc-AMP1) of the common ice plant, to establish an ecological tool for plant-microbe studies in the field. Transgenic plants showed in planta activity against plant-beneficial bacteria and were phenotyped within the plants´ natural habitat regarding growth, fitness and the resistance against herbivores. Multiple field experiments, conducted over 3 years, indicated no differences compared to isogenic controls. Pyrosequencing analysis of the root-associated microbial communities showed no major alterations but marginal effects at the genus level. Experimental infiltrations revealed a high heterogeneity in peptide tolerance among native isolates and suggests that the diversity of natural microbial communities can be a major obstacle for microbiome manipulations in nature. PMID:29661271

Antimicrobial peptide expression in a wild tobacco plant reveals the limits of host-microbe-manipulations in the field.

PubMed

Weinhold, Arne; Karimi Dorcheh, Elham; Li, Ran; Rameshkumar, Natarajan; Baldwin, Ian T

2018-04-17

Plant-microbe associations are thought to be beneficial for plant growth and resistance against biotic or abiotic stresses, but for natural ecosystems, the ecological analysis of microbiome function remains in its infancy. We used transformed wild tobacco plants ( Nicotiana attenuata ) which constitutively express an antimicrobial peptide (Mc-AMP1) of the common ice plant, to establish an ecological tool for plant-microbe studies in the field. Transgenic plants showed in planta activity against plant-beneficial bacteria and were phenotyped within the plants´ natural habitat regarding growth, fitness and the resistance against herbivores. Multiple field experiments, conducted over 3 years, indicated no differences compared to isogenic controls. Pyrosequencing analysis of the root-associated microbial communities showed no major alterations but marginal effects at the genus level. Experimental infiltrations revealed a high heterogeneity in peptide tolerance among native isolates and suggests that the diversity of natural microbial communities can be a major obstacle for microbiome manipulations in nature. © 2018, Weinhold et al.

Co-occurrence Networks Among Bacteria and Microbial Eukaryotes of Lake Baikal During a Spring Phytoplankton Bloom.

PubMed

Mikhailov, Ivan S; Zakharova, Yulia R; Bukin, Yuri S; Galachyants, Yuri P; Petrova, Darya P; Sakirko, Maria V; Likhoshway, Yelena V

2018-06-07

The pelagic zone of Lake Baikal is an ecological niche where phytoplankton bloom causes increasing microbial abundance in spring which plays a key role in carbon turnover in the freshwater lake. Co-occurrence patterns revealed among different microbes can be applied to predict interactions between the microbes and environmental conditions in the ecosystem. We used 454 pyrosequencing of 16S rRNA and 18S rRNA genes to study bacterial and microbial eukaryotic communities and their co-occurrence patterns at the pelagic zone of Lake Baikal during a spring phytoplankton bloom. We found that microbes within one domain mostly correlated positively with each other and are highly interconnected. The highly connected taxa in co-occurrence networks were operational taxonomic units (OTUs) of Actinobacteria, Bacteroidetes, Alphaproteobacteria, and autotrophic and unclassified Eukaryota which might be analogous to microbial keystone taxa. Constrained correspondence analysis revealed the relationships of bacterial and microbial eukaryotic communities with geographical location.

A pyrosequencing assay for the quantitative methylation analysis of the PCDHB gene cluster, the major factor in neuroblastoma methylator phenotype.

PubMed

Banelli, Barbara; Brigati, Claudio; Di Vinci, Angela; Casciano, Ida; Forlani, Alessandra; Borzì, Luana; Allemanni, Giorgio; Romani, Massimo

2012-03-01

Epigenetic alterations are hallmarks of cancer and powerful biomarkers, whose clinical utilization is made difficult by the absence of standardization and of common methods of data interpretation. The coordinate methylation of many loci in cancer is defined as 'CpG island methylator phenotype' (CIMP) and identifies clinically distinct groups of patients. In neuroblastoma (NB), CIMP is defined by a methylation signature, which includes different loci, but its predictive power on outcome is entirely recapitulated by the PCDHB cluster only. We have developed a robust and cost-effective pyrosequencing-based assay that could facilitate the clinical application of CIMP in NB. This assay permits the unbiased simultaneous amplification and sequencing of 17 out of 19 genes of the PCDHB cluster for quantitative methylation analysis, taking into account all the sequence variations. As some of these variations were at CpG doublets, we bypassed the data interpretation conducted by the methylation analysis software to assign the corrected methylation value at these sites. The final result of the assay is the mean methylation level of 17 gene fragments in the protocadherin B cluster (PCDHB) cluster. We have utilized this assay to compare the methylation levels of the PCDHB cluster between high-risk and very low-risk NB patients, confirming the predictive value of CIMP. Our results demonstrate that the pyrosequencing-based assay herein described is a powerful instrument for the analysis of this gene cluster that may simplify the data comparison between different laboratories and, in perspective, could facilitate its clinical application. Furthermore, our results demonstrate that, in principle, pyrosequencing can be efficiently utilized for the methylation analysis of gene clusters with high internal homologies.

Clinical Implications of Quantitative JAK2 V617F Analysis using Droplet Digital PCR in Myeloproliferative Neoplasms

PubMed Central

Lee, Eunyoung; Lee, Kyoung Joo; Park, Hyein; Chung, Jin Young; Lee, Mi-Na; Chang, Myung Hee; Yoo, Jongha; Lee, Hyewon

2018-01-01

Background JAK2 V617F is the most common mutation in myeloproliferative neoplasms (MPNs) and is a major diagnostic criterion. Mutation quantification is useful for classifying patients with MPN into subgroups and for prognostic prediction. Droplet digital PCR (ddPCR) can provide accurate and reproducible quantitative analysis of DNA. This study was designed to verify the correlation of ddPCR with pyrosequencing results in the diagnosis of MPN and to investigate clinical implications of the mutational burden. Methods Peripheral blood or bone marrow samples were obtained from 56 patients newly diagnosed with MPN or previously diagnosed with MPN but not yet indicated for JAK2 inhibitor treatment between 2012 and 2016. The JAK2 V617F mutation was detected by pyrosequencing as a diagnostic work-up. The same samples were used for ddPCR to determine the correlation between assays and establish a detection sensitivity cut-off. Clinical and hematologic aspects were reviewed. Results Forty-two (75%) and 46 (82.1%) patients were positive for JAK2 V617F by pyrosequencing and ddPCR, respectively. The mean mutated allele frequency at diagnosis was 37.5±30.1% and was 40.7±31.2% with ddPCR, representing a strong correlation (r=0.9712, P<0.001). Follow-up samples were available for 12 patients, including eight that were JAK2 V617F-positive. Of these, mutational burden reduction after treatment was observed in six patients (75%), consistent with trends of hematologic improvement. Conclusions Quantitative analysis of the JAK2 V617F mutation using ddPCR was highly correlated with pyrosequencing data and may reflect the clinical response to treatment. PMID:29214759

A molecular gram stain using broad range PCR and pyrosequencing technology: a potentially useful tool for diagnosing orthopaedic infections.

PubMed

Kobayashi, Naomi; Bauer, Thomas W; Togawa, Daisuke; Lieberman, Isador H; Sakai, Hiroshige; Fujishiro, Takaaki; Tuohy, Marion J; Procop, Gary W

2005-06-01

The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins

PubMed Central

2011-01-01

Abstract Background Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway. Results One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with β-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with β-AS in various B. chinense tissues. Conclusions A collection of high-quality ESTs for B. chinense obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of B. chinense and other Bupleurum species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the P450s and UGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins. PMID:22047182

Dogs Leaving the ICU Carry a Very Large Multi-Drug Resistant Enterococcal Population with Capacity for Biofilm Formation and Horizontal Gene Transfer

PubMed Central

Ghosh, Anuradha; Dowd, Scot E.; Zurek, Ludek

2011-01-01

The enterococcal community from feces of seven dogs treated with antibiotics for 2–9 days in the veterinary intensive care unit (ICU) was characterized. Both, culture-based approach and culture-independent 16S rDNA amplicon 454 pyrosequencing, revealed an abnormally large enterococcal community: 1.4±0.8×108 CFU gram−1 of feces and 48.9±11.5% of the total 16,228 sequences, respectively. The diversity of the overall microbial community was very low which likely reflects a high selective antibiotic pressure. The enterococcal diversity based on 210 isolates was also low as represented by Enterococcus faecium (54.6%) and Enterococcus faecalis (45.4%). E. faecium was frequently resistant to enrofloxacin (97.3%), ampicillin (96.5%), tetracycline (84.1%), doxycycline (60.2%), erythromycin (53.1%), gentamicin (48.7%), streptomycin (42.5%), and nitrofurantoin (26.5%). In E. faecalis, resistance was common to tetracycline (59.6%), erythromycin (56.4%), doxycycline (53.2%), and enrofloxacin (31.9%). No resistance was detected to vancomycin, tigecycline, linezolid, and quinupristin/dalfopristin in either species. Many isolates carried virulence traits including gelatinase, aggregation substance, cytolysin, and enterococcal surface protein. All E. faecalis strains were biofilm formers in vitro and this phenotype correlated with the presence of gelE and/or esp. In vitro intra-species conjugation assays demonstrated that E. faecium were capable of transferring tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin resistance traits to human clinical strains. Multi-locus variable number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE) of E. faecium strains showed very low genotypic diversity. Interestingly, three E. faecium clones were shared among four dogs suggesting their nosocomial origin. Furthermore, multi-locus sequence typing (MLST) of nine representative MLVA types revealed that six sequence types (STs) originating from five dogs were identical or closely related to STs of human clinical isolates and isolates from hospital outbreaks. It is recommended to restrict close physical contact between pets released from the ICU and their owners to avoid potential health risks. PMID:21811613

Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island

PubMed Central

2010-01-01

Background The Gram-positive bacterium Enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients. Results We present a pyrosequencing-based comparative genome analysis of seven E. faecium strains that were isolated from various sources. In the genomes of clinical isolates several antibiotic resistance genes were identified, including the vanA transposon that confers resistance to vancomycin in two strains. A functional comparison between E. faecium and the related opportunistic pathogen E. faecalis based on differences in the presence of protein families, revealed divergence in plant carbohydrate metabolic pathways and oxidative stress defense mechanisms. The E. faecium pan-genome was estimated to be essentially unlimited in size, indicating that E. faecium can efficiently acquire and incorporate exogenous DNA in its gene pool. One of the most prominent sources of genomic diversity consists of bacteriophages that have integrated in the genome. The CRISPR-Cas system, which contributes to immunity against bacteriophage infection in prokaryotes, is not present in the sequenced strains. Three sequenced isolates carry the esp gene, which is involved in urinary tract infections and biofilm formation. The esp gene is located on a large pathogenicity island (PAI), which is between 64 and 104 kb in size. Conjugation experiments showed that the entire esp PAI can be transferred horizontally and inserts in a site-specific manner. Conclusions Genes involved in environmental persistence, colonization and virulence can easily be aquired by E. faecium. This will make the development of successful treatment strategies targeted against this organism a challenge for years to come. PMID:20398277

New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis.

PubMed

Smith, Michael G; Gianoulis, Tara A; Pukatzki, Stefan; Mekalanos, John J; Ornston, L Nicholas; Gerstein, Mark; Snyder, Michael

2007-03-01

Acinetobacter baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections including pneumonia, meningitis, septicemia, and urinary tract infections. We explored the pathogenic content of this harmful pathogen using a combination of DNA sequencing and insertional mutagenesis. The genome of this organism was sequenced using a strategy involving high-density pyrosequencing, a novel, rapid method of high-throughput sequencing. Excluding the rDNA repeats, the assembled genome is 3,976,746 base pairs (bp) and has 3830 ORFs. A significant fraction of ORFs (17.2%) are located in 28 putative alien islands, indicating that the genome has acquired a large amount of foreign DNA. Consistent with its role in pathogenesis, a remarkable number of the islands (16) contain genes implicated in virulence, indicating the organism devotes a considerable portion of its genes to pathogenesis. The largest island contains elements homologous to the Legionella/Coxiella Type IV secretion apparatus. Type IV secretion systems have been demonstrated to be important for virulence in other organisms and thus are likely to help mediate pathogenesis of A. baumannii. Insertional mutagenesis generated avirulent isolates of A. baumannii and verified that six of the islands contain virulence genes, including two novel islands containing genes that lacked homology with others in the databases. The DNA sequencing approach described in this study allows the rapid elucidation of the DNA sequence of any microbe and, when combined with genetic screens, can identify many novel genes important for microbial pathogenesis.

Biogeography and Photosynthetic Biomass of Arctic Marine Pico-Eukaroytes during Summer of the Record Sea Ice Minimum 2012

PubMed Central

Metfies, Katja; von Appen, Wilken-Jon; Kilias, Estelle; Nicolaus, Anja; Nöthig, Eva-Maria

2016-01-01

Information on recent photosynthetic biomass distribution and biogeography of Arctic marine pico-eukaryotes (0.2–3 μm) is needed to better understand consequences of environmental change for Arctic marine ecosystems. We analysed pico-eukaryote biomass and community composition in Fram Strait and large parts of the Central Arctic Ocean (Nansen Basin, Amundsen Basin) using chlorophyll a (Chl a) measurements, automated ribosomal intergenic spacer analysis (ARISA) and 454-pyrosequencing. Samples were collected during summer 2012, the year with the most recent record sea ice minimum. Chl a concentrations were highest in eastern Fram Strait and pico-plankton accounted for 60–90% of Chl a biomass during the observation period. ARISA-patterns and 454-pyrosequencing revealed that pico-eukaryote distribution is closely related to water mass distribution in the euphotic zone of the Arctic Ocean. Phaeocystaceae, Micromonas sp., Dinophyceae and Syndiniales constitute a high proportion of sequence reads, while sequence abundance of autotrophic Phaeocystaceae and mixotrophic Micromonas sp. was inversely correlated. Highest sequence abundances of Phaeocystaceae were observed in the warm Atlantic Waters in Fram Strait, while Micromonas sp. dominated the abundant biosphere in the arctic halocline. Our results are of particular interest considering existing hypotheses that environmental conditions in Nansen Basin might become more similar to the current conditions in Fram Strait. We propose that in response, biodiversity and biomass of pico-eukaryotes in Nansen Basin could resemble those currently observed in Fram Strait in the future. This would significantly alter biogeochemical cycles in a large part of the Central Arctic Ocean. PMID:26895333

Phylogenetically Distinct Phylotypes Modulate Nitrification in a Paddy Soil

PubMed Central

Zhao, Jun; Wang, Baozhan

2015-01-01

Paddy fields represent a unique ecosystem in which regular flooding occurs, allowing for rice cultivation. However, the taxonomic identity of the microbial functional guilds that catalyze soil nitrification remains poorly understood. In this study, we provide molecular evidence for distinctly different phylotypes of nitrifying communities in a neutral paddy soil using high-throughput pyrosequencing and DNA-based stable isotope probing (SIP). Following urea addition, the levels of soil nitrate increased significantly, accompanied by an increase in the abundance of the bacterial and archaeal amoA gene in microcosms subjected to SIP (SIP microcosms) during a 56-day incubation period. High-throughput fingerprints of the total 16S rRNA genes in SIP microcosms indicated that nitrification activity positively correlated with the abundance of Nitrosospira-like ammonia-oxidizing bacteria (AOB), soil group 1.1b-like ammonia-oxidizing archaea (AOA), and Nitrospira-like nitrite-oxidizing bacteria (NOB). Pyrosequencing of 13C-labeled DNA further revealed that 13CO2 was assimilated by these functional groups to a much greater extent than by marine group 1.1a-associated AOA and Nitrobacter-like NOB. Phylogenetic analysis demonstrated that active AOB communities were closely affiliated with Nitrosospira sp. strain L115 and the Nitrosospira multiformis lineage and that the 13C-labeled AOA were related to phylogenetically distinct groups, including the moderately thermophilic “Candidatus Nitrososphaera gargensis,” uncultured fosmid 29i4, and acidophilic “Candidatus Nitrosotalea devanaterra” lineages. These results suggest that a wide variety of microorganisms were involved in soil nitrification, implying physiological diversification of soil nitrifying communities that are constantly exposed to environmental fluctuations in paddy fields. PMID:25724959

Microbial diversity in Paris polyphylla var. yunnanensis rhizomes of varying ages.

PubMed

Yang, Y; Yang, S C; Zhao, J; Udikeri, S; Liu, T

2015-12-21

Endophyte microorganisms live inside plants without causing them any apparent damage. Recently, endophytic microorganisms have attracted attention because they can produce bioactive compounds of biotechnological interest. The endophytic microorganisms in Paris polyphylla var. yunnanensis (Liliaceae) - a species used since antiquity in traditional Chinese medicine - are under scrutiny because they may be responsible for producing the bioactive metabolites associated with the plant. The levels of bioactive metabolites in the rhizomes of P. polyphylla increase with rhizome age. To elucidate the roles played by endophytes in the accumulation of bioactive metabolites, we investigated the community structure and diversity of the endophytic microorganisms in P. polyphylla rhizomes of different ages (4, 6, and 8 years) using 16S rRNA and internal transcribed spacer (ITS) sequence analysis. 16S rDNA amplicon pyrosequencing revealed that the number of operational taxonomic units was lower in the 8-year-old samples than in the other samples. A total of 28 phyla were observed in the P. polyphylla samples and the predominant bacteria were of the Cyanobacteria and Proteobacteria phyla. Moreover, the percentage of Cyanobacteria increased with rhizome age. Similarly, ITS1 amplicon pyrosequencing identified developmental changes in the most abundant fungal classes; some classes were more prevalent in the 8-year-old rhizomes than in younger rhizomes, indicating the importance in secondary metabolism in older rhizomes. Our study showed that endophyte microorganism diversity and prevalence depend on P. polyphylla rhizome age. There was also an indication that some endophyte microorganisms contribute to the higher saponin content in older P. polyphylla specimens.

Discovery of novel MHC-class I alleles and haplotypes in Filipino cynomolgus macaques (Macaca fascicularis) by pyrosequencing and Sanger sequencing: Mafa-class I polymorphism.

PubMed

Shiina, Takashi; Yamada, Yukiho; Aarnink, Alice; Suzuki, Shingo; Masuya, Anri; Ito, Sayaka; Ido, Daisuke; Yamanaka, Hisashi; Iwatani, Chizuru; Tsuchiya, Hideaki; Ishigaki, Hirohito; Itoh, Yasushi; Ogasawara, Kazumasa; Kulski, Jerzy K; Blancher, Antoine

2015-10-01

Although the low polymorphism of the major histocompatibility complex (MHC) transplantation genes in the Filipino cynomolgus macaque (Macaca fascicularis) is expected to have important implications in the selection and breeding of animals for medical research, detailed polymorphism information is still lacking for many of the duplicated class I genes. To better elucidate the degree and types of MHC polymorphisms and haplotypes in the Filipino macaque population, we genotyped 127 unrelated animals by the Sanger sequencing method and high-resolution pyrosequencing and identified 112 different alleles, 28 at cynomolgus macaque MHC (Mafa)-A, 54 at Mafa-B, 12 at Mafa-I, 11 at Mafa-E, and seven at Mafa-F alleles, of which 56 were newly described. Of them, the newly discovered Mafa-A8*01:01 lineage allele had low nucleotide similarities (<86%) with primate MHC class I genes, and it was also conserved in the Vietnamese and Indonesian populations. In addition, haplotype estimations revealed 17 Mafa-A, 23 Mafa-B, and 12 Mafa-E haplotypes integrated with 84 Mafa-class I haplotypes and Mafa-F alleles. Of these, the two Mafa-class I haplotypes, F/A/E/B-Hp1 and F/A/E/B-Hp2, had the highest haplotype frequencies at 10.6 and 10.2%, respectively. This suggests that large scale genetic screening of the Filipino macaque population would identify these and other high-frequency Mafa-class I haplotypes that could be used as MHC control animals for the benefit of biomedical research.

Low-complexity microbiota in the duodenum of children with newly diagnosed ulcerative colitis.

PubMed

Sjöberg, Fei; Barkman, Cecilia; Nookaew, Intawat; Östman, Sofia; Adlerberth, Ingegerd; Saalman, Robert; Wold, Agnes E

2017-01-01

Inflammatory bowel disease (IBD) is characterized by gut dysbiosis. To date, the large bowel microbiota has been in focus. However, the microbiota of the small intestine may also be of importance, as the small bowel is a site for the induction and control of mucosal immune responses, which can be modulated by constituents of the local microbiota. Duodenal fluids were collected during diagnostic work-up of treatment-naïve children who were suspected of having IBD. The duodenal fluids were analyzed by pyrosequencing (average of 32,000 reads/sample, read length of 500 nucleotides). After diagnosis, the duodenal microbiota of subjects with ulcerative colitis (N = 8) or Crohn's disease (N = 5), and non-IBD controls (N = 8) were compared. Pyrosequencing revealed that the duodenal microbiota of children with ulcerative colitis contained fewer Operational Taxonomic Units (OTUs) per individual than the duodenal microbiota of the controls (P = 0.005). This reduction in richness of the duodenal microbiota was seen for three major phyla: Firmicutes, Actinobacteria, and Bacteroidetes. Several bacterial genera were detected less frequently in the children with ulcerative colitis than in the non-IBD controls, including Collinsella (P = 0.001), Lactobacillus (P = 0.007), and Bacillus (P = 0.007), as well as a non-identified member of the order Sphingobacteriales (P = 0.007). In this pilot study, we show that the duodenal microbiota of children with ulcerative colitis exhibits reduced overall richness, despite the fact that the inflammation is primarily localized to the colon. These results should be corroborated in a larger study.

Fungal Community Structure in Disease Suppressive Soils Assessed by 28S LSU Gene Sequencing

PubMed Central

Penton, C. Ryan; Gupta, V. V. S. R.; Tiedje, James M.; Neate, Stephen M.; Ophel-Keller, Kathy; Gillings, Michael; Harvey, Paul; Pham, Amanda; Roget, David K.

2014-01-01

Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. Soil microbe and phytopathogen interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soil-borne disease. Fungi were analyzed in co-located soils ‘suppressive’ or ‘non-suppressive’ for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. DNA was extracted from a minimum of 125 g of soil per replicate to reduce the micro-scale community variability, and from soil samples taken at sowing and from the rhizosphere at 7 weeks to cover the peak Rhizoctonia infection period. A total of ∼994,000 reads were classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Statistical analyses and community ordinations revealed significant differences in fungal community composition between suppressive and non-suppressive soil and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites such as Xylaria spp. Non-suppressive soils were dominated by Alternaria, Gibberella and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and identified candidate taxa that may influence pathogen-plant interactions in stable disease suppression. PMID:24699870

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing

PubMed Central

Green-Engert, Rebecca; Hoelzle, Katharina; Zeller, Ellen; Seifert, Jana; Hoelzle, Ludwig E.; Rodehutscord, Markus

2015-01-01

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information. PMID:26588075

Spatial Variation of the Gut Microbiota in Broiler Chickens as Affected by Dietary Available Phosphorus and Assessed by T-RFLP Analysis and 454 Pyrosequencing.

PubMed

Witzig, Maren; Carminha-Silva, Amelia; Camarinha da Silva, Amelia; Green-Engert, Rebecca; Hoelzle, Katharina; Zeller, Ellen; Seifert, Jana; Hoelzle, Ludwig E; Rodehutscord, Markus

2015-01-01

Molecular fingerprinting and sequencing based techniques have been widely used to characterize microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) and 454-pyrosequencing were used to determine the microorganisms present in the different sections of the chicken gastrointestinal tract (GIT) (crop, jejunum, ileum and caeca). Broilers fed with diets differing in phosphorous (P) and calcium (Ca) as well as in phytase levels were used to study the microbiota of the upper and lower part of the GIT. A database with terminal restriction fragments (T-RF) of the most important organism present in the different gastrointestinal sections was constructed. The analysis revealed a distinct microbial assemblage on each section. Regardless of the diet, crop, jejunum and ileum were mainly colonized by Lactobacillaceae, and caeca were the most diverse site. The correlation between Lactobacillus crispatus and L. reuteri was positive in the crop, but negative in the jejunum. In crop samples, higher P and Ca levels led to a shift in the abundance of L. reuteri and L. crispatus to L. salivarius and L. taiwanensis whereas in the ileum supplementation of phytase favored L. salivarius and L. taiwanensis but resulted in decreased abundance of L. crispatus. Both methods were correlating significantly, being T-RFLP a reliable fingerprinting method to rapidly analyze large numbers of samples in a cost-effective and rapid manner. Results are easy to interpret with no need of deep bioinformatics knowledge and can be integrated with taxonomic information.

Bacterial community structure in two permafrost wetlands on the Tibetan Plateau and Sanjiang Plain, China.

PubMed

Yun, Juanli; Ju, Yiwen; Deng, Yongcui; Zhang, Hongxun

2014-08-01

Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10(10) 16S rRNA gene copies per gram of wet soil in both wetlands, with 10(8) pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.

Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours.

PubMed

Kajiura, Koichiro; Takizawa, Hiromitsu; Morimoto, Yuki; Masuda, Kiyoshi; Tsuboi, Mitsuhiro; Kishibuchi, Reina; Wusiman, Nuliamina; Sawada, Toru; Kawakita, Naoya; Toba, Hiroaki; Yoshida, Mitsuteru; Kawakami, Yukikiyo; Naruto, Takuya; Imoto, Issei; Tangoku, Akira; Kondo, Kazuya

2017-09-01

Aberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. Genome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. We identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. RASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploration of bacterial species associated with the salivary microbiome of individuals with a low susceptibility to dental caries.

PubMed

Yasunaga, Haruna; Takeshita, Toru; Shibata, Yukie; Furuta, Michiko; Shimazaki, Yoshihiro; Akifusa, Sumio; Ninomiya, Toshiharu; Kiyohara, Yutaka; Takahashi, Ichiro; Yamashita, Yoshihisa

2017-11-01

Dental caries is caused by acidogenic plaque microbiota formed on saliva-bathed tooth surfaces, in which multiple organisms act collectively to initiate and expand a cavity. We explored bacterial species associated with the salivary microbiome of individuals with low susceptibility to dental caries. The bacterial composition of saliva from 19 young adults was analyzed using barcoded pyrosequencing of the 16S rRNA gene; we compared 10 caries-experienced (CE) and nine caries-free (CF) individuals. A quantitative PCR assay of saliva from 139 orally healthy adults aged 40-59 years was carried out to confirm the result obtained by pyrosequencing analysis. The microbiomes of CF individuals showed more diverse communities with a significantly greater proportion of the genus Porphyromonas. Among operational taxonomic units (OTUs) corresponding to the genus Porphyromonas, the OTU corresponding to P. pasteri was the most predominant and its relative abundance in CF individuals was significantly greater than in CE individuals (P < 0.001, Wilcoxon rank sum test). A quantitative PCR assay of saliva confirmed that the amounts of P. pasteri were significantly higher in individuals with lower caries experience (filled teeth <15, n = 67) than in those with higher caries experience (filled teeth ≥15, n = 72) (P < 0.001, Student's t test). These results revealed an association between a greater abundance of P. pasteri and lower susceptibility to dental caries. P. pasteri may be a bacterial species that could potentially be used as a marker for maintaining a healthy oral microbiome against dental caries.

MAOA promoter methylation and susceptibility to carotid atherosclerosis: role of familial factors in a monozygotic twin sample

PubMed Central

2012-01-01

Background Atherosclerosis is a complex process involving both genetic and epigenetic factors. The monoamine oxidase A (MAOA) gene regulates the metabolism of key neurotransmitters and has been associated with cardiovascular risk factors. This study investigates whether MAOA promoter methylation is associated with atherosclerosis, and whether this association is confounded by familial factors in a monozygotic (MZ) twin sample. Methods We studied 84 monozygotic (MZ) twin pairs drawn from the Vietnam Era Twin Registry. Carotid intima-media thickness (IMT) was measured by ultrasound. DNA methylation in the MAOA promoter region was quantified by bisulfite pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. The association between DNA methylation and IMT was first examined by generalized estimating equation, followed by matched pair analyses to determine whether the association was confounded by familial factors. Results When twins were analyzed as individuals, increased methylation level was associated with decreased IMT at four of the seven studied CpG sites. However, this association substantially reduced in the matched pair analyses. Further adjustment for MAOA genotype also considerably attenuated this association. Conclusions The association between MAOA promoter methylation and carotid IMT is largely explained by familial factors shared by the twins. Because twins reared together share early life experience, which may leave a long-lasting epigenetic mark, aberrant MAOA methylation may represent an early biomarker for unhealthy familial environment. Clarification of familial factors associated with DNA methylation and early atherosclerosis will provide important information to uncover clinical correlates of disease. PMID:23116433

Visceral hypersensitive rats share common dysbiosis features with irritable bowel syndrome patients.

PubMed

Zhou, Xiao-Yan; Li, Ming; Li, Xia; Long, Xin; Zuo, Xiu-Li; Hou, Xiao-Hua; Cong, Ying-Zi; Li, Yan-Qing

2016-06-14

To evaluate gut microbial dysbiosis in two visceral hypersensitive models in comparison with irritable bowel syndrome (IBS) patients and to explore the extent to which these models capture the dysbiosis of IBS patients. Visceral hypersensitivity was developed using the maternal separation (MS) rat model and post-inflammatory rat model. The visceral sensitivity of the model groups and control group was evaluated using the abdominal withdraw reflex score and electromyography in response to graded colorectal distention. The 16S ribosomal RNA gene from fecal samples was pyrosequenced and analyzed. The correlation between dysbiosis in the microbiota and visceral hypersensitivity was calculated. Positive findings were compared to sequencing data from a published human IBS cohort. Dysbiosis triggered by neonatal maternal separation was lasting but not static. Both MS and post-inflammatory rat fecal microbiota deviated from that of the control rats to an extent that was larger than the co-housing effect. Two short chain fatty acid producing genera, Fusobacterium and Clostridium XI, were shared by the human IBS cohort and by the maternal separation rats and post-inflammatory rats, respectively, to different extents. Fusobacterium was significantly increased in the MS group, and its abundance positively correlated with the degree of visceral hypersensitivity. Porphyromonadaceae was a protective biomarker for both the rat control group and healthy human controls. The dysbiosis MS rat model and the post-inflammatory rat model captured some of the dysbiosis features of IBS patients. Fusobacterium, Clostridium XI and Porphyromonadaceae were identified as targets for future mechanistic research.

MAOA promoter methylation and susceptibility to carotid atherosclerosis: role of familial factors in a monozygotic twin sample.

PubMed

Zhao, Jinying; Forsberg, Christopher W; Goldberg, Jack; Smith, Nicholas L; Vaccarino, Viola

2012-11-02

Atherosclerosis is a complex process involving both genetic and epigenetic factors. The monoamine oxidase A (MAOA) gene regulates the metabolism of key neurotransmitters and has been associated with cardiovascular risk factors. This study investigates whether MAOA promoter methylation is associated with atherosclerosis, and whether this association is confounded by familial factors in a monozygotic (MZ) twin sample. We studied 84 monozygotic (MZ) twin pairs drawn from the Vietnam Era Twin Registry. Carotid intima-media thickness (IMT) was measured by ultrasound. DNA methylation in the MAOA promoter region was quantified by bisulfite pyrosequencing using genomic DNA isolated from peripheral blood leukocytes. The association between DNA methylation and IMT was first examined by generalized estimating equation, followed by matched pair analyses to determine whether the association was confounded by familial factors. When twins were analyzed as individuals, increased methylation level was associated with decreased IMT at four of the seven studied CpG sites. However, this association substantially reduced in the matched pair analyses. Further adjustment for MAOA genotype also considerably attenuated this association. The association between MAOA promoter methylation and carotid IMT is largely explained by familial factors shared by the twins. Because twins reared together share early life experience, which may leave a long-lasting epigenetic mark, aberrant MAOA methylation may represent an early biomarker for unhealthy familial environment. Clarification of familial factors associated with DNA methylation and early atherosclerosis will provide important information to uncover clinical correlates of disease.

Droplet-based pyrosequencing using digital microfluidics.

PubMed

Boles, Deborah J; Benton, Jonathan L; Siew, Germaine J; Levy, Miriam H; Thwar, Prasanna K; Sandahl, Melissa A; Rouse, Jeremy L; Perkins, Lisa C; Sudarsan, Arjun P; Jalili, Roxana; Pamula, Vamsee K; Srinivasan, Vijay; Fair, Richard B; Griffin, Peter B; Eckhardt, Allen E; Pollack, Michael G

2011-11-15

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.

Massively parallel rRNA gene sequencing exacerbates the potential for biased community diversity comparisons due to variable library sizes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gihring, Thomas; Green, Stefan; Schadt, Christopher Warren

2011-01-01

Technologies for massively parallel sequencing are revolutionizing microbial ecology and are vastly increasing the scale of ribosomal RNA (rRNA) gene studies. Although pyrosequencing has increased the breadth and depth of possible rRNA gene sampling, one drawback is that the number of reads obtained per sample is difficult to control. Pyrosequencing libraries typically vary widely in the number of sequences per sample, even within individual studies, and there is a need to revisit the behaviour of richness estimators and diversity indices with variable gene sequence library sizes. Multiple reports and review papers have demonstrated the bias in non-parametric richness estimators (e.g.more » Chao1 and ACE) and diversity indices when using clone libraries. However, we found that biased community comparisons are accumulating in the literature. Here we demonstrate the effects of sample size on Chao1, ACE, CatchAll, Shannon, Chao-Shen and Simpson's estimations specifically using pyrosequencing libraries. The need to equalize the number of reads being compared across libraries is reiterated, and investigators are directed towards available tools for making unbiased diversity comparisons.« less

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

PubMed

Gilling, Damian H; Luna, Vicki Ann; Pflugradt, Cori

2014-01-01

The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.

Pyrosequencing-based assessment of microbial community shifts in leachate from animal carcass burial lysimeter.

PubMed

Kim, Hyun Young; Seo, Jiyoung; Kim, Tae-Hun; Shim, Bomi; Cha, Seok Mun; Yu, Seungho

2017-06-01

This study examined the use of microbial community structure as a bio-indicator of decomposition levels. High-throughput pyrosequencing technology was used to assess the shift in microbial community of leachate from animal carcass lysimeter. The leachate samples were collected monthly for one year and a total of 164,639 pyrosequencing reads were obtained and used in the taxonomic classification and operational taxonomy units (OTUs) distribution analysis based on sequence similarity. Our results show considerable changes in the phylum-level bacterial composition, suggesting that the microbial community is a sensitive parameter affected by the burial environment. The phylum classification results showed that Proteobacteria (Pseudomonas) were the most influential taxa in earlier decomposition stage whereas Firmicutes (Clostridium, Sporanaerobacter, and Peptostreptococcus) were dominant in later stage under anaerobic conditions. The result of this study can provide useful information on a time series of leachate profiles of microbial community structures and suggest patterns of microbial diversity in livestock burial sites. In addition, this result can be applicable to predict the decomposition stages under clay loam based soil conditions of animal livestock. Copyright © 2017 Elsevier B.V. All rights reserved.

Droplet-Based Pyrosequencing Using Digital Microfluidics

PubMed Central

Boles, Deborah J.; Benton, Jonathan L.; Siew, Germaine J.; Levy, Miriam H.; Thwar, Prasanna K.; Sandahl, Melissa A.; Rouse, Jeremy L.; Perkins, Lisa C.; Sudarsan, Arjun P.; Jalili, Roxana; Pamula, Vamsee K.; Srinivasan, Vijay; Fair, Richard B.; Griffin, Peter B.; Eckhardt, Allen E.; Pollack, Michael G.

2013-01-01

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., “sample-to-sequence” capability) could eventually be achieved using this low-cost platform. PMID:21932784

The Gut Microbiomes of Two Pachysoma MacLeay Desert Dung Beetle Species (Coleoptera: Scarabaeidae: Scarabaeinae) Feeding on Different Diets.

PubMed

Franzini, Philippa Z N; Ramond, Jean-Baptiste; Scholtz, Clarke H; Sole, Catherine L; Ronca, Sandra; Cowan, Don A

2016-01-01

Micro-organisms inhabiting animal guts benefit from a protected and nutrient-rich environment while assisting the host with digestion and nutrition. In this study we compare, for the first time, the bacterial and fungal gut communities of two species of the small desert dung beetle genus Pachysoma feeding on different diets: the detritivorous P. endroedyi and the dry-dung-feeding P. striatum. Whole-gut microbial communities from 5 individuals of each species were assessed using 454 pyrosequencing of the bacterial 16S rRNA gene and fungal ITS gene regions. The two bacterial communities were significantly different, with only 3.7% of operational taxonomic units shared, and displayed intra-specific variation. The number of bacterial phyla present within the guts of P. endroedyi and P. striatum individuals ranged from 6-11 and 4-7, respectively. Fungal phylotypes could only be detected within the gut of P. striatum. Although the role of host phylogeny in Pachysoma microbiome assembly remains unknown, evidence presented in this study suggests that host diet may be a deterministic factor.

Bacterial community analysis of Tatsoi cultivated by hydroponics.

PubMed

Koo, Ok K; Kim, Hun; Kim, Hyun J; Baker, Christopher A; Ricke, Steven C

2016-07-02

Tatsoi (Brassica narinosa) is a popular Asian salad green that is mostly consumed as a source of fresh produce. The purpose of this study was to assess the microbial diversity of Tatsoi cultivated in a hydroponic system and of its ecosystem. Tatsoi leaves, nutrient solution, and perlite/earth samples from a trickle feed system (TFS) and an ebb-and-flow system (EFS) were collected and their microbial communities were analyzed by pyrosequencing analysis. The results showed that most bacteria in the leaves from the TFS contained genus Sporosarcina (99.6%), while Rhizobium (60.4%) was dominant in the leaves from the EFS. Genus Paucibacter (18.21%) and Pelomonas (12.37%) were the most abundant microbiota in the nutrient solution samples of the TFS. In the EFS, the nutrient solution samples contained mostly genus Rhodococcus and Acinetobacter. Potential microbial transfer between the leaves and the ecosystem was observed in the EFS, while samples in the TFS were found to share only one species between the leaves, nutrient solution, and earth. Together, these results show that the bacterial populations in Tatsoi and in its ecosystem are highly diverse based on the cultivation system.

The microbiome of neotropical ticks parasitizing on passerine migratory birds.

PubMed

Budachetri, Khemraj; Williams, Jaclyn; Mukherjee, Nabanita; Sellers, Michael; Moore, Frank; Karim, Shahid

2017-01-01

Seasonal migration of passerine birds between temperate North America and tropical Central and South America is an ecological phenomenon. Migration of birds has been associated with the introduction of ectoparasites like ticks or tick-borne pathogens across the avian migration routes. In this study, the microbial diversity was determined in the ticks and bird DNA samples using 454 pyrosequencing of bacterial 16S rRNA gene. Tick DNA samples showed the dominance of genera Lactococcus, Francisella, Raoultella, Wolbachia and Rickettsia across all the ticks, but birds DNA did not share common microbial diversity with ticks. Furthermore, "Candidatus Rickettsia amblyommii" infection in the 91 ticks collected off the songbirds was also quantified by qPCR assay. Interestingly, "Candidatus R. amblyommii" was tested positive in 24 ticks (26% infection), and infection varied from as low as three copies to thousands of copies, but bird blood samples showed no amplification. Our results provide evidence that songbirds serve as transport carrier for immature ticks, and less likely to be a reservoir for "Candidatus R. amblyommii". Copyright © 2016 Elsevier GmbH. All rights reserved.

Fungal and bacterial successions in the process of co-composting of organic wastes as revealed by 454 pyrosequencing.

PubMed

Galitskaya, Polina; Biktasheva, Liliya; Saveliev, Anatoly; Grigoryeva, Tatiana; Boulygina, Eugenia; Selivanovskaya, Svetlana

2017-01-01

Composting is viewed as one of the primary methods to treat organic wastes. Co-composting may improve the efficiency of this treatment by establishing the most suitable conditions for decomposers than those present in the individual wastes. Given that bacteria and fungi are the driving agents of composting, information about the composition of their communities and dynamics during composting may improve reproducibility, performance and quality of the final compost as well as help to evaluate the potential human health risk and the choice of the most appropriate application procedure. In this study, the co-composting of mixtures containing two similar components (organic fraction of municipal solid waste and sawdust polluted by oil) and one discriminate component (sewage sludges of different origin) were investigated. Bacterial and fungal community successions in the two mixtures were analyzed during the composting process by determining the change in their structural dynamics using qPCR and 454 pyrosequencing methods in a lab experiment for a period of 270 days. During the initial composting stage, the number of 16S bacterial copies was (3.0±0.2) x 106 and (0.4±0.0) x 107 g-1, and the Rhodospiralles and Lactobacialles orders dominated. Fungal communities had (2.9±0.0) x105 and (6.1±0.2) x105 ITS copies g-1, and the Saccharomycetales order dominated. At the end of the thermophilic stage on the 30th day of composting, bacterial and fungal communities underwent significant changes: dominants changed and their relative abundance decreased. Typical compost residents included Flavobacteriales, Chitinophagaceae and Bacterioidetes for bacteria and Microascaceae, Dothideomycetes, Eurotiomycetes, Sordariomycetes, and Agaricomycetes for fungi. During the later composting stages, the dominating taxa of both bacterial and fungal communities remained, while their relative abundance decreased. In accordance with the change in the dominating OTUs, it was concluded that the dynamics of the bacterial and fungal communities were not similar. Analysis by non-metric multidimensional scaling (NMDS) revealed that the bacterial communities of the two composts became progressively more similar; a similar trend was followed by the fungal community.

Fungal and bacterial successions in the process of co-composting of organic wastes as revealed by 454 pyrosequencing

PubMed Central

Galitskaya, Polina; Saveliev, Anatoly; Grigoryeva, Tatiana; Boulygina, Eugenia; Selivanovskaya, Svetlana

2017-01-01

Composting is viewed as one of the primary methods to treat organic wastes. Co-composting may improve the efficiency of this treatment by establishing the most suitable conditions for decomposers than those present in the individual wastes. Given that bacteria and fungi are the driving agents of composting, information about the composition of their communities and dynamics during composting may improve reproducibility, performance and quality of the final compost as well as help to evaluate the potential human health risk and the choice of the most appropriate application procedure. In this study, the co-composting of mixtures containing two similar components (organic fraction of municipal solid waste and sawdust polluted by oil) and one discriminate component (sewage sludges of different origin) were investigated. Bacterial and fungal community successions in the two mixtures were analyzed during the composting process by determining the change in their structural dynamics using qPCR and 454 pyrosequencing methods in a lab experiment for a period of 270 days. During the initial composting stage, the number of 16S bacterial copies was (3.0±0.2) x 106 and (0.4±0.0) x 107 g-1, and the Rhodospiralles and Lactobacialles orders dominated. Fungal communities had (2.9±0.0) x105 and (6.1±0.2) x105 ITS copies g-1, and the Saccharomycetales order dominated. At the end of the thermophilic stage on the 30th day of composting, bacterial and fungal communities underwent significant changes: dominants changed and their relative abundance decreased. Typical compost residents included Flavobacteriales, Chitinophagaceae and Bacterioidetes for bacteria and Microascaceae, Dothideomycetes, Eurotiomycetes, Sordariomycetes, and Agaricomycetes for fungi. During the later composting stages, the dominating taxa of both bacterial and fungal communities remained, while their relative abundance decreased. In accordance with the change in the dominating OTUs, it was concluded that the dynamics of the bacterial and fungal communities were not similar. Analysis by non-metric multidimensional scaling (NMDS) revealed that the bacterial communities of the two composts became progressively more similar; a similar trend was followed by the fungal community. PMID:29059245

Oligotyping reveals stronger relationship of organic soil bacterial community structure with N-amendments and soil chemistry in comparison to that of mineral soil at Harvard Forest, MA, USA.

PubMed

Turlapati, Swathi A; Minocha, Rakesh; Long, Stephanie; Ramsdell, Jordan; Minocha, Subhash C

2015-01-01

The impact of chronic nitrogen amendments on bacterial communities was evaluated at Harvard Forest, Petersham, MA, USA. Thirty soil samples (3 treatments × 2 soil horizons × 5 subplots) were collected in 2009 from untreated (control), low nitrogen-amended (LN; 50 kg NH4NO3 ha(-1) yr(-1)) and high nitrogen-amended (HN; 150 kg NH4NO3 ha(-1) yr(-1)) plots. PCR-amplified partial 16S rRNA gene sequences made from soil DNA were subjected to pyrosequencing (Turlapati et al., 2013) and analyses using oligotyping. The parameters M (the minimum count of the most abundant unique sequence in an oligotype) and s (the minimum number of samples in which an oligotype is expected to be present) had to be optimized for forest soils because of high diversity and the presence of rare organisms. Comparative analyses of the pyrosequencing data by oligotyping and operational taxonomic unit clustering tools indicated that the former yields more refined units of taxonomy with sequence similarity of ≥99.5%. Sequences affiliated with four new phyla and 73 genera were identified in the present study as compared to 27 genera reported earlier from the same data (Turlapati et al., 2013). Significant rearrangements in the bacterial community structure were observed with N-amendments revealing the presence of additional genera in N-amended plots with the absence of some that were present in the control plots. Permutational MANOVA analyses indicated significant variation associated with soil horizon and N treatment for a majority of the phyla. In most cases soil horizon partitioned more variation relative to treatment and treatment effects were more evident for the organic (Org) horizon. Mantel test results for Org soil showed significant positive correlations between bacterial communities and most soil parameters including NH4 and NO3. In mineral soil, correlations were seen only with pH, NH4, and NO3. Regardless of the pipeline used, a major hindrance for such a study remains to be the lack of reference databases for forest soils.

Pyrosequencing the Manduca sexta larval midgut transcriptome: messages for digestion, detoxification and defence.

PubMed

Pauchet, Y; Wilkinson, P; Vogel, H; Nelson, D R; Reynolds, S E; Heckel, D G; ffrench-Constant, R H

2010-02-01

The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing

PubMed Central

Gilling, Damian H.; Luna, Vicki Ann; Pflugradt, Cori

2014-01-01

The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future. PMID:27350960

Rapid detection and subtyping of human influenza A viruses and reassortants by pyrosequencing.

PubMed

Deng, Yi-Mo; Caldwell, Natalie; Barr, Ian G

2011-01-01

Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches.

Rapid Detection and Subtyping of Human Influenza A Viruses and Reassortants by Pyrosequencing

PubMed Central

Deng, Yi-Mo; Caldwell, Natalie; Barr, Ian G.

2011-01-01

Background Given the continuing co-circulation of the 2009 H1N1 pandemic influenza A viruses with seasonal H3N2 viruses, rapid and reliable detection of newly emerging influenza reassortant viruses is important to enhance our influenza surveillance. Methodology/Principal Findings A novel pyrosequencing assay was developed for the rapid identification and subtyping of potential human influenza A virus reassortants based on all eight gene segments of the virus. Except for HA and NA genes, one universal set of primers was used to amplify and subtype each of the six internal genes. With this method, all eight gene segments of 57 laboratory isolates and 17 original specimens of seasonal H1N1, H3N2 and 2009 H1N1 pandemic viruses were correctly matched with their corresponding subtypes. In addition, this method was shown to be capable of detecting reassortant viruses by correctly identifying the source of all 8 gene segments from three vaccine production reassortant viruses and three H1N2 viruses. Conclusions/Significance In summary, this pyrosequencing assay is a sensitive and specific procedure for screening large numbers of viruses for reassortment events amongst the commonly circulating human influenza A viruses, which is more rapid and cheaper than using conventional sequencing approaches. PMID:21886790

Analysis of Gastric Microbiota by Pyrosequencing: Minor Role of Bacteria Other Than Helicobacter pylori in the Gastric Carcinogenesis.

PubMed

Jo, Hyun Jin; Kim, Jaeyeon; Kim, Nayoung; Park, Ji Hyun; Nam, Ryoung Hee; Seok, Yeong-Jae; Kim, Yeon-Ran; Kim, Joo Sung; Kim, Jung Mogg; Kim, Jung Min; Lee, Dong Ho; Jung, Hyun Chae

2016-10-01

Little is known about the role of gastric microbiota except for Helicobacter pylori (HP) in human health and disease. We compared the differences of human gastric microbiota according to gastric cancer or control and HP infection status and assessed the role of bacteria other than HP. Gastric microbiota of 63 antral mucosal and 18 corpus mucosal samples were analyzed by bar-coded 454 pyrosequencing of the 16S rRNA gene. Antral samples were divided into four subgroups based on HP positivity in pyrosequencing and the presence of cancer. The analysis was focused on bacteria other than HP, especially nitrosating or nitrate-reducing bacteria (NB). The changes of NB in antral mucosa of 16 subjects were followed up. The number of NB other than HP (non-HP-NB) was two times higher in the cancer groups than in the control groups, but it did not reach statistical significance. The number of non-HP-NB tends to increase over time, but this phenomenon was prevented by HP eradication in the HP-positive control group, but not in the HP-positive cancer group. We could not find the significant role of bacteria other than HP in the gastric carcinogenesis. © 2016 John Wiley & Sons Ltd.

Evaluation of two real time PCR assays for the detection of bacterial DNA in amniotic fluid.

PubMed

Girón de Velasco-Sada, Patricia; Falces-Romero, Iker; Quiles-Melero, Inmaculada; García-Perea, Adela; Mingorance, Jesús

2018-01-01

The aim of this study was to evaluate two non-commercial Real-Time PCR assays for the detection of microorganisms in amniotic fluid followed by identification by pyrosequencing. We collected 126 amniotic fluids from 2010 to 2015 for the evaluation of two Real-Time PCR assays for detection of bacterial DNA in amniotic fluid (16S Universal PCR and Ureaplasma spp. specific PCR). The method was developed in the Department of Microbiology of the University Hospital La Paz. Thirty-seven samples (29.3%) were positive by PCR/pyrosequencing and/or culture, 4 of them were mixed cultures with Ureaplasma urealyticum. The Universal 16S Real-Time PCR was compared with the standard culture (81.8% sensitivity, 97.4% specificity, 75% positive predictive value, 98% negative predictive value). The Ureaplasma spp. specific Real-Time PCR was compared with the Ureaplasma/Mycoplasma specific culture (92.3% sensitivity, 89.4% specificity, 50% positive predictive value, 99% negative predictive value) with statistically significant difference (p=0.005). Ureaplasma spp. PCR shows a rapid response time (5h from DNA extraction until pyrosequencing) when comparing with culture (48h). So, the response time of bacteriological diagnosis in suspected chorioamnionitis is reduced. Copyright © 2017 Elsevier B.V. All rights reserved.

High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes

PubMed Central

Pereiro, Patricia; Balseiro, Pablo; Romero, Alejandro; Dios, Sonia; Forn-Cuni, Gabriel; Fuste, Berta; Planas, Josep V.; Beltran, Sergi; Novoa, Beatriz; Figueras, Antonio

2012-01-01

Background Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations. Methodology/Principal Findings Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways. Conclusions/Significance To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms. PMID:22629298

Transcriptome Exploration in Leymus chinensis under Saline-Alkaline Treatment Using 454 Pyrosequencing

PubMed Central

Sun, Yepeng; Wang, Fawei; Wang, Nan; Dong, Yuanyuan; Liu, Qi; Zhao, Lei; Chen, Huan; Liu, Weican; Yin, Hailong; Zhang, Xiaomei; Yuan, Yanxi; Li, Haiyan

2013-01-01

Background Leymus chinensis (Trin.) Tzvel. is a high saline-alkaline tolerant forage grass genus of the tribe Gramineae family, which also plays an important role in protection of natural environment. To date, little is known about the saline-alkaline tolerance of L. chinensis on the molecular level. To better understand the molecular mechanism of saline-alkaline tolerance in L. chinensis, 454 pyrosequencing was used for the transcriptome study. Results We used Roche-454 massive parallel pyrosequencing technology to sequence two different cDNA libraries that were built from the two samples of control and under saline-alkaline treatment (optimal stress concentration-Hoagland solution with 100 mM NaCl and 200 mM NaHCO3). A total of 363,734 reads in control group and 526,267 reads in treatment group with an average length of 489 bp and 493 bp were obtained, respectively. The reads were assembled into 104,105 unigenes with MIRA sequence assemable software, among which, 73,665 unigenes were in control group, 88,016 unigenes in treatment group and 57,576 unigenes in both groups. According to the comparative expression analysis between the two groups with the threshold of “log2 Ratio ≥1”, there were 36,497 up-regulated unegenes and 18,218 down-regulated unigenes predicted to be the differentially expressed genes. After gene annotation and pathway enrichment analysis, most of them were involved in stress and tolerant function, signal transduction, energy production and conversion, and inorganic ion transport. Furthermore, 16 of these differentially expressed genes were selected for real-time PCR validation, and they were successfully confirmed with the results of 454 pyrosequencing. Conclusions This work is the first time to study the transcriptome of L. chinensis under saline-alkaline treatment based on the 454-FLX massively parallel DNA sequencing platform. It also deepened studies on molecular mechanisms of saline-alkaline in L. chinensis, and constituted a database for future studies. PMID:23365637

Detection of KRAS G12D in colorectal cancer stool by droplet digital PCR

PubMed Central

Olmedillas-López, Susana; Lévano-Linares, Dennis César; Alexandre, Carmen Laura Aúz; Vega-Clemente, Luz; Sánchez, Edurne León; Villagrasa, Alejandro; Ruíz-Tovar, Jaime; García-Arranz, Mariano; García-Olmo, Damián

2017-01-01

AIM To assess KRAS G12D mutation detection by droplet digital PCR (ddPCR) in stool-derived DNA from colorectal cancer (CRC) patients. METHODS In this study, tumor tissue and stool samples were collected from 70 patients with stage I-IV CRC diagnosed by preoperative biopsy. KRAS mutational status was determined by pyrosequencing analysis of DNA obtained from formalin-fixed paraffin-embedded (FFPE) tumor tissues. The KRAS G12D mutation was then analyzed by ddPCR in FFPE tumors and stool-derived DNA from patients with this point mutation. Wild-type (WT) tumors, as determined by pyrosequencing, were included as controls; analysis of FFPE tissue and stool-derived DNA by ddPCR was performed for these patients as well. RESULTS Among the total 70 patients included, KRAS mutations were detected by pyrosequencing in 32 (45.71%), whereas 38 (54.29%) had WT tumors. The frequency of KRAS mutations was higher in left-sided tumors (11 located in the right colon, 15 in the left, and 6 in the rectum). The predominant point mutation was KRAS G12D (14.29%, n = 10), which was more frequent in early-stage tumors (I-IIA, n = 7). In agreement with pyrosequencing results, the KRAS G12D mutation was detected by ddPCR in FFPE tumor-derived DNA, and only a residual number of mutated copies was found in WT controls. The KRAS G12D mutation was also detected in stool-derived DNA in 80% of all fecal samples from CRC patients with this point mutation. CONCLUSION ddPCR is a reliable and sensitive method to analyze KRAS G12D mutation in stool-derived DNA from CRC patients, especially at early stages. This non-invasive approach is potentially applicable to other relevant biomarkers for CRC management. PMID:29093617

Evaluation of six primer pairs targeting the nuclear rRNA operon for characterization of arbuscular mycorrhizal fungal (AMF) communities using 454 pyrosequencing.

PubMed

Van Geel, Maarten; Busschaert, Pieter; Honnay, Olivier; Lievens, Bart

2014-11-01

In the last few years, 454 pyrosequencing-based analysis of arbuscular mycorrhizal fungal (AMF; Glomeromycota) communities has tremendously increased our knowledge of the distribution and diversity of AMF. Nonetheless, comparing results between different studies is difficult, as different target genes (or regions thereof) and primer combinations, with potentially dissimilar specificities and efficacies, are being utilized. In this study we evaluated six primer pairs that have previously been used in AMF studies (NS31-AM1, AMV4.5NF-AMDGR, AML1-AML2, NS31-AML2, FLR3-LSUmBr and Glo454-NDL22) for their use in 454 pyrosequencing based on both an in silico approach and 454 pyrosequencing of AMF communities from apple tree roots. Primers were evaluated in terms of (i) in silico coverage of Glomeromycota fungi, (ii) the number of high-quality sequences obtained, (iii) selectivity for AMF species, (iv) reproducibility and (v) ability to accurately describe AMF communities. We show that primer pairs AMV4.5NF-AMDGR, AML1-AML2 and NS31-AML2 outperformed the other tested primer pairs in terms of number of Glomeromycota reads (AMF specificity and coverage). Additionally, these primer pairs were found to have no or only few mismatches to AMF sequences and were able to consistently describe AMF communities from apple roots. However, whereas most high-quality AMF sequences were obtained for AMV4.5NF-AMDGR, our results also suggest that this primer pair favored amplification of Glomeraceae sequences at the expense of Ambisporaceae, Claroideoglomeraceae and Paraglomeraceae sequences. Furthermore, we demonstrate the complementary specificity of AMV4.5NF-AMDGR with AML1-AML2, and of AMV4.5NF-AMDGR with NS31-AML2, making these primer combinations highly suitable for tandem use in covering the diversity of AMF communities. Copyright © 2014 Elsevier B.V. All rights reserved.

Shotgun Pyrosequencing Metagenomic Analyses of Dusts from Swine Confinement and Grain Facilities

PubMed Central

Boissy, Robert J.; Romberger, Debra J.; Roughead, William A.; Weissenburger-Moser, Lisa; Poole, Jill A.; LeVan, Tricia D.

2014-01-01

Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance). The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70%) at the phylum level, Clostridia (44%) at the Class level, and Clostridiales at the Order level (41%). In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the bioinformatic analyses used for such studies must be carefully designed to avoid the potential contribution of non-microbial DNA, e.g. from resident mammals. PMID:24748147

Shotgun pyrosequencing metagenomic analyses of dusts from swine confinement and grain facilities.

PubMed

Boissy, Robert J; Romberger, Debra J; Roughead, William A; Weissenburger-Moser, Lisa; Poole, Jill A; LeVan, Tricia D

2014-01-01

Inhalation of agricultural dusts causes inflammatory reactions and symptoms such as headache, fever, and malaise, which can progress to chronic airway inflammation and associated diseases, e.g. asthma, chronic bronchitis, chronic obstructive pulmonary disease, and hypersensitivity pneumonitis. Although in many agricultural environments feed particles are the major constituent of these dusts, the inflammatory responses that they provoke are likely attributable to particle-associated bacteria, archaebacteria, fungi, and viruses. In this study, we performed shotgun pyrosequencing metagenomic analyses of DNA from dusts from swine confinement facilities or grain elevators, with comparisons to dusts from pet-free households. DNA sequence alignment showed that 19% or 62% of shotgun pyrosequencing metagenomic DNA sequence reads from swine facility or household dusts, respectively, were of swine or human origin, respectively. In contrast only 2% of such reads from grain elevator dust were of mammalian origin. These metagenomic shotgun reads of mammalian origin were excluded from our analyses of agricultural dust microbiota. The ten most prevalent bacterial taxa identified in swine facility compared to grain elevator or household dust were comprised of 75%, 16%, and 42% gram-positive organisms, respectively. Four of the top five swine facility dust genera were assignable (Clostridium, Lactobacillus, Ruminococcus, and Eubacterium, ranging from 4% to 19% relative abundance). The relative abundances of these four genera were lower in dust from grain elevators or pet-free households. These analyses also highlighted the predominance in swine facility dust of Firmicutes (70%) at the phylum level, Clostridia (44%) at the Class level, and Clostridiales at the Order level (41%). In summary, shotgun pyrosequencing metagenomic analyses of agricultural dusts show that they differ qualitatively and quantitatively at the level of microbial taxa present, and that the bioinformatic analyses used for such studies must be carefully designed to avoid the potential contribution of non-microbial DNA, e.g. from resident mammals.

Analysis of oral microbiota in children with dental caries by PCR-DGGE and barcoded pyrosequencing.

PubMed

Ling, Zongxin; Kong, Jianming; Jia, Peng; Wei, Chaochun; Wang, Yuezhu; Pan, Zhiwen; Huang, Wujing; Li, Lanjuan; Chen, Hui; Xiang, Charlie

2010-10-01

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.

Determinants of orofacial clefting I: Effects of 5-Aza-2'-deoxycytidine on cellular processes and gene expression during development of the first branchial arch.

PubMed

Mukhopadhyay, Partha; Seelan, Ratnam S; Rezzoug, Francine; Warner, Dennis R; Smolenkova, Irina A; Brock, Guy; Pisano, M Michele; Greene, Robert M

2017-01-01

In this study, we identify gene targets and cellular events mediating the teratogenic action(s) of 5-Aza-2'-deoxycytidine (AzaD), an inhibitor of DNA methylation, on secondary palate development. Exposure of pregnant mice (on gestation day (GD) 9.5) to AzaD for 12h resulted in the complete penetrance of cleft palate (CP) in fetuses. Analysis of cells of the embryonic first branchial arch (1-BA), in fetuses exposed to AzaD, revealed: 1) significant alteration in expression of genes encoding several morphogenetic factors, cell cycle inhibitors and regulators of apoptosis; 2) a decrease in cell proliferation; and, 3) an increase in apoptosis. Pyrosequencing of selected genes, displaying pronounced differential expression in AzaD-exposed 1-BAs, failed to reveal significant alterations in CpG methylation levels in their putative promoters or gene bodies. CpG methylation analysis suggested that the effects of AzaD on gene expression were likely indirect. Copyright © 2016 Elsevier Inc. All rights reserved.

Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts.

PubMed

Webster, Nicole S; Taylor, Michael W; Behnam, Faris; Lücker, Sebastian; Rattei, Thomas; Whalan, Stephen; Horn, Matthias; Wagner, Michael

2010-08-01

Marine sponges contain complex bacterial communities of considerable ecological and biotechnological importance, with many of these organisms postulated to be specific to sponge hosts. Testing this hypothesis in light of the recent discovery of the rare microbial biosphere, we investigated three Australian sponges by massively parallel 16S rRNA gene tag pyrosequencing. Here we show bacterial diversity that is unparalleled in an invertebrate host, with more than 250,000 sponge-derived sequence tags being assigned to 23 bacterial phyla and revealing up to 2996 operational taxonomic units (95% sequence similarity) per sponge species. Of the 33 previously described 'sponge-specific' clusters that were detected in this study, 48% were found exclusively in adults and larvae - implying vertical transmission of these groups. The remaining taxa, including 'Poribacteria', were also found at very low abundance among the 135,000 tags retrieved from surrounding seawater. Thus, members of the rare seawater biosphere may serve as seed organisms for widely occurring symbiont populations in sponges and their host association might have evolved much more recently than previously thought. © 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.

Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts

PubMed Central

Webster, Nicole S; Taylor, Michael W; Behnam, Faris; Lücker, Sebastian; Rattei, Thomas; Whalan, Stephen; Horn, Matthias; Wagner, Michael

2010-01-01

Marine sponges contain complex bacterial communities of considerable ecological and biotechnological importance, with many of these organisms postulated to be specific to sponge hosts. Testing this hypothesis in light of the recent discovery of the rare microbial biosphere, we investigated three Australian sponges by massively parallel 16S rRNA gene tag pyrosequencing. Here we show bacterial diversity that is unparalleled in an invertebrate host, with more than 250 000 sponge-derived sequence tags being assigned to 23 bacterial phyla and revealing up to 2996 operational taxonomic units (95% sequence similarity) per sponge species. Of the 33 previously described ‘sponge-specific’ clusters that were detected in this study, 48% were found exclusively in adults and larvae – implying vertical transmission of these groups. The remaining taxa, including ‘Poribacteria’, were also found at very low abundance among the 135 000 tags retrieved from surrounding seawater. Thus, members of the rare seawater biosphere may serve as seed organisms for widely occurring symbiont populations in sponges and their host association might have evolved much more recently than previously thought. PMID:21966903

Comparison of the Oral Microbiomes of Canines and Their Owners Using Next-Generation Sequencing.

PubMed

Oh, Changin; Lee, Kunkyu; Cheong, Yeotaek; Lee, Sang-Won; Park, Seung-Yong; Song, Chang-Seon; Choi, In-Soo; Lee, Joong-Bok

2015-01-01

The oral microbiome, which is closely associated with many diseases, and the resident pathogenic oral bacteria, which can be transferred by close physical contact, are important public health considerations. Although the dog is the most common companion animal, the composition of the canine oral microbiome, which may include human pathogenic bacteria, and its relationship with that of their owners are unclear. In this study, 16S rDNA pyrosequencing was used to compare the oral microbiomes of 10 dogs and their owners and to identify zoonotic pathogens. Pyrosequencing revealed 246 operational taxonomic units in the 10 samples, representing 57 genera from eight bacterial phyla. Firmicutes (57.6%), Proteobacteria (21.6%), Bacteroidetes (9.8%), Actinobacteria (7.1%), and Fusobacteria (3.9%) were the predominant phyla in the human oral samples, whereas Proteobacteria (25.7%), Actinobacteria (21%), Bacteroidetes (19.7%), Firmicutes (19.3%), and Fusobacteria (12.3%) were predominant in the canine oral samples. The predominant genera in the human samples were Streptococcus (43.9%), Neisseria (10.3%), Haemophilus (9.6%), Prevotella (8.4%), and Veillonella (8.1%), whereas the predominant genera in the canine samples were Actinomyces (17.2%), Unknown (16.8), Porphyromonas (14.8), Fusobacterium (11.8), and Neisseria (7.2%). The oral microbiomes of dogs and their owners were appreciably different, and similarity in the microbiomes of canines and their owners was not correlated with residing in the same household. Oral-to-oral transfer of Neisseria shayeganii, Porphyromonas canigingivalis, Tannerella forsythia, and Streptococcus minor from dogs to humans was suspected. The finding of potentially zoonotic and periodontopathic bacteria in the canine oral microbiome may be a public health concern.

Genome-wide analysis of short interspersed nuclear elements SINES revealed high sequence conservation, gene association and retrotranspositional activity in wheat

PubMed Central

Ben-David, Smadar; Yaakov, Beery; Kashkush, Khalil

2013-01-01

Short interspersed nuclear elements (SINEs) are non-autonomous non-LTR retroelements that are present in most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, they are poorly studied in plants, especially in wheat (Triticum aestivum). We used quantitative PCR of various wheat species to determine the copy number of a wheat SINE family, termed Au SINE, combined with computer-assisted analyses of the publicly available 454 pyrosequencing database of T. aestivum. In addition, we utilized site-specific PCR on 57 Au SINE insertions, transposon methylation display and transposon display on newly formed wheat polyploids to assess retrotranspositional activity, epigenetic status and genetic rearrangements in Au SINE, respectively. We retrieved 3706 different insertions of Au SINE from the 454 pyrosequencing database of T. aestivum, and found that most of the elements are inserted in A/T-rich regions, while approximately 38% of the insertions are associated with transcribed regions, including known wheat genes. We observed typical retrotransposition of Au SINE in the second generation of a newly formed wheat allohexaploid, and massive hypermethylation in CCGG sites surrounding Au SINE in the third generation. Finally, we observed huge differences in the copy numbers in diploid Triticum and Aegilops species, and a significant increase in the copy numbers in natural wheat polyploids, but no significant increase in the copy number of Au SINE in the first four generations for two of three newly formed allopolyploid species used in this study. Our data indicate that SINEs may play a prominent role in the genomic evolution of wheat through stress-induced activation. PMID:23855320

Genome-wide analysis of short interspersed nuclear elements SINES revealed high sequence conservation, gene association and retrotranspositional activity in wheat.

PubMed

Ben-David, Smadar; Yaakov, Beery; Kashkush, Khalil

2013-10-01

Short interspersed nuclear elements (SINEs) are non-autonomous non-LTR retroelements that are present in most eukaryotic species. While SINEs have been intensively investigated in humans and other animal systems, they are poorly studied in plants, especially in wheat (Triticum aestivum). We used quantitative PCR of various wheat species to determine the copy number of a wheat SINE family, termed Au SINE, combined with computer-assisted analyses of the publicly available 454 pyrosequencing database of T. aestivum. In addition, we utilized site-specific PCR on 57 Au SINE insertions, transposon methylation display and transposon display on newly formed wheat polyploids to assess retrotranspositional activity, epigenetic status and genetic rearrangements in Au SINE, respectively. We retrieved 3706 different insertions of Au SINE from the 454 pyrosequencing database of T. aestivum, and found that most of the elements are inserted in A/T-rich regions, while approximately 38% of the insertions are associated with transcribed regions, including known wheat genes. We observed typical retrotransposition of Au SINE in the second generation of a newly formed wheat allohexaploid, and massive hypermethylation in CCGG sites surrounding Au SINE in the third generation. Finally, we observed huge differences in the copy numbers in diploid Triticum and Aegilops species, and a significant increase in the copy numbers in natural wheat polyploids, but no significant increase in the copy number of Au SINE in the first four generations for two of three newly formed allopolyploid species used in this study. Our data indicate that SINEs may play a prominent role in the genomic evolution of wheat through stress-induced activation. © 2013 Ben-Gurion University The Plant Journal © 2013 John Wiley & Sons Ltd.

Response of ammonia oxidizing archaea and bacteria to decabromodiphenyl ether and copper contamination in river sediments.

PubMed

Wang, Linqiong; Li, Yi; Niu, Lihua; Zhang, Wenlong; Zhang, Huanjun; Wang, Longfei; Wang, Peifang

2018-01-01

Ammonia oxidation plays a fundamental role in river nitrogen cycling ecosystems, which is normally governed by both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB). Co-contamination of typical emerging pollutant Polybrominated diphenyl ethers (PBDEs) and heavy metal on AOA and AOB communities in river sediments remains unknown. In this study, multiple analytical tools, including high-throughput pyrosequencing and real-time quantitative PCR (qPCR), were used to reveal the ammonia monooxygenase (AMO) activity, subunit alpha (amoA) gene abundance, and community structures of AOA and AOB in river sediments. It was found that the inhibition of AMO activities was increased with the increase of decabromodiphenyl ether (BDE 209, 1-100 mg kg -1 ) and copper (Cu, 50-500 mg kg -1 ) concentrations. Moreover, the synergic effects of BDE 209 and Cu resulted in a higher AMO activity reduction than the individual pollutant BDE 209. The AOA amoA copy number declined by 75.9% and 83.2% and AOB amoA gene abundance declined 82.8% and 90.0% at 20 and 100 mg kg -1 BDE 209 with a 100 mg kg -1 Cu co-contamination, respectively. The pyrosequencing results showed that both AOB and AOA community structures were altered, with a higher change of AOB than that of AOA. The results demonstrated that the AOB microbial community may be better adapted to BDE 209 and Cu pollution, while AOA might possess a greater capacity for stress resistance. Our study provides a better understanding of the ecotoxicological effects of heavy metal and micropollutant combined exposure on AOA and AOB in river sediments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Bacterial community development in experimental gingivitis.

PubMed

Kistler, James O; Booth, Veronica; Bradshaw, David J; Wade, William G

2013-01-01

Current knowledge of the microbial composition of dental plaque in early gingivitis is based largely on microscopy and cultural methods, which do not provide a comprehensive description of oral microbial communities. This study used 454-pyrosequencing of the V1-V3 region of 16S rRNA genes (approximately 500 bp), and bacterial culture, to characterize the composition of plaque during the transition from periodontal health to gingivitis. A total of 20 healthy volunteers abstained from oral hygiene for two weeks, allowing plaque to accumulate and gingivitis to develop. Plaque samples were analyzed at baseline, and after one and two weeks. In addition, plaque samples from 20 chronic periodontitis patients were analyzed for cross-sectional comparison to the experimental gingivitis cohort. All of the healthy volunteers developed gingivitis after two weeks. Pyrosequencing yielded a final total of 344,267 sequences after filtering, with a mean length of 354 bases, that were clustered into an average of 299 species-level Operational Taxonomic Units (OTUs) per sample. Principal coordinates analysis (PCoA) plots revealed significant shifts in the bacterial community structure of plaque as gingivitis was induced, and community diversity increased significantly after two weeks. Changes in the relative abundance of OTUs during the transition from health to gingivitis were correlated to bleeding on probing (BoP) scores and resulted in the identification of new health- and gingivitis-associated taxa. Comparison of the healthy volunteers to the periodontitis patients also confirmed the association of a number of putative periodontal pathogens with chronic periodontitis. Taxa associated with gingivitis included Fusobacterium nucleatum subsp. polymorphum, Lachnospiraceae [G-2] sp. HOT100, Lautropia sp. HOTA94, and Prevotella oulorum, whilst Rothia dentocariosa was associated with periodontal health. Further study of these taxa is warranted and may lead to new therapeutic approaches to prevent periodontal disease.

Microbial diversity in hummock and hollow soils of three wetlands on the Qinghai-Tibetan Plateau revealed by 16S rRNA pyrosequencing.

PubMed

Deng, Yongcui; Cui, Xiaoyong; Hernández, Marcela; Dumont, Marc G

2014-01-01

The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo) on the Qinghai-Tibetan Plateau, the world's highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance), Actinobacteria (17.3%) and Bacteroidetes (11%) had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1-10%). In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales). Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta) and methanotrophs (mainly Methylocystis) compared with the other two wetlands. Principal coordinate analysis (PCoA) indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of the Qinghai-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem.

International Space Station environmental microbiome - microbial inventories of ISS filter debris.

PubMed

Venkateswaran, Kasthuri; Vaishampayan, Parag; Cisneros, Jessica; Pierson, Duane L; Rogers, Scott O; Perry, Jay

2014-01-01

Despite an expanding array of molecular approaches for detecting microorganisms in a given sample, rapid and robust means of assessing the differential viability of the microbial cells, as a function of phylogenetic lineage, remain elusive. A propidium monoazide (PMA) treatment coupled with downstream quantitative polymerase chain reaction (qPCR) and pyrosequencing analyses was carried out to better understand the frequency, diversity, and distribution of viable microorganisms associated with debris collected from the crew quarters of the International Space Station (ISS). The cultured bacterial counts were more in the ISS samples than cultured fungal population. The rapid molecular analyses targeted to estimate viable population exhibited 5-fold increase in bacterial (qPCR-PMA assay) and 25-fold increase in microbial (adenosine triphosphate assay) burden than the cultured bacterial population. The ribosomal nucleic acid-based identification of cultivated strains revealed the presence of only four to eight bacterial species in the ISS samples, however, the viable bacterial diversity detected by the PMA-pyrosequencing method was far more diverse (12 to 23 bacterial taxa) with the majority consisting of members of actinobacterial genera (Propionibacterium, Corynebacterium) and Staphylococcus. Sample fractions not treated with PMA (inclusive of both live and dead cells) yielded a great abundance of highly diverse bacterial (94 to 118 taxa) and fungal lineages (41 taxa). Even though deep sequencing capability of the molecular analysis widened the understanding about the microbial diversity, the cultivation assay also proved to be essential since some of the spore-forming microorganisms were detected only by the culture-based method. Presented here are the findings of the first comprehensive effort to assess the viability of microbial cells associated with ISS surfaces, and correlate differential viability with phylogenetic affiliation.

Analysis of microbial community adaptation in mesophilic hydrogen fermentation from food waste by tagged 16S rRNA gene pyrosequencing.

PubMed

Laothanachareon, Thanaporn; Kanchanasuta, Suwimon; Mhuanthong, Wuttichai; Phalakornkule, Chantaraporn; Pisutpaisal, Nipon; Champreda, Verawat

2014-11-01

Dark fermentation is an attractive process for generation of biohydrogen, which involves complex microbial processes on decomposition of organic wastes and subsequent conversion of metabolic intermediates to hydrogen. The microbes present in an upflow anaerobic sludge blanket (UASB) reactor for waste water treatment were tested for application in batch dark fermentation of food waste at varying ratios of feedstock to heat-treated microbial inoculum (F/M) of 1-8 (g TVS/g TVS). Biohydrogen yields between 0.39 and 2.68 mol H2/mol hexose were obtained, indicating that the yields were highly dependent on the starting F/M ratio. The highest H2 purity of 66% was obtained from the first 8 h of fermentation at the F/M ratio of 2, whereas the highest H2 production was obtained after 35 h of fermentation at the F/M ratio of 5. Tagged 16S rRNA gene pyrosequencing showed that the seed culture comprised largely of uncultured bacteria with various Proteobacteria, Bacteroidetes, and Firmicutes, while the starting food waste contained mainly lactic acid bacteria. Enrichment of Firmicutes, particularly Clostridia and lactic acid bacteria occurred within 8 h of the dark fermentation and the H2 producing microcosm at 35 h was dominated >80% by Clostridium spp. The major H2 producer was identified as a Clostridial strain related to Clostridium frigidicarnis. This work demonstrated the adaption of the microbial community during the dark fermentation of complex food waste and revealed the major roles of Clostridia in both substrate degradation and biohydrogen production. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of the Oral Microbiomes of Canines and Their Owners Using Next-Generation Sequencing

PubMed Central

Oh, Changin; Lee, Kunkyu; Cheong, Yeotaek; Lee, Sang-Won; Park, Seung-Yong; Song, Chang-Seon; Choi, In-Soo; Lee, Joong-Bok

2015-01-01

The oral microbiome, which is closely associated with many diseases, and the resident pathogenic oral bacteria, which can be transferred by close physical contact, are important public health considerations. Although the dog is the most common companion animal, the composition of the canine oral microbiome, which may include human pathogenic bacteria, and its relationship with that of their owners are unclear. In this study, 16S rDNA pyrosequencing was used to compare the oral microbiomes of 10 dogs and their owners and to identify zoonotic pathogens. Pyrosequencing revealed 246 operational taxonomic units in the 10 samples, representing 57 genera from eight bacterial phyla. Firmicutes (57.6%), Proteobacteria (21.6%), Bacteroidetes (9.8%), Actinobacteria (7.1%), and Fusobacteria (3.9%) were the predominant phyla in the human oral samples, whereas Proteobacteria (25.7%), Actinobacteria (21%), Bacteroidetes (19.7%), Firmicutes (19.3%), and Fusobacteria (12.3%) were predominant in the canine oral samples. The predominant genera in the human samples were Streptococcus (43.9%), Neisseria (10.3%), Haemophilus (9.6%), Prevotella (8.4%), and Veillonella (8.1%), whereas the predominant genera in the canine samples were Actinomyces (17.2%), Unknown (16.8), Porphyromonas (14.8), Fusobacterium (11.8), and Neisseria (7.2%). The oral microbiomes of dogs and their owners were appreciably different, and similarity in the microbiomes of canines and their owners was not correlated with residing in the same household. Oral-to-oral transfer of Neisseria shayeganii, Porphyromonas canigingivalis, Tannerella forsythia, and Streptococcus minor from dogs to humans was suspected. The finding of potentially zoonotic and periodontopathic bacteria in the canine oral microbiome may be a public health concern. PMID:26134411

454 pyrosequencing reveals a shift in fecal microbiota of healthy adult men consuming polydextrose or soluble corn fiber.

PubMed

Hooda, Seema; Boler, Brittany M Vester; Serao, Mariana C Rossoni; Brulc, Jennifer M; Staeger, Michael A; Boileau, Thomas W; Dowd, Scot E; Fahey, George C; Swanson, Kelly S

2012-07-01

The relative contribution of novel fibers such as polydextrose and soluble corn fiber (SCF) to the human gut microbiome and its association with host physiology has not been well studied. This study was conducted to test the impact of polydextrose and SCF on the composition of the human gut microbiota using 454 pyrosequencing and to identify associations among fecal microbiota and fermentative end-products. Healthy adult men (n = 20) with a mean dietary fiber (DF) intake of 14 g/d were enrolled in a randomized, double-blind, placebo-controlled crossover study. Participants consumed 3 treatment snack bars/d during each 21-d period that contained no supplemental fiber (NFC), polydextrose (PDX; 21 g/d), or SCF (21 g/d) for 21 d. There were no washout periods. Fecal samples were collected on d 16-21 of each period; DNA was extracted, followed by amplification of the V4-V6 region of the 16S rRNA gene using barcoded primers. PDX and SCF significantly affected the relative abundance of bacteria at the class, genus, and species level. The consumption of PDX and SCF led to greater fecal Clostridiaceae and Veillonellaceae and lower Eubacteriaceae compared with a NFC. The abundance of Faecalibacterium, Phascolarctobacterium, and Dialister was greater (P < 0.05) in response to PDX and SCF intake, whereas Lactobacillus was greater (P < 0.05) only after SCF intake. Faecalibacterium prausnitzii, well known for its antiinflammatory properties, was greater (P < 0.05) after fiber consumption. Principal component analysis clearly indicated a distinct clustering of individuals consuming supplemental fibers. Our data demonstrate a beneficial shift in the gut microbiome of adults consuming PDX and SCF, with potential application as prebiotics.

Bacterial Community Development in Experimental Gingivitis

PubMed Central

Kistler, James O.; Booth, Veronica; Bradshaw, David J.; Wade, William G.

2013-01-01

Current knowledge of the microbial composition of dental plaque in early gingivitis is based largely on microscopy and cultural methods, which do not provide a comprehensive description of oral microbial communities. This study used 454-pyrosequencing of the V1–V3 region of 16S rRNA genes (approximately 500 bp), and bacterial culture, to characterize the composition of plaque during the transition from periodontal health to gingivitis. A total of 20 healthy volunteers abstained from oral hygiene for two weeks, allowing plaque to accumulate and gingivitis to develop. Plaque samples were analyzed at baseline, and after one and two weeks. In addition, plaque samples from 20 chronic periodontitis patients were analyzed for cross-sectional comparison to the experimental gingivitis cohort. All of the healthy volunteers developed gingivitis after two weeks. Pyrosequencing yielded a final total of 344 267 sequences after filtering, with a mean length of 354 bases, that were clustered into an average of 299 species-level Operational Taxonomic Units (OTUs) per sample. Principal coordinates analysis (PCoA) plots revealed significant shifts in the bacterial community structure of plaque as gingivitis was induced, and community diversity increased significantly after two weeks. Changes in the relative abundance of OTUs during the transition from health to gingivitis were correlated to bleeding on probing (BoP) scores and resulted in the identification of new health- and gingivitis-associated taxa. Comparison of the healthy volunteers to the periodontitis patients also confirmed the association of a number of putative periodontal pathogens with chronic periodontitis. Taxa associated with gingivitis included Fusobacterium nucleatum subsp. polymorphum, Lachnospiraceae [G-2] sp. HOT100, Lautropia sp. HOTA94, and Prevotella oulorum, whilst Rothia dentocariosa was associated with periodontal health. Further study of these taxa is warranted and may lead to new therapeutic approaches to prevent periodontal disease. PMID:23967169

Mangrove Bacterial Diversity and the Impact of Oil Contamination Revealed by Pyrosequencing: Bacterial Proxies for Oil Pollution

PubMed Central

dos Santos, Henrique Fragoso; Cury, Juliano Carvalho; do Carmo, Flávia Lima; dos Santos, Adriana Lopes; Tiedje, James; van Elsas, Jan Dirk; Rosado, Alexandre Soares; Peixoto, Raquel Silva

2011-01-01

Background Mangroves are transitional coastal ecosystems in tropical and sub-tropical regions and represent biologically important and productive ecosystems. Despite their great ecological and economic importance, mangroves are often situated in areas of high anthropogenic influence, being exposed to pollutants, such as those released by oil spills. Methodology/Principal Findings A microcosm experiment was conducted, which simulated an oil spill in previously pristine mangrove sediment. The effect of the oil spill on the extant microbial community was studied using direct pyrosequencing. Extensive bacterial diversity was observed in the pristine mangrove sediment, even after oil contamination. The number of different OTUs only detected in contaminated samples was significantly higher than the number of OTUs only detected in non-contaminated samples. The phylum Proteobacteria, in particular the classes Gammaproteobacteria and Deltaproteobacteria, were prevalent before and after the simulated oil spill. On the other hand, the order Chromatiales and the genus Haliea decreased upon exposure to 2 and 5% oil, these are proposed as sensitive indicators of oil contamination. Three other genera, Marinobacterium, Marinobacter and Cycloclasticus increased their prevalence when confronted with oil. These groups are possible targets for the biomonitoring of the impact of oil in mangrove settings. Conclusions/Significance We suggest the use of sequences of the selected genera as proxies for oil pollution, using qPCR assessments. The quantification of these genera in distinct mangrove systems in relation to the local oil levels would permit the evaluation of the level of perturbance of mangroves, being useful in field monitoring. Considering the importance of mangroves to many other environments and the susceptibility of such areas to oil spills this manuscript will be of broad interest. PMID:21399677

Relationships between and formation dynamics of the microbiota of consumers, producers, and the environment in an abalone aquatic system

PubMed Central

Zhao, Wang; Liu, Guang-Feng; Wang, Jiang-Yong

2017-01-01

An ecosystem is a community comprising living and nonliving components of the environment. Microbes are ubiquitous elements in each of these components. The dynamics of microbiota formation in an ecosystem is important to elucidate, because how the different components of a system exchange microbes, and how the microbes control ecological processes remain unresolved. In this study, an abalone, Haliotis diversicolor, seed-nursing pond was used as a model system. We first examined changes in bacterial communities during the seedling cultivation of this herbivorous juvenile aquatic invertebrate animal. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to analyze bacterial community dynamics and spatio-temporal interactions of different system components: consumers (abalone), producers (algae or a substrate), and the environment (water). DGGE fingerprints revealed that the developmental stages of abalone influences bacterial communities of both the abalone and substrate. Although the communities in water fluctuated daily, they could be divided into two clusters that coincided with abalone stages, reflecting the transition from larva to juvenile at around day 21. Pyrosequencing showed that the microbiota in the abalone and substrate had more operational taxonomic units in common than that of either with water. The Bray-Curtis similarity index was used to quantify the formation dynamics of microbiota among the various components of the system. The bacterial communities in producers and consumers showed similar changes. These communities were unstable at the beginning and then slowly stabilized over time. The environmental bacterial community was more stable than the bacterial communities in consumers and producers, and may have been the basis for stability in the system. Our research provides insights into the dynamics of microbiota formation in various biotic elements of a system that will contribute to predictive systems modeling. PMID:28787009

Comparison of Bacterial Community Composition of Primary and Persistent Endodontic Infections Using Pyrosequencing.

PubMed

Tzanetakis, Giorgos N; Azcarate-Peril, M Andrea; Zachaki, Sophia; Panopoulos, Panos; Kontakiotis, Evangelos G; Madianos, Phoebus N; Divaris, Kimon

2015-08-01

Elucidating the microbial ecology of endodontic infections (EIs) is a necessary step in developing effective intracanal antimicrobials. The aim of the present study was to investigate the bacterial composition of symptomatic and asymptomatic primary and persistent infections in a Greek population using high-throughput sequencing methods. 16S amplicon pyrosequencing of 48 root canal bacterial samples was conducted, and sequencing data were analyzed using an oral microbiome-specific and a generic (Greengenes) database. Bacterial abundance and diversity were examined by EI type (primary or persistent), and statistical analysis was performed by using non-parametric and parametric tests accounting for clustered data. Bacteroidetes was the most abundant phylum in both infection groups. Significant, albeit weak associations of bacterial diversity were found, as measured by UniFrac distances with infection type (analyses of similarity, R = 0.087, P = .005) and symptoms (analyses of similarity, R = 0.055, P = .047). Persistent infections were significantly enriched for Proteobacteria and Tenericutes compared with primary ones; at the genus level, significant differences were noted for 14 taxa, including increased enrichment of persistent infections for Lactobacillus, Streptococcus, and Sphingomonas. More but less abundant phyla were identified using the Greengenes database; among those, Cyanobacteria (0.018%) and Acidobacteria (0.007%) were significantly enriched among persistent infections. Persistent infections showed higher phylogenetic diversity (PD) (asymptomatic: PD = 9.2, standard error [SE] = 1.3; symptomatic: PD = 8.2, SE = 0.7) compared with primary infections (asymptomatic: PD = 5.9, SE = 0.8; symptomatic: PD = 7.4, SE = 1.0). The present study revealed a high bacterial diversity of EI and suggests that persistent infections may have more diverse bacterial communities than primary infections. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Microbial Diversity in Hummock and Hollow Soils of Three Wetlands on the Qinghai-Tibetan Plateau Revealed by 16S rRNA Pyrosequencing

PubMed Central

Deng, Yongcui; Cui, Xiaoyong; Hernández, Marcela; Dumont, Marc G.

2014-01-01

The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo) on the Qinghai-Tibetan Plateau, the world’s highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance), Actinobacteria (17.3%) and Bacteroidetes (11%) had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1–10%). In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales). Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta) and methanotrophs (mainly Methylocystis) compared with the other two wetlands. Principal coordinate analysis (PCoA) indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of the Qinghai-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem. PMID:25078273

Horizon-Specific Bacterial Community Composition of German Grassland Soils, as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes ▿ †

PubMed Central

Will, Christiane; Thürmer, Andrea; Wollherr, Antje; Nacke, Heiko; Herold, Nadine; Schrumpf, Marion; Gutknecht, Jessica; Wubet, Tesfaye; Buscot, François; Daniel, Rolf

2010-01-01

The diversity of bacteria in soil is enormous, and soil bacterial communities can vary greatly in structure. Here, we employed a pyrosequencing-based analysis of the V2-V3 16S rRNA gene region to characterize the overall and horizon-specific (A and B horizons) bacterial community compositions in nine grassland soils, which covered three different land use types. The entire data set comprised 752,838 sequences, 600,544 of which could be classified below the domain level. The average number of sequences per horizon was 41,824. The dominant taxonomic groups present in all samples and horizons were the Acidobacteria, Betaproteobacteria, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Chloroflexi, Firmicutes, and Bacteroidetes. Despite these overarching dominant taxa, the abundance, diversity, and composition of bacterial communities were horizon specific. In almost all cases, the estimated bacterial diversity (H′) was higher in the A horizons than in the corresponding B horizons. In addition, the H′ was positively correlated with the organic carbon content, the total nitrogen content, and the C-to-N ratio, which decreased with soil depth. It appeared that lower land use intensity results in higher bacterial diversity. The majority of sequences affiliated with the Actinobacteria, Bacteroidetes, Cyanobacteria, Fibrobacteres, Firmicutes, Spirochaetes, Verrucomicrobia, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria were derived from A horizons, whereas the majority of the sequences related to Acidobacteria, Chloroflexi, Gemmatimonadetes, Nitrospira, TM7, and WS3 originated from B horizons. The distribution of some bacterial phylogenetic groups and subgroups in the different horizons correlated with soil properties such as organic carbon content, total nitrogen content, or microbial biomass. PMID:20729324

Relationships between and formation dynamics of the microbiota of consumers, producers, and the environment in an abalone aquatic system.

PubMed

Jiang, Jing-Zhe; Zhao, Wang; Liu, Guang-Feng; Wang, Jiang-Yong

2017-01-01

An ecosystem is a community comprising living and nonliving components of the environment. Microbes are ubiquitous elements in each of these components. The dynamics of microbiota formation in an ecosystem is important to elucidate, because how the different components of a system exchange microbes, and how the microbes control ecological processes remain unresolved. In this study, an abalone, Haliotis diversicolor, seed-nursing pond was used as a model system. We first examined changes in bacterial communities during the seedling cultivation of this herbivorous juvenile aquatic invertebrate animal. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to analyze bacterial community dynamics and spatio-temporal interactions of different system components: consumers (abalone), producers (algae or a substrate), and the environment (water). DGGE fingerprints revealed that the developmental stages of abalone influences bacterial communities of both the abalone and substrate. Although the communities in water fluctuated daily, they could be divided into two clusters that coincided with abalone stages, reflecting the transition from larva to juvenile at around day 21. Pyrosequencing showed that the microbiota in the abalone and substrate had more operational taxonomic units in common than that of either with water. The Bray-Curtis similarity index was used to quantify the formation dynamics of microbiota among the various components of the system. The bacterial communities in producers and consumers showed similar changes. These communities were unstable at the beginning and then slowly stabilized over time. The environmental bacterial community was more stable than the bacterial communities in consumers and producers, and may have been the basis for stability in the system. Our research provides insights into the dynamics of microbiota formation in various biotic elements of a system that will contribute to predictive systems modeling.

Pyrosequencing of mcrA and Archaeal 16S rRNA Genes Reveals Diversity and Substrate Preferences of Methanogen Communities in Anaerobic Digesters

PubMed Central

Wilkins, David; Lu, Xiao-Ying; Shen, Zhiyong; Chen, Jiapeng

2014-01-01

Methanogenic archaea play a key role in biogas-producing anaerobic digestion and yet remain poorly taxonomically characterized. This is in part due to the limitations of low-throughput Sanger sequencing of a single (16S rRNA) gene, which in the past may have undersampled methanogen diversity. In this study, archaeal communities from three sludge digesters in Hong Kong and one wastewater digester in China were examined using high-throughput pyrosequencing of the methyl coenzyme M reductase (mcrA) and 16S rRNA genes. Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in each digester, indicating that both hydrogenotrophic and acetoclastic methanogenesis was occurring. Two sludge digesters had similar community structures, likely due to their similar design and feedstock. Taxonomic classification of the mcrA genes suggested that these digesters were dominated by acetoclastic methanogens, particularly Methanosarcinales, while the other digesters were dominated by hydrogenotrophic Methanomicrobiales. The proposed euryarchaeotal order Methanomassiliicoccales and the uncultured WSA2 group were detected with the 16S rRNA gene, and potential mcrA genes for these groups were identified. 16S rRNA gene sequencing also recovered several crenarchaeotal groups potentially involved in the initial anaerobic digestion processes. Overall, the two genes produced different taxonomic profiles for the digesters, while greater methanogen richness was detected using the mcrA gene, supporting the use of this functional gene as a complement to the 16S rRNA gene to better assess methanogen diversity. A significant positive correlation was detected between methane production and the abundance of mcrA transcripts in digesters treating sludge and wastewater samples, supporting the mcrA gene as a biomarker for methane yield. PMID:25381241

A pyrosequencing-based metagenomic study of methane-producing microbial community in solid-state biogas reactor

PubMed Central

2013-01-01

Background A solid-state anaerobic digestion method is used to produce biogas from various solid wastes in China but the efficiency of methane production requires constant improvement. The diversity and abundance of relevant microorganisms play important roles in methanogenesis of biomass. The next-generation high-throughput pyrosequencing platform (Roche/454 GS FLX Titanium) provides a powerful tool for the discovery of novel microbes within the biogas-generating microbial communities. Results To improve the power of our metagenomic analysis, we first evaluated five different protocols for extracting total DNA from biogas-producing mesophilic solid-state fermentation materials and then chose two high-quality protocols for a full-scale analysis. The characterization of both sequencing reads and assembled contigs revealed that the most prevalent microbes of the fermentation materials are derived from Clostridiales (Firmicutes), which contribute to degrading both protein and cellulose. Other important bacterial species for decomposing fat and carbohydrate are Bacilli, Gammaproteobacteria, and Bacteroidetes (belonging to Firmicutes, Proteobacteria, and Bacteroidetes, respectively). The dominant bacterial species are from six genera: Clostridium, Aminobacterium, Psychrobacter, Anaerococcus, Syntrophomonas, and Bacteroides. Among them, abundant Psychrobacter species, which produce low temperature-adaptive lipases, and Anaerococcus species, which have weak fermentation capabilities, were identified for the first time in biogas fermentation. Archaea, represented by genera Methanosarcina, Methanosaeta and Methanoculleus of Euryarchaeota, constitute only a small fraction of the entire microbial community. The most abundant archaeal species include Methanosarcina barkeri fusaro, Methanoculleus marisnigri JR1, and Methanosaeta theromphila, and all are involved in both acetotrophic and hydrogenotrophic methanogenesis. Conclusions The identification of new bacterial genera and species involved in biogas production provides insights into novel designs of solid-state fermentation under mesophilic or low-temperature conditions. PMID:23320936

Fungal communities associated with the biodegradation of polyester polyurethane buried under compost at different temperatures.

PubMed

Zafar, Urooj; Houlden, Ashley; Robson, Geoffrey D

2013-12-01

Plastics play an essential role in the modern world due to their low cost and durability. However, accumulation of plastic waste in the environment causes wide-scale pollution with long-lasting effects, making plastic waste management expensive and problematic. Polyurethanes (PUs) are heteropolymers that made up ca. 7% of the total plastic production in Europe in 2011. Polyester PUs in particular have been extensively reported as susceptible to microbial biodegradation in the environment, particularly by fungi. In this study, we investigated the impact of composting on PUs, as composting is a microbially rich process that is increasingly being used for the processing of green waste and food waste as an economically viable alternative to landfill disposal. PU coupons were incubated for 12 weeks in fresh compost at 25°C, 45°C, and 50°C to emulate the thermophilic and maturation stages of the composting process. Incubation at all temperatures caused significant physical deterioration of the polyester PU coupons and was associated with extensive fungal colonization. Terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of the fungal communities on the PU surface and in the surrounding compost revealed that the population on the surface of PU was different from the surrounding compost community, suggesting enrichment and selection. The most dominant fungi identified from the surfaces of PU coupons by pyrosequencing was Fusarium solani at 25°C, while at both 45°C and 50°C, Candida ethanolica was the dominant species. The results of this preliminary study suggest that the composting process has the potential to biodegrade PU waste if optimized further in the future.

Characterization of the bacterial biodiversity in Pico cheese (an artisanal Azorean food).

PubMed

Riquelme, Cristina; Câmara, Sandra; Dapkevicius, Maria de Lurdes N Enes; Vinuesa, Pablo; da Silva, Célia Costa Gomes; Malcata, F Xavier; Rego, Oldemiro A

2015-01-02

This work presents the first study on the bacterial communities in Pico cheese, a traditional cheese of the Azores (Portugal), made from raw cow's milk. Pyrosequencing of tagged amplicons of the V3-V4 regions of the 16S rDNA and Operational Taxonomic Unit-based (OTU-based) analysis were applied to obtain an overall idea of the microbiota in Pico cheese and to elucidate possible differences between cheese-makers (A, B and C) and maturation times. Pyrosequencing revealed a high bacterial diversity in Pico cheese. Four phyla (Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes) and 54 genera were identified. The predominant genus was Lactococcus (77% of the sequences). Sequences belonging to major cheese-borne pathogens were not found. Staphylococcus accounted for 0.5% of the sequences. Significant differences in bacterial community composition were observed between cheese-maker B and the other two units that participated in the study. However, OTU analysis identified a set of taxa (Lactococcus, Streptococcus, Acinetobacter, Enterococcus, Lactobacillus, Staphylococcus, Rothia, Pantoea and unclassified genera belonging to the Enterobacteriaceae family) that would represent the core components of artisanal Pico cheese microbiota. A diverse bacterial community was present at early maturation, with an increase in the number of phylotypes up to 2 weeks, followed by a decrease at the end of ripening. The most remarkable trend in abundance patterns throughout ripening was an increase in the number of sequences belonging to the Lactobacillus genus, with a concomitant decrease in Acinetobacter, and Stenotrophomonas. Microbial rank abundance curves showed that Pico cheese's bacterial communities are characterized by a few dominant taxa and many low-abundance, highly diverse taxa that integrate the so-called "rare biosphere". Copyright © 2014 Elsevier B.V. All rights reserved.

Prerequisites for amplicon pyrosequencing of microbial methanol utilizers in the environment

PubMed Central

Kolb, Steffen; Stacheter, Astrid

2013-01-01

The commercial availability of next generation sequencing (NGS) technologies facilitated the assessment of functional groups of microorganisms in the environment with high coverage, resolution, and reproducibility. Soil methylotrophs were among the first microorganisms in the environment that were assessed with molecular tools, and nowadays, as well with NGS technologies. Studies in the past years re-attracted notice to the pivotal role of methylotrophs in global conversions of methanol, which mainly originates from plants, and is involved in oxidative reactions and ozone formation in the atmosphere. Aerobic methanol utilizers belong to Bacteria, yeasts, Ascomycota, and molds. Numerous bacterial methylotrophs are facultatively aerobic, and also contribute to anaerobic methanol oxidation in the environment, whereas strict anaerobic methanol utilizers belong to methanogens and acetogens. The diversity of enzymes catalyzing the initial oxidation of methanol is considerable, and comprises at least five different enzyme types in aerobes, and one in strict anaerobes. Only the gene of the large subunit of pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH; mxaF) has been analyzed by environmental pyrosequencing. To enable a comprehensive assessment of methanol utilizers in the environment, new primers targeting genes of the PQQ MDH in Methylibium (mdh2), of the nicotinamide adenine dinucleotide-dependent MDH (mdh), of the methanol oxidoreductase of Actinobacteria (mdo), of the fungal flavin adenine nucleotide-dependent alcohol oxidase (mod1, mod2, and homologs), and of the gene of the large subunit of the methanol:corrinoid methyltransferases (mtaC) in methanogens and acetogens need to be developed. Combined stable isotope probing of nucleic acids or proteins with amplicon-based NGS are straightforward approaches to reveal insights into functions of certain methylotrophic taxa in the global methanol cycle. PMID:24046766

Microbial Successions Are Associated with Changes in Chemical Profiles of a Model Refrigerated Fresh Pork Sausage during an 80-Day Shelf Life Study

PubMed Central

David, Jairus R. D.; Gilbreth, Stefanie Evans; Smith, Gordon; Nietfeldt, Joseph; Legge, Ryan; Kim, Jaehyoung; Sinha, Rohita; Duncan, Christopher E.; Ma, Junjie; Singh, Indarpal

2014-01-01

Fresh pork sausage is produced without a microbial kill step and therefore chilled or frozen to control microbial growth. In this report, the microbiota in a chilled fresh pork sausage model produced with or without an antimicrobial combination of sodium lactate and sodium diacetate was studied using a combination of traditional microbiological methods and deep pyrosequencing of 16S rRNA gene amplicons. In the untreated system, microbial populations rose from 102 to 106 CFU/g within 15 days of storage at 4°C, peaking at nearly 108 CFU/g by day 30. Pyrosequencing revealed a complex community at day 0, with taxa belonging to the Bacilli, Gammaproteobacteria, Betaproteobacteria, Actinobacteria, Bacteroidetes, and Clostridia. During storage at 4°C, the untreated system displayed a complex succession, with species of Weissella and Leuconostoc that dominate the product at day 0 being displaced by species of Pseudomonas (P. lini and P. psychrophila) within 15 days. By day 30, a second wave of taxa (Lactobacillus graminis, Carnobacterium divergens, Buttiauxella brennerae, Yersinia mollaretti, and a taxon of Serratia) dominated the population, and this succession coincided with significant chemical changes in the matrix. Treatment with lactate-diacetate altered the dynamics dramatically, yielding a monophasic growth curve of a single species of Lactobacillus (L. graminis), followed by a uniform selective die-off of the majority of species in the population. Of the six species of Lactobacillus that were routinely detected, L. graminis became the dominant member in all samples, and its origins were traced to the spice blend used in the formulation. PMID:24928886

Relationship of subseafloor microbial diversity to sediment age and organic carbon content

NASA Astrophysics Data System (ADS)

Walsh, E. A.; Kirkpatrick, J. B.; Sogin, M. L.; D'Hondt, S. L.

2013-12-01

Our tag pyrosequencing investigation of four globally distant sites reveals sediment age and total organic carbon content to be significant components in understanding subseafloor diversity. Our sampling locations include two sites from high-productivity regions (Indian Ocean and Bering Sea) and two from moderate-productivity (eastern and central equatorial Pacific Ocean). Sediment from the high-productivity sites has much higher TOC than sediment from the moderate-productivity equatorial sites. We applied a high-resolution 16S V4-V6 tag pyrosequencing approach to 24 bacterial and 17 archaeal samples, totaling 602,502 reads. We identified1,291 archaeal and 15,910 bacterial OTUs (97%) from these reads. We analyzed bacterial samples from all four sites in addition to archaeal samples from our high productivity sites. These high productivity, high TOC sites have a pronounced methane-rich sulfate-free zone at depth from which archaea have been previously considered to dominate (Biddle et al., 2006). At all four locations, microbial diversity is highest near the seafloor and drops rapidly to low but stable values with increasing sediment depth. The depth at which diversity stabilizes varies greatly from site to site, but the age at which it stabilizes is relatively constant. At all four sites, diversity reaches low stable values a few hundred thousand years after sediment deposition. The sites with high total organic carbon (high productivity sites) generally exhibit higher diversity at each sediment age than the sites with lower total organic carbon (moderate-productivity sites). Archaeal diversity is lower than bacterial diversity at every sampled depth. Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sørensen, K.B., Anderson, R. et al. (2006) Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru. PNAS 103: 3846-3851.

Evidence for trade-offs in detoxification and chemosensation gene signatures in Plutella xylostella.

PubMed

Bautista, Ma Anita M; Bhandary, Binny; Wijeratne, Asela J; Michel, Andrew P; Hoy, Casey W; Mittapalli, Omprakash

2015-03-01

Detoxification genes have been associated with insecticide adaptation in the diamondback moth, Plutella xylostella. The link between chemosensation genes and adaptation, however, remains unexplored. To gain a better understanding of the involvement of these genes in insecticide adaptation, the authors exposed lines of P. xylostella to either high uniform (HU) or low heterogeneous (LH) concentrations of permethrin, expecting primarily physiological or behavioral selection respectively. Initially, 454 pyrosequencing was applied, followed by an examination of expression profiles of candidate genes that responded to selection [cytochrome P450 (CYP), glutathione S-transferase (GST), carboxylesterase (CarE), chemosensory protein (CSP) and odorant-binding protein (OBP)] by quantitative PCR in the larvae. Toxicity and behavioral assays were also conducted to document the effects of the two forms of exposure. Pyrosequencing of the P. xylostella transcriptome from adult heads and third instars produced 198,753 reads with 52,752,486 bases. Quantitative PCR revealed overexpression of CYP4M14, CYP305B1 and CSP8 in HU larvae. OBP13, however, was highest in LH. Larvae from LH and HU lines had up to five- and 752-fold resistance levels respectively, which could be due to overexpression of P450s. However, the behavioral responses of all lines to a series of permethrin concentrations did not vary significantly in any of the generations examined, in spite of the observed upregulation of CSP8 and OBP13. Expression patterns from the target genes provide insights into behavioral and physiological responses to permethrin and suggest a new avenue of research on the role of chemosensation genes in insect adaptation to toxins. © 2014 Society of Chemical Industry.

Molecular analysis of bacterial communities and detection of potential pathogens in a recirculating aquaculture system for Scophthalmus maximus and Solea senegalensis.

PubMed

Martins, Patrícia; Cleary, Daniel F R; Pires, Ana C C; Rodrigues, Ana Maria; Quintino, Victor; Calado, Ricardo; Gomes, Newton C M

2013-01-01

The present study combined a DGGE and barcoded 16S rRNA pyrosequencing approach to assess bacterial composition in the water of a recirculating aquaculture system (RAS) with a shallow raceway system (SRS) for turbot (Scophthalmus maximus) and sole (Solea senegalensis). Barcoded pyrosequencing results were also used to determine the potential pathogen load in the RAS studied. Samples were collected from the water supply pipeline (Sup), fish production tanks (Pro), sedimentation filter (Sed), biofilter tank (Bio), and protein skimmer (Ozo; also used as an ozone reaction chamber) of twin RAS operating in parallel (one for each fish species). Our results revealed pronounced differences in bacterial community composition between turbot and sole RAS, suggesting that in the systems studied there is a strong species-specific effect on water bacterial communities. Proteobacteria was the most abundant phylum in the water supply and all RAS compartments. Other important taxonomic groups included the phylum Bacteriodetes. The saltwater supplied displayed a markedly lower richness and appeared to have very little influence on bacterial composition. The following potentially pathogenic species were detected: Photobacterium damselae in turbot (all compartments), Tenacibaculum discolor in turbot and sole (all compartments), Tenacibaculum soleae in turbot (all compartments) and sole (Pro, Sed and Bio), and Serratia marcescens in turbot (Sup, Sed, Bio and Ozo) and sole (only Sed) RAS. Despite the presence of these pathogens, no symptomatic fish were observed. Although we were able to identify potential pathogens, this approach should be employed with caution when monitoring aquaculture systems, as the required phylogenetic resolution for reliable identification of pathogens may not always be possible to achieve when employing 16S rRNA gene fragments.

Molecular Analysis of Bacterial Communities and Detection of Potential Pathogens in a Recirculating Aquaculture System for Scophthalmus maximus and Solea senegalensis

PubMed Central

Martins, Patrícia; Cleary, Daniel F. R.; Pires, Ana C. C.; Rodrigues, Ana Maria; Quintino, Victor; Calado, Ricardo; Gomes, Newton C. M.

2013-01-01

The present study combined a DGGE and barcoded 16S rRNA pyrosequencing approach to assess bacterial composition in the water of a recirculating aquaculture system (RAS) with a shallow raceway system (SRS) for turbot (Scophthalmus maximus) and sole (Solea senegalensis). Barcoded pyrosequencing results were also used to determine the potential pathogen load in the RAS studied. Samples were collected from the water supply pipeline (Sup), fish production tanks (Pro), sedimentation filter (Sed), biofilter tank (Bio), and protein skimmer (Ozo; also used as an ozone reaction chamber) of twin RAS operating in parallel (one for each fish species). Our results revealed pronounced differences in bacterial community composition between turbot and sole RAS, suggesting that in the systems studied there is a strong species-specific effect on water bacterial communities. Proteobacteria was the most abundant phylum in the water supply and all RAS compartments. Other important taxonomic groups included the phylum Bacteriodetes. The saltwater supplied displayed a markedly lower richness and appeared to have very little influence on bacterial composition. The following potentially pathogenic species were detected: Photobacterium damselae in turbot (all compartments), Tenacibaculum discolor in turbot and sole (all compartments), Tenacibaculum soleae in turbot (all compartments) and sole (Pro, Sed and Bio), and Serratia marcescens in turbot (Sup, Sed, Bio and Ozo) and sole (only Sed) RAS. Despite the presence of these pathogens, no symptomatic fish were observed. Although we were able to identify potential pathogens, this approach should be employed with caution when monitoring aquaculture systems, as the required phylogenetic resolution for reliable identification of pathogens may not always be possible to achieve when employing 16S rRNA gene fragments. PMID:24278329

Fungal Communities Associated with the Biodegradation of Polyester Polyurethane Buried under Compost at Different Temperatures

PubMed Central

Zafar, Urooj; Houlden, Ashley

2013-01-01

Plastics play an essential role in the modern world due to their low cost and durability. However, accumulation of plastic waste in the environment causes wide-scale pollution with long-lasting effects, making plastic waste management expensive and problematic. Polyurethanes (PUs) are heteropolymers that made up ca. 7% of the total plastic production in Europe in 2011. Polyester PUs in particular have been extensively reported as susceptible to microbial biodegradation in the environment, particularly by fungi. In this study, we investigated the impact of composting on PUs, as composting is a microbially rich process that is increasingly being used for the processing of green waste and food waste as an economically viable alternative to landfill disposal. PU coupons were incubated for 12 weeks in fresh compost at 25°C, 45°C, and 50°C to emulate the thermophilic and maturation stages of the composting process. Incubation at all temperatures caused significant physical deterioration of the polyester PU coupons and was associated with extensive fungal colonization. Terminal restriction fragment length polymorphism (TRFLP) analysis and pyrosequencing of the fungal communities on the PU surface and in the surrounding compost revealed that the population on the surface of PU was different from the surrounding compost community, suggesting enrichment and selection. The most dominant fungi identified from the surfaces of PU coupons by pyrosequencing was Fusarium solani at 25°C, while at both 45°C and 50°C, Candida ethanolica was the dominant species. The results of this preliminary study suggest that the composting process has the potential to biodegrade PU waste if optimized further in the future. PMID:24056469

Urease gene-containing Archaea dominate autotrophic ammonia oxidation in two acid soils.

PubMed

Lu, Lu; Jia, Zhongjun

2013-06-01

The metabolic traits of ammonia-oxidizing archaea (AOA) and bacteria (AOB) interacting with their environment determine the nitrogen cycle at the global scale. Ureolytic metabolism has long been proposed as a mechanism for AOB to cope with substrate paucity in acid soil, but it remains unclear whether urea hydrolysis could afford AOA greater ecological advantages. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, here we show that autotrophic ammonia oxidation in two acid soils was predominately driven by AOA that contain ureC genes encoding the alpha subunit of a putative archaeal urease. In urea-amended SIP microcosms of forest soil (pH 5.40) and tea orchard soil (pH 3.75), nitrification activity was stimulated significantly by urea fertilization when compared with water-amended soils in which nitrification resulted solely from the oxidation of ammonia generated through mineralization of soil organic nitrogen. The stimulated activity was paralleled by changes in abundance and composition of archaeal amoA genes. Time-course incubations indicated that archaeal amoA genes were increasingly labelled by (13) CO2 in both microcosms amended with water and urea. Pyrosequencing revealed that archaeal populations were labelled to a much greater extent in soils amended with urea than water. Furthermore, archaeal ureC genes were successfully amplified in the (13) C-DNA, and acetylene inhibition suggests that autotrophic growth of urease-containing AOA depended on energy generation through ammonia oxidation. The sequences of AOB were not detected, and active AOA were affiliated with the marine Group 1.1a-associated lineage. The results suggest that ureolytic N metabolism could afford AOA greater advantages for autotrophic ammonia oxidation in acid soil, but the mechanism of how urea activates AOA cells remains unclear. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform

PubMed Central

Park, Sung Yong; Goeken, Nolan; Lee, Hyo Jin; Bolan, Robert; Dubé, Michael P.

2014-01-01

ABSTRACT Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10−4 per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys. IMPORTANCE Annual HIV incidence, the number of newly infected individuals within a year, is the key measure of monitoring the epidemic's rise and decline. Developing reliable assays differentiating recent from chronic infections has been a long-standing quest in the HIV community. Over the past 15 years, these assays have traditionally measured various HIV-specific antibodies, but recent technological advancements have expanded the diversity of proposed accurate, user-friendly, and financially viable tools. Here we designed a high-throughput genomic HIV incidence assay based on the signature imprinted in the HIV gene sequence population. By combining next-generation sequencing techniques with bioinformatics analysis, we demonstrated that genomic fingerprints are capable of distinguishing recently infected patients from chronically infected patients with high precision. Our high-throughput platform is expected to allow us to process many patients' samples from a single experiment, permitting the assay to be cost-effective for routine surveillance. PMID:24371062

Rapid and accurate pyrosequencing of angiosperm plastid genomes

PubMed Central

Moore, Michael J; Dhingra, Amit; Soltis, Pamela S; Shaw, Regina; Farmerie, William G; Folta, Kevin M; Soltis, Douglas E

2006-01-01

Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid genome sequence was generated for a significant reduction in time and cost over traditional shotgun-based genome sequencing techniques, although with approximately half the coverage of previously reported GS 20 de novo genome sequence. The GS 20 should be broadly applicable to angiosperm plastid genome sequencing, and therefore promises to expand the scale of plant genetic and phylogenetic research dramatically. PMID:16934154

Visceral hypersensitive rats share common dysbiosis features with irritable bowel syndrome patients

PubMed Central

Zhou, Xiao-Yan; Li, Ming; Li, Xia; Long, Xin; Zuo, Xiu-Li; Hou, Xiao-Hua; Cong, Ying-Zi; Li, Yan-Qing

2016-01-01

AIM: To evaluate gut microbial dysbiosis in two visceral hypersensitive models in comparison with irritable bowel syndrome (IBS) patients and to explore the extent to which these models capture the dysbiosis of IBS patients. METHODS: Visceral hypersensitivity was developed using the maternal separation (MS) rat model and post-inflammatory rat model. The visceral sensitivity of the model groups and control group was evaluated using the abdominal withdraw reflex score and electromyography in response to graded colorectal distention. The 16S ribosomal RNA gene from fecal samples was pyrosequenced and analyzed. The correlation between dysbiosis in the microbiota and visceral hypersensitivity was calculated. Positive findings were compared to sequencing data from a published human IBS cohort. RESULTS: Dysbiosis triggered by neonatal maternal separation was lasting but not static. Both MS and post-inflammatory rat fecal microbiota deviated from that of the control rats to an extent that was larger than the co-housing effect. Two short chain fatty acid producing genera, Fusobacterium and Clostridium XI, were shared by the human IBS cohort and by the maternal separation rats and post-inflammatory rats, respectively, to different extents. Fusobacterium was significantly increased in the MS group, and its abundance positively correlated with the degree of visceral hypersensitivity. Porphyromonadaceae was a protective biomarker for both the rat control group and healthy human controls. CONCLUSION: The dysbiosis MS rat model and the post-inflammatory rat model captured some of the dysbiosis features of IBS patients. Fusobacterium, Clostridium XI and Porphyromonadaceae were identified as targets for future mechanistic research. PMID:27298564

The structural approach to shared knowledge: an application to engineering design teams.

PubMed

Avnet, Mark S; Weigel, Annalisa L

2013-06-01

We propose a methodology for analyzing shared knowledge in engineering design teams. Whereas prior work has focused on shared knowledge in small teams at a specific point in time, the model presented here is both scalable and dynamic. By quantifying team members' common views of design drivers, we build a network of shared mental models to reveal the structure of shared knowledge at a snapshot in time. Based on a structural comparison of networks at different points in time, a metric of change in shared knowledge is computed. Analysis of survey data from 12 conceptual space mission design sessions reveals a correlation between change in shared knowledge and each of several system attributes, including system development time, system mass, and technological maturity. From these results, we conclude that an early period of learning and consensus building could be beneficial to the design of engineered systems. Although we do not examine team performance directly, we demonstrate that shared knowledge is related to the technical design and thus provide a foundation for improving design products by incorporating the knowledge and thoughts of the engineering design team into the process.

Development of an ELA-DRA gene typing method based on pyrosequencing technology.

PubMed

Díaz, S; Echeverría, M G; It, V; Posik, D M; Rogberg-Muñoz, A; Pena, N L; Peral-García, P; Vega-Pla, J L; Giovambattista, G

2008-11-01

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.

Biodiversity of Trichoderma Community in the Tidal Flats and Wetland of Southeastern China.

PubMed

Saravanakumar, Kandasamy; Yu, Chuanjin; Dou, Kai; Wang, Meng; Li, Yaqian; Chen, Jie

2016-01-01

To investigate the biodiversity of Trichoderma (Hypocreaceae) and their relation to sediment physical and chemical properties, we collected a total of 491 sediment samples from coastal wetlands (tidal flat and wetland) in Southeast China. Further, we applied two types of molecular approaches such as culture dependent and independent methods for identification of Trichoderma spp. A total of 254 isolates were obtained and identified to 13 species such as T. aureoviride, T. asperellum, T. harzianum, T. atroviride, T. koningiopsis, T. longibrachiatum, T. koningii. T. tawa, T. viridescens, T. virens, T. hamatum, T. viride, and T. velutinum by the culture-dependent (CD) method of these, T. tawa was newly described in China. Subsequently, the culture indepented method of 454 pyrosequencing analysis revealed a total of six species such as T. citrinoviride, T. virens, T. polysporum, T. harzianum/Hypocrea lixii and two unknown species. Notably, T. citrinoviride and T. polysporum were not found by the CD method. Therefore, this work revealed that the combination of these two methods could show the higher biodiversity of Trichoderma spp., than either of this method alone. Among the sampling sites, Hangzhou, Zhejiang Province, exhibited rich biodiversity and low in Fengxian. Correlation and Redundancy discriminant analysis (RDA) revealed that sediment properties of temperature, redox potential (Eh) and pH significantly influenced the biodiversity of Trichoderma spp.

Language influences music harmony perception: effects of shared syntactic integration resources beyond attention

PubMed Central

Willems, Roel M.; Hagoort, Peter

2016-01-01

Many studies have revealed shared music–language processing resources by finding an influence of music harmony manipulations on concurrent language processing. However, the nature of the shared resources has remained ambiguous. They have been argued to be syntax specific and thus due to shared syntactic integration resources. An alternative view regards them as related to general attention and, thus, not specific to syntax. The present experiments evaluated these accounts by investigating the influence of language on music. Participants were asked to provide closure judgements on harmonic sequences in order to assess the appropriateness of sequence endings. At the same time participants read syntactic garden-path sentences. Closure judgements revealed a change in harmonic processing as the result of reading a syntactically challenging word. We found no influence of an arithmetic control manipulation (experiment 1) or semantic garden-path sentences (experiment 2). Our results provide behavioural evidence for a specific influence of linguistic syntax processing on musical harmony judgements. A closer look reveals that the shared resources appear to be needed to hold a harmonic key online in some form of syntactic working memory or unification workspace related to the integration of chords and words. Overall, our results support the syntax specificity of shared music–language processing resources. PMID:26998339

Pyrosequencing analysis yields comprehensive assessment of microbial communities in pilot-scale two-stage membrane biofilm reactors.

PubMed

Ontiveros-Valencia, Aura; Tang, Youneng; Zhao, He-Ping; Friese, David; Overstreet, Ryan; Smith, Jennifer; Evans, Patrick; Rittmann, Bruce E; Krajmalnik-Brown, Rosa

2014-07-01

We studied the microbial community structure of pilot two-stage membrane biofilm reactors (MBfRs) designed to reduce nitrate (NO3(-)) and perchlorate (ClO4(-)) in contaminated groundwater. The groundwater also contained oxygen (O2) and sulfate (SO4(2-)), which became important electron sinks that affected the NO3(-) and ClO4(-) removal rates. Using pyrosequencing, we elucidated how important phylotypes of each "primary" microbial group, i.e., denitrifying bacteria (DB), perchlorate-reducing bacteria (PRB), and sulfate-reducing bacteria (SRB), responded to changes in electron-acceptor loading. UniFrac, principal coordinate analysis (PCoA), and diversity analyses documented that the microbial community of biofilms sampled when the MBfRs had a high acceptor loading were phylogenetically distant from and less diverse than the microbial community of biofilm samples with lower acceptor loadings. Diminished acceptor loading led to SO4(2-) reduction in the lag MBfR, which allowed Desulfovibrionales (an SRB) and Thiothrichales (sulfur-oxidizers) to thrive through S cycling. As a result of this cooperative relationship, they competed effectively with DB/PRB phylotypes such as Xanthomonadales and Rhodobacterales. Thus, pyrosequencing illustrated that while DB, PRB, and SRB responded predictably to changes in acceptor loading, a decrease in total acceptor loading led to important shifts within the "primary" groups, the onset of other members (e.g., Thiothrichales), and overall greater diversity.

Using pyrosequencing to shed light on deep mine microbial ecology

PubMed Central

Edwards, Robert A; Rodriguez-Brito, Beltran; Wegley, Linda; Haynes, Matthew; Breitbart, Mya; Peterson, Dean M; Saar, Martin O; Alexander, Scott; Alexander, E Calvin; Rohwer, Forest

2006-01-01

Background Contrasting biological, chemical and hydrogeological analyses highlights the fundamental processes that shape different environments. Generating and interpreting the biological sequence data was a costly and time-consuming process in defining an environment. Here we have used pyrosequencing, a rapid and relatively inexpensive sequencing technology, to generate environmental genome sequences from two sites in the Soudan Mine, Minnesota, USA. These sites were adjacent to each other, but differed significantly in chemistry and hydrogeology. Results Comparisons of the microbes and the subsystems identified in the two samples highlighted important differences in metabolic potential in each environment. The microbes were performing distinct biochemistry on the available substrates, and subsystems such as carbon utilization, iron acquisition mechanisms, nitrogen assimilation, and respiratory pathways separated the two communities. Although the correlation between much of the microbial metabolism occurring and the geochemical conditions from which the samples were isolated could be explained, the reason for the presence of many pathways in these environments remains to be determined. Despite being physically close, these two communities were markedly different from each other. In addition, the communities were also completely different from other microbial communities sequenced to date. Conclusion We anticipate that pyrosequencing will be widely used to sequence environmental samples because of the speed, cost, and technical advantages. Furthermore, subsystem comparisons rapidly identify the important metabolisms employed by the microbes in different environments. PMID:16549033

De Novo Transcriptome of the Hemimetabolous German Cockroach (Blattella germanica)

PubMed Central

Zhou, Xiaojie; Qian, Kun; Tong, Ying; Zhu, Junwei Jerry; Qiu, Xinghui; Zeng, Xiaopeng

2014-01-01

Background The German cockroach, Blattella germanica, is an important insect pest that transmits various pathogens mechanically and causes severe allergic diseases. This insect has long served as a model system for studies of insect biology, physiology and ecology. However, the lack of genome or transcriptome information heavily hinder our further understanding about the German cockroach in every aspect at a molecular level and on a genome-wide scale. To explore the transcriptome and identify unique sequences of interest, we subjected the B. germanica transcriptome to massively parallel pyrosequencing and generated the first reference transcriptome for B. germanica. Methodology/Principal Findings A total of 1,365,609 raw reads with an average length of 529 bp were generated via pyrosequencing the mixed cDNA library from different life stages of German cockroach including maturing oothecae, nymphs, adult females and males. The raw reads were de novo assembled to 48,800 contigs and 3,961 singletons with high-quality unique sequences. These sequences were annotated and classified functionally in terms of BLAST, GO and KEGG, and the genes putatively coding detoxification enzyme systems, insecticide targets, key components in systematic RNA interference, immunity and chemoreception pathways were identified. A total of 3,601 SSRs (Simple Sequence Repeats) loci were also predicted. Conclusions/Significance The whole transcriptome pyrosequencing data from this study provides a usable genetic resource for future identification of potential functional genes involved in various biological processes. PMID:25265537

Barcoded pyrosequencing analysis of the microbial community in a simulator of the human gastrointestinal tract showed a colon region-specific microbiota modulation for two plant-derived polysaccharide blends.

PubMed

Marzorati, Massimo; Maignien, Lois; Verhelst, An; Luta, Gabriela; Sinnott, Robert; Kerckhof, Frederiek Maarten; Boon, Nico; Van de Wiele, Tom; Possemiers, Sam

2013-02-01

The combination of a Simulator of the Human Intestinal Microbial Ecosystem with ad hoc molecular techniques (i.e. pyrosequencing, denaturing gradient gel electrophoresis and quantitative PCR) allowed an evaluation of the extent to which two plant polysaccharide supplements could modify a complex gut microbial community. The presence of Aloe vera gel powder and algae extract in product B as compared to the standard blend (product A) improved its fermentation along the entire simulated colon. The potential extended effect of product B in the simulated distal colon, as compared to product A, was confirmed by: (i) the separate clustering of the samples before and after the treatment in the phylogenetic-based dendrogram and OTU-based PCoA plot only for product B; (ii) a higher richness estimator (+33 vs. -36 % of product A); and (iii) a higher dynamic parameter (21 vs. 13 %). These data show that the combination of well designed in vitro simulators with barcoded pyrosequencing is a powerful tool for characterizing changes occurring in the gut microbiota following a treatment. However, for the quantification of low-abundance species-of interest because of their relationship to potential positive health effects (i.e. bifidobacteria or lactobacilli)-conventional molecular ecological approaches, such as PCR-DGGE and qPCR, still remain a very useful complementary tool.

Gene discovery using next-generation pyrosequencing to develop ESTs for Phalaenopsis orchids

PubMed Central

2011-01-01

Background Orchids are one of the most diversified angiosperms, but few genomic resources are available for these non-model plants. In addition to the ecological significance, Phalaenopsis has been considered as an economically important floriculture industry worldwide. We aimed to use massively parallel 454 pyrosequencing for a global characterization of the Phalaenopsis transcriptome. Results To maximize sequence diversity, we pooled RNA from 10 samples of different tissues, various developmental stages, and biotic- or abiotic-stressed plants. We obtained 206,960 expressed sequence tags (ESTs) with an average read length of 228 bp. These reads were assembled into 8,233 contigs and 34,630 singletons. The unigenes were searched against the NCBI non-redundant (NR) protein database. Based on sequence similarity with known proteins, these analyses identified 22,234 different genes (E-value cutoff, e-7). Assembled sequences were annotated with Gene Ontology, Gene Family and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Among these annotations, over 780 unigenes encoding putative transcription factors were identified. Conclusion Pyrosequencing was effective in identifying a large set of unigenes from Phalaenopsis. The informative EST dataset we developed constitutes a much-needed resource for discovery of genes involved in various biological processes in Phalaenopsis and other orchid species. These transcribed sequences will narrow the gap between study of model organisms with many genomic resources and species that are important for ecological and evolutionary studies. PMID:21749684

Assessment of replicate bias in 454 pyrosequencing and a multi-purpose read-filtering tool.

PubMed

Jérôme, Mariette; Noirot, Céline; Klopp, Christophe

2011-05-26

Roche 454 pyrosequencing platform is often considered the most versatile of the Next Generation Sequencing technology platforms, permitting the sequencing of large genomes, the analysis of variations or the study of transcriptomes. A recent reported bias leads to the production of multiple reads for a unique DNA fragment in a random manner within a run. This bias has a direct impact on the quality of the measurement of the representation of the fragments using the reads. Other cleaning steps are usually performed on the reads before assembly or alignment. PyroCleaner is a software module intended to clean 454 pyrosequencing reads in order to ease the assembly process. This program is a free software and is distributed under the terms of the GNU General Public License as published by the Free Software Foundation. It implements several filters using criteria such as read duplication, length, complexity, base-pair quality and number of undetermined bases. It also permits to clean flowgram files (.sff) of paired-end sequences generating on one hand validated paired-ends file and the other hand single read file. Read cleaning has always been an important step in sequence analysis. The pyrocleaner python module is a Swiss knife dedicated to 454 reads cleaning. It includes commonly used filters as well as specialised ones such as duplicated read removal and paired-end read verification.

Teacher Strategies in Shared Reading for Children with Hearing Impairment

ERIC Educational Resources Information Center

Girgin, Ümit

2013-01-01

Problem Statement: Utilization of shared reading practice in hearing impaired children's literacy instruction may yield quite beneficial. However an investigation of Turkish literature revealed no studies regarding application of shared reading within elementary settings for hearing or hearing-impaired children. Furthermore international…

Job-sharing: an innovative approach for administration.

PubMed

Foster, D; Wilcox, C; Gibson, H

1992-01-01

A job-sharing arrangement for the Assistant Directors of Physiotherapy at the Royal Jubilee Hospital proved to be an innovative and successful experience demonstrating the feasibility of job-sharing at administrative levels in rehabilitation. Physiotherapy is traditionally a female dominated profession. By the time therapists are most highly skilled and clinically experienced, they have arrived at prime marriage and child-bearing years. Many valuable members are lost to the profession each year as therapists leave the work force to take care of their families, continue their education and participate in recreational activities. Alternative employment opportunities are needed to retain and return therapists to the work force. Convenience of work time is often important. Financial expectations may become a secondary consideration. A search of the literature revealed that while job-sharing has much to recommend it, it is not yet generally accepted in most health professional situations. A few anecdotal references described job-sharing in nursing. An industry-wide literature search revealed few references to the application of job-sharing at administrative levels.

Variation in the Gut Microbiota of Termites (Tsaitermes ampliceps) Against Different Diets.

PubMed

Su, Lijuan; Yang, Lele; Huang, Shi; Li, Yan; Su, Xiaoquan; Wang, Fengqin; Bo, Cunpei; Wang, En Tao; Song, Andong

2017-01-01

Termites are well recognized for their thriving on recalcitrant lignocellulosic diets through nutritional symbioses with gut-dwelling microbiota; however, the effects of diet changes on termite gut microbiota are poorly understood, especially for the lower termites. In this study, we employed high-throughput 454 pyrosequencing of 16S V1-V3 amplicons to compare gut microbiotas of Tsaitermes ampliceps fed with lignin-rich and lignin-poor cellulose diets after a 2-week-feeding period. As a result, the majority of bacterial taxa were shared across the treatments with different diets, but their relative abundances were modified. In particular, the relative abundance was reduced for Spirochaetes and it was increased for Proteobacteria and Bacteroides by feeding the lignin-poor diet. The evenness of gut microbiota exhibited a significant difference in response to the diet type (filter paper diets < corn stover diets < wood diets), while their richness was constant, which may be related to the lower recalcitrance of this biomass to degradation. These results have important implications for sampling and analysis strategies to probe the lignocellulose degradation features of termite gut microbiota and suggest that the dietary lignocellulose composition could cause shifting rapidly in the termite gut microbiota.

Hidden profiles and concealed information: strategic information sharing and use in group decision making.

PubMed

Toma, Claudia; Butera, Fabrizio

2009-06-01

Two experiments investigated the differential impact of cooperation and competition on strategic information sharing and use in a three-person group decision-making task. Information was distributed in order to create a hidden profile so that disconfirmation of group members' initial preferences was required to solve the task. Experiment 1 revealed that competition, compared to cooperation, led group members to withhold unshared information, a difference that was not significant for shared information. In competition, compared to cooperation, group members were also more reluctant to disconfirm their initial preferences. Decision quality was lower in competition than in cooperation, this effect being mediated by disconfirmation use and not by information sharing. Experiment 2 replicated these findings and revealed the role of mistrust in predicting strategic information sharing and use in competition. These results support a motivated information processing approach of group decision making.

Characterization, Microbial Community Structure, and Pathogen Occurrence in Urban Faucet Biofilms in South China

PubMed Central

Lin, Huirong; Zhang, Shuting; Gong, Song; Zhang, Shenghua; Yu, Xin

2015-01-01

The composition and microbial community structure of the drinking water system biofilms were investigated using microstructure analysis and 454 pyrosequencing technique in Xiamen city, southeast of China. SEM (scanning electron microscope) results showed different features of biofilm morphology in different fields of PVC pipe. Extracellular matrix material and sparse populations of bacteria (mainly rod-shaped and coccoid) were observed. CLSM (confocal laser scanning microscope) revealed different distributions of attached cells, extracellular proteins, α-polysaccharides, and β-polysaccharides. The biofilms had complex bacterial compositions. Differences in bacteria diversity and composition from different tap materials and ages were observed. Proteobacteria was the common and predominant group in all biofilms samples. Some potential pathogens (Legionellales, Enterobacteriales, Chromatiales, and Pseudomonadales) and corrosive microorganisms were also found in the biofilms. This study provides the information of characterization and visualization of the drinking water biofilms matrix, as well as the microbial community structure and opportunistic pathogens occurrence. PMID:26273617

Insights into the respiratory tract microbiota of patients with cystic fibrosis during early Pseudomonas aeruginosa colonization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keravec, Marlène; Mounier, Jérôme; Prestat, Emmanuel

Pseudomonas aeruginosa plays a major role in cystic fibrosis (CF) progression. Therefore, it is important to understand the initial steps of P. aeruginosa infection. The structure and dynamics of CF respiratory tract microbial communities during the early stages of P. aeruginosa colonization were characterized by pyrosequencing and cloning-sequencing. The respiratory microbiota showed high diversity, related to the young age of the CF cohort (mean age 10 years). Wide inter- and intra-individual variations were revealed. A common core microbiota of 5 phyla and 13 predominant genera was found, the majority of which were obligate anaerobes. A few genera were significantly moremore » prevalent in patients never infected by P. aeruginosa. Persistence of an anaerobic core microbiota regardless of P. aeruginosa status suggests a major role of certain anaerobes in the pathophysiology of lung infections in CF. Some genera may be potential biomarkers of pulmonary infection state.« less

Diversity of fungi associated with roots of Calanthe orchid species in Korea.

PubMed

Park, Myung Soo; Eimes, John A; Oh, Sang Hoon; Suh, Hwa Jung; Oh, Seung-Yoon; Lee, Seobihn; Park, Ki Hyeong; Kwon, Hyuk Joon; Kim, Soo-Young; Lim, Young Woon

2018-01-01

While symbiotic fungi play a key role in the growth of endangered Calanthe orchid species, the relationship between fungal diversity and Calanthe species remains unclear. Here, we surveyed root associated fungal diversity of six Calanthe orchid species by sequencing the internal transcribed spacer (ITS) region using 454 pyrosequencing. Our results revealed that Paraboeremia and Coprinopsis are dominant fungal genera among Calanthe species. In terms of overall relative abundance, Paraboeremia was the most common fungal genus associated with Calanthe roots, followed by Coprinopsis. Overall fungal diversity showed a significant degree of variation depending on both location and Calanthe species. In terms of number of different fungal genera detected within Calanthe species, C. discolor had the most diverse fungal community, with 10 fungal genera detected. This study will contribute toward a better understanding of those fungi that are required for successful cultivation and conservation of Korean Calanthe species.

Insights into the respiratory tract microbiota of patients with cystic fibrosis during early Pseudomonas aeruginosa colonization

DOE PAGES

Keravec, Marlène; Mounier, Jérôme; Prestat, Emmanuel; ...

2015-08-09

Pseudomonas aeruginosa plays a major role in cystic fibrosis (CF) progression. Therefore, it is important to understand the initial steps of P. aeruginosa infection. The structure and dynamics of CF respiratory tract microbial communities during the early stages of P. aeruginosa colonization were characterized by pyrosequencing and cloning-sequencing. The respiratory microbiota showed high diversity, related to the young age of the CF cohort (mean age 10 years). Wide inter- and intra-individual variations were revealed. A common core microbiota of 5 phyla and 13 predominant genera was found, the majority of which were obligate anaerobes. A few genera were significantly moremore » prevalent in patients never infected by P. aeruginosa. Persistence of an anaerobic core microbiota regardless of P. aeruginosa status suggests a major role of certain anaerobes in the pathophysiology of lung infections in CF. Some genera may be potential biomarkers of pulmonary infection state.« less

Biological support media influence the bacterial biofouling community in reverse osmosis water reclamation demonstration plants.

PubMed

Ferrera, Isabel; Mas, Jordi; Taberna, Elisenda; Sanz, Joan; Sánchez, Olga

2015-01-01

The diversity of the bacterial community developed in different stages of two reverse osmosis (RO) water reclamation demonstration plants designed in a wastewater treatment plant (WWTP) in Tarragona (Spain) was characterized by applying 454-pyrosequencing of the 16S rRNA gene. The plants were fed by secondary treated effluent to a conventional pretreatment train prior to the two-pass RO system. Plants differed in the material used in the filtration process, which was sand in one demonstration plant and Scandinavian schists in the second plant. The results showed the presence of a highly diverse and complex community in the biofilms, mainly composed of members of the Betaproteobacteria and Bacteroidetes in all stages, with the presence of some typical wastewater bacteria, suggesting a feed water origin. Community similarities analyses revealed that samples clustered according to filter type, highlighting the critical influence of the biological supporting medium in biofilm community structure.

Preliminary characterization of the oral microbiota of Chinese adults with and without gingivitis

PubMed Central

2011-01-01

Background Microbial communities inhabiting human mouth are associated with oral health and disease. Previous studies have indicated the general prevalence of adult gingivitis in China to be high. The aim of this study was to characterize in depth the oral microbiota of Chinese adults with or without gingivitis, by defining the microbial phylogenetic diversity and community-structure using highly paralleled pyrosequencing. Methods Six non-smoking Chinese, three with and three without gingivitis (age range 21-39 years, 4 females and 2 males) were enrolled in the present cross-sectional study. Gingival parameters of inflammation and bleeding on probing were characterized by a clinician using the Mazza Gingival Index (MGI). Plaque (sampled separately from four different oral sites) and salivary samples were obtained from each subject. Sequences and relative abundance of the bacterial 16 S rDNA PCR-amplicons were determined via pyrosequencing that produced 400 bp-long reads. The sequence data were analyzed via a computational pipeline customized for human oral microbiome analyses. Furthermore, the relative abundances of selected microbial groups were validated using quantitative PCR. Results The oral microbiomes from gingivitis and healthy subjects could be distinguished based on the distinct community structures of plaque microbiomes, but not the salivary microbiomes. Contributions of community members to community structure divergence were statistically accessed at the phylum, genus and species-like levels. Eight predominant taxa were found associated with gingivitis: TM7, Leptotrichia, Selenomonas, Streptococcus, Veillonella, Prevotella, Lautropia, and Haemophilus. Furthermore, 98 species-level OTUs were identified to be gingivitis-associated, which provided microbial features of gingivitis at a species resolution. Finally, for the two selected genera Streptococcus and Fusobacterium, Real-Time PCR based quantification of relative bacterial abundance validated the pyrosequencing-based results. Conclusions This methods study suggests that oral samples from this patient population of gingivitis can be characterized via plaque microbiome by pyrosequencing the 16 S rDNA genes. Further studies that characterize serial samples from subjects (longitudinal study design) with a larger population size may provide insight into the temporal and ecological features of oral microbial communities in clinically-defined states of gingivitis. PMID:22152152

Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

PubMed

Arellano, Shawn M; Lee, On On; Lafi, Feras F; Yang, Jiangke; Wang, Yong; Young, Craig M; Qian, Pei-Yuan

2013-02-01

The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

Maternal Bias and Escape from X Chromosome Imprinting in the Midgestation Mouse Placenta

PubMed Central

Finn, Elizabeth H; Smith, Cheryl L; Rodriguez, Jesse; Sidow, Arend; Baker, Julie C

2014-01-01

To investigate the epigenetic landscape at the interface between mother and fetus, we provide a comprehensive analysis of parent-of-origin bias in the mouse placenta. Using F1 interspecies hybrids between mus musculus (C57BL/6J) and mus musculus castaneus, we sequenced RNA from 23 individual midgestation placentas, five late stage placentas, and two yolk sac samples and then used SNPs to determine whether transcripts were preferentially generated from the maternal or paternal allele. In the placenta, we find 103 genes that show significant and reproducible parent-of-origin bias, of which 78 are novel candidates. Most (96%) show a strong maternal bias which we demonstrate, via multiple mathematical models, pyrosequencing, and FISH, is not due to maternal decidual contamination. Analysis of the X chromosome also reveals paternal expression of Xist and several genes that escape inactivation, most significantly Alas2, Fhl1, and Slc38a5. Finally, sequencing individual placentas allowed us to reveal notable expression similarity between littermates. In all, we observe a striking preference for maternal transcription in the midgestation mouse placenta and a dynamic imprinting landscape in extraembryonic tissues, reflecting the complex nature of epigenetic pathways in the placenta. PMID:24594094

Pyrosequencing analysis of the microbiota of kusaya gravy obtained from Izu Islands.

PubMed

Fujii, Tateo; Kyoui, Daisuke; Takahashi, Hajime; Kuda, Takashi; Kimura, Bon; Washizu, Yukio; Emoto, Eiji; Hiramoto, Tadahiro

2016-12-05

Kusaya is a salted, dried fish product traditionally produced on the Izu Islands in Japan. Fish are added to kusaya gravy repeatedly and intermittently, and used over several hundred years, which makes unique microbiota and unique flavors. In this study, we performed a metagenomic analysis to compare the composition of the microbiota of kusaya gravy between different islands. Twenty samples obtained from a total of 13 manufacturers on three islands (Hachijojima, Niijima, and Oshima Islands) were analyzed. The statistical analysis revealed that the microbiota in kusaya gravy maintain a stable composition regardless of the production steps, and that the microbiota are characteristic to the particular islands. The bacterial taxa common to all of the samples were not necessarily the dominant ones. On the other hand, the genera Halanaerobium and Tissierella were found to be characteristic to the microbiota of one or two islands. Because these genera are known to be present in the natural environment, it is likely that the bacterial strains peculiar to an island had colonized kusaya gravy for many years. The results of this study revealed an influence of geographical conditions on the microbiota in fermented food. Copyright © 2016 Elsevier B.V. All rights reserved.

Potential for direct interspecies electron transfer in an electric-anaerobic system to increase methane production from sludge digestion.

PubMed

Zhao, Zhiqiang; Zhang, Yaobin; Wang, Liying; Quan, Xie

2015-06-09

Direct interspecies electron transfer (DIET) between Geobacter species and Methanosaeta species is an alternative to interspecies hydrogen transfer (IHT) in anaerobic digester, which however has not been established in anaerobic sludge digestion as well as in bioelectrochemical systems yet. In this study, it was found that over 50% of methane production of an electric-anaerobic sludge digester was resulted from unknown pathway. Pyrosequencing analysis revealed that Geobacter species were significantly enriched with electrodes. Fluorescence in situ hybridization (FISH) further confirmed that the dominant Geobacter species enriched belonged to Geobacter metallireducens. Together with Methanosaeta species prevailing in the microbial communities, the direct electron exchange between Geobacter species and Methanosaeta species might be an important reason for the "unknown" increase of methane production. Conductivity of the sludge in this electric-anaerobic digester was about 30% higher than that of the sludge in a control digester without electrodes. This study not only revealed for the first time that DIET might be the important mechanism on the methanogenesis of bioelectrochemical system, but also provided a new method to enhance DIET by means of bioelectric enrichment of Geobacter species.

Potential for direct interspecies electron transfer in an electric-anaerobic system to increase methane production from sludge digestion

PubMed Central

Zhao, Zhiqiang; Zhang, Yaobin; Wang, Liying; Quan, Xie

2015-01-01

Direct interspecies electron transfer (DIET) between Geobacter species and Methanosaeta species is an alternative to interspecies hydrogen transfer (IHT) in anaerobic digester, which however has not been established in anaerobic sludge digestion as well as in bioelectrochemical systems yet. In this study, it was found that over 50% of methane production of an electric-anaerobic sludge digester was resulted from unknown pathway. Pyrosequencing analysis revealed that Geobacter species were significantly enriched with electrodes. Fluorescence in situ hybridization (FISH) further confirmed that the dominant Geobacter species enriched belonged to Geobacter metallireducens. Together with Methanosaeta species prevailing in the microbial communities, the direct electron exchange between Geobacter species and Methanosaeta species might be an important reason for the “unknown” increase of methane production. Conductivity of the sludge in this electric-anaerobic digester was about 30% higher than that of the sludge in a control digester without electrodes. This study not only revealed for the first time that DIET might be the important mechanism on the methanogenesis of bioelectrochemical system, but also provided a new method to enhance DIET by means of bioelectric enrichment of Geobacter species. PMID:26057581

Seasonal Variations and Resilience of Bacterial Communities in a Sewage Polluted Urban River

PubMed Central

Ouattara, Nouho Koffi; Anzil, Adriana; Verbanck, Michel A.; Brion, Natacha; Servais, Pierre

2014-01-01

The Zenne River in Brussels (Belgium) and effluents of the two wastewater treatment plants (WWTPs) of Brussels were chosen to assess the impact of disturbance on bacterial community composition (BCC) of an urban river. Organic matters, nutrients load and oxygen concentration fluctuated highly along the river and over time because of WWTPs discharge. Tag pyrosequencing of bacterial 16S rRNA genes revealed the significant effect of seasonality on the richness, the bacterial diversity (Shannon index) and BCC. The major grouping: -winter/fall samples versus spring/summer samples- could be associated with fluctuations of in situ bacterial activities (dissolved and particulate organic carbon biodegradation associated with oxygen consumption and N transformation). BCC of the samples collected upstream from the WWTPs discharge were significantly different from BCC of downstream samples and WWTPs effluents, while no significant difference was found between BCC of WWTPs effluents and the downstream samples as revealed by ANOSIM. Analysis per season showed that allochthonous bacteria brought by WWTPs effluents triggered the changes in community composition, eventually followed by rapid post-disturbance return to the original composition as observed in April (resilience), whereas community composition remained altered after the perturbation by WWTPs effluents in the other seasons. PMID:24667680

Low expression of N-myc downstream-regulated gene 2 (NDRG2) correlates with poor prognosis in hepatoblastoma.

PubMed

Gödeke, Jan; Luxenburger, Elke; Trippel, Franziska; Becker, Kristina; Häberle, Beate; Müller-Höcker, Josef; von Schweinitz, Dietrich; Kappler, Roland

2016-03-01

Despite tremendous progress in therapy, about 30% of patients with hepatoblastoma still succumb to the disease. Thus, the development of improved therapies as well as the identification of prognostic factors are urgently needed. In the present study, expression and promoter methylation of the N-myc downstream-regulated gene (NDRG2), a tumor suppressor gene contributing to the regulation of the Wnt signalling pathway, was analysed in 38 hepatoblastoma samples by real-time reverse transcription-PCR and pyrosequencing, respectively. The NDRG2 gene was highly expressed in normal pediatric liver tissue, but was significantly downregulated in heptoblastoma primary tumors. Detailed methylation analysis of CpG sites in the NDRG2 promoter region revealed a general high degree of DNA methylation in hepatoblastoma, which correlated with the suppression of NDRG2. By analyzing clinicopathological features we could demonstrate a strong association between low NDRG2 expression and tumor metastasis. Importantly, the overall survival analysis by Kaplan-Meier revealed that high NDRG2 expression was correlated with a higher survival rate in hepatoblastoma patients. Our data show that downregulation of NDRG2 may play an important role in advanced hepatoblastomas.

Coupled cryoconite ecosystem structure-function relationships are revealed by comparing bacterial communities in alpine and Arctic glaciers.

PubMed

Edwards, Arwyn; Mur, Luis A J; Girdwood, Susan E; Anesio, Alexandre M; Stibal, Marek; Rassner, Sara M E; Hell, Katherina; Pachebat, Justin A; Post, Barbara; Bussell, Jennifer S; Cameron, Simon J S; Griffith, Gareth Wyn; Hodson, Andrew J; Sattler, Birgit

2014-08-01

Cryoconite holes are known as foci of microbial diversity and activity on polar glacier surfaces, but are virtually unexplored microbial habitats in alpine regions. In addition, whether cryoconite community structure reflects ecosystem functionality is poorly understood. Terminal restriction fragment length polymorphism and Fourier transform infrared metabolite fingerprinting of cryoconite from glaciers in Austria, Greenland and Svalbard demonstrated cryoconite bacterial communities are closely correlated with cognate metabolite fingerprints. The influence of bacterial-associated fatty acids and polysaccharides was inferred, underlining the importance of bacterial community structure in the properties of cryoconite. Thus, combined application of T-RFLP and FT-IR metabolite fingerprinting promises high throughput, and hence, rapid assessment of community structure-function relationships. Pyrosequencing revealed Proteobacteria were particularly abundant, with Cyanobacteria likely acting as ecosystem engineers in both alpine and Arctic cryoconite communities. However, despite these generalities, significant differences in bacterial community structures, compositions and metabolomes are found between alpine and Arctic cryoconite habitats, reflecting the impact of local and regional conditions on the challenges of thriving in glacial ecosystems. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Egypt's Red Sea coast: phylogenetic analysis of cultured microbial consortia in industrialized sites.

PubMed

Mustafa, Ghada A; Abd-Elgawad, Amr; Abdel-Haleem, Alyaa M; Siam, Rania

2014-01-01

The Red Sea possesses a unique geography, and its shores are rich in mangrove, macro-algal and coral reef ecosystems. Various sources of pollution affect Red Sea biota, including microbial life. We assessed the effects of industrialization on microbes along the Egyptian Red Sea coast at eight coastal sites and two lakes. The bacterial communities of sediment samples were analyzed using bacterial 16S rDNA pyrosequencing of V6-V4 hypervariable regions. The taxonomic assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled sites: Proteobacteria (68%), Firmicutes (13%), Fusobacteria (12%), Bacteriodetes (6%), and Spirochetes (0.03%). Further analysis revealed distinct bacterial consortia that primarily included (1) marine Vibrio spp.-suggesting a "marine Vibrio phenomenon"; (2) potential human pathogens; and (3) oil-degrading bacteria. We discuss two divergent microbial consortia that were sampled from Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; these consortia contained the highest abundance of human pathogens and no pathogens, respectively. Our results draw attention to the effects of industrialization on the Red Sea and suggest the need for further analysis to overcome the hazardous effects observed at the impacted sites.

Transformation of chlorpyrifos in integrated recirculating constructed wetlands (IRCWs) as revealed by compound-specific stable isotope (CSIA) and microbial community structure analysis.

PubMed

Tang, Xiaoyan; Yang, Yang; Huang, Wenda; McBride, Murray B; Guo, Jingjing; Tao, Ran; Dai, Yunv

2017-06-01

Carbon isotope analysis and 454 pyrosequencing methods were used to investigate in situ biodegradation of chlorpyrifos during its transport through three model integrated recirculating constructed wetlands (IRCWs). Results show that plant and Fe-impregnated biochar promoted degradation of chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP). Carbon isotope ratios in the IRCWs shifted to -31.24±0.58‰ (IRCW1, plant free), -26.82±0.60‰ (IRCW2, with plant) and -24.76±0.94‰ (IRCW3, with plant and Fe-biochar). The enrichment factors (Ɛ bulk,c ) were determined as -0.69±0.06‰ (IRCW1), -0.91±0.07‰ (IRCW2) and -1.03±0.09‰ (IRCW3). Microbial community analysis showed that IRCW3 was dominated by members of Bacillus, which can utilize and degrade chlorpyrifos. These results reveal that plant and Fe-biochar can induce carbon isotope fractionation and have a positive impact on the chlorpyrifos degradation efficiency by influencing the development of beneficial microbial communities. Copyright © 2017. Published by Elsevier Ltd.

FT-IR spectroscopy: A powerful tool for studying the inter- and intraspecific biodiversity of cultivable non-Saccharomyces yeasts isolated from grape must.

PubMed

Grangeteau, Cédric; Gerhards, Daniel; Terrat, Sebastien; Dequiedt, Samuel; Alexandre, Hervé; Guilloux-Benatier, Michèle; von Wallbrunn, Christian; Rousseaux, Sandrine

2016-02-01

The efficiency of the FT-IR technique for studying the inter- and intra biodiversity of cultivable non-Saccharomyces yeasts (NS) present in different must samples was examined. In first, the capacity of the technique FT-IR to study the global diversity of a given sample was compared to the pyrosequencing method, used as a reference technique. Seven different genera (Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Issatchenkia, Metschnikowia and Pichia) were identified by FT-IR and also by pyrosequencing. Thirty-eight other genera were identified by pyrosequencing, but together they represented less than 6% of the average total population of 6 musts. Among the species identified, some of them present organoleptic potentials in winemaking, particularly Starmerella bacillaris (synonym Candidazemplinina). So in a second time, we evaluated the capacity of the FT-IR technique to discriminate the isolates of this species because few techniques were able to study intraspecific NS yeast biodiversity. The results obtained were validated by using a classic method as ITS sequencing. Biodiversity at strain level was high: 19 different strains were identified from 58 isolates. So, FT-IR spectroscopy seems to be an accurate and reliable method for identifying major genera present in the musts. The two biggest advantages of the FT-IR are the capacity to characterize intraspecific biodiversity of non-Saccharomyces yeasts and the possibility to discriminate a lot of strains. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid quantitation of neuraminidase inhibitor drug resistance in influenza virus quasispecies.

PubMed

Lackenby, Angie; Democratis, Jane; Siqueira, Marilda M; Zambon, Maria C

2008-01-01

Emerging resistance of influenza viruses to neuraminidase inhibitors is a concern, both in surveillance of global circulating strains and in treatment of individual patients. Current methodologies to detect resistance rely on the use of cultured virus, thus taking time to complete or lacking the sensitivity to detect mutations in viral quasispecies. Methodology for rapid detection of clinically meaningful resistance is needed to assist individual patient management and to track the transmission of resistant viruses in the community. We have developed a pyrosequencing methodology to detect and quantitate influenza neuraminidase inhibitor resistance mutations in cultured virus and directly in clinical material. Our assays target polymorphisms associated with drug resistance in the neuraminidase genes of human influenza A H1N1 as well as human and avian H5N1 viruses. Quantitation can be achieved using viral RNA extracted directly from respiratory or tissue samples, thus eliminating the need for virus culture and allowing the assay of highly pathogenic viruses such as H5N1 without high containment laboratory facilities. Antiviral-resistant quasispecies are detected and quantitated accurately when present in the total virus population at levels as low as 10%. Pyrosequencing is a real-time assay; therefore, results can be obtained within a clinically relevant timeframe and provide information capable of informing individual patient or outbreak management. Pyrosequencing is ideally suited for early identification of emerging antiviral resistance in human and avian influenza infection and is a useful tool for laboratory surveillance and pandemic preparedness.

Impact of subacute ruminal acidosis (SARA) adaptation on rumen microbiota in dairy cattle using pyrosequencing.

PubMed

Mao, S Y; Zhang, R Y; Wang, D S; Zhu, W Y

2013-12-01

The objective of this study was to evaluate the changes in bacterial populations in the rumen of dairy cattle following adaptation to subacute ruminal acidosis (SARA) using 16S rRNA gene pyrosequencing. Rumen contents were collected from four cattle adapted to either a 40% (control diet, COD) or 70% (SARA induction diet, SAID) concentrate feeds. DNA was extracted from each of the samples. Bacterial 16S rRNA genes of ruminal DNA extracts were PCR amplified with 2 bar coded primer sets and sequenced by 454 pyrosequencing. At a high taxonomic level, the percentage of Proteobacteria and Bacteroidetes were reduced by SAID feeding, whereas Firmicutes and Actinobacteria were more abundant in the SAID than in the COD group. At the genus level, as compared with the COD group, the abundances of Prevotella, Treponema, Anaeroplasma, Papillibacter, Acinetobacter and unclassified populations including unclassified Lentisphaerae, and unclassified bacteria were lower (P < 0.05), while the percentages of Ruminococcus, Atopobium, unclassified Clostridiales and Bifidobacterium were increased (P < 0.05) in the SAID group. Feeding of SAID reduced (P < 0.001) the diversity of the rumen microbial community. Taken together, our findings provide a comprehensive picture of current knowledge of the community structure of the rumen bacterial ecosystem during SARA, and enhance our understanding about the ruminal microbial ecology that may be useful in the prevention of ruminal acidosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparisons of the fungal and protistan communities among different marine sponge holobionts by pyrosequencing.

PubMed

He, Liming; Liu, Fang; Karuppiah, Valliappan; Ren, Yi; Li, Zhiyong

2014-05-01

To date, the knowledge of eukaryotic communities associated with sponges remains limited compared with prokaryotic communities. In a manner similar to prokaryotes, it could be hypothesized that sponge holobionts have phylogenetically diverse eukaryotic symbionts, and the eukaryotic community structures in different sponge holobionts were probably different. In order to test this hypothesis, the communities of eukaryota associated with 11 species of South China Sea sponges were compared with the V4 region of 18S ribosomal ribonucleic acid gene using 454 pyrosequencing. Consequently, 135 and 721 unique operational taxonomic units (OTUs) of fungi and protists were obtained at 97 % sequence similarity, respectively. These sequences were assigned to 2 phyla of fungi (Ascomycota and Basidiomycota) and 9 phyla of protists including 5 algal phyla (Chlorophyta, Haptophyta, Streptophyta, Rhodophyta, and Stramenopiles) and 4 protozoal phyla (Alveolata, Cercozoa, Haplosporidia, and Radiolaria) including 47 orders (12 fungi, 35 protists). Entorrhizales of fungi and 18 orders of protists were detected in marine sponges for the first time. Particularly, Tilletiales of fungi and Chlorocystidales of protists were detected for the first time in marine habitats. Though Ascomycota, Alveolata, and Radiolaria were detected in all the 11 sponge species, sponge holobionts have different fungi and protistan communities according to OTU comparison and principal component analysis at the order level. This study provided the first insights into the fungal and protistan communities associated with different marine sponge holobionts using pyrosequencing, thus further extending the knowledge on sponge-associated eukaryotic diversity.

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay.

PubMed

Lee, Sung Hak; Chung, Arthur Minwoo; Lee, Ahwon; Oh, Woo Jin; Choi, Yeong Jin; Lee, Youn-Soo; Jung, Eun Sun

2017-01-01

Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti-epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment. We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing. KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing-454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%. The cobas test is an accurate and sensitive test for detecting KRAS -activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC.

Diversity patterns and activity of uncultured marine heterotrophic flagellates unveiled with pyrosequencing

PubMed Central

Logares, Ramiro; Audic, Stephane; Santini, Sebastien; Pernice, Massimo C; de Vargas, Colomban; Massana, Ramon

2012-01-01

Flagellated heterotrophic microeukaryotes have key roles for the functioning of marine ecosystems as they channel large amounts of organic carbon to the upper trophic levels and control the population sizes of bacteria and archaea. Still, we know very little on the diversity patterns of most groups constituting this evolutionary heterogeneous assemblage. Here, we investigate 11 groups of uncultured flagellates known as MArine STramenopiles (MASTs). MASTs are ecologically very important and branch at the base of stramenopiles. We explored the diversity patterns of MASTs using pyrosequencing (18S rDNA) in coastal European waters. We found that MAST groups range from highly to lowly diversified. Pyrosequencing (hereafter ‘454') allowed us to approach to the limits of taxonomic diversity for all MAST groups, which varied in one order of magnitude (tens to hundreds) in terms of operational taxonomic units (98% similarity). We did not evidence large differences in activity, as indicated by ratios of DNA:RNA-reads. Most groups were strictly planktonic, although we found some groups that were active in sediments and even in anoxic waters. The proportion of reads per size fraction indicated that most groups were composed of very small cells (∼2–5 μm). In addition, phylogenetically different assemblages appeared to be present in different size fractions, depths and geographic zones. Thus, MAST diversity seems to be highly partitioned in spatial scales. Altogether, our results shed light on these ecologically very important but poorly known groups of uncultured marine flagellates. PMID:22534609

Pyrosequencing of prey DNA in reptile faeces: analysis of earthworm consumption by slow worms.

PubMed

Brown, David S; Jarman, Simon N; Symondson, William O C

2012-03-01

Little quantitative ecological information exists on the diets of most invertebrate feeding reptiles, particularly nocturnal or elusive species that are difficult to observe. In the UK and elsewhere, reptiles are legally required to be relocated before land development can proceed, but without knowledge of their dietary requirements, the suitability of receptor sites cannot be known. Here, we tested the ability of non-invasive DNA-based molecular diagnostics (454 pyrosequencing) to analyse reptile diets, with the specific aims of determining which earthworm species are exploited by slow worms (the legless lizard Anguis fragilis) and whether they feed on the deeper-living earthworm species that only come to the surface at night. Slow worm faecal samples from four different habitats were analysed using earthworm-specific PCR primers. We found that 86% of slow worms (N=80) had eaten earthworms. In lowland heath and marshy/acid grassland, Lumbricus rubellus, a surface-dwelling epigeic species, dominated slow worm diet. In two other habitats, riverside pasture and calciferous coarse grassland, diet was dominated by deeper-living anecic and endogeic species. We conclude that all species of earthworm are exploited by these reptiles and lack of specialization allows slow worms to thrive in a wide variety of habitats. Pyrosequencing of prey DNA in faeces showed promise as a practical, rapid and relatively inexpensive means of obtaining detailed and valuable ecological information on the diets of reptiles. © 2011 Blackwell Publishing Ltd.

Massively parallel pyrosequencing-based transcriptome analyses of small brown planthopper (Laodelphax striatellus), a vector insect transmitting rice stripe virus (RSV)

PubMed Central

2010-01-01

Background The small brown planthopper (Laodelphax striatellus) is an important agricultural pest that not only damages rice plants by sap-sucking, but also acts as a vector that transmits rice stripe virus (RSV), which can cause even more serious yield loss. Despite being a model organism for studying entomology, population biology, plant protection, molecular interactions among plants, viruses and insects, only a few genomic sequences are available for this species. To investigate its transcriptome and determine the differences between viruliferous and naïve L. striatellus, we employed 454-FLX high-throughput pyrosequencing to generate EST databases of this insect. Results We obtained 201,281 and 218,681 high-quality reads from viruliferous and naïve L. striatellus, respectively, with an average read length as 230 bp. These reads were assembled into contigs and two EST databases were generated. When all reads were combined, 16,885 contigs and 24,607 singletons (a total of 41,492 unigenes) were obtained, which represents a transcriptome of the insect. BlastX search against the NCBI-NR database revealed that only 6,873 (16.6%) of these unigenes have significant matches. Comparison of the distribution of GO classification among viruliferous, naïve, and combined EST databases indicated that these libraries are broadly representative of the L. striatellus transcriptomes. Functionally diverse transcripts from RSV, endosymbiotic bacteria Wolbachia and yeast-like symbiotes were identified, which reflects the possible lifestyles of these microbial symbionts that live in the cells of the host insect. Comparative genomic analysis revealed that L. striatellus encodes similar innate immunity regulatory systems as other insects, such as RNA interference, JAK/STAT and partial Imd cascades, which might be involved in defense against viral infection. In addition, we determined the differences in gene expression between vector and naïve samples, which generated a list of candidate genes that are potentially involved in the symbiosis of L. striatellus and RSV. Conclusions To our knowledge, the present study is the first description of a genomic project for L. striatellus. The identification of transcripts from RSV, Wolbachia, yeast-like symbiotes and genes abundantly expressed in viruliferous insect, provided a starting-point for investigating the molecular basis of symbiosis among these organisms. PMID:20462456

Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak

PubMed Central

2010-01-01

Background The Fagaceae family comprises about 1,000 woody species worldwide. About half belong to the Quercus family. These oaks are often a source of raw material for biomass wood and fiber. Pedunculate and sessile oaks, are among the most important deciduous forest tree species in Europe. Despite their ecological and economical importance, very few genomic resources have yet been generated for these species. Here, we describe the development of an EST catalogue that will support ecosystem genomics studies, where geneticists, ecophysiologists, molecular biologists and ecologists join their efforts for understanding, monitoring and predicting functional genetic diversity. Results We generated 145,827 sequence reads from 20 cDNA libraries using the Sanger method. Unexploitable chromatograms and quality checking lead us to eliminate 19,941 sequences. Finally a total of 125,925 ESTs were retained from 111,361 cDNA clones. Pyrosequencing was also conducted for 14 libraries, generating 1,948,579 reads, from which 370,566 sequences (19.0%) were eliminated, resulting in 1,578,192 sequences. Following clustering and assembly using TGICL pipeline, 1,704,117 EST sequences collapsed into 69,154 tentative contigs and 153,517 singletons, providing 222,671 non-redundant sequences (including alternative transcripts). We also assembled the sequences using MIRA and PartiGene software and compared the three unigene sets. Gene ontology annotation was then assigned to 29,303 unigene elements. Blast search against the SWISS-PROT database revealed putative homologs for 32,810 (14.7%) unigene elements, but more extensive search with Pfam, Refseq_protein, Refseq_RNA and eight gene indices revealed homology for 67.4% of them. The EST catalogue was examined for putative homologs of candidate genes involved in bud phenology, cuticle formation, phenylpropanoids biosynthesis and cell wall formation. Our results suggest a good coverage of genes involved in these traits. Comparative orthologous sequences (COS) with other plant gene models were identified and allow to unravel the oak paleo-history. Simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were searched, resulting in 52,834 SSRs and 36,411 SNPs. All of these are available through the Oak Contig Browser http://genotoul-contigbrowser.toulouse.inra.fr:9092/Quercus_robur/index.html. Conclusions This genomic resource provides a unique tool to discover genes of interest, study the oak transcriptome, and develop new markers to investigate functional diversity in natural populations. PMID:21092232

Pyrosequencing Reveals the Predominance of Pseudomonadaceae in Gut Microbiome of a Gall Midge

PubMed Central

Bansal, Raman; Hulbert, Scot H.; Reese, John C.; Whitworth, Robert J.; Stuart, Jeffrey J.; Chen, Ming-Shun

2014-01-01

Gut microbes are known to play various roles in insects such as digestion of inaccessible nutrients, synthesis of deficient amino acids, and interaction with ecological environments, including host plants. Here, we analyzed the gut microbiome in Hessian fly, a serious pest of wheat. A total of 3,654 high quality sequences of the V3 hypervariable region of the 16S rRNA gene were obtained through 454-pyrosequencing. From these sequences, 311 operational taxonomic units (OTUs) were obtained at the ≥97% similarity cutoff. In the gut of 1st instar, otu01, a member of Pseudomonas, was predominant, representing 90.2% of total sequences. otu13, an unidentified genus in the Pseudomonadaceae family, represented 1.9% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 2nd instar, otu01 and otu13 decreased to 85.5% and 1.5%, respectively. otu04, a member of Buttiauxella, represented 9.7% of total sequences. The remaining OTUs were each less than 1%. In the gut of the 3rd instar, otu01 and otu13 further decreased to 29.0% and 0%, respectively. otu06, otu08, and otu16, also three members of the Pseudomonadaceae family were 13.2%, 8.6%, and 2.3%, respectively. In addition, otu04 and otu14, two members of the Enterobacteriaceae family, were 4.7% and 2.5%; otu18 and otu20, two members of the Xanthomonadaceae family, were 1.3% and 1.2%, respectively; otu12, a member of Achromobacter, was 4.2%; otu19, a member of Undibacterium, was 1.4%; and otu9, otu10, and otu15, members of various families, were 6.1%, 6.3%, and 1.9%, respectively. The investigation into dynamics of Pseudomonas, the most abundant genera, revealed that its population level was at peak in freshly hatched or 1 day larvae as well as in later developmental stages, thus suggesting a prominent role for this bacterium in Hessian fly development and in its interaction with host plants. This study is the first comprehensive survey on bacteria associated with the gut of a gall midge, and provides a foundation for future studies to elucidate the roles of gut microbes in Hessian fly virulence and biology. PMID:25437809

Knowledge-Sharing Intention among Information Professionals in Nigeria: A Statistical Analysis

ERIC Educational Resources Information Center

Tella, Adeyinka

2016-01-01

In this study, the researcher administered a survey and developed and tested a statistical model to examine the factors that determine the intention of information professionals in Nigeria to share knowledge with their colleagues. The result revealed correlations between the overall score for intending to share knowledge and other…

Biodiversity of Trichoderma Community in the Tidal Flats and Wetland of Southeastern China

PubMed Central

Saravanakumar, Kandasamy; Yu, Chuanjin; Dou, Kai; Wang, Meng; Li, Yaqian; Chen, Jie

2016-01-01

To investigate the biodiversity of Trichoderma (Hypocreaceae) and their relation to sediment physical and chemical properties, we collected a total of 491 sediment samples from coastal wetlands (tidal flat and wetland) in Southeast China. Further, we applied two types of molecular approaches such as culture dependent and independent methods for identification of Trichoderma spp. A total of 254 isolates were obtained and identified to 13 species such as T. aureoviride, T. asperellum, T. harzianum, T. atroviride, T. koningiopsis, T. longibrachiatum, T. koningii. T. tawa, T. viridescens, T. virens, T. hamatum, T. viride, and T. velutinum by the culture-dependent (CD) method of these, T. tawa was newly described in China. Subsequently, the culture indepented method of 454 pyrosequencing analysis revealed a total of six species such as T. citrinoviride, T. virens, T. polysporum, T. harzianum/Hypocrea lixii and two unknown species. Notably, T. citrinoviride and T. polysporum were not found by the CD method. Therefore, this work revealed that the combination of these two methods could show the higher biodiversity of Trichoderma spp., than either of this method alone. Among the sampling sites, Hangzhou, Zhejiang Province, exhibited rich biodiversity and low in Fengxian. Correlation and Redundancy discriminant analysis (RDA) revealed that sediment properties of temperature, redox potential (Eh) and pH significantly influenced the biodiversity of Trichoderma spp. PMID:28002436

Pyrosequencing reveals the microbial communities in the Red Sea sponge Carteriospongia foliascens and their impressive shifts in abnormal tissues.

PubMed

Gao, Zhao-Ming; Wang, Yong; Lee, On On; Tian, Ren-Mao; Wong, Yue Him; Bougouffa, Salim; Batang, Zenon; Al-Suwailem, Abdulaziz; Lafi, Feras F; Bajic, Vladimir B; Qian, Pei-Yuan

2014-10-01

Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species "Candidatus Synechococcus spongiarum," were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.

The microbiome of Brazilian mangrove sediments as revealed by metagenomics.

PubMed

Andreote, Fernando Dini; Jiménez, Diego Javier; Chaves, Diego; Dias, Armando Cavalcante Franco; Luvizotto, Danice Mazzer; Dini-Andreote, Francisco; Fasanella, Cristiane Cipola; Lopez, Maryeimy Varon; Baena, Sandra; Taketani, Rodrigo Gouvêa; de Melo, Itamar Soares

2012-01-01

Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H(2)S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments.

The Microbiome of Brazilian Mangrove Sediments as Revealed by Metagenomics

PubMed Central

Andreote, Fernando Dini; Jiménez, Diego Javier; Chaves, Diego; Dias, Armando Cavalcante Franco; Luvizotto, Danice Mazzer; Dini-Andreote, Francisco; Fasanella, Cristiane Cipola; Lopez, Maryeimy Varon; Baena, Sandra; Taketani, Rodrigo Gouvêa; de Melo, Itamar Soares

2012-01-01

Here we embark in a deep metagenomic survey that revealed the taxonomic and potential metabolic pathways aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H2S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments. PMID:22737213

Molecular assessment of the fecal microbiota in healthy cats and dogs before and during supplementation with fructo-oligosaccharides (FOS) and inulin using high-throughput 454-pyrosequencing

PubMed Central

Barcenas-Walls, Jose R.; Suchodolski, Jan S.; Steiner, Jörg M.

2017-01-01

Prebiotics are selectively fermentable dietary compounds that result in changes in the composition and/or activity of the intestinal microbiota, thus conferring benefits upon host health. In veterinary medicine, commercially available products containing prebiotics have not been well studied with regard to the changes they trigger on the composition of the gut microbiota. This study evaluated the effect of a commercially available nutraceutical containing fructo-oligosaccharides (FOS) and inulin on the fecal microbiota of healthy cats and dogs when administered for 16 days. Fecal samples were collected at two time points before and at two time points during prebiotic administration. Total genomic DNA was obtained from fecal samples and 454-pyrosequencing was used for 16S rRNA gene bacterial profiling. The linear discriminant analysis (LDA) effect size (LEfSe) method was used for detecting bacterial taxa that may respond (i.e., increase or decrease in its relative abundance) to prebiotic administration. Prebiotic administration was associated with a good acceptance and no side effects (e.g., diarrhea) were reported by the owners. A low dose of prebiotics (50 mL total regardless of body weight with the end product containing 0.45% of prebiotics) revealed a lower abundance of Gammaproteobacteria and a higher abundance of Veillonellaceae during prebiotic administration in cats, while Staphylococcaceae showed a higher abundance during prebiotic administration in dogs. These differences were not sufficient to separate bacterial communities as shown by analysis of weighted UniFrac distance metrics. A predictive approach of the fecal bacterial metagenome using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) also did not reveal differences between the period before and during prebiotic administration. A second trial using a higher dose of prebiotics (3.2 mL/kg body weight with the end product containing 3.1% of prebiotics) was tested in dogs and revealed a lower abundance of Dorea (family Clostridiaceae) and a higher abundance of Megamonas and other (unknown) members of Veillonellaceae during prebiotic administration. Again, these changes were not sufficient to separate bacterial communities or predicted metabolic profiles according to treatment. A closer analysis of bacterial communities at all time-points revealed highly individualized patterns of variation. This study shows a high interindividual variation of fecal bacterial communities from pet cats and dogs, that these communities are relatively stable over time, and that some of this variation can be attributable to prebiotic administration, a phenomenon that may be affected by the amount of the prebiotic administered in the formulation. This study also provides insights into the response of gut bacterial communities in pet cats and dogs during administration of commercially available products containing prebiotics. More studies are needed to explore potentially beneficial effects on host health beyond changes in bacterial communities. PMID:28439463

Data sharing for public health research: A qualitative study of industry and academia.

PubMed

Saunders, Pamela A; Wilhelm, Erin E; Lee, Sinae; Merkhofer, Elizabeth; Shoulson, Ira

2014-01-01

Data sharing is a key biomedical research theme for the 21st century. Biomedical data sharing is the exchange of data among (non)affiliated parties under mutually agreeable terms to promote scientific advancement and the development of safe and effective medical products. Wide sharing of research data is important for scientific discovery, medical product development, and public health. Data sharing enables improvements in development of medical products, more attention to rare diseases, and cost-efficiencies in biomedical research. We interviewed 11 participants about their attitudes and beliefs about data sharing. Using a qualitative, thematic analysis approach, our analysis revealed a number of themes including: experiences, approaches, perceived challenges, and opportunities for sharing data.

Problem-Solving Test: Pyrosequencing

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2013-01-01

Terms to be familiar with before you start to solve the test: Maxam-Gilbert sequencing, Sanger sequencing, gel electrophoresis, DNA synthesis reaction, polymerase chain reaction, template, primer, DNA polymerase, deoxyribonucleoside triphosphates, orthophosphate, pyrophosphate, nucleoside monophosphates, luminescence, acid anhydride bond,…

Fluorogenic DNA Sequencing in PDMS Microreactors

PubMed Central

Sims, Peter A.; Greenleaf, William J.; Duan, Haifeng; Xie, X. Sunney

2012-01-01

We have developed a multiplex sequencing-by-synthesis method combining terminal-phosphate labeled fluorogenic nucleotides (TPLFNs) and resealable microreactors. In the presence of phosphatase, the incorporation of a non-fluorescent TPLFN into a DNA primer by DNA polymerase results in a fluorophore. We immobilize DNA templates within polydimethylsiloxane (PDMS) microreactors, sequentially introduce one of the four identically labeled TPLFNs, seal the microreactors, allow template-directed TPLFN incorporation, and measure the signal from the fluorophores trapped in the microreactors. This workflow allows sequencing in a manner akin to pyrosequencing but without constant monitoring of each microreactor. With cycle times of <10 minutes, we demonstrate 30 base reads with ∼99% raw accuracy. “Fluorogenic pyrosequencing” combines benefits of pyrosequencing, such as rapid turn-around, native DNA generation, and single-color detection, with benefits of fluorescence-based approaches, such as highly sensitive detection and simple parallelization. PMID:21666670

Novel multiplex qualitative detection using universal primer-multiplex-PCR combined with pyrosequencing.

PubMed

Shang, Ying; Xu, Wentao; Wang, Yong; Xu, Yuancong; Huang, Kunlun

2017-12-15

This study described a novel multiplex qualitative detection method using pyrosequencing. Based on the principle of the universal primer-multiplex-PCR, only one sequencing primer was employed to realize the detection of the multiple targets. Samples containing three genetically modified (GM) crops in different proportions were used to validate the method. The dNTP dispensing order was designed based on the product sequences. Only 12 rounds (ATCTGATCGACT) of dNTPs addition and, often, as few as three rounds (CAT) under ideal conditions, were required to detect the GM events qualitatively, and sensitivity was as low as 1% of a mixture. However, when considering a mixture, calculating signal values allowed the proportion of each GM to be estimated. Based on these results, we concluded that our novel method not only realized detection but also allowed semi-quantitative detection of individual events. Copyright © 2017. Published by Elsevier Ltd.

Evaluation of feed grade sodium bisulfate impact on gastrointestinal tract microbiota ecology in broilers via a pyrosequencing platform.

PubMed

Park, Si Hong; Dowd, Scot E; McReynolds, Jack L; Byrd, James A; Nisbet, David J; Ricke, Steven C

2015-12-01

The gastrointestinal microbial community in broiler chickens consists of many different species of bacteria, and the overall microbiota can vary from bird to bird. To control pathogenic bacteria in broilers and improve gut health, numerous potential dietary amendments have been used. In this study, we used a pyrosequencing platform to evaluate the effect of sodium bisulfate on microbiota of the crop, cecum, and ileum of broiler chickens grown over several weeks. The diversity information in each digestive organ sample exhibited considerable variation and was clearly separable, suggesting distinct bacterial populations. Although no apparent microbial clustering occurred between the control and the dietary treatments, we did observe shifts in overall microbiota populations in the crop, ileum, and ceca as well as changes in specific microorganisms such as Bacteroides, Clostridium, and Lactobacillus species that were identified as birds became older. © 2015 Poultry Science Association Inc.

Performances of two biotrickling filters in treating H₂S-containing waste gases and analysis of corresponding bacterial communities by pyrosequencing.

PubMed

Li, Jianjun; Ye, Guangyun; Sun, Duanfang; Sun, Guoping; Zeng, Xiaowei; Xu, Jian; Liang, Shizhong

2012-09-01

Two identical biotrickling filters named BTFa and BTFb were run in parallel to examine their performances in removing hydrogen sulfide. BTFa was filled with ceramic granules, and BTFb was filled with volcanic rocks. The results showed that BTFb was more robust than BTFa under acidic conditions. At empty bed residence times (EBRTs) of 20 and 15 s, the removal efficiency of BTFa was close to 100%. At EBRTs of 10 and 5 s, the removal efficiency of BTFa slightly decreased. The removal efficiencies of BTFa decreased by different degrees at the end of each stage, dropping to 94%, 81%, 60%, and 71%, respectively. However, the H(2)S removal efficiency in BTFb consistently reached 99% throughout the experiment. Pyrosequencing analyses indicated that members of Thiomonas dominated in both BTFs, but the relative abundance of Acidithiobacillus was higher in BTFb than in BTFa.

Forensic discrimination of vaginal epithelia by DNA methylation analysis through pyrosequencing.

PubMed

Antunes, Joana; Silva, Deborah S B S; Balamurugan, Kuppareddi; Duncan, George; Alho, Clarice S; McCord, Bruce

2016-10-01

The accurate identification of body fluids from crime scenes can aid in the discrimination between criminal and innocent intent. This research aimed to determine if the levels of DNA methylation in the locus PFN3A could be used to discriminate vaginal epithelia from other body fluids. In this work we bisulfite-modified and amplified DNA samples from blood, saliva, semen, and vaginal epithelia using primers for PFN3A. Through pyrosequencing we were able to show that vaginal epithelia present distinct methylation levels when compared to other body fluids. Mixtures of different body fluids present methylation values that correlate with single-source body fluid samples and the primers for PFN3A are specific for primates. This report successfully demonstrated that the analysis of methylation in the PFN3A locus can be used for vaginal epithelia discrimination in forensic samples. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and pyrosequencing analysis of an in-vitro oral biofilm model.

PubMed

Kistler, James O; Pesaro, Manuel; Wade, William G

2015-02-10

Dental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers. Pyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms. Diverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.

Arbuscular Mycorhizal Fungi Associated with the Olive Crop across the Andalusian Landscape: Factors Driving Community Differentiation

PubMed Central

Montes-Borrego, Miguel; Metsis, Madis; Landa, Blanca B.

2014-01-01

Background In the last years, many olive plantations in southern Spain have been mediated by the use of self-rooted planting stocks, which have incorporated commercial AMF during the nursery period to facilitate their establishment. However, this was practised without enough knowledge on the effect of cropping practices and environment on the biodiversity of AMF in olive orchards in Spain. Methodology/Principal Findings Two culture-independent molecular methods were used to study the AMF communities associated with olive in a wide-region analysis in southern Spain including 96 olive locations. The use of T-RFLP and pyrosequencing analysis of rDNA sequences provided the first evidence of an effect of agronomic and climatic characteristics, and soil physicochemical properties on AMF community composition associated with olive. Thus, the factors most strongly associated to AMF distribution varied according to the technique but included among the studied agronomic characteristics the cultivar genotype and age of plantation and the irrigation regimen but not the orchard management system or presence of a cover crop to prevent soil erosion. Soil physicochemical properties and climatic characteristics most strongly associated to the AMF community composition included pH, textural components and nutrient contents of soil, and average evapotranspiration, rainfall and minimum temperature of the sampled locations. Pyrosequencing analysis revealed 33 AMF OTUs belonging to five families, with Archaeospora spp., Diversispora spp. and Paraglomus spp., being first records in olive. Interestingly, two of the most frequent OTUs included a diverse group of Claroideoglomeraceae and Glomeraceae sequences, not assigned to any known AMF species commonly used as inoculants in olive during nursery propagation. Conclusions/Significance Our data suggests that AMF can exert higher host specificity in olive than previously thought, which may have important implications for redirecting the olive nursery process in the future as well as to take into consideration the specific soils and environments where the mycorrhized olive trees will be established. PMID:24797669

Fiber supplementation influences phylogenetic structure and functional capacity of the human intestinal microbiome: follow-up of a randomized controlled trial.

PubMed

Holscher, Hannah D; Caporaso, J Gregory; Hooda, Seema; Brulc, Jennifer M; Fahey, George C; Swanson, Kelly S

2015-01-01

In our published randomized, double-blind, placebo-controlled, 3-period crossover trial, healthy adult men (n = 21) consumed bars containing no supplemental fiber (placebo; NFC), polydextrose (21 g/d), and soluble corn fiber (SCF; 21 g/d) for 21 d each. Fecal specimens were collected between days 16 and 21 for fermentative end-product analysis and 16S ribosomal RNA bacterial gene amplification for bacterial taxa identification. Fiber supplementation decreased fecal putrefaction compounds and shifted abundances of several bacterial taxa. The objective was to perform whole-genome shotgun 454 pyrosequencing on the same fecal specimens collected in that clinical trial to obtain comprehensive fecal bacterial genome sequencing coverage and explore the full range of bacterial genetic information in the fecal microbiome, thereby using a systematic approach to study the impact of dietary fiber supplementation on fecal metabolites, bacterial taxa, and bacterial metagenomes. Fecal samples were subjected to whole-genome shotgun 454 pyrosequencing to identify both fecal bacterial populations present and their functional genetic capacity. Whole-genome shotgun sequencing results revealed that fiber consumption shifted the Bacteroidetes:Firmicutes ratio, increasing the relative abundance of Bacteroidetes 12 ± 2% and 13 ± 2% with polydextrose and SCF, respectively, compared with NFC. Bivariate correlations showed a positive correlation between the Bacteroidetes:Firmicutes ratio and total dietary fiber intake but not body mass index. Principal coordinates analysis of Bray-Curtis distances indicated that bacterial gene composition was more similar in participants consuming fibers (polydextrose and SCF combined) in comparison with NFC. Shifts in bacterial gene abundances after polydextrose and SCF supplementation included genes associated with carbohydrate, amino acid, and lipid metabolism, as well as metabolism of cofactors and vitamins. This study conveys novel information about the impact of dietary fiber supplementation on the phylogenetic structure and functional capacity of the fecal microbiome of healthy adults. © 2015 American Society for Nutrition.

Distinct Soil Bacterial Communities Revealed under a Diversely Managed Agroecosystem

PubMed Central

Shange, Raymon S.; Ankumah, Ramble O.; Ibekwe, Abasiofiok M.; Zabawa, Robert; Dowd, Scot E.

2012-01-01

Land-use change and management practices are normally enacted to manipulate environments to improve conditions that relate to production, remediation, and accommodation. However, their effect on the soil microbial community and their subsequent influence on soil function is still difficult to quantify. Recent applications of molecular techniques to soil biology, especially the use of 16S rRNA, are helping to bridge this gap. In this study, the influence of three land-use systems within a demonstration farm were evaluated with a view to further understand how these practices may impact observed soil bacterial communities. Replicate soil samples collected from the three land-use systems (grazed pine forest, cultivated crop, and grazed pasture) on a single soil type. High throughput 16S rRNA gene pyrosequencing was used to generate sequence datasets. The different land use systems showed distinction in the structure of their bacterial communities with respect to the differences detected in cluster analysis as well as diversity indices. Specific taxa, particularly Actinobacteria, Acidobacteria, and classes of Proteobacteria, showed significant shifts across the land-use strata. Families belonging to these taxa broke with notions of copio- and oligotrphy at the class level, as many of the less abundant groups of families of Actinobacteria showed a propensity for soil environments with reduced carbon/nutrient availability. Orders Actinomycetales and Solirubrobacterales showed their highest abundance in the heavily disturbed cultivated system despite the lowest soil organic carbon (SOC) values across the site. Selected soil properties ([SOC], total nitrogen [TN], soil texture, phosphodiesterase [PD], alkaline phosphatase [APA], acid phosphatase [ACP] activity, and pH) also differed significantly across land-use regimes, with SOM, PD, and pH showing variation consistent with shifts in community structure and composition. These results suggest that use of pyrosequencing along with traditional analysis of soil physiochemical properties may provide insight into the ecology of descending taxonomic groups in bacterial communities. PMID:22844402

16SrDNA Pyrosequencing of the Mediterranean Gorgonian Paramuricea clavata Reveals a Link among Alterations in Bacterial Holobiont Members, Anthropogenic Influence and Disease Outbreaks

PubMed Central

Vezzulli, Luigi; Pezzati, Elisabetta; Huete-Stauffer, Carla; Pruzzo, Carla; Cerrano, Carlo

2013-01-01

Mass mortality events of benthic invertebrates in the Mediterranean Sea are becoming an increasing concern with catastrophic effects on the coastal marine environment. Sea surface temperature anomalies leading to physiological stress, starvation and microbial infections were identified as major factors triggering animal mortality. However the highest occurrence of mortality episodes in particular geographic areas and occasionally in low temperature deep environments suggest that other factors play a role as well. We conducted a comparative analysis of bacterial communities associated with the purple gorgonian Paramuricea clavata, one of the most affected species, collected at different geographic locations and depth, showing contrasting levels of anthropogenic disturbance and health status. Using massive parallel 16SrDNA gene pyrosequencing we showed that the bacterial community associated with healthy P. clavata in pristine locations was dominated by a single genus Endozoicomonas within the order Oceanospirillales which represented ∼90% of the overall bacterial community. P. clavata samples collected in human impacted areas and during disease events had higher bacterial diversity and abundance of disease-related bacteria, such as vibrios, than samples collected in pristine locations whilst showed a reduced dominance of Endozoicomonas spp. In contrast, bacterial symbionts exhibited remarkable stability in P. clavata collected both at euphotic and mesophotic depths in pristine locations suggesting that fluctuations in environmental parameters such as temperature have limited effect in structuring the bacterial holobiont. Interestingly the coral pathogen Vibrio coralliilyticus was not found on diseased corals collected during a deep mortality episode suggesting that neither temperature anomalies nor recognized microbial pathogens are solely sufficient to explain for the events. Overall our data suggest that anthropogenic influence may play a significant role in determining the coral health status by affecting the composition of the associated microbial community. Environmental stressful events and microbial infections may thus be superimposed to compromise immunity and trigger mortality outbreaks. PMID:23840768

Distinct soil bacterial communities revealed under a diversely managed agroecosystem.

PubMed

Shange, Raymon S; Ankumah, Ramble O; Ibekwe, Abasiofiok M; Zabawa, Robert; Dowd, Scot E

2012-01-01

Land-use change and management practices are normally enacted to manipulate environments to improve conditions that relate to production, remediation, and accommodation. However, their effect on the soil microbial community and their subsequent influence on soil function is still difficult to quantify. Recent applications of molecular techniques to soil biology, especially the use of 16S rRNA, are helping to bridge this gap. In this study, the influence of three land-use systems within a demonstration farm were evaluated with a view to further understand how these practices may impact observed soil bacterial communities. Replicate soil samples collected from the three land-use systems (grazed pine forest, cultivated crop, and grazed pasture) on a single soil type. High throughput 16S rRNA gene pyrosequencing was used to generate sequence datasets. The different land use systems showed distinction in the structure of their bacterial communities with respect to the differences detected in cluster analysis as well as diversity indices. Specific taxa, particularly Actinobacteria, Acidobacteria, and classes of Proteobacteria, showed significant shifts across the land-use strata. Families belonging to these taxa broke with notions of copio- and oligotrphy at the class level, as many of the less abundant groups of families of Actinobacteria showed a propensity for soil environments with reduced carbon/nutrient availability. Orders Actinomycetales and Solirubrobacterales showed their highest abundance in the heavily disturbed cultivated system despite the lowest soil organic carbon (SOC) values across the site. Selected soil properties ([SOC], total nitrogen [TN], soil texture, phosphodiesterase [PD], alkaline phosphatase [APA], acid phosphatase [ACP] activity, and pH) also differed significantly across land-use regimes, with SOM, PD, and pH showing variation consistent with shifts in community structure and composition. These results suggest that use of pyrosequencing along with traditional analysis of soil physiochemical properties may provide insight into the ecology of descending taxonomic groups in bacterial communities.

Bacterial abundance and composition in marine sediments beneath the Ross Ice Shelf, Antarctica.

PubMed

Carr, S A; Vogel, S W; Dunbar, R B; Brandes, J; Spear, J R; Levy, R; Naish, T R; Powell, R D; Wakeham, S G; Mandernack, K W

2013-07-01

Marine sediments of the Ross Sea, Antarctica, harbor microbial communities that play a significant role in the decomposition, mineralization, and recycling of organic carbon (OC). In this study, the cell densities within a 153-cm sediment core from the Ross Sea were estimated based on microbial phospholipid fatty acid (PLFA) concentrations and acridine orange direct cell counts. The resulting densities were as high as 1.7 × 10⁷ cells mL⁻¹ in the top ten centimeters of sediments. These densities are lower than those calculated for most near-shore sites but consistent with deep-sea locations with comparable sedimentation rates. The δ¹³C measurements of PLFAs and sedimentary and dissolved carbon sources, in combination with ribosomal RNA (SSU rRNA) gene pyrosequencing, were used to infer microbial metabolic pathways. The δ¹³C values of dissolved inorganic carbon (DIC) in porewaters ranged downcore from -2.5‰ to -3.7‰, while δ¹³C values for the corresponding sedimentary particulate OC (POC) varied from -26.2‰ to -23.1‰. The δ¹³C values of PLFAs ranged between -29‰ and -35‰ throughout the sediment core, consistent with a microbial community dominated by heterotrophs. The SSU rRNA gene pyrosequencing revealed that members of this microbial community were dominated by β-, δ-, and γ-Proteobacteria, Actinobacteria, Chloroflexi and Bacteroidetes. Among the sequenced organisms, many appear to be related to known heterotrophs that utilize OC sources such as amino acids, oligosaccharides, and lactose, consistent with our interpretation from δ¹³CPLFA analysis. Integrating phospholipids analyses with porewater chemistry, δ¹³CDIC and δ¹³CPOC values and SSU rRNA gene sequences provides a more comprehensive understanding of microbial communities and carbon cycling in marine sediments, including those of this unique ice shelf environment. © 2013 John Wiley & Sons Ltd.

Assessing CpG island methylator phenotype, 1p/19q codeletion, and MGMT promoter methylation from epigenome-wide data in the biomarker cohort of the NOA-04 trial

PubMed Central

Wiestler, Benedikt; Capper, David; Hovestadt, Volker; Sill, Martin; Jones, David T.W.; Hartmann, Christian; Felsberg, Joerg; Platten, Michael; Feiden, Wolfgang; Keyvani, Kathy; Pfister, Stefan M.; Wiestler, Otmar D.; Meyermann, Richard; Reifenberger, Guido; Pietsch, Thorsten; von Deimling, Andreas; Weller, Michael; Wick, Wolfgang

2014-01-01

Background Molecular biomarkers including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation may improve prognostication and guide treatment decisions for patients with World Health Organization (WHO) anaplastic gliomas. At present, each marker is individually tested by distinct assays. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) enable the determination of large-scale methylation profiles and genome-wide DNA copy number changes. Algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion, and MGMT promoter methylation using a single assay. Methods Here, we retrospectively investigated the diagnostic and prognostic performance of these algorithms in comparison to individual marker testing and patient outcome in the biomarker cohort (n = 115 patients) of the NOA-04 trial. Results Concordance for IDH and 1p/19q status was very high: In 92% of samples, the HM450 and reference data agreed. In discordant samples, survival analysis by Kaplan-Meier and Cox regression analyses suggested a more accurate assessment of biological phenotype by the HM450 analysis. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of samples than conventional methylation-specific PCR, with 87 of 91 G-CIMP tumors predicted as MGMT promoter-methylated. Pyrosequencing of discordant samples confirmed the HM450 assessment in 14 of 17 cases. Conclusions G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and are associated with prognosis in the NOA-04 trial. For MGMT, HM450 suggests promoter methylation in the vast majority of G-CIMP tumors, which is supported by pyrosequencing. PMID:25028501

Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: a multicohort European case-control study using centralized ultrasensitive 454 pyrosequencing.

PubMed

Cozzi-Lepri, Alessandro; Noguera-Julian, Marc; Di Giallonardo, Francesca; Schuurman, Rob; Däumer, Martin; Aitken, Sue; Ceccherini-Silberstein, Francesca; D'Arminio Monforte, Antonella; Geretti, Anna Maria; Booth, Clare L; Kaiser, Rolf; Michalik, Claudia; Jansen, Klaus; Masquelier, Bernard; Bellecave, Pantxika; Kouyos, Roger D; Castro, Erika; Furrer, Hansjakob; Schultze, Anna; Günthard, Huldrych F; Brun-Vezinet, Francoise; Paredes, Roger; Metzner, Karin J

2015-03-01

It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART. This Europe-wide case-control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%-25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression. Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35-5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76-6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12-5.18, P = 0.024). A dose-effect relationship between virological failure and mutational load was found. Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based ART. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

Pyrosequencing analysis of bacterial diversity in dental unit waterlines.

PubMed

Costa, Damien; Mercier, Anne; Gravouil, Kevin; Lesobre, Jérôme; Delafont, Vincent; Bousseau, Anne; Verdon, Julien; Imbert, Christine

2015-09-15

Some infections cases due to exposure to output water from dental unit waterlines (DUWL) have been reported in the literature. However, this type of healthcare-associated risk has remained unclear and up until now the overall bacterial composition of DUWL has been poorly documented. In this study, 454 high-throughput pyrosequencing was used to investigate the bacterial community in seven dental offices (N = 7) and to identify potential bacterial pathogenic sequences. Dental unit waters (DUW) were collected from the tap water supplying units (Incoming Water; IW) to the output exposure point of the turbine handpiece (Output water; OW) following a stagnation period (OWS), and immediately after the last patient of the sampling day (OWA). A high bacterial diversity was revealed in DUW with 394 operational taxonomic units detected at the genus level. In addition to the inter-unit variability observed, results showed increased total bacterial cell concentration and shifts in bacterial community composition and abundance at the genus level, mainly within the Gamma- and Alpha-Proteobacteria class, as water circulated in the dental unit (DU). Results showed that 96.7%, 96.8% and 97.4% of the total sequences from IW, OWS and OWA respectively were common to the 3 defined water groups, thereby highlighting a common core microbiome. Results also suggested that stagnation and DU maintenance practices were critical to composition of the bacterial community. The presence of potentially pathogenic genera was detected, including Pseudomonas and Legionella spp. Emerging and opportunistic pathogenic genera such as Mycobacterium, Propionibacterium and Stenotrophomonas were likewise recovered in DUW. For the first time, an exhaustive evaluation of the bacterial communities present in DUW was performed taking into account the circulation of water within the DU. This study highlights an ignored diversity of the DUWL bacterial community. Our findings also contribute to a better appreciation of the potential infectious risk associated with dental care and suggest the importance of better managing microbial quality in DUW. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Restoration of microbial ammonia oxidizers in air-dried forest soils upon wetting].

PubMed

Zhou, Xue; Huang, Rong; Song, Ge; Pan, Xianzhang; Jia, Zhongjun

2014-11-04

This study was aimed to investigate the abundance and community shift of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in air-dried forest soils in response to water addition, to explore the applicability of air-dried soil for microbial ecology study, and to elucidate whether AOA within the marine group 1. 1a dominate ammonia oxidizers communities in the acidic forest soils in China. Soil samples were collected from 10 forest sites of the China Ecosystem Research Network (CERN) and kept under air-drying conditions in 2010. In 2013 the air-dried soil samples were adjusted to 60% of soil maximum water holding capacity for a 28-day incubation at 28 degrees C in darkness. DGGE fingerprinting, clone library construction, pyrosequencing and quantitative PCR of amoA genes were performed to assess community change of ammonia oxidizers in air-dried and re-wetted soils. After incubation for 28 days, the abundance of bacteria and archaea increased significantly, up to 3,230 and 568 times, respectively. AOA increased significantly in 8 samples, and AOB increased significantly in 5 of 10 samples. However, pyrosequencing of amoA genes reveals insignificant changes in composition of AOA and AOB communities. Phylogenetic analysis of amoA genes indicates that archaeal ammonia oxidizers were predominated by AOA within the soil group 1. 1b lineage, while the Nitrosospira-like AOB dominate bacteria ammonia oxidizer communities. There was a significantly positive correlation between AOA/AOB ratio and total nitrogen (r2 = 0.54, P < 0.05), implying that soil ammonia oxidation might be dominated by AOA in association with ammonium released from soil mineralization. Phylogenetic analysis suggest that AOA members within the soil group 1. 1b lineage were not restricted to non-acidic soils as previously thought. The abundance rather than composition of AOA and AOB changed in response to water addition. This indicates that air-dried soil could be of help for microbial biogeography study.

An Image Secret Sharing Method

DTIC Science & Technology

2006-07-01

the secret image in lossless manner and (2) any or fewer image shares cannot get sufficient information to reveal the ... secret image. It is an effective, reliable and secure method to prevent the secret image from being lost, stolen or corrupted. In comparison with...other image secret sharing methods, this approach’s advantages are its large compression rate on the size of the image shares, its strong protection of the secret image and its ability for real-time

Implicit and explicit social mentalizing: dual processes driven by a shared neural network

PubMed Central

Van Overwalle, Frank; Vandekerckhove, Marie

2013-01-01

Recent social neuroscientific evidence indicates that implicit and explicit inferences on the mind of another person (i.e., intentions, attributions or traits), are subserved by a shared mentalizing network. Under both implicit and explicit instructions, ERP studies reveal that early inferences occur at about the same time, and fMRI studies demonstrate an overlap in core mentalizing areas, including the temporo-parietal junction (TPJ) and the medial prefrontal cortex (mPFC). These results suggest a rapid shared implicit intuition followed by a slower explicit verification processes (as revealed by additional brain activation during explicit vs. implicit inferences). These data provide support for a default-adjustment dual-process framework of social mentalizing. PMID:24062663

Transcriptomic analysis of grain amaranth (Amaranthus hypochondriacus) using 454 pyrosequencing: comparison with A. tuberculatus, expression profiling in stems and in response to biotic and abiotic stress

PubMed Central

2011-01-01

Background Amaranthus hypochondriacus, a grain amaranth, is a C4 plant noted by its ability to tolerate stressful conditions and produce highly nutritious seeds. These possess an optimal amino acid balance and constitute a rich source of health-promoting peptides. Although several recent studies, mostly involving subtractive hybridization strategies, have contributed to increase the relatively low number of grain amaranth expressed sequence tags (ESTs), transcriptomic information of this species remains limited, particularly regarding tissue-specific and biotic stress-related genes. Thus, a large scale transcriptome analysis was performed to generate stem- and (a)biotic stress-responsive gene expression profiles in grain amaranth. Results A total of 2,700,168 raw reads were obtained from six 454 pyrosequencing runs, which were assembled into 21,207 high quality sequences (20,408 isotigs + 799 contigs). The average sequence length was 1,064 bp and 930 bp for isotigs and contigs, respectively. Only 5,113 singletons were recovered after quality control. Contigs/isotigs were further incorporated into 15,667 isogroups. All unique sequences were queried against the nr, TAIR, UniRef100, UniRef50 and Amaranthaceae EST databases for annotation. Functional GO annotation was performed with all contigs/isotigs that produced significant hits with the TAIR database. Only 8,260 sequences were found to be homologous when the transcriptomes of A. tuberculatus and A. hypochondriacus were compared, most of which were associated with basic house-keeping processes. Digital expression analysis identified 1,971 differentially expressed genes in response to at least one of four stress treatments tested. These included several multiple-stress-inducible genes that could represent potential candidates for use in the engineering of stress-resistant plants. The transcriptomic data generated from pigmented stems shared similarity with findings reported in developing stems of Arabidopsis and black cottonwood (Populus trichocarpa). Conclusions This study represents the first large-scale transcriptomic analysis of A. hypochondriacus, considered to be a highly nutritious and stress-tolerant crop. Numerous genes were found to be induced in response to (a)biotic stress, many of which could further the understanding of the mechanisms that contribute to multiple stress-resistance in plants, a trait that has potential biotechnological applications in agriculture. PMID:21752295

He Asked Me What!?--Using Shared Online Accounts as Training Tools for Distance Learning Librarians

ERIC Educational Resources Information Center

Robinson, Kelly; Casey, Anne Marie; Citro, Kathleen

2017-01-01

This study explores the idea of creating a knowledge base from shared online accounts to use in training librarians who perform distance reference services. Through a survey, follow-up interviews and a case study, the investigators explored current and potential use of shared online accounts as training tools. This study revealed that the…

Knowledge sharing within organizations: linking art, theory, scenarios and professional experience

NASA Technical Reports Server (NTRS)

Burton, Y. C.; Bailey, T.

2000-01-01

In this presentation, Burton and Bailey, discuss the challenges and opportunities in developing knowledge sharing systems in organizations. Bailey provides a tool using imagery and collage for identifying and utilizing the diverse values and beliefs of individuals and groups. Burton reveals findings from a business research study that examines how social construction influences knowledge sharing among task oriented groups.

Comprehensive Gene expression meta-analysis and integrated bioinformatic approaches reveal shared signatures between thrombosis and myeloproliferative disorders

PubMed Central

Jha, Prabhash Kumar; Vijay, Aatira; Sahu, Anita; Ashraf, Mohammad Zahid

2016-01-01

Thrombosis is a leading cause of morbidity and mortality in patients with myeloproliferative disorders (MPDs), particularly polycythemia vera (PV) and essential thrombocythemia (ET). Despite the attempts to establish a link between them, the shared biological mechanisms are yet to be characterized. An integrated gene expression meta-analysis of five independent publicly available microarray data of the three diseases was conducted to identify shared gene expression signatures and overlapping biological processes. Using INMEX bioinformatic tool, based on combined Effect Size (ES) approaches, we identified a total of 1,157 differentially expressed genes (DEGs) (697 overexpressed and 460 underexpressed genes) shared between the three diseases. EnrichR tool’s rich library was used for comprehensive functional enrichment and pathway analysis which revealed “mRNA Splicing” and “SUMO E3 ligases SUMOylate target proteins” among the most enriched terms. Network based meta-analysis identified MYC and FN1 to be the most highly ranked hub genes. Our results reveal that the alterations in biomarkers of the coagulation cascade like F2R, PROS1, SELPLG and ITGB2 were common between the three diseases. Interestingly, the study has generated a novel database of candidate genetic markers, pathways and transcription factors shared between thrombosis and MPDs, which might aid in the development of prognostic therapeutic biomarkers. PMID:27892526

Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies.

PubMed

Lamy, Pierre-Jean; Castan, Florence; Lozano, Nicolas; Montélion, Cécile; Audran, Patricia; Bibeau, Frédéric; Roques, Sylvie; Montels, Frédéric; Laberenne, Anne-Claire

2015-07-01

The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedded cutaneous melanoma biopsies. We compared the results obtained by dPCR with high-resolution melting curve analysis and pyrosequencing or with one of the allele-specific PCR methods available on the market. dPCR showed the lowest limit of detection. dPCR and allele-specific amplification detected the highest number of mutated samples. For the BRAF mutation load quantification both dPCR and pyrosequencing gave similar results with strong disparities in allele frequencies in the 47 tumor samples under study (from 0.7% to 79% of BRAF V600E mutations/sample). In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A comprehensive survey of soil acidobacterial diversity using pyrosequencing and clone library analyses

PubMed Central

Jones, Ryan T; Robeson, Michael S; Lauber, Christian L; Hamady, Micah; Knight, Rob; Fierer, Noah

2010-01-01

Acidobacteria are ubiquitous and abundant members of soil bacterial communities. However, an ecological understanding of this important phylum has remained elusive because its members have been difficult to culture and few molecular investigations have focused exclusively on this group. We generated an unprecedented number of acidobacterial DNA sequence data using pyrosequencing and clone libraries (39 707 and 1787 sequences, respectively) to characterize the relative abundance, diversity and composition of acidobacterial communities across a range of soil types. To gain insight into the ecological characteristics of acidobacterial taxa, we investigated the large-scale biogeographic patterns exhibited by acidobacterial communities, and related soil and site characteristics to acidobacterial community assemblage patterns. The 87 soils analyzed by pyrosequencing contained more than 8600 unique acidobacterial phylotypes (at the 97% sequence similarity level). One phylotype belonging to Acidobacteria subgroup 1, but not closely related to any cultured representatives, was particularly abundant, accounting for 7.4% of bacterial sequences and 17.6% of acidobacterial sequences, on average, across the soils. The abundance of Acidobacteria relative to other bacterial taxa was highly variable across the soils examined, but correlated strongly with soil pH (R = −0.80, P<0.001). Soil pH was also the best predictor of acidobacterial community composition, regardless of how the communities were characterized, and the relative abundances of the dominant Acidobacteria subgroups were readily predictable. Acidobacterial communities were more phylogenetically clustered as soil pH departed from neutrality, suggesting that pH is an effective habitat filter, restricting community membership to progressively more narrowly defined lineages as pH deviates from neutrality. PMID:19129864

Maternal blood contamination of collected cord blood can be identified using DNA methylation at three CpGs.

PubMed

Morin, Alexander M; Gatev, Evan; McEwen, Lisa M; MacIsaac, Julia L; Lin, David T S; Koen, Nastassja; Czamara, Darina; Räikkönen, Katri; Zar, Heather J; Koenen, Karestan; Stein, Dan J; Kobor, Michael S; Jones, Meaghan J

2017-01-01

Cord blood is a commonly used tissue in environmental, genetic, and epigenetic population studies due to its ready availability and potential to inform on a sensitive period of human development. However, the introduction of maternal blood during labor or cross-contamination during sample collection may complicate downstream analyses. After discovering maternal contamination of cord blood in a cohort study of 150 neonates using Illumina 450K DNA methylation (DNAm) data, we used a combination of linear regression and random forest machine learning to create a DNAm-based screening method. We identified a panel of DNAm sites that could discriminate between contaminated and non-contaminated samples, then designed pyrosequencing assays to pre-screen DNA prior to being assayed on an array. Maternal contamination of cord blood was initially identified by unusual X chromosome DNA methylation patterns in 17 males. We utilized our DNAm panel to detect contaminated male samples and a proportional amount of female samples in the same cohort. We validated our DNAm screening method on an additional 189 sample cohort using both pyrosequencing and DNAm arrays, as well as 9 publically available cord blood 450K data sets. The rate of contamination varied from 0 to 10% within these studies, likely related to collection specific methods. Maternal blood can contaminate cord blood during sample collection at appreciable levels across multiple studies. We have identified a panel of markers that can be used to identify this contamination, either post hoc after DNAm arrays have been completed, or in advance using a targeted technique like pyrosequencing.

Investigation of postpartum dairy cows' uterine microbial diversity using metagenomic pyrosequencing of the 16S rRNA gene.

PubMed

Machado, V S; Oikonomou, G; Bicalho, M L S; Knauer, W A; Gilbert, R; Bicalho, R C

2012-10-12

The objective of this study was the use of metagenomic pyrosequencing of the 16S rRNA gene for the investigation of postpartum dairy cows' uterine bacterial diversity. The effect of subcutaneous supplementation of a trace mineral supplement containing Zn, Mn, Se, and Cu (Multimin North America, Inc., Fort Collins, CO) at 230 days of gestation and 260 days of gestation on dairy cows' uterine microbiota was also evaluated. Uterine lavage samples were collected at 35 DIM and were visually scored for the presence of purulent or mucopurulent secretion. The same samples were also used for the acquisition of bacterial DNA. The 16S rRNA genes were individually amplified from each sample. Pyrosequencing of the samples was carried at the Cornell University Life Sciences Core Laboratories Center using Roche 454 GS-FLX System Titanium Chemistry. The Ribosomal Database Project online tools were used for the analysis of the obtained sequences library. Bacteroides spp., Ureaplasma spp., Fusobacterium spp., Peptostreptococcus spp., Sneathia spp., Prevotella spp. and Arcanobacterium spp. prevalence was significantly (P<0.05) higher in samples derived from cows that had a higher uterine lavage sample score. Bacteroides spp., Ureaplasma spp., Fusobacterium spp., and Arcanobacterium spp. prevalence was significantly (P<0.05) higher in samples derived from cows that were not pregnant by 200 DIM. Anaerococcus spp., Peptostreptococcus spp., Parabacteroides spp., and Propionibacterium spp. prevalence was significantly (P<0.05) lower in samples derived from cows that were trace mineral supplemented. Copyright © 2012 Elsevier B.V. All rights reserved.

KRAS mutation testing in colorectal cancer: comparison of the results obtained using 3 different methods for the analysis of codons G12 and G13.

PubMed

Bihl, Michel P; Hoeller, Sylvia; Andreozzi, Maria Carla; Foerster, Anja; Rufle, Alexander; Tornillo, Luigi; Terracciano, Luigi

2012-03-01

Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.

Differential resistance of drinking water bacterial populations to monochloramine disinfection.

PubMed

Chiao, Tzu-Hsin; Clancy, Tara M; Pinto, Ameet; Xi, Chuanwu; Raskin, Lutgarde

2014-04-01

The impact of monochloramine disinfection on the complex bacterial community structure in drinking water systems was investigated using culture-dependent and culture-independent methods. Changes in viable bacterial diversity were monitored using culture-independent methods that distinguish between live and dead cells based on membrane integrity, providing a highly conservative measure of viability. Samples were collected from lab-scale and full-scale drinking water filters exposed to monochloramine for a range of contact times. Culture-independent detection of live cells was based on propidium monoazide (PMA) treatment to selectively remove DNA from membrane-compromised cells. Quantitative PCR (qPCR) and pyrosequencing of 16S rRNA genes was used to quantify the DNA of live bacteria and characterize the bacterial communities, respectively. The inactivation rate determined by the culture-independent PMA-qPCR method (1.5-log removal at 664 mg·min/L) was lower than the inactivation rate measured by the culture-based methods (4-log removal at 66 mg·min/L). Moreover, drastic changes in the live bacterial community structure were detected during monochloramine disinfection using PMA-pyrosequencing, while the community structure appeared to remain stable when pyrosequencing was performed on samples that were not subject to PMA treatment. Genera that increased in relative abundance during monochloramine treatment include Legionella, Escherichia, and Geobacter in the lab-scale system and Mycobacterium, Sphingomonas, and Coxiella in the full-scale system. These results demonstrate that bacterial populations in drinking water exhibit differential resistance to monochloramine, and that the disinfection process selects for resistant bacterial populations.

Effect of postharvest practices including degreening on citrus carpoplane microbial biomes.

PubMed

Gomba, A; Chidamba, L; Korsten, L

2017-04-01

To investigate the effect of commercial citrus packhouse processing steps on the fruit surface microbiome of Clementines and Palmer navel oranges. Viable bacteria, yeast and fungi counts, and the pyrosequencing analysis of the 16S rRNA and ITS were used to evaluate the community structure and population dynamics of phylloepiphytic bacteria and fungi associated with commercial postharvest processing. Drenching significantly reduced microbial counts in all cases except for yeasts on navels, while the extent of degreening effects varied between the citrus varieties. Pyrosequencing analysis showed a total of 4409 bacteria and 5792 fungi nonchimeric unique sequences with an average of 1102 bacteria and 1448 fungi reads per sample. Dominant phyla on the citrus carpoplane were Proteobacteria (53·5%), Actinobacteria (19·9%), Bacteroidetes (5·6%) and Deinococcus-Thermus (5·4%) for bacteria and Ascomycota (80·5%) and Basidiomycota (9·8%) for fungi. Beginning with freshly harvested fruit fungal diversity declined significantly after drenching, but had little effect on bacteria and populations recovered during degreening treatments, including those for Penicillium sp. Packhouse processing greatly influences microbial communities on the citrus carpoplane. A broad orange biome was described with pyrosequencing and gave insight into the likely survival and persistence of pathogens, especially as they may affect the quality and safety of the packed product. A close examination of the microbiota of fruit and the impact of intervention strategies on the ecological balance may provide a more durable approach to reduce losses and spoilage. © 2017 The Society for Applied Microbiology.

Challenges in Whole-Genome Annotation of Pyrosequenced Eukaryotic Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, Alan; Grigoriev, Igor

2009-04-17

Pyrosequencing technologies such as 454/Roche and Solexa/Illumina vastly lower the cost of nucleotide sequencing compared to the traditional Sanger method, and thus promise to greatly expand the number of sequenced eukaryotic genomes. However, the new technologies also bring new challenges such as shorter reads and new kinds and higher rates of sequencing errors, which complicate genome assembly and gene prediction. At JGI we are deploying 454 technology for the sequencing and assembly of ever-larger eukaryotic genomes. Here we describe our first whole-genome annotation of a purely 454-sequenced fungal genome that is larger than a yeast (>30 Mbp). The pezizomycotine (filamentousmore » ascomycote) Aspergillus carbonarius belongs to the Aspergillus section Nigri species complex, members of which are significant as platforms for bioenergy and bioindustrial technology, as members of soil microbial communities and players in the global carbon cycle, and as agricultural toxigens. Application of a modified version of the standard JGI Annotation Pipeline has so far predicted ~;;10k genes. ~;;12percent of these preliminary annotations suffer a potential frameshift error, which is somewhat higher than the ~;;9percent rate in the Sanger-sequenced and conventionally assembled and annotated genome of fellow Aspergillus section Nigri member A. niger. Also,>90percent of A. niger genes have potential homologs in the A. carbonarius preliminary annotation. Weconclude, and with further annotation and comparative analysis expect to confirm, that 454 sequencing strategies provide a promising substrate for annotation of modestly sized eukaryotic genomes. We will also present results of annotation of a number of other pyrosequenced fungal genomes of bioenergy interest.« less

Pyrosequencing analysis of oral microbiota shifting in various caries states in childhood.

PubMed

Jiang, Wen; Ling, Zongxin; Lin, Xiaolong; Chen, Yadong; Zhang, Jie; Yu, Jinjin; Xiang, Charlie; Chen, Hui

2014-05-01

Dental caries is one of the most prevalent childhood diseases worldwide, but little is known about the dynamic characteristics of oral microbiota in the development of dental caries. To investigate the shifting bacterial profiles in different caries states, 60 children (3-7-year-old) were enrolled in this study, including 30 caries-free subjects and 30 caries-active subjects. Supragingival plaques were collected from caries-active subjects on intact enamel, white spot lesions and carious dentin lesions. Plaques from caries-free subjects were used as a control. All samples were analyzed by 454 pyrosequencing based on 16S rRNA gene V1-V3 hypervariable regions. A total of 572,773 pyrosequencing reads passed the quality control and 25,444 unique phylotypes were identified, which represented 18 phyla and 145 genera. Reduced bacterial diversity in the cavitated dentin was observed as compared with the other groups. Thirteen genera (including Capnocytophaga, Fusobacterium, Porphyromonas, Abiotrophia, Comamonas, Tannerella, Eikenella, Paludibacter, Treponema, Actinobaculum, Stenotrophomonas, Aestuariimicrobium, and Peptococcus) were found to be associated with dental health, and the bacterial profiles differed considerably depending on caries status. Eight genera (including Cryptobacterium, Lactobacillus, Megasphaera, Olsenella, Scardovia, Shuttleworthia, Cryptobacterium, and Streptococcus) were increased significantly in cavitated dentin lesions, and Actinomyces and Corynebacterium were present at significant high levels in white spot lesions (P < 0.05), while Flavobacterium, Neisseria, Bergeyella, and Derxia were enriched in the intact surfaces of caries individuals (P < 0.05). Our results showed that oral bacteria were specific at different stages of caries progression, which contributes to informing the prevention and treatment of childhood dental caries.

Microbial diversity and dynamics throughout manufacturing and ripening of surface ripened semi-hard Danish Danbo cheeses investigated by culture-independent techniques.

PubMed

Ryssel, Mia; Johansen, Pernille; Al-Soud, Waleed Abu; Sørensen, Søren; Arneborg, Nils; Jespersen, Lene

2015-12-23

Microbial successions on the surface and in the interior of surface ripened semi-hard Danish Danbo cheeses were investigated by culture-dependent and -independent techniques. Culture-independent detection of microorganisms was obtained by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing, using amplicons of 16S and 26S rRNA genes for prokaryotes and eukaryotes, respectively. With minor exceptions, the results from the culture-independent analyses correlated to the culture-dependent plating results. Even though the predominant microorganisms detected with the two culture-independent techniques correlated, a higher number of genera were detected by pyrosequencing compared to DGGE. Additionally, minor parts of the microbiota, i.e. comprising <10.0% of the operational taxonomic units (OTUs), were detected by pyrosequencing, resulting in more detailed information on the microbial succession. As expected, microbial profiles of the surface and the interior of the cheeses diverged. During cheese production pyrosequencing determined Lactococcus as the dominating genus on cheese surfaces, representing on average 94.7%±2.1% of the OTUs. At day 6 Lactococcus spp. declined to 10.0% of the OTUs, whereas Staphylococcus spp. went from 0.0% during cheese production to 75.5% of the OTUs at smearing. During ripening, i.e. from 4 to 18 weeks, Corynebacterium was the dominant genus on the cheese surface (55.1%±9.8% of the OTUs), with Staphylococcus (17.9%±11.2% of the OTUs) and Brevibacterium (10.4%±8.3% of the OTUs) being the second and third most abundant genera. Other detected bacterial genera included Clostridiisalibacter (5.0%±4.0% of the OTUs), as well as Pseudoclavibacter, Alkalibacterium and Marinilactibacillus, which represented <2% of the OTUs. At smearing, yeast counts were low with Debaryomyces being the dominant genus accounting for 46.5% of the OTUs. During ripening the yeast counts increased significantly with Debaryomyces being the predominant genus, on average accounting for 96.7%±4.1% of the OTUs. The interior of the cheeses was dominated by Lactococcus spp. comprising on average 93.9%±7.8% of the OTUs throughout the cheese processing. The microbial dynamics described at genus level in this study add to a comprehensive understanding of the complex microbiota existing especially on surface ripened semi-hard cheeses. Copyright © 2015 Elsevier B.V. All rights reserved.

Archaeal Community Changes Associated with Cultivation of Amazon Forest Soil with Oil Palm

PubMed Central

Tupinambá, Daiva Domenech; Cantão, Maurício Egídio; Costa, Ohana Yonara Assis; Bergmann, Jessica Carvalho; Kruger, Ricardo Henrique; Kyaw, Cynthia Maria; Barreto, Cristine Chaves; Quirino, Betania Ferraz

2016-01-01

This study compared soil archaeal communities of the Amazon forest with that of an adjacent area under oil palm cultivation by 16S ribosomal RNA gene pyrosequencing. Species richness and diversity were greater in native forest soil than in the oil palm-cultivated area, and 130 OTUs (13.7%) were shared between these areas. Among the classified sequences, Thaumarchaeota were predominant in the native forest, whereas Euryarchaeota were predominant in the oil palm-cultivated area. Archaeal species diversity was 1.7 times higher in the native forest soil, according to the Simpson diversity index, and the Chao1 index showed that richness was five times higher in the native forest soil. A phylogenetic tree of unclassified Thaumarchaeota sequences showed that most of the OTUs belong to Miscellaneous Crenarchaeotic Group. Several archaeal genera involved in nutrient cycling (e.g., methanogens and ammonia oxidizers) were identified in both areas, but significant differences were found in the relative abundances of Candidatus Nitrososphaera and unclassified Soil Crenarchaeotic Group (prevalent in the native forest) and Candidatus Nitrosotalea and unclassified Terrestrial Group (prevalent in the oil palm-cultivated area). More studies are needed to culture some of these Archaea in the laboratory so that their metabolism and physiology can be studied. PMID:27006640

Selection in the host structures the microbiota associated with developing cod larvae (Gadus morhua).

PubMed

Bakke, Ingrid; Coward, Eivind; Andersen, Tom; Vadstein, Olav

2015-10-01

Marine fish larvae are immature upon hatching, and share their environment with high numbers of bacteria. The microbial communities associated with developing fish larvae might be structured by other factors than those important in developing terrestrial animals. Here, we analysed the beta (β)-diversity of the microbiota associated with developing cod larvae and compared it with the bacterial communities in water and live feed by applying pyrosequencing of bar coded v4 16S rDNA amplicons. A total of 15 phyla were observed in the cod larval microbiota. Proteobacteria was the most abundant, followed by Firmicutes, Bacteroidetes and Actinobacteria. The composition and diversity of the cod larval microbiota changed considerably with age. The temporal and spatial patterns of β-diversity could not be explained by stochastic processes, and did not coincide with changes in the rearing conditions. Furthermore, the larval microbiota was highly distinct from the water and the live feed microbiota, particularly at early developmental stages. However, the similarity between larval and water microbiota increased with age. This study suggests that strong selection in the host structures the cod larval microbiota. The changes in community structure observed with increasing age can be explained by altered selection pressure due to development of the intestinal system. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Sex ratio of mirid populations shifts in response to hostplant co-infestation or altered cytokinin signaling

PubMed Central

Adam, Nora; Erler, Theresa; Kallenbach, Mario; Kaltenpoth, Martin; Kunert, Grit; Baldwin, Ian T.; Schuman, Meredith C.

2016-01-01

Herbivore species sharing a host plant often compete. In this study, we show that host plant-mediated interaction between two insect herbivores – a generalist and a specialist – results in a sex ratio shift of the specialist’s offspring. We studied demographic parameters of the specialist Tupiocoris notatus (Hemiptera: Miridae) when co-infesting the host plant Nicotiana attenuata (Solanaceae) with the generalist leafhopper Empoasca sp. (Hemiptera: Cicadellidae). We show that the usually female-biased sex ratio of T. notatus shifts toward a higher male proportion in the offspring on plants co-infested by Empoasca sp. This sex ratio change did not occur after oviposition, nor is it due differential mortality of female and male nymphs. Based on pyrosequencing and PCR of bacterial 16S rRNA amplicons, we concluded that sex ratio shifts were unlikely to be due to infection with Wolbachia or other known sex ratio- distorting endosymbionts. Finally, we used transgenic lines of N. attenuata to evaluate if the sex ratio shift could be mediated by changes in general or specialized host plant metabolites. We found that the sex ratio shift occurred on plants deficient in two cytokinin receptors (irCHK2/3). Thus, cytokinin-regulated traits can alter the offspring sex ratio of the specialist T. notatus. PMID:27862998

Comparison of Deep-Water Viromes from the Atlantic Ocean and the Mediterranean Sea

PubMed Central

Winter, Christian; Garcia, Juan A. L.; Weinbauer, Markus G.; DuBow, Michael S.; Herndl, Gerhard J.

2014-01-01

The aim of this study was to compare the composition of two deep-sea viral communities obtained from the Romanche Fracture Zone in the Atlantic Ocean (collected at 5200 m depth) and the southwest Mediterranean Sea (from 2400 m depth) using a pyro-sequencing approach. The results are based on 18.7% and 6.9% of the sequences obtained from the Atlantic Ocean and the Mediterranean Sea, respectively, with hits to genomes in the non-redundant viral RefSeq database. The identifiable richness and relative abundance in both viromes were dominated by archaeal and bacterial viruses accounting for 92.3% of the relative abundance in the Atlantic Ocean and for 83.6% in the Mediterranean Sea. Despite characteristic differences in hydrographic features between the sampling sites in the Atlantic Ocean and the Mediterranean Sea, 440 virus genomes were found in both viromes. An additional 431 virus genomes were identified in the Atlantic Ocean and 75 virus genomes were only found in the Mediterranean Sea. The results indicate that the rather contrasting deep-sea environments of the Atlantic Ocean and the Mediterranean Sea share a common core set of virus types constituting the majority of both virus communities in terms of relative abundance (Atlantic Ocean: 81.4%; Mediterranean Sea: 88.7%). PMID:24959907

Elevational characteristics of the archaeal community in full-scale activated sludge wastewater treatment plants at a 3,660-meter elevational scale.

PubMed

Niu, Lihua; Zhang, Xue; Li, Yi; Wang, Peifang; Zhang, Wenlong; Wang, Chao; Wang, Qing

2017-07-01

Due to the important roles of archaea in wastewater treatment processes, archaeal communities have been studied extensively in various anaerobic reactors, but the knowledge of archaeal communities in full-scale activated sludge wastewater treatment plants (WWTPs) remains quite poor. In this study, 454-pyrosequencing was for the first time employed to investigate archaeal communities from 20 full-scale activated sludge WWTPs distributed at a 3,660-meter elevational scale in China. Results showed that archaeal communities from WWTPs were dominated by Methanosarcinales (84.6%). A core archaeal population (94.5%) composed of Methanosaeta, Methanosarcina, Methanogenium and Methanobrevibacter was shared among WWTPs. The elevational pattern of archaeal communities was observed in WWTPs, with an elevational threshold associated with archaeal community richness and structures at approximately 1,500 meters above sea level (masl). A declining trend in community richness with increasing elevation was observed at higher elevations, whereas no trend was presented at lower elevations. Spearman correlation analysis indicated that the archaeal community richness at higher elevations was associated with more environmental variables than that at lower elevations. Redundancy analysis indicated that wastewater variables were the dominant contributors to the variation of community structures at higher elevations, followed by operational variables and elevation.

Gut microbiota can transfer fiber characteristics and lipid metabolic profiles of skeletal muscle from pigs to germ-free mice.

PubMed

Yan, Honglin; Diao, Hui; Xiao, Yi; Li, Wenxia; Yu, Bing; He, Jun; Yu, Jie; Zheng, Ping; Mao, Xiangbing; Luo, Yuheng; Zeng, Benhua; Wei, Hong; Chen, Daiwen

2016-08-22

Obesity causes changes in microbiota composition, and an altered gut microbiota can transfer obesity-associated phenotypes from donors to recipients. Obese Rongchang pigs (RP) exhibited distinct fiber characteristics and lipid metabolic profiles in their muscle compared with lean Yorkshire pigs (YP). However, whether RP have a different gut microbiota than YP and whether there is a relationship between the microbiota and muscle properties are poorly understood. The present study was conducted to test whether the muscle properties can be transferred from pigs to germ-free (GF) mice. High-throughput pyrosequencing confirms the presence of distinct core microbiota between pig breeds, with alterations in taxonomic distribution and modulations in β diversity. RP displayed a significant higher Firmicutes/Bacteroidetes ratio and apparent genera differences compared with YP. Transplanting the porcine microbiota into GF mice replicated the phenotypes of the donors. RP and their GF mouse recipients exhibited a higher body fat mass, a higher slow-contracting fiber proportion, a decreased fiber size and fast IIb fiber percentage, and enhanced lipogenesis in the gastrocnemius muscle. Furthermore, the gut microbiota composition of colonized mice shared high similarity with their donor pigs. Taken together, the gut microbiota of obese pigs intrinsically influences skeletal muscle development and the lipid metabolic profiles.

Geomicrobiology of Fe-rich crusts in Lake Superior sediment

NASA Astrophysics Data System (ADS)

Dittrich, M.; Monreau, L.; Quazi, S.; Raoof, B.; Chesnyuk, A.; Katsev, S.; Fulthorpe, R.

2012-04-01

The limnological puzzles of Lake Superior are increasingly attracting scientists, and very little is known about the sediments and their associated microflora. The sediments are organic poor (less than 5%C) and the lake is deep oligotrophic, with water temperatures at the bottom around 3C. Previous studies reveal Fe-rich layers in the sediments at multiple loccations around the lake. The origin and mechanisms of formation of this layer remain unknown. In this study we investigated geochemical and microbiological processes that may lead to the formation of a two cm thick iron layer about 10 cm below the sediment surface. Sediment cores from two stations (EM, 230m water depth and ED, 310m water depth) in the East Basin were used. We monitored oxygen and pH depth profiles with microsensors, porewater and sediment solid matter were analyzed for nutrient and metal contents. Furthermore, phosphorus and iron sequantial extractions of sediment cores have been perfomed. The total cell count was determined using DAPI epifluoresence microscopy. DNA was extracted from the sediment samples and 16S ribosonal RNA amplicons were analyzed with denaturing gradient gel electrophoresis (DGGE). For a more in depth analysis, DNA samples from 8-10 cm and 10-12 cm were sent to the Research and Testing Lab (Texas) for pyrosequencing of 16S rRNA gene amplicons amplified using barcoded universal primers 27f-519r. The scanning electron microscope (SEM) images from the iron layer 10-12cm show filaments that were encrusted with spheres ca. 20 nm in diameter. SEM observations of thin sections also indicate the presence of very fine particles showing various morphologies. Analyses of the deposit material by SEM and energy dispersive X-ray spectroscopy (EDS) indicate that bacteria cells surfaces served as nucleation surfaces for Fe-oxide formation. EDS line-scans through bacterial cells covered with precipitates reveal phosphorus and carbon peaks at interface between cell surface and Fe-particles. The cluster analysis performed on the DGGE separation of ribosomal RNA gene fragments revealed that the two iron layers were not highly similar to each other. We obtained a total of 26,062 16S rRNA gene sequence reads from the two iron layers and the layers directly above them, which were clustered into operational taxonomic units sharing 80% similarity or more. 64-70% of these clusters could not be classified below the phylum level. While the 8-10 cm sediment layers were dominated (46.5% of reads) by relatives of Paenisporosarcina, the iron layers contained far fewer gram positive organisms, far more proteobacteria, and an a high proportion of Nitrospira species which show relatively high similarity to organisms found in an iron II rich seep.

How Do Astronomers Share Data? Reliability and Persistence of Datasets Linked in AAS Publications and a Qualitative Study of Data Practices among US Astronomers

PubMed Central

Pepe, Alberto; Goodman, Alyssa; Muench, August; Crosas, Merce; Erdmann, Christopher

2014-01-01

We analyze data sharing practices of astronomers over the past fifteen years. An analysis of URL links embedded in papers published by the American Astronomical Society reveals that the total number of links included in the literature rose dramatically from 1997 until 2005, when it leveled off at around 1500 per year. The analysis also shows that the availability of linked material decays with time: in 2011, 44% of links published a decade earlier, in 2001, were broken. A rough analysis of link types reveals that links to data hosted on astronomers' personal websites become unreachable much faster than links to datasets on curated institutional sites. To gauge astronomers' current data sharing practices and preferences further, we performed in-depth interviews with 12 scientists and online surveys with 173 scientists, all at a large astrophysical research institute in the United States: the Harvard-Smithsonian Center for Astrophysics, in Cambridge, MA. Both the in-depth interviews and the online survey indicate that, in principle, there is no philosophical objection to data-sharing among astronomers at this institution. Key reasons that more data are not presently shared more efficiently in astronomy include: the difficulty of sharing large data sets; over reliance on non-robust, non-reproducible mechanisms for sharing data (e.g. emailing it); unfamiliarity with options that make data-sharing easier (faster) and/or more robust; and, lastly, a sense that other researchers would not want the data to be shared. We conclude with a short discussion of a new effort to implement an easy-to-use, robust, system for data sharing in astronomy, at theastrodata.org, and we analyze the uptake of that system to-date. PMID:25165807

How do astronomers share data? Reliability and persistence of datasets linked in AAS publications and a qualitative study of data practices among US astronomers.

PubMed

Pepe, Alberto; Goodman, Alyssa; Muench, August; Crosas, Merce; Erdmann, Christopher

2014-01-01

We analyze data sharing practices of astronomers over the past fifteen years. An analysis of URL links embedded in papers published by the American Astronomical Society reveals that the total number of links included in the literature rose dramatically from 1997 until 2005, when it leveled off at around 1500 per year. The analysis also shows that the availability of linked material decays with time: in 2011, 44% of links published a decade earlier, in 2001, were broken. A rough analysis of link types reveals that links to data hosted on astronomers' personal websites become unreachable much faster than links to datasets on curated institutional sites. To gauge astronomers' current data sharing practices and preferences further, we performed in-depth interviews with 12 scientists and online surveys with 173 scientists, all at a large astrophysical research institute in the United States: the Harvard-Smithsonian Center for Astrophysics, in Cambridge, MA. Both the in-depth interviews and the online survey indicate that, in principle, there is no philosophical objection to data-sharing among astronomers at this institution. Key reasons that more data are not presently shared more efficiently in astronomy include: the difficulty of sharing large data sets; over reliance on non-robust, non-reproducible mechanisms for sharing data (e.g. emailing it); unfamiliarity with options that make data-sharing easier (faster) and/or more robust; and, lastly, a sense that other researchers would not want the data to be shared. We conclude with a short discussion of a new effort to implement an easy-to-use, robust, system for data sharing in astronomy, at theastrodata.org, and we analyze the uptake of that system to-date.

How Do Astronomers Share Data? Reliability and Persistence of Datasets Linked in AAS Publications and a Qualitative Study of Data Practices among US Astronomers

NASA Astrophysics Data System (ADS)

Pepe, Alberto; Goodman, Alyssa; Muench, August; Crosas, Merce; Erdmann, Christopher

2014-08-01

We analyze data sharing practices of astronomers over the past fifteen years. An analysis of URL links embedded in papers published by the American Astronomical Society reveals that the total number of links included in the literature rose dramatically from 1997 until 2005, when it leveled off at around 1500 per year. The analysis also shows that the availability of linked material decays with time: in 2011, 44% of links published a decade earlier, in 2001, were broken. A rough analysis of link types reveals that links to data hosted on astronomers' personal websites become unreachable much faster than links to datasets on curated institutional sites. To gauge astronomers' current data sharing practices and preferences further, we performed in-depth interviews with 12 scientists and online surveys with 173 scientists, all at a large astrophysical research institute in the United States: the Harvard-Smithsonian Center for Astrophysics, in Cambridge, MA. Both the in-depth interviews and the online survey indicate that, in principle, there is no philosophical objection to data-sharing among astronomers at this institution. Key reasons that more data are not presently shared more efficiently in astronomy include: the difficulty of sharing large data sets; over reliance on non-robust, non-reproducible mechanisms for sharing data (e.g. emailing it); unfamiliarity with options that make data-sharing easier (faster) and/or more robust; and, lastly, a sense that other researchers would not want the data to be shared. We conclude with a short discussion of a new effort to implement an easy-to-use, robust, system for data sharing in astronomy, at theastrodata.org, and we analyze the uptake of that system to-date.

Mineralogical controls on surface colonization by sulfur-metabolizing microbial communities

NASA Astrophysics Data System (ADS)

Jones, A. A.; Bennett, P.

2012-12-01

When characterizing microbial diversity and the microbial ecosystem of the shallow subsurface the mineral matrix is generally assumed to be homogenous and unreactive. We report here experimental evidence that microorganisms colonize rock surfaces according to the rock's chemistry and the organism's metabolic requirements and tolerances. We investigated this phenomenon using laboratory biofilm reactors with both a pure culture of sulfur-oxidizing Thiothrix unzii and a mixed environmental sulfur-metabolizing community from Lower Kane, Cave, WY, USA. Reactors contained rock and mineral chips (calcite, albite, microcline, quartz, chert, Madison Limestone (ML), Madison Dolostone (MD), and basalt) amended with one of the two inoculants. Biomass of attached microorganisms on each mineral surface was quantified. The 16S rRNA of attached microbial communities were compared using Roche FLX and Titanium 454 next generation pyrosequencing. A primary controlling factor on taxonomy of attached microorganisms in both pure and mixed culture experiments was mineral buffering capacity. In mixed culture experiments acid-buffering carbonates were preferentially colonized by neutrophilic sulfur-oxidizing microorganisms (~18% to ~27% of microorganisms), while acidophilic sulfur-oxidizing microorganisms colonized non-buffering quartz exclusively (~46% of microorganisms). The nutrient content of the rock was a controlling factor on biomass accumulation, with neutrophilic organisms selecting between carbonate surfaces of equivalent buffer capacities according to the availability of phosphate. Dry biomass on ML was 17.8 ± 2.3 mg/cm2 and MD was 20.6 ± 6.8 mg/cm2; while nutrient poor calcite accumulated 2.4 ± 0.3 mg/cm2. Biomass accumulation was minimal on non-buffering nutrient-limited surfaces. These factors are countered by the competitive exclusion of some populations. A pure culture of T. unzii preferentially colonizes carbonates while a very closely related Thiothrix spp is excluded from these same rock samples in a mixed culture. Diversity analysis reveals that ML, MD, and calcite have >98% of sequences belonging to shared OTUs. The carbonates have <3% of sequences belonging to OTUs shared with any silicate mineral surface with the exception of basalt (~85% similarity). These four surfaces were host to the least diverse microbial communities, suggesting that competitive exclusion of microorganisms not adapted to these surfaces is a controlling variable on taxonomy. Furthermore, the microorganisms on basalt reveal an unique association between Thiothrix unzii (often found in mid-ocean ridge environments) and basalt, where it excludes other sulfur oxidizers and accumulates the highest non-carbonate biomass in both pure (3.5 ± 1.0 mg/cm2) and mixed culture (5.4 ± 1.4 mg/cm2) experiments. This association suggests that adaptations to specific rocks may be retained even when the organism is displaced from an ancestral rock/mineral surface habitat. Combined, these variables (buffering capacity, nutrient availability, competitive exclusion, tolerance of surface geochemistry, and latent adaptations) affect biomass density, local diversity, and global diversity of the attached communities on mineral and rock surfaces and suggest that different populations are more tolerant of, and more competitive on, specific rock/mineral types.

Nitrogen removal performance and loading capacity of a novel single-stage nitritation-anammox system with syntrophic micro-granules.

PubMed

Wang, Shaopo; Liu, Yuan; Niu, Qigui; Ji, Jiayuan; Hojo, Toshimasa; Li, Yu-You

2017-07-01

The operation performance of a novel micro-granule based syntrophic system of nitritation and anammox was studied by controlling the oxygen concentration and maintaining a constant temperature of 25°C. With the oxygen concentration of around 0.11 (<0.15)mg/L, the single-stage nitritation-anammox system was startup successfully at a nitrogen loading rate (NLR) of 1.5kgN/m 3 /d. The reactor was successfully operated at volumetric N loadings ranging from 0.5 to 2.5kgN/m 3 /d with a high nitrogen removal of 82%. The microbial community was composed by ammonia oxidizing bacteria (AOB) and anammox bacteria forming micro-granules with an average diameter of 0.8mm and good settleability. Results from pyrosequencing analysis revealed that Ca. Kuenenia and Nitrosomonas were selected and enriched in the community over the startup period, and these were identified as the dominant anammox bacteria and AOB species, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reactor performance and microbial community dynamics during anaerobic co-digestion of municipal wastewater sludge with restaurant grease waste at steady state and overloading stages.

PubMed

Razaviarani, Vahid; Buchanan, Ian D

2014-11-01

Linkage between reactor performance and microbial community dynamics was investigated during mesophilic anaerobic co-digestion of restaurant grease waste (GTW) with municipal wastewater sludge (MWS) using 10L completely mixed reactors and a 20day SRT. Test reactors received a mixture of GTW and MWS while control reactors received only MWS. Addition of GTW to the test reactors enhanced the biogas production and methane yield by up to 65% and 120%, respectively. Pyrosequencing revealed that Methanosaeta and Methanomicrobium were the dominant acetoclastic and hydrogenotrophic methanogen genera, respectively, during stable reactor operation. The number of Methanosarcina and Methanomicrobium sequences increased and that of Methanosaeta declined when the proportion of GTW in the feed was increased to cause an overload condition. Under this overload condition, the pH, alkalinity and methane production decreased and VFA concentrations increased dramatically. Candidatus cloacamonas, affiliated within phylum Spirochaetes, were the dominant bacterial genus at all reactor loadings. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effects of Hypoxia on the Phylogenetic Composition and Species Distribution of Protists in a Subtropical Harbor.

PubMed

Rocke, Emma; Jing, Hongmei; Xia, Xiaomin; Liu, Hongbin

2016-07-01

Tolo Harbor, a subtropical semi-enclosed coastal water body, is surrounded by an expanding urban community, which contributes to large concentrations of nutrient runoff, leading to algal blooms and localized hypoxic episodes. Present knowledge of protist distributions in subtropical waters during hypoxic conditions is very limited. In this study, therefore, we combined parallel 454 pyrosequencing technology and denaturing gradient gel electrophoresis (DGGE) fingerprint analyses to reveal the protist community shifts before, during, and after a 2-week hypoxic episode during the summer of 2011. Hierarchical clustering for DGGE demonstrated similar grouping of hypoxic samples separately from oxic samples. Dissolved oxygen (DO) concentration and dissolved inorganic nitrogen:phosphate (DIN:PO4) concentrations significantly affected OTU distribution in 454 sequenced samples, and a shift toward a ciliate and marine alveolate clade II (MALV II) species composition occurred as waters shifted from oxic to hypoxic. These results suggest that protist community shifts toward heterotrophic and parasitic tendencies as well as decreased diversity and richness in response to hypoxic outbreaks.

Enhanced phenol removal in an innovative lignite activated coke-assisted biological process.

PubMed

Zhang, Chen; Li, Jianfeng; Cheng, Fangqin; Liu, Yu

2018-07-01

In this study, a lignite activated coke (LAC)-assisted activated sludge (AS) process was developed for enhancing biodegradation of phenol, while the effects of LAC on sludge properties and microbial community structure were investigated. It was found that more than 90% of phenol was removed within 1 h in the LAC/AS, which was 3 times higher than the conventional AS process. Moreover, the floc size and settleability were also significantly improved in the LAC/AS. These results suggested that LAC could serve as the nucleating agent to promote the formation of compact floc, which was beneficial for toxicity mitigation and system stability. The microbial community analysis by 16S high-throughput pyrosequencing technology further revealed a more abundant bacterial richness and diversity in the LAC/AS process loaded with phenol, while some phenol degraders, such as Propionibacteriaceae were enriched. Engineering implications further suggests the LAC-assisted AS process is technically sound and economically viable. Copyright © 2018 Elsevier Ltd. All rights reserved.

Novel Rickettsiella Bacterium in the Leafhopper Orosius albicinctus (Hemiptera: Cicadellidae)

PubMed Central

Iasur-Kruh, Lilach; Weintraub, Phyllis G.; Mozes-Daube, Netta; Robinson, Wyatt E.; Perlman, Steve J.

2013-01-01

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens. PMID:23645190

Insights into the respiratory tract microbiota of patients with cystic fibrosis during early Pseudomonas aeruginosa colonization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keravec, Marlene; Mounier, Jerome; Prestat , Emmanuel

Abstract Pseudomonas aeruginosa plays a major role in cystic fibrosis (CF) progression. Therefore, it is important to understand the initial steps of P. aeruginosa infection. The structure and dynamics of CF respiratory tract microbial communities during the early stages of P. aeruginosa colonization were characterized by pyrosequencing and cloning-sequencing. The respiratory microbiota showed high diversity, related to the young age of the CF cohort (mean age 10 years). Wide inter- and intra-individual variations were revealed. A common core microbiota of 5 phyla and 13 predominant genera was found, the majority of which were obligate anaerobes. A few genera were significantlymore » more prevalent in patients never infected by P. aeruginosa. Persistence of an anaerobic core microbiota regardless of P. aeruginosa status suggests a major role of certain anaerobes in the pathophysiology of lung infections in CF. Some genera may be potential biomarkers of pulmonary infection state.« less

The Sequence of Two Bacteriophages with Hypermodified Bases Reveals Novel Phage-Host Interactions.

PubMed

Kropinski, Andrew M; Turner, Dann; Nash, John H E; Ackermann, Hans-Wolfgang; Lingohr, Erika J; Warren, Richard A; Ehrlich, Kenneth C; Ehrlich, Melanie

2018-04-24

Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas ) acidovorans , respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this is that the physico-chemical properties of the DNA are radically altered (melting temperature (Tm), buoyant density and susceptibility to restriction endonucleases). Using 454 pyrosequencing technology, we sequenced the genomes of both viruses. Phage ΦW-14 possesses a 157-kb genome (56.3% GC) specifying 236 proteins, while SP-15 is larger at 222 kb (38.6 mol % G + C) and encodes 318 proteins. In both cases, the phages can be considered genomic singletons since they do not possess BLASTn homologs. While no obvious genes were identified as being responsible for the modified base in ΦW-14, SP-15 contains a cluster of genes obviously involved in carbohydrate metabolism.

Associations between MHC genes and Puumala virus infection in Myodes glareolus are detected in wild populations, but not from experimental infection data.

PubMed

Guivier, Emmanuel; Galan, Maxime; Malé, Pierre-Jean G; Kallio, Eva R; Voutilainen, Liina; Henttonen, Heikki; Olsson, Gert E; Lundkvist, Ake; Tersago, Katrien; Augot, Denis; Cosson, Jean-François; Charbonnel, Nathalie

2010-10-01

We analysed the influence of MHC class II Dqa and Drb genes on Puumala virus (PUUV) infection in bank voles (Myodes glareolus). We considered voles sampled in five European localities or derived from a previous experiment that showed variable infection success of PUUV. The genetic variation observed in the Dqa and Drb genes was assessed by using single-strand conformation polymorphism and pyrosequencing methods, respectively. Patterns were compared with those obtained from 13 microsatellites. We revealed significant genetic differentiation between PUUV-seronegative and -seropositive bank voles sampled in wild populations, at the Drb gene only. The absence of genetic differentiation observed at neutral microsatellites confirmed the important role of selective pressures in shaping these Drb patterns. Also, we found no significant associations between infection success and MHC alleles among laboratory-colonized bank voles, which is explained by a loss of genetic variability that occurred during the captivity of these voles.

Changes in the bacterial community in the fermentation process of kôso, a Japanese sugar-vegetable fermented beverage.

PubMed

Chiou, Tai-Ying; Suda, Wataru; Oshima, Kenshiro; Hattori, Masahira; Takahashi, Tomoya

2017-02-01

Kôso is a Japanese fermented beverage made with over 20 kinds of vegetables, mushrooms, and sugars. The changes in the bacterial population of kôso during fermentation at 25 °C over a period of 10 days were studied using 454 pyrosequencing of the 16S rRNA gene. The analysis detected 224 operational taxonomic units (OTUs) clustered from 8 DNA samples collected on days 0, 3, 7, and 10 from two fermentation batches. Proteobacteria were the dominant phylum in the starting community, but were replaced by Firmicutes within three days. Seventy-eight genera were identified from the 224 OTUs, in which Bifidobacterium, Leuconostoc, Lactococcus, and Lactobacillus dominated, accounting for over 96% of the total bacterial population after three days' fermentation. UniFrac-Principal Coordinate Analysis of longitudinal fermented samples revealed dramatic changes in the bacterial community in kôso, resulting in significantly low diversity at the end of fermentation as compared with the complex starting community.

Bacterial communities on food court tables and cleaning equipment in a shopping mall.

PubMed

Dingsdag, S; Coleman, N V

2013-08-01

The food court at a shopping mall is a potential transfer point for pathogenic microbes, but to date, this environment has not been the subject of detailed molecular microbiological study. We used a combination of culture-based and culture-independent approaches to investigate the types and numbers of bacteria present on food court tables, and on a food court cleaning cloth. Bacteria were found at 10²-10⁵ c.f.u./m² on food court tables and 10¹⁰ c.f.u./m² on the cleaning cloth. Tag-pyrosequencing of amplified 16S rRNA genes revealed that the dominant bacterial types on the cleaning cloth were genera known to include pathogenic species (Stenotrophomonas, Aeromonas), and that these genera were also evident at lower levels on table surfaces, suggesting possible cross-contamination. The evidence suggests a public health threat is posed by bacteria in the food court, and that this may be due to cross-contamination between cleaning equipment and table surfaces.

Anammox biofilm in activated sludge swine wastewater treatment plants.

PubMed

Suto, Ryu; Ishimoto, Chikako; Chikyu, Mikio; Aihara, Yoshito; Matsumoto, Toshimi; Uenishi, Hirohide; Yasuda, Tomoko; Fukumoto, Yasuyuki; Waki, Miyoko

2017-01-01

We investigated anammox with a focus on biofilm in 10 wastewater treatment plants (WWTPs) that use activated sludge treatment of swine wastewater. In three plants, we found red biofilms in aeration tanks or final sedimentation tanks. The biofilm had higher anammox 16S rRNA gene copy numbers (up to 1.35 × 10 12 copies/g-VSS) and higher anammox activity (up to 295 μmoL/g-ignition loss/h) than suspended solids in the same tank. Pyrosequencing analysis revealed that Planctomycetes accounted for up to 17.7% of total reads in the biofilm. Most of them were related to Candidatus Brocadia or Ca. Jettenia. The highest copy number and the highest proportion of Planctomycetes were comparable to those of enriched anammox sludge. Thus, swine WWTPs that use activated sludge treatment can fortuitously acquire anammox biofilm. Thus, concentrated anammox can be detected by focusing on red biofilm. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of Nitrification Efficiency and Microbial Community in a Membrane Bioreactor Fed with Low COD/N-Ratio Wastewater

PubMed Central

Ma, Jinxing; Wang, Zhiwei; Zhu, Chaowei; Liu, Shumeng; Wang, Qiaoying; Wu, Zhichao

2013-01-01

In this study, an approach using influent COD/N ratio reduction was employed to improve process performance and nitrification efficiency in a membrane bioreactor (MBR). Besides sludge reduction, membrane fouling alleviation was observed during 330 d operation, which was attributed to the decreased production of soluble microbial products (SMP) and efficient carbon metabolism in the autotrophic nitrifying community. 454 high-throughput 16S rRNA gene pyrosequencing revealed that the diversity of microbial sequences was mainly determined by the feed characteristics, and that microbes could derive energy by switching to a more autotrophic metabolism to resist the environmental stress. The enrichment of nitrifiers in an MBR with a low COD/N-ratio demonstrated that this condition stimulated nitrification, and that the community distribution of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) resulted in faster nitrite uptake rates. Further, ammonia oxidation was the rate-limiting step during the full nitrification. PMID:23667573

Effect of haylage and monensin supplementation on ruminal bacterial communities of feedlot cattle.

PubMed

Kim, Minseok; Felix, Tara L; Loerch, Steve C; Yu, Zhongtang

2014-08-01

The objective of this study was to investigate the ruminal bacterial communities as affected by monensin, haylage, and their interaction of feedlot cattle fed 60 % dried distillers grains with solubles in a replicated 4 × 4 Latin square design. Pyrosequencing analysis of the V1-V3 region (about 500 bp) of 16S rRNA gene from the four dietary treatments (3 treatment plus one control diets) collectively revealed 51 genera of bacteria within 11 phyla. Firmicutes and Bacteroidetes were the first and the second most predominant phyla, respectively, irrespective of the dietary treatments. Monensin supplementation decreased the proportion of Gram-positive Firmicutes while increasing that of Gram-negative Bacteroidetes. However, the monensin supplementation did not reduce the proportion of all genera of Gram-positive bacteria placed within Firmicutes and lowered that of some genera of Gram-negative bacteria placed within Bacteroidetes. Haylage supplementation appeared to attenuate inhibition of monensin on some genera of bacteria. Factors other than monensin and haylage could affect ruminal bacterial communities.

A comparison of microbial characteristics between the thermophilic and mesophilic anaerobic digesters exposed to elevated food waste loadings.

PubMed

Guo, Xiaohui; Wang, Cheng; Sun, Faqian; Zhu, Weijing; Wu, Weixiang

2014-01-01

Thermophilic and mesophilic anaerobic digestion reactors (TR and MR) using food waste as substrate were compared with emphasis on microbial responses to increasing organic loading rate (OLR). At OLR ranging from 1.0 to 2.5 g VS L(-1) d(-1), MR exhibited more stable performance compared to TR in terms of methane yield. Amplicons pyrosequencing results revealed the distinct microbial dynamics in the two reactors. Primarily, MR had greater richness and evenness of bacteria species. With OLR elevated, larger shifts of bacterial phylogeny were observed in MR; Methanosaeta dominated in archaeal community in MR while Methanothermobacter and Methanoculleus were favored in TR. The high functional redundancy in bacterial community integrated with acetoclastic methanogenesis in MR resulted in its better performance; whereas delicate interactions between hydrogen-producer and hydrogenotrophic methanogens in TR were much more prone to disruption. These results are conductive to understanding the microbial mechanisms of low methane yield during food waste anaerobic digestion. Copyright © 2013 Elsevier Ltd. All rights reserved.

The first genome-level transcriptome of the wood-degrading fungus Phanerochaete chrysosporium grown on red oak.

PubMed

Sato, Shin; Feltus, F Alex; Iyer, Prashanti; Tien, Ming

2009-06-01

As part of an effort to determine all the gene products involved in wood degradation, we have performed massively parallel pyrosequencing on an expression library from the white rot fungus Phanerochaete chrysosporium grown in shallow stationary cultures with red oak as the carbon source. Approximately 48,000 high quality sequence tags (246 bp average length) were generated. 53% of the sequence tags aligned to 4,262 P. chrysosporium gene models, and an additional 18.5% of the tags reliably aligned to the P. chrysosporium genome providing evidence for 961 putative novel fragmented gene models. Due to their role in lignocellulose degradation, the secreted proteins were focused upon. Our results show that the four enzymes required for cellulose degradation: endocellulase, exocellulase CBHI, exocellulase CBHII, and beta-glucosidase are all produced. For hemicellulose degradation, not all known enzymes were produced, but endoxylanases, acetyl xylan esterases and mannosidases were detected. For lignin degradation, the role of peroxidases has been questioned; however, our results show that lignin peroxidase is highly expressed along with the H(2)O(2) generating enzyme, alcohol oxidase. The transcriptome snapshot reveals that H(2)O(2) generation and utilization are central in wood degradation. Our results also reveal new transcripts that encode extracellular proteins with no known function.

Molecular markers reveal infestation dynamics of the bed bug (Hemiptera: Cimicidae) within apartment buildings.

PubMed

Booth, Warren; Saenz, Virna L; Santangelo, Richard G; Wang, Changlu; Schal, Coby; Vargo, Edward L

2012-05-01

The bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), has experienced an extraordinary global resurgence in recent years, the reasons for which remain poorly understood. Once considered a pest of lower socioeconomic classes, bed bugs are now found extensively across all residential settings, with widespread infestations established in multiapartment buildings. Within such buildings, understanding the population genetic structure and patterns of dispersal may prove critical to the development of effective control strategies. Here, we describe the development of 24 high-resolution microsatellite markers through next generation 454 pyrosequencing and their application to elucidate infestation dynamics within three multistory apartment buildings in the United States. Results reveal contrasting characteristics potentially representative of geographic or locale differences. In Raleigh, NC, an infestation within an apartment building seemed to have started from a single introduction followed by extensive spread. In Jersey City, NJ, two or more introductions followed by spread are evident in two buildings. Populations within single apartments in all buildings were characterized by high levels of relatedness and low levels of diversity, indicative of foundation from small, genetically depauperate propagules. Regardless of the number of unique introductions, genetic data indicate that spread within buildings is extensive, supporting both active and human-mediated dispersal within and between adjacent rooms or apartments spanning multiple floors.

Egypt's Red Sea coast: phylogenetic analysis of cultured microbial consortia in industrialized sites

PubMed Central

Mustafa, Ghada A.; Abd-Elgawad, Amr; Abdel-Haleem, Alyaa M.; Siam, Rania

2014-01-01

The Red Sea possesses a unique geography, and its shores are rich in mangrove, macro-algal and coral reef ecosystems. Various sources of pollution affect Red Sea biota, including microbial life. We assessed the effects of industrialization on microbes along the Egyptian Red Sea coast at eight coastal sites and two lakes. The bacterial communities of sediment samples were analyzed using bacterial 16S rDNA pyrosequencing of V6-V4 hypervariable regions. The taxonomic assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled sites: Proteobacteria (68%), Firmicutes (13%), Fusobacteria (12%), Bacteriodetes (6%), and Spirochetes (0.03%). Further analysis revealed distinct bacterial consortia that primarily included (1) marine Vibrio spp.—suggesting a “marine Vibrio phenomenon”; (2) potential human pathogens; and (3) oil-degrading bacteria. We discuss two divergent microbial consortia that were sampled from Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; these consortia contained the highest abundance of human pathogens and no pathogens, respectively. Our results draw attention to the effects of industrialization on the Red Sea and suggest the need for further analysis to overcome the hazardous effects observed at the impacted sites. PMID:25157243

Mapping and determinism of soil microbial community distribution across an agricultural landscape

PubMed Central

Constancias, Florentin; Terrat, Sébastien; Saby, Nicolas P A; Horrigue, Walid; Villerd, Jean; Guillemin, Jean-Philippe; Biju-Duval, Luc; Nowak, Virginie; Dequiedt, Samuel; Ranjard, Lionel; Chemidlin Prévost-Bouré, Nicolas

2015-01-01

Despite the relevance of landscape, regarding the spatial patterning of microbial communities and the relative influence of environmental parameters versus human activities, few investigations have been conducted at this scale. Here, we used a systematic grid to characterize the distribution of soil microbial communities at 278 sites across a monitored agricultural landscape of 13 km². Molecular microbial biomass was estimated by soil DNA recovery and bacterial diversity by 16S rRNA gene pyrosequencing. Geostatistics provided the first maps of microbial community at this scale and revealed a heterogeneous but spatially structured distribution of microbial biomass and diversity with patches of several hundreds of meters. Variance partitioning revealed that both microbial abundance and bacterial diversity distribution were highly dependent of soil properties and land use (total variance explained ranged between 55% and 78%). Microbial biomass and bacterial richness distributions were mainly explained by soil pH and texture whereas bacterial evenness distribution was mainly related to land management. Bacterial diversity (richness, evenness, and Shannon index) was positively influenced by cropping intensity and especially by soil tillage, resulting in spots of low microbial diversity in soils under forest management. Spatial descriptors also explained a small but significant portion of the microbial distribution suggesting that landscape configuration also shapes microbial biomass and bacterial diversity. PMID:25833770

The vertical distribution of prokaryotes in the surface sediment of Jiaolong cold seep at the northern South China Sea.

PubMed

Wu, Yuzhi; Qiu, Jian-Wen; Qian, Pei-Yuan; Wang, Yong

2018-05-01

In deep-sea cold seeps, microbial communities are shaped by geochemical components in seepage solutions. In the present study, we report the composition of microbial communities and potential metabolic activities in the surface sediment of Jiaolong cold seep at the northern South China Sea. Pyrosequencing of 16S rRNA gene amplicons revealed that a majority of the microbial inhabitants of the surface layers (0-6 cm) were sulfur oxidizer bacteria Sulfurimonas and archaeal methane consumer ANME-1, while sulfate reducer bacteria SEEP-SRB1, ANME-1 and ANME-2 dominated the bottom layers (8-14 cm). The potential ecological roles of the microorganisms were further supported by the presence of functional genes for methane oxidation, sulfur oxidation, sulfur reduction and nitrate reduction in the metagenomes. Metagenomic analysis revealed a significant correlation between coverage of 16S rRNA gene of sulfur oxidizer bacteria, functional genes involved in sulfur oxidation and nitrate reduction in different layers, indicating that sulfur oxidizing may be coupled to nitrate reducing at the surface layers of Jiaolong seeping site. This is probably related to the sulfur oxidizers of Sulfurimonas and Sulfurovum, which may be the capacity of nitrate reduction or associated with unidentified syntrophic nitrate-reducing microbes in the surface of the cold seep.

Analysis of microbial community composition in a lab-scale membrane distillation bioreactor.

PubMed

Zhang, Q; Shuwen, G; Zhang, J; Fane, A G; Kjelleberg, S; Rice, S A; McDougald, D

2015-04-01

Membrane distillation bioreactors (MDBR) have potential for industrial applications where wastewater is hot or waste heat is available, but the role of micro-organisms in MDBRs has never been determined, and thus was the purpose of this study. Microbial communities were characterized by bacterial and archaeal 16S and eukaryotic 18S rRNA gene tag-encoded pyrosequencing of DNA obtained from sludge. Taxonomy-independent analysis revealed that bacterial communities had a relatively low richness and diversity, and community composition strongly correlated with conductivity, total nitrogen and bound extracellular polymeric substances (EPS). Taxonomy-dependent analysis revealed that Rubrobacter and Caldalkalibacillus were abundant members of the bacterial community, but no archaea were detected. Eukaryotic communities had a relatively high richness and diversity, and both changes in community composition and abundance of the dominant genus, Candida, correlated with bound EPS. Thermophilic MDBR communities were comprised of a low diversity bacterial community and a highly diverse eukaryotic community with no archea detected. Communities exhibited low resilience to changes in operational parameters. Specifically, retenatate nutrient composition and concentration was strongly correlated with the dominant species. This study provides an understanding of microbial community diversity in an MDBR, which is fundamental to the optimization of reactor performance. © 2015 The Authors published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.

The transcriptome recipe for the venom cocktail of Tityus bahiensis scorpion.

PubMed

de Oliveira, Ursula Castro; Candido, Denise Maria; Dorce, Valquíria Abrão Coronado; Junqueira-de-Azevedo, Inácio de Loiola Meirelles

2015-03-01

Scorpion venom is a mixture of peptides, including antimicrobial, bradykinin-potentiating and anionic peptides and small to medium proteins, such as ion channel toxins, metalloproteinases and phospholipases that together cause severe clinical manifestation. Tityus bahiensis is the second most medically important scorpion species in Brazil and it is widely distributed in the country with the exception of the North Region. Here we sequenced and analyzed the transcripts from the venom glands of T. bahiensis, aiming at identifying and annotating venom gland expressed genes. A total of 116,027 long reads were generated by pyrosequencing and assembled in 2891 isotigs. An annotation process identified transcripts by similarity to known toxins, revealing that putative venom components represent 7.4% of gene expression. The major toxins identified are potassium and sodium channel toxins, whereas metalloproteinases showed an unexpected high abundance. Phylogenetic analysis of deduced metalloproteinases from T. bahiensis and other scorpions revealed a pattern of ancient and intraspecific gene expansions. Other venom molecules identified include antimicrobial, anionic and bradykinin-potentiating peptides, besides several putative new venom components. This report provides the first attempt to massively identify the venom components of this species and constitutes one of the few transcriptomic efforts on the genus Tityus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Subgingival microbiome in patients with healthy and ailing dental implants

PubMed Central

Zheng, Hui; Xu, Lixin; Wang, Zicheng; Li, Lianshuo; Zhang, Jieni; Zhang, Qian; Chen, Ting; Lin, Jiuxiang; Chen, Feng

2015-01-01

Dental implants are commonly used to replace missing teeth. However, the dysbiotic polymicrobial communities of peri-implant sites are responsible for peri-implant diseases, such as peri-implant mucositis and peri-implantitis. In this study, we analyzed the microbial characteristics of oral plaque from peri-implant pockets or sulci of healthy implants (n = 10), peri-implant mucositis (n = 8) and peri-implantitis (n = 6) sites using pyrosequencing of the 16S rRNA gene. An increase in microbial diversity was observed in subgingival sites of ailing implants, compared with healthy implants. Microbial co-occurrence analysis revealed that periodontal pathogens, such as Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, were clustered into modules in the peri-implant mucositis network. Putative pathogens associated with peri-implantitis were present at a moderate relative abundance in peri-implant mucositis, suggesting that peri-implant mucositis an important early transitional phase during the development of peri-implantitis. Furthermore, the relative abundance of Eubacterium was increased at peri-implantitis locations, and co-occurrence analysis revealed that Eubacterium minutum was correlated with Prevotella intermedia in peri-implantitis sites, which suggests the association of Eubacterium with peri-implantitis. This study indicates that periodontal pathogens may play important roles in the shifting of healthy implant status to peri-implant disease. PMID:26077225

Molecular Characterization of Arbuscular Mycorrhizal Fungi in an Agroforestry System Reveals the Predominance of Funneliformis spp. Associated with Colocasia esculenta and Pterocarpus officinalis Adult Trees and Seedlings.

PubMed

Geoffroy, Alexandre; Sanguin, Hervé; Galiana, Antoine; Bâ, Amadou

2017-01-01

Pterocarpus officinalis (Jacq.) is a leguminous forestry tree species endemic to Caribbean swamp forests. In Guadeloupe, smallholder farmers traditionally cultivate flooded taro ( Colocasia esculenta ) cultures under the canopy of P. officinalis stands. The role of arbuscular mycorrhizal (AM) fungi in the sustainability of this traditional agroforestry system has been suggested but the composition and distribution of AM fungi colonizing the leguminous tree and/or taro are poorly characterized. An in-depth characterization of root-associated AM fungal communities from P. officinalis adult trees and seedlings and taro cultures, sampled in two localities of Guadeloupe, was performed by pyrosequencing (GS FLX+) of partial 18S rRNA gene. The AM fungal community was composed of 215 operational taxonomic units (OTUs), belonging to eight fungal families dominated by Glomeraceae, Acaulosporaceae, and Gigasporaceae. Results revealed a low AM fungal community membership between P. officinalis and C. esculenta . However, certain AM fungal community taxa (10% of total community) overlapped between P. officinalis and C. esculenta , notably predominant Funneliformis OTUs. These findings provide new perspectives in deciphering the significance of Funneliformis in nutrient exchange between P. officinalis and C. esculenta by forming a potential mycorrhizal network.

Molecular Characterization of Arbuscular Mycorrhizal Fungi in an Agroforestry System Reveals the Predominance of Funneliformis spp. Associated with Colocasia esculenta and Pterocarpus officinalis Adult Trees and Seedlings

PubMed Central

Geoffroy, Alexandre; Sanguin, Hervé; Galiana, Antoine; Bâ, Amadou

2017-01-01

Pterocarpus officinalis (Jacq.) is a leguminous forestry tree species endemic to Caribbean swamp forests. In Guadeloupe, smallholder farmers traditionally cultivate flooded taro (Colocasia esculenta) cultures under the canopy of P. officinalis stands. The role of arbuscular mycorrhizal (AM) fungi in the sustainability of this traditional agroforestry system has been suggested but the composition and distribution of AM fungi colonizing the leguminous tree and/or taro are poorly characterized. An in-depth characterization of root-associated AM fungal communities from P. officinalis adult trees and seedlings and taro cultures, sampled in two localities of Guadeloupe, was performed by pyrosequencing (GS FLX+) of partial 18S rRNA gene. The AM fungal community was composed of 215 operational taxonomic units (OTUs), belonging to eight fungal families dominated by Glomeraceae, Acaulosporaceae, and Gigasporaceae. Results revealed a low AM fungal community membership between P. officinalis and C. esculenta. However, certain AM fungal community taxa (10% of total community) overlapped between P. officinalis and C. esculenta, notably predominant Funneliformis OTUs. These findings provide new perspectives in deciphering the significance of Funneliformis in nutrient exchange between P. officinalis and C. esculenta by forming a potential mycorrhizal network. PMID:28804479

Comparative Genome Analysis of Ciprofloxacin-Resistant Pseudomonas aeruginosa Reveals Genes Within Newly Identified High Variability Regions Associated With Drug Resistance Development

PubMed Central

Su, Hsun-Cheng; Khatun, Jainab; Kanavy, Dona M.

2013-01-01

The alarming rise of ciprofloxacin-resistant Pseudomonas aeruginosa has been reported in several clinical studies. Though the mutation of resistance genes and their role in drug resistance has been researched, the process by which the bacterium acquires high-level resistance is still not well understood. How does the genomic evolution of P. aeruginosa affect resistance development? Could the exposure of antibiotics to the bacteria enrich genomic variants that lead to the development of resistance, and if so, how are these variants distributed through the genome? To answer these questions, we performed 454 pyrosequencing and a whole genome analysis both before and after exposure to ciprofloxacin. The comparative sequence data revealed 93 unique resistance strain variation sites, which included a mutation in the DNA gyrase subunit A gene. We generated variation-distribution maps comparing the wild and resistant types, and isolated 19 candidates from three discrete resistance-associated high variability regions that had available transposon mutants, to perform a ciprofloxacin exposure assay. Of these region candidates with transposon disruptions, 79% (15/19) showed a reduction in the ability to gain high-level resistance, suggesting that genes within these high variability regions might enrich for certain functions associated with resistance development. PMID:23808957

Bacterial Diversity Analysis during the Fermentation Processing of Traditional Chinese Yellow Rice Wine Revealed by 16S rDNA 454 Pyrosequencing.

PubMed

Fang, Ruo-si; Dong, Ya-chen; Chen, Feng; Chen, Qi-he

2015-10-01

Rice wine is a traditional Chinese fermented alcohol drink. Spontaneous fermentation with the use of the Chinese starter and wheat Qu lead to the growth of various microorganisms during the complete brewing process. It's of great importance to fully understand the composition of bacteria diversity in rice wine in order to improve the quality and solve safety problems. In this study, a more comprehensive bacterial description was shown with the use of bacteria diversity analysis, which enabled us to have a better understanding. Rarefaction, rank abundance, alpha Diversity, beta diversity and principal coordinates analysis simplified their complex bacteria components and provide us theoretical foundation for further investigation. It has been found bacteria diversity is more abundant at mid-term and later stage of brewing process. Bacteria community analysis reveals there is a potential safety hazard existing in the fermentation, since most of the sequence reads are assigned to Enterobacter (7900 at most) and Pantoea (7336 at most), followed by Staphylococcus (2796 at most) and Pseudomonas (1681 at most). Lactic acid bacteria are rare throughout the fermentation process which is not in accordance with other reports. This work may offer us an opportunity to investigate micro ecological fermentation system in food industry. © 2015 Institute of Food Technologists®

Analysis of microbial community composition in a lab-scale membrane distillation bioreactor

PubMed Central

Zhang, Q; Shuwen, G; Zhang, J; Fane, AG; Kjelleberg, S; Rice, SA; McDougald, D

2015-01-01

Aims Membrane distillation bioreactors (MDBR) have potential for industrial applications where wastewater is hot or waste heat is available, but the role of micro-organisms in MDBRs has never been determined, and thus was the purpose of this study. Methods and Results Microbial communities were characterized by bacterial and archaeal 16S and eukaryotic 18S rRNA gene tag-encoded pyrosequencing of DNA obtained from sludge. Taxonomy-independent analysis revealed that bacterial communities had a relatively low richness and diversity, and community composition strongly correlated with conductivity, total nitrogen and bound extracellular polymeric substances (EPS). Taxonomy-dependent analysis revealed that Rubrobacter and Caldalkalibacillus were abundant members of the bacterial community, but no archaea were detected. Eukaryotic communities had a relatively high richness and diversity, and both changes in community composition and abundance of the dominant genus, Candida, correlated with bound EPS. Conclusions Thermophilic MDBR communities were comprised of a low diversity bacterial community and a highly diverse eukaryotic community with no archea detected. Communities exhibited low resilience to changes in operational parameters. Specifically, retenatate nutrient composition and concentration was strongly correlated with the dominant species. Significance and Impact of the Study This study provides an understanding of microbial community diversity in an MDBR, which is fundamental to the optimization of reactor performance. PMID:25604265

Treatment of oil sands process-affected water (OSPW) using a membrane bioreactor with a submerged flat-sheet ceramic microfiltration membrane.

PubMed

Xue, Jinkai; Zhang, Yanyan; Liu, Yang; Gamal El-Din, Mohamed

2016-01-01

The release of oil sands process-affected water (OSPW) into the environment is a concern because it contains persistent organic pollutants that are toxic to aquatic life. A modified Ludzack-Ettinger membrane bioreactor (MLE-MBR) with a submerged ceramic membrane was continuously operated for 425 days to evaluate its feasibility on OSPW treatment. A stabilized biomass concentration of 3730 mg mixed liquor volatile suspended solids per litre and a naphthenic acid (NA) removal of 24.7% were observed in the reactor after 361 days of operation. Ultra Performance Liquid Chromatography/High Resolution Mass Spectrometry analysis revealed that the removal of individual NA species declined with increased ring numbers. Pyrosequencing analysis revealed that Betaproteobacteria were dominant in sludge samples from the MLE-MBR, with microorganisms such as Rhodocyclales and Sphingobacteriales capable of degrading hydrocarbon and aromatic compounds. During 425 days of continuous operation, no severe membrane fouling was observed as the transmembrane pressure (TMP) of the MLE-MBR never exceeded -20 kPa given that the manufacturer's suggested critical TMP for chemical cleaning is -35 kPa. Our results indicated that the proposed MLE-MBR has a good potential for removing recalcitrant organics in OSPW. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library.

PubMed

Nguyen, Nhung Hong; Maruset, Lalita; Uengwetwanit, Tanaporn; Mhuantong, Wuttichai; Harnpicharnchai, Piyanun; Champreda, Verawat; Tanapongpipat, Sutipa; Jirajaroenrat, Kanya; Rakshit, Sudip K; Eurwilaichitr, Lily; Pongpattanakitshote, Somchai

2012-01-01

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.

Microbiote shift in the Medicago sativa rhizosphere in response to cyanotoxins extract exposure.

PubMed

El Khalloufi, Fatima; Oufdou, Khalid; Bertrand, Marie; Lahrouni, Majida; Oudra, Brahim; Ortet, Philippe; Barakat, Mohamed; Heulin, Thierry; Achouak, Wafa

2016-01-01

The bloom-containing water bodies may have an impact due to cyanotoxins production on other microorganisms and aquatic plants. Where such water is being used for crops irrigation, the presence of cyanotoxins may also have a toxic impact on terrestrial plants and their rhizosphere microbiota. For that purpose, PCR-based 454 pyrosequencing was applied to phylogenetically characterize the bacterial community of Medicago sativa rhizosphere in response to cyanotoxins extract. This analysis revealed a wide diversity at species level, which decreased from unplanted soil to root tissues indicating that only some populations were able to compete for nutrients and niches in this selective habitat. Gemmatimonas, Actinobacteria, Deltaproteobacteria and Opitutae mainly inhabited the bulk soil, whereas, the root-adhering soil and the root tissues were inhabited by Gammaproteobacteria and Alphaproteobacteria. The proportion of these populations fluctuated in response to cyanotoxins extract exposure. Betaproteobacteria proportion increased in the three studied compartments, whereas Gammaproteobacteria proportion decreased except in the bulk soil. This study revealed the potential toxicity of cyanotoxins extract towards Actinobacteria, Gemmatimonas, Deltaproteobacteria, and Gammaproteobacteria, however Clostridia, Opitutae and bacteria related with Betaproteobacteria, were stimulated denoting their tolerance. Altogether, these data indicate that crop irrigation using cyanotoxins containing water might alter the rhizosphere functioning. Copyright © 2015 Elsevier B.V. All rights reserved.

Y-chromosomal analysis of Greek Cypriots reveals a primarily common pre-Ottoman paternal ancestry with Turkish Cypriots

PubMed Central

Fernández-Domínguez, Eva; Bertoncini, Stefania; Chimonas, Marios; Christofi, Vasilis; King, Jonathan; Budowle, Bruce; Manoli, Panayiotis

2017-01-01

Genetics can provide invaluable information on the ancestry of the current inhabitants of Cyprus. A Y-chromosome analysis was performed to (i) determine paternal ancestry among the Greek Cypriot (GCy) community in the context of the Central and Eastern Mediterranean and the Near East; and (ii) identify genetic similarities and differences between Greek Cypriots (GCy) and Turkish Cypriots (TCy). Our haplotype-based analysis has revealed that GCy and TCy patrilineages derive primarily from a single gene pool and show very close genetic affinity (low genetic differentiation) to Calabrian Italian and Lebanese patrilineages. In terms of more recent (past millennium) ancestry, as indicated by Y-haplotype sharing, GCy and TCy share much more haplotypes between them than with any surrounding population (7–8% of total haplotypes shared), while TCy also share around 3% of haplotypes with mainland Turks, and to a lesser extent with North Africans. In terms of Y-haplogroup frequencies, again GCy and TCy show very similar distributions, with the predominant haplogroups in both being J2a-M410, E-M78, and G2-P287. Overall, GCy also have a similar Y-haplogroup distribution to non-Turkic Anatolian and Southwest Caucasian populations, as well as Cretan Greeks. TCy show a slight shift towards Turkish populations, due to the presence of Eastern Eurasian (some of which of possible Ottoman origin) Y-haplogroups. Overall, the Y-chromosome analysis performed, using both Y-STR haplotype and binary Y-haplogroup data puts Cypriot in the middle of a genetic continuum stretching from the Levant to Southeast Europe and reveals that despite some differences in haplotype sharing and haplogroup structure, Greek Cypriots and Turkish Cypriots share primarily a common pre-Ottoman paternal ancestry. PMID:28622394

Y-chromosomal analysis of Greek Cypriots reveals a primarily common pre-Ottoman paternal ancestry with Turkish Cypriots.

PubMed

Heraclides, Alexandros; Bashiardes, Evy; Fernández-Domínguez, Eva; Bertoncini, Stefania; Chimonas, Marios; Christofi, Vasilis; King, Jonathan; Budowle, Bruce; Manoli, Panayiotis; Cariolou, Marios A

2017-01-01

Genetics can provide invaluable information on the ancestry of the current inhabitants of Cyprus. A Y-chromosome analysis was performed to (i) determine paternal ancestry among the Greek Cypriot (GCy) community in the context of the Central and Eastern Mediterranean and the Near East; and (ii) identify genetic similarities and differences between Greek Cypriots (GCy) and Turkish Cypriots (TCy). Our haplotype-based analysis has revealed that GCy and TCy patrilineages derive primarily from a single gene pool and show very close genetic affinity (low genetic differentiation) to Calabrian Italian and Lebanese patrilineages. In terms of more recent (past millennium) ancestry, as indicated by Y-haplotype sharing, GCy and TCy share much more haplotypes between them than with any surrounding population (7-8% of total haplotypes shared), while TCy also share around 3% of haplotypes with mainland Turks, and to a lesser extent with North Africans. In terms of Y-haplogroup frequencies, again GCy and TCy show very similar distributions, with the predominant haplogroups in both being J2a-M410, E-M78, and G2-P287. Overall, GCy also have a similar Y-haplogroup distribution to non-Turkic Anatolian and Southwest Caucasian populations, as well as Cretan Greeks. TCy show a slight shift towards Turkish populations, due to the presence of Eastern Eurasian (some of which of possible Ottoman origin) Y-haplogroups. Overall, the Y-chromosome analysis performed, using both Y-STR haplotype and binary Y-haplogroup data puts Cypriot in the middle of a genetic continuum stretching from the Levant to Southeast Europe and reveals that despite some differences in haplotype sharing and haplogroup structure, Greek Cypriots and Turkish Cypriots share primarily a common pre-Ottoman paternal ancestry.

Shared decision-making in medical encounters regarding breast cancer treatment: the contribution of methodological triangulation.

PubMed

Durif-Bruckert, C; Roux, P; Morelle, M; Mignotte, H; Faure, C; Moumjid-Ferdjaoui, N

2015-07-01

The aim of this study on shared decision-making in the doctor-patient encounter about surgical treatment for early-stage breast cancer, conducted in a regional cancer centre in France, was to further the understanding of patient perceptions on shared decision-making. The study used methodological triangulation to collect data (both quantitative and qualitative) about patient preferences in the context of a clinical consultation in which surgeons followed a shared decision-making protocol. Data were analysed from a multi-disciplinary research perspective (social psychology and health economics). The triangulated data collection methods were questionnaires (n = 132), longitudinal interviews (n = 47) and observations of consultations (n = 26). Methodological triangulation revealed levels of divergence and complementarity between qualitative and quantitative results that suggest new perspectives on the three inter-related notions of decision-making, participation and information. Patients' responses revealed important differences between shared decision-making and participation per se. The authors note that subjecting patients to a normative behavioural model of shared decision-making in an era when paradigms of medical authority are shifting may undermine the patient's quest for what he or she believes is a more important right: a guarantee of the best care available. © 2014 John Wiley & Sons Ltd.

Transcriptome assembly and digital gene expression atlas of the rainbow trout

USDA-ARS?s Scientific Manuscript database

Background: Transcriptome analysis is a preferred method for gene discovery, marker development and gene expression profiling in non-model organisms. Previously, we sequenced a transcriptome reference using Sanger-based and 454-pyrosequencing, however, a transcriptome assembly is still incomplete an...

Functional characterization of two concrete biofilms using pyrosequencing data

EPA Science Inventory

Phylogenetic studies of concrete biofilms using 16SrRNA-based approaches have demonstrated that concrete surfaces harbor a diverse microbial community. These approaches can provide information on the general taxonomical groups present in a sample but cannot shed light on the func...