Analysis of bacterial communities and bacterial pathogens in a biogas plant by the combination of ethidium monoazide, PCR and Ion Torrent sequencing.

PubMed

Luo, Gang; Angelidaki, Irini

2014-09-01

The present study investigated the changes of bacterial community composition including bacterial pathogens along a biogas plant, i.e. from the influent, to the biogas reactor and to the post-digester. The effects of post-digestion temperature and time on the changes of bacterial community composition and bacterial pathogens were also studied. Microbial analysis was made by Ion Torrent sequencing of the PCR amplicons from ethidium monoazide treated samples, and ethidium monoazide was used to cleave DNA from dead cells and exclude it from PCR amplification. Both similarity and taxonomic analysis showed that the bacterial community composition in the influent was changed after anaerobic digestion. Firmicutes were dominant in all the samples, while Proteobacteria decreased in the biogas reactor compared with the influent. Variations of bacterial community composition in the biogas reactor with time were also observed. This could be attributed to varying composition of the influent. Batch experiments showed that the methane recovery from the digested residues (obtained from biogas reactor) was mainly related with post-digestion temperature. However, post-digestion time rather than temperature had a significant effect on the changes of bacterial community composition. The changes of bacterial community composition were also reflected in the changes of relative abundance of bacterial pathogens. The richness and relative abundance of bacterial pathogens were reduced after anaerobic digestion in the biogas reactor. It was found in batch experiments that bacterial pathogens showed the highest relative abundance and richness after 30 days' post-digestion. Streptococcus bovis was found in all the samples. Our results showed that special attention should be paid to the post-digestion since the increase in relative abundance of bacterial pathogens after post-digestion might reflect regrowth of bacterial pathogens and limit biosolids disposal vectors. Copyright © 2014 Elsevier

Gene calling and bacterial genome annotation with BG7.

PubMed

Tobes, Raquel; Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Kovach, Evdokim; Alekhin, Alexey; Pareja, Eduardo

2015-01-01

New massive sequencing technologies are providing many bacterial genome sequences from diverse taxa but a refined annotation of these genomes is crucial for obtaining scientific findings and new knowledge. Thus, bacterial genome annotation has emerged as a key point to investigate in bacteria. Any efficient tool designed specifically to annotate bacterial genomes sequenced with massively parallel technologies has to consider the specific features of bacterial genomes (absence of introns and scarcity of nonprotein-coding sequence) and of next-generation sequencing (NGS) technologies (presence of errors and not perfectly assembled genomes). These features make it convenient to focus on coding regions and, hence, on protein sequences that are the elements directly related with biological functions. In this chapter we describe how to annotate bacterial genomes with BG7, an open-source tool based on a protein-centered gene calling/annotation paradigm. BG7 is specifically designed for the annotation of bacterial genomes sequenced with NGS. This tool is sequence error tolerant maintaining their capabilities for the annotation of highly fragmented genomes or for annotating mixed sequences coming from several genomes (as those obtained through metagenomics samples). BG7 has been designed with scalability as a requirement, with a computing infrastructure completely based on cloud computing (Amazon Web Services).

Potential use of bacterial community succession for estimating post-mortem interval as revealed by high-throughput sequencing

PubMed Central

Guo, Juanjuan; Fu, Xiaoliang; Liao, Huidan; Hu, Zhenyu; Long, Lingling; Yan, Weitao; Ding, Yanjun; Zha, Lagabaiyila; Guo, Yadong; Yan, Jie; Chang, Yunfeng; Cai, Jifeng

2016-01-01

Decomposition is a complex process involving the interaction of both biotic and abiotic factors. Microbes play a critical role in the process of carrion decomposition. In this study, we analysed bacterial communities from live rats and rat remains decomposed under natural conditions, or excluding sarcosaphagous insect interference, in China using Illumina MiSeq sequencing of 16S rRNA gene amplicons. A total of 1,394,842 high-quality sequences and 1,938 singleton operational taxonomic units were obtained. Bacterial communities showed notable variation in relative abundance and became more similar to each other across body sites during the decomposition process. As decomposition progressed, Proteobacteria (mostly Gammaproteobacteria) became the predominant phylum in both the buccal cavity and rectum, while Firmicutes and Bacteroidetes in the mouth and rectum, respectively, gradually decreased. In particular, the arrival and oviposition of sarcosaphagous insects had no obvious influence on bacterial taxa composition, but accelerated the loss of biomass. In contrast to the rectum, the microbial community structure in the buccal cavity of live rats differed considerably from that of rats immediately after death. Although this research indicates that bacterial communities can be used as a “microbial clock” for the estimation of post-mortem interval, further work is required to better understand this concept. PMID:27052375

The bias associated with amplicon sequencing does not affect the quantitative assessment of bacterial community dynamics.

PubMed

Ibarbalz, Federico M; Pérez, María Victoria; Figuerola, Eva L M; Erijman, Leonardo

2014-01-01

The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1-V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1-V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1-V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa-time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1-V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point.

The Bias Associated with Amplicon Sequencing Does Not Affect the Quantitative Assessment of Bacterial Community Dynamics

PubMed Central

Figuerola, Eva L. M.; Erijman, Leonardo

2014-01-01

The performance of two sets of primers targeting variable regions of the 16S rRNA gene V1–V3 and V4 was compared in their ability to describe changes of bacterial diversity and temporal turnover in full-scale activated sludge. Duplicate sets of high-throughput amplicon sequencing data of the two 16S rRNA regions shared a collection of core taxa that were observed across a series of twelve monthly samples, although the relative abundance of each taxon was substantially different between regions. A case in point was the changes in the relative abundance of filamentous bacteria Thiothrix, which caused a large effect on diversity indices, but only in the V1–V3 data set. Yet the relative abundance of Thiothrix in the amplicon sequencing data from both regions correlated with the estimation of its abundance determined using fluorescence in situ hybridization. In nonmetric multidimensional analysis samples were distributed along the first ordination axis according to the sequenced region rather than according to sample identities. The dynamics of microbial communities indicated that V1–V3 and the V4 regions of the 16S rRNA gene yielded comparable patterns of: 1) the changes occurring within the communities along fixed time intervals, 2) the slow turnover of activated sludge communities and 3) the rate of species replacement calculated from the taxa–time relationships. The temperature was the only operational variable that showed significant correlation with the composition of bacterial communities over time for the sets of data obtained with both pairs of primers. In conclusion, we show that despite the bias introduced by amplicon sequencing, the variable regions V1–V3 and V4 can be confidently used for the quantitative assessment of bacterial community dynamics, and provide a proper qualitative account of general taxa in the community, especially when the data are obtained over a convenient time window rather than at a single time point. PMID:24923665

Sequences of multiple bacterial genomes and a Chlamydia trachomatis genotype from direct sequencing of DNA derived from a vaginal swab diagnostic specimen.

PubMed

Andersson, P; Klein, M; Lilliebridge, R A; Giffard, P M

2013-09-01

Ultra-deep Illumina sequencing was performed on whole genome amplified DNA derived from a Chlamydia trachomatis-positive vaginal swab. Alignment of reads with reference genomes allowed robust SNP identification from the C. trachomatis chromosome and plasmid. This revealed that the C. trachomatis in the specimen was very closely related to the sequenced urogenital, serovar F, clade T1 isolate F-SW4. In addition, high genome-wide coverage was obtained for Prevotella melaninogenica, Gardnerella vaginalis, Clostridiales genomosp. BVAB3 and Mycoplasma hominis. This illustrates the potential of metagenome data to provide high resolution bacterial typing data from multiple taxa in a diagnostic specimen. ©2013 The Authors Clinical Microbiology and Infection ©2013 European Society of Clinical Microbiology and Infectious Diseases.

A Bacterial Analysis Platform: An Integrated System for Analysing Bacterial Whole Genome Sequencing Data for Clinical Diagnostics and Surveillance.

PubMed

Thomsen, Martin Christen Frølund; Ahrenfeldt, Johanne; Cisneros, Jose Luis Bellod; Jurtz, Vanessa; Larsen, Mette Voldby; Hasman, Henrik; Aarestrup, Frank Møller; Lund, Ole

2016-01-01

Recent advances in whole genome sequencing have made the technology available for routine use in microbiological laboratories. However, a major obstacle for using this technology is the availability of simple and automatic bioinformatics tools. Based on previously published and already available web-based tools we developed a single pipeline for batch uploading of whole genome sequencing data from multiple bacterial isolates. The pipeline will automatically identify the bacterial species and, if applicable, assemble the genome, identify the multilocus sequence type, plasmids, virulence genes and antimicrobial resistance genes. A short printable report for each sample will be provided and an Excel spreadsheet containing all the metadata and a summary of the results for all submitted samples can be downloaded. The pipeline was benchmarked using datasets previously used to test the individual services. The reported results enable a rapid overview of the major results, and comparing that to the previously found results showed that the platform is reliable and able to correctly predict the species and find most of the expected genes automatically. In conclusion, a combined bioinformatics platform was developed and made publicly available, providing easy-to-use automated analysis of bacterial whole genome sequencing data. The platform may be of immediate relevance as a guide for investigators using whole genome sequencing for clinical diagnostics and surveillance. The platform is freely available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 and it is the intention that it will continue to be expanded with new features as these become available.

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

High-throughput nucleotide sequence analysis of diverse bacterial communities in leachates of decomposing pig carcasses

PubMed Central

Yang, Seung Hak; Lim, Joung Soo; Khan, Modabber Ahmed; Kim, Bong Soo; Choi, Dong Yoon; Lee, Eun Young; Ahn, Hee Kwon

2015-01-01

The leachate generated by the decomposition of animal carcass has been implicated as an environmental contaminant surrounding the burial site. High-throughput nucleotide sequencing was conducted to investigate the bacterial communities in leachates from the decomposition of pig carcasses. We acquired 51,230 reads from six different samples (1, 2, 3, 4, 6 and 14 week-old carcasses) and found that sequences representing the phylum Firmicutes predominated. The diversity of bacterial 16S rRNA gene sequences in the leachate was the highest at 6 weeks, in contrast to those at 2 and 14 weeks. The relative abundance of Firmicutes was reduced, while the proportion of Bacteroidetes and Proteobacteria increased from 3–6 weeks. The representation of phyla was restored after 14 weeks. However, the community structures between the samples taken at 1–2 and 14 weeks differed at the bacterial classification level. The trend in pH was similar to the changes seen in bacterial communities, indicating that the pH of the leachate could be related to the shift in the microbial community. The results indicate that the composition of bacterial communities in leachates of decomposing pig carcasses shifted continuously during the study period and might be influenced by the burial site. PMID:26500442

Molecular Characterization of Epiphytic Bacterial Communities on Charophycean Green Algae

PubMed Central

Fisher, Madeline M.; Wilcox, Lee W.; Graham, Linda E.

1998-01-01

Epiphytic bacterial communities within the sheath material of three filamentous green algae, Desmidium grevillii, Hyalotheca dissiliens, and Spondylosium pulchrum (class Charophyceae, order Zygnematales), collected from a Sphagnum bog were characterized by PCR amplification, cloning, and sequencing of 16S ribosomal DNA. A total of 20 partial sequences and nine different sequence types were obtained, and one sequence type was recovered from the bacterial communities on all three algae. By phylogenetic analysis, the cloned sequences were placed into several major lineages of the Bacteria domain: the Flexibacter/Cytophaga/Bacteroides phylum and the α, β, and γ subdivisions of the phylum Proteobacteria. Analysis at the subphylum level revealed that the majority of our sequences were not closely affiliated with those of known, cultured taxa, although the estimated evolutionary distances between our sequences and their nearest neighbors were always less than 0.1 (i.e., greater than 90% similar). This result suggests that the majority of sequences obtained in this study represent as yet phenotypically undescribed bacterial species and that the range of bacterial-algal interactions that occur in nature has not yet been fully described. PMID:9797295

SSPACE-LongRead: scaffolding bacterial draft genomes using long read sequence information

PubMed Central

2014-01-01

Background The recent introduction of the Pacific Biosciences RS single molecule sequencing technology has opened new doors to scaffolding genome assemblies in a cost-effective manner. The long read sequence information is promised to enhance the quality of incomplete and inaccurate draft assemblies constructed from Next Generation Sequencing (NGS) data. Results Here we propose a novel hybrid assembly methodology that aims to scaffold pre-assembled contigs in an iterative manner using PacBio RS long read information as a backbone. On a test set comprising six bacterial draft genomes, assembled using either a single Illumina MiSeq or Roche 454 library, we show that even a 50× coverage of uncorrected PacBio RS long reads is sufficient to drastically reduce the number of contigs. Comparisons to the AHA scaffolder indicate our strategy is better capable of producing (nearly) complete bacterial genomes. Conclusions The current work describes our SSPACE-LongRead software which is designed to upgrade incomplete draft genomes using single molecule sequences. We conclude that the recent advances of the PacBio sequencing technology and chemistry, in combination with the limited computational resources required to run our program, allow to scaffold genomes in a fast and reliable manner. PMID:24950923

Identification of the bacterial etiology of culture-negative endocarditis by amplification and sequencing of a small ribosomal RNA gene.

PubMed

Khulordava, Irakli; Miller, Geraldine; Haas, David; Li, Haijing; McKinsey, Joel; Vanderende, Daniel; Tang, Yi-Wei

2003-05-01

We report two cases of culture-negative bacterial endocarditis in which the organisms were identified by amplification and sequencing of the bacterial 16S rRNA gene. These results support an important role for polymerase chain reaction followed by direct sequencing to determine the etiology of culture-negative bacterial endocarditis and to guide appropriate antimicrobial therapy.

Bacterial diversity of the American sand fly Lutzomyia intermedia using high-throughput metagenomic sequencing.

PubMed

Monteiro, Carolina Cunha; Villegas, Luis Eduardo Martinez; Campolina, Thais Bonifácio; Pires, Ana Clara Machado Araújo; Miranda, Jose Carlos; Pimenta, Paulo Filemon Paolucci; Secundino, Nagila Francinete Costa

2016-08-31

Parasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector. We performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota. Bacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups. The metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing

Bacterial community comparisons by taxonomy-supervised analysis independent of sequence alignment and clustering

PubMed Central

Sul, Woo Jun; Cole, James R.; Jesus, Ederson da C.; Wang, Qiong; Farris, Ryan J.; Fish, Jordan A.; Tiedje, James M.

2011-01-01

High-throughput sequencing of 16S rRNA genes has increased our understanding of microbial community structure, but now even higher-throughput methods to the Illumina scale allow the creation of much larger datasets with more samples and orders-of-magnitude more sequences that swamp current analytic methods. We developed a method capable of handling these larger datasets on the basis of assignment of sequences into an existing taxonomy using a supervised learning approach (taxonomy-supervised analysis). We compared this method with a commonly used clustering approach based on sequence similarity (taxonomy-unsupervised analysis). We sampled 211 different bacterial communities from various habitats and obtained ∼1.3 million 16S rRNA sequences spanning the V4 hypervariable region by pyrosequencing. Both methodologies gave similar ecological conclusions in that β-diversity measures calculated by using these two types of matrices were significantly correlated to each other, as were the ordination configurations and hierarchical clustering dendrograms. In addition, our taxonomy-supervised analyses were also highly correlated with phylogenetic methods, such as UniFrac. The taxonomy-supervised analysis has the advantages that it is not limited by the exhaustive computation required for the alignment and clustering necessary for the taxonomy-unsupervised analysis, is more tolerant of sequencing errors, and allows comparisons when sequences are from different regions of the 16S rRNA gene. With the tremendous expansion in 16S rRNA data acquisition underway, the taxonomy-supervised approach offers the potential to provide more rapid and extensive community comparisons across habitats and samples. PMID:21873204

Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems

PubMed Central

Shaw, Jennifer L. A.; Weyrich, Laura S.; Sawade, Emma; Drikas, Mary; Cooper, Alan J.

2015-01-01

Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. PMID:26162884

Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons.

PubMed

Połka, Justyna; Rebecchi, Annalisa; Pisacane, Vincenza; Morelli, Lorenzo; Puglisi, Edoardo

2015-04-01

The bacterial diversity involved in food fermentations is one of the most important factors shaping the final characteristics of traditional foods. Knowledge about this diversity can be greatly improved by the application of high-throughput sequencing technologies (HTS) coupled to the PCR amplification of the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were carried out in order to be compared with HTS results. A total of 722,196 high quality sequences were obtained after trimming, paired-reads assembly and quality screening of raw reads obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable regions V3 and V4; manual curation of 16S database allowed a correct taxonomical classification at the species for 99.5% of these reads. Results confirmed the presence of main bacterial species involved in the fermentation of salami as assessed by PCR-DGGE, but with a greater extent of resolution and quantitative assessments that are not possible by the mere analyses of gel banding patterns. Thirty-two different Staphylococcus and 33 Lactobacillus species where identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the observed diversity. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating that HTS has the potential

Amplicon sequencing of bacterial microbiota in abortion material from cattle.

PubMed

Vidal, Sara; Kegler, Kristel; Posthaus, Horst; Perreten, Vincent; Rodriguez-Campos, Sabrina

2017-10-10

Abortions in cattle have a significant economic impact on animal husbandry and require prompt diagnosis for surveillance of epizootic infectious agents. Since most abortions are not epizootic but sporadic with often undetected etiologies, this study examined the bacterial community present in the placenta (PL, n = 32) and fetal abomasal content (AC, n = 49) in 64 cases of bovine abortion by next generation sequencing (NGS) of the 16S rRNA gene. The PL and AC from three fetuses of dams that died from non-infectious reasons were included as controls. All samples were analyzed by bacterial culture, and 17 were examined by histopathology. We observed 922 OTUs overall and 267 taxa at the genus level. No detectable bacterial DNA was present in the control samples. The microbial profiles of the PL and AC differed significantly, both in their composition (PERMANOVA), species richness and Chao-1 (Mann-Whitney test). In both organs, Pseudomonas was the most abundant genus. The combination of NGS and culture identified opportunistic pathogens of interest in placentas with lesions, such as Vibrio metschnikovii, Streptococcus uberis, Lactococcus lactis and Escherichia coli. In placentas with lesions where culturing was unsuccessful, Pseudomonas and unidentified Aeromonadaceae were identified by NGS displaying high number of reads. Three cases with multiple possible etiologies and placentas presenting lesions were detected by NGS. Amplicon sequencing has the potential to uncover unknown etiological agents. These new insights on cattle abortion extend our focus to previously understudied opportunistic abortive bacteria.

Characterization of bacterial communities associating with larval development of Yesso Scallop ( Patinopecten yessoensisis Jay, 1857) by high-throughput sequencing

NASA Astrophysics Data System (ADS)

Sun, Xueying; Liu, Jichen; Li, Ming; Zhao, Xuewei; Liang, Jun; Sun, Pihai; Ma, Yuexin

2016-12-01

Bacterial community presumably plays an essential role in inhibiting pathogen colonization and maintaining the health of scallop larvae, but limiting data are available for Yesso scallop ( Patinopecten yessoensisis Jay, 1857) larval development stages. The aim of this study was to characterize and compare the bacterial communities associating with Yesso scallop larval development at fertilized egg S1, trochophora S2, D-shaped larvae S3, umbo larvae S4, and juvenile scallop S5 stages by Illumina high-throughput sequencing. Genomic DNA was extracted from the larvae and their associating bactera, and a gene segment covering V3-V4 region of 16S rRNA gene was amplified and sequenced using an Illumina Miseq sequencer. Overall, 106760 qualified sequences with an average length of 449 bp were obtained. Sequences were compared with those retrieved from 16S rRNA gene databases, and 4 phyla, 7 classes, 15 orders, 21 families, 31 genera were identified. Proteobacteria was predominant phylum, accounting for more than 99%, at all 5 larval development stages. At genus level, Pseudomonas was dominant at stages S1 (80.60%), S2 (87.77%) and S5 (68.71%), followed by Photobacterium (17.06%) and Aeromonas (1.64%) at stage S1, Serratia (6.94%), Stenotrophomonas (3.08%) and Acinetobacter (1.2%) at stage S2, Shewanella (25.95%) and Pseudoalteromonas (4.57%) at stage S5. Moreover, genus Pseudoalteromonas became dominant at stages S3 (44.85%) and S4 (56.02%), followed by Photobacterium (29.82%), Pseudomonas (11.86%), Aliivibrio (8.60%) and Shewanella (3.39%) at stage S3, Pseudomonas (18.16%), Aliivibrio (14.29%), Shewanella (4.11%), Psychromonas (4.04%) and Psychrobacter (1.81%) at stage S4. From the results, we concluded that the bacterial community changed significantly at different development stages of Yesso Scallop larvae.

Are commercial providers a viable option for clinical bacterial sequencing?

PubMed

Raven, Kathy; Blane, Beth; Churcher, Carol; Parkhill, Julian; Peacock, Sharon J

2018-04-05

Bacterial whole-genome sequencing in the clinical setting has the potential to bring major improvements to infection control and clinical practice. Sequencing instruments are not currently available in the majority of routine microbiology laboratories worldwide, but an alternative is to use external sequencing providers. To foster discussion around this we investigated whether send-out services were a viable option. Four providers offering MiSeq sequencing were selected based on cost and evaluated based on the service provided and sequence data quality. DNA was prepared from five methicillin-resistant Staphylococcus aureus (MRSA) isolates, four of which were investigated during a previously published outbreak in the UK together with a reference MRSA isolate (ST22 HO 5096 0412). Cost of sequencing per isolate ranged from £155 to £342 and turnaround times from DNA postage to arrival of sequence data ranged from 12 to 63 days. Comparison of commercially generated genomes against the original sequence data demonstrated very high concordance, with no more than one single nucleotide polymorphism (SNP) difference on core genome mapping between the original sequences and the new sequence for all four providers. Multilocus sequence type could not be assigned based on assembly for the two cheapest sequence providers due to fragmented assemblies probably caused by a lower output of sequence data per isolate. Our results indicate that external providers returned highly accurate genome data, but that improvements are required in turnaround time to make this a viable option for use in clinical practice.

RNA-Seq for Bacterial Gene Expression.

PubMed

Poulsen, Line Dahl; Vinther, Jeppe

2018-06-01

RNA sequencing (RNA-seq) has become the preferred method for global quantification of bacterial gene expression. With the continued improvements in sequencing technology and data analysis tools, the most labor-intensive and expensive part of an RNA-seq experiment is the preparation of sequencing libraries, which is also essential for the quality of the data obtained. Here, we present a straightforward and inexpensive basic protocol for preparation of strand-specific RNA-seq libraries from bacterial RNA as well as a computational pipeline for the data analysis of sequencing reads. The protocol is based on the Illumina platform and allows easy multiplexing of samples and the removal of sequencing reads that are PCR duplicates. © 2018 by John Wiley & Sons, Inc. © 2018 John Wiley & Sons, Inc.

Using Amplicon Sequencing To Characterize and Monitor Bacterial Diversity in Drinking Water Distribution Systems.

PubMed

Shaw, Jennifer L A; Monis, Paul; Weyrich, Laura S; Sawade, Emma; Drikas, Mary; Cooper, Alan J

2015-09-01

Drinking water assessments use a variety of microbial, physical, and chemical indicators to evaluate water treatment efficiency and product water quality. However, these indicators do not allow the complex biological communities, which can adversely impact the performance of drinking water distribution systems (DWDSs), to be characterized. Entire bacterial communities can be studied quickly and inexpensively using targeted metagenomic amplicon sequencing. Here, amplicon sequencing of the 16S rRNA gene region was performed alongside traditional water quality measures to assess the health, quality, and efficiency of two distinct, full-scale DWDSs: (i) a linear DWDS supplied with unfiltered water subjected to basic disinfection before distribution and (ii) a complex, branching DWDS treated by a four-stage water treatment plant (WTP) prior to disinfection and distribution. In both DWDSs bacterial communities differed significantly after disinfection, demonstrating the effectiveness of both treatment regimes. However, bacterial repopulation occurred further along in the DWDSs, and some end-user samples were more similar to the source water than to the postdisinfection water. Three sample locations appeared to be nitrified, displaying elevated nitrate levels and decreased ammonia levels, and nitrifying bacterial species, such as Nitrospira, were detected. Burkholderiales were abundant in samples containing large amounts of monochloramine, indicating resistance to disinfection. Genera known to contain pathogenic and fecal-associated species were also identified in several locations. From this study, we conclude that metagenomic amplicon sequencing is an informative method to support current compliance-based methods and can be used to reveal bacterial community interactions with the chemical and physical properties of DWDSs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

LLNL Genomic Assessment: Viral and Bacterial Sequencing Needs for TMTI, Tier 1 Report

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

Slezak, T; Borucki, M; Lenhoff, R

2009-09-29

The Lawrence Livermore National Lab Bioinformatics group has recently taken on a role in DTRA's Transformation Medical Technologies Initiative (TMTI). The high-level goal of TMTI is to accelerate the development of broad-spectrum countermeasures. To achieve those goals, TMTI has a near term need to obtain more sequence information across a large range of pathogens, near neighbors, and across a broad geographical and host range. Our role in this project is to research available sequence data for the organisms of interest and identify critical microbial sequence and knowledge gaps that need to be filled to meet TMTI objectives. This effort includes:more » (1) assessing current genomic sequence for each agent including phylogenetic and geographical diversity, host range, date of isolation range, virulence, sequence availability of key near neighbors, and other characteristics; (2) identifying Subject Matter Experts (SME's) and potential holders of isolate collections, contacting appropriate SME's with known expertise and isolate collections to obtain information on isolate availability and specific recommendations; (3) identifying sequence as well as knowledge gaps (eg virulence, host range, and antibiotic resistance determinants); (4) providing specific recommendations as to the most valuable strains to be placed on the DTRA sequencing queue. We acknowledge that criteria for prioritization of isolates for sequencing falls into two categories aligning with priority queues 1 and 2 as described in the summary. (Priority queue 0 relates to DTRA operational isolates whose availability is not predictable in advance.) 1. Selection of isolates that appear to have likelihood to provide information on virulence and antibiotic resistance. This will include sequence of known virulent strains. Particularly valuable would be virulent strains that have genetically similar yet avirulent, or non human transmissible, counterparts that can be used for comparison to help identify key

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

Bacterial Landscape of Bloodstream Infections in Neutropenic Patients via High Throughput Sequencing

PubMed Central

Gyarmati, Peter; Kalin, Mats; Öhrmalm, Lars; Giske, Christian G.

2015-01-01

Background Bloodstream infection (BSI) is a common and potentially life-threatening complication in patients with hematological malignancies and therapy-induced neutropenia. Administration of broad spectrum antibiotics has substantially decreased the mortality rate in febrile neutropenia, but bacterial infection is documented in only one-third or fewer of the cases. BSI is typically diagnosed by blood culture; however, this method can detect only culturable pathogens. Methods In the present study, a total of 130 blood samples from hematological patients receiving dose-intensive antitumoural treatment were subjected to 16S rRNA PCR and 62 of them were cultured. PCR positive samples were processed to high throughput sequencing by amplifying the V1-V3 regions of the 16S rRNA gene to obtain a full spectrum of bacteria present in BSI. Results Five phyla and 30 genera were identified with sequencing compared to 2 phyla and 4 genera with culture. The largest proportion of bacteria detected by sequencing belonged to Proteobacteria (55.2%), Firmicutes (33.4%) and Actinobacteria (8.6%), while Fusobacteria (0.4%) and Bacteroidetes (0.1%) were also detected. Ninety-eight percent of the bacteria identified by sequencing were opportunistic human pathogens and 65% belonged to the normal human microbiota. Conclusions The present study indicates that BSIs in neutropenic hosts contain a much broader diversity of bacteria, likely with host origin, than previously realized. The elevated ratio of Proteobacteria in BSI corroborates the results found in other systemic inflammatory diseases, such as inflammatory bowel disease or mucosal infections. This knowledge may become of value for tailoring antimicrobial drug administration. PMID:26270467

Bacterial Artificial Chromosome Libraries for Mouse Sequencing and Functional Analysis

PubMed Central

Osoegawa, Kazutoyo; Tateno, Minako; Woon, Peng Yeong; Frengen, Eirik; Mammoser, Aaron G.; Catanese, Joseph J.; Hayashizaki, Yoshihide; de Jong, Pieter J.

2000-01-01

Bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9–14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, ∼1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research. [The sequence data described in this paper have been submitted to the GenBank data library under accession numbers AQ797173–AQ797398.] PMID:10645956

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease

PubMed Central

Owen, Joseph R.; Noyes, Noelle; Young, Amy E.; Prince, Daniel J.; Blanchard, Patricia C.; Lehenbauer, Terry W.; Aly, Sharif S.; Davis, Jessica H.; O’Rourke, Sean M.; Abdo, Zaid; Belk, Keith; Miller, Michael R.; Morley, Paul; Van Eenennaam, Alison L.

2017-01-01

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease–associated bacterial isolates (Histophilus somni, Mycoplasma bovis, Mannheimia haemolytica, and Pasteurella multocida) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis. While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni, M. haemolytica, and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% (P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease

AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES

EPA Science Inventory

This book chapter offers an overview of the use of ribosomal RNA sequences. A history of the technology traces the evolution of techniques to measure bacterial phylogenetic relationships and recent advances in obtaining rRNA sequence information. The manual also describes procedu...

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.

IonGAP: integrative bacterial genome analysis for Ion Torrent sequence data.

PubMed

Baez-Ortega, Adrian; Lorenzo-Diaz, Fabian; Hernandez, Mariano; Gonzalez-Vila, Carlos Ignacio; Roda-Garcia, Jose Luis; Colebrook, Marcos; Flores, Carlos

2015-09-01

We introduce IonGAP, a publicly available Web platform designed for the analysis of whole bacterial genomes using Ion Torrent sequence data. Besides assembly, it integrates a variety of comparative genomics, annotation and bacterial classification routines, based on the widely used FASTQ, BAM and SRA file formats. Benchmarking with different datasets evidenced that IonGAP is a fast, powerful and simple-to-use bioinformatics tool. By releasing this platform, we aim to translate low-cost bacterial genome analysis for microbiological prevention and control in healthcare, agroalimentary and pharmaceutical industry applications. IonGAP is hosted by the ITER's Teide-HPC supercomputer and is freely available on the Web for non-commercial use at http://iongap.hpc.iter.es. mcolesan@ull.edu.es or cflores@ull.edu.es Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Efficient generation of complete sequences of MDR-encoding plasmids by rapid assembly of MinION barcoding sequencing data.

PubMed

Li, Ruichao; Xie, Miaomiao; Dong, Ning; Lin, Dachuan; Yang, Xuemei; Wong, Marcus Ho Yin; Chan, Edward Wai-Chi; Chen, Sheng

2018-03-01

Multidrug resistance (MDR)-encoding plasmids are considered major molecular vehicles responsible for transmission of antibiotic resistance genes among bacteria of the same or different species. Delineating the complete sequences of such plasmids could provide valuable insight into the evolution and transmission mechanisms underlying bacterial antibiotic resistance development. However, due to the presence of multiple repeats of mobile elements, complete sequencing of MDR plasmids remains technically complicated, expensive, and time-consuming. Here, we demonstrate a rapid and efficient approach to obtaining multiple MDR plasmid sequences through the use of the MinION nanopore sequencing platform, which is incorporated in a portable device. By assembling the long sequencing reads generated by a single MinION run according to a rapid barcoding sequencing protocol, we obtained the complete sequences of 20 plasmids harbored by multiple bacterial strains. Importantly, single long reads covering a plasmid end-to-end were recorded, indicating that de novo assembly may be unnecessary if the single reads exhibit high accuracy. This workflow represents a convenient and cost-effective approach for systematic assessment of MDR plasmids responsible for treatment failure of bacterial infections, offering the opportunity to perform detailed molecular epidemiological studies to probe the evolutionary and transmission mechanisms of MDR-encoding elements.

Whole-Genome Sequencing and Concordance Between Antimicrobial Susceptibility Genotypes and Phenotypes of Bacterial Isolates Associated with Bovine Respiratory Disease.

PubMed

Owen, Joseph R; Noyes, Noelle; Young, Amy E; Prince, Daniel J; Blanchard, Patricia C; Lehenbauer, Terry W; Aly, Sharif S; Davis, Jessica H; O'Rourke, Sean M; Abdo, Zaid; Belk, Keith; Miller, Michael R; Morley, Paul; Van Eenennaam, Alison L

2017-09-07

Extended laboratory culture and antimicrobial susceptibility testing timelines hinder rapid species identification and susceptibility profiling of bacterial pathogens associated with bovine respiratory disease, the most prevalent cause of cattle mortality in the United States. Whole-genome sequencing offers a culture-independent alternative to current bacterial identification methods, but requires a library of bacterial reference genomes for comparison. To contribute new bacterial genome assemblies and evaluate genetic diversity and variation in antimicrobial resistance genotypes, whole-genome sequencing was performed on bovine respiratory disease-associated bacterial isolates ( Histophilus somni , Mycoplasma bovis , Mannheimia haemolytica , and Pasteurella multocida ) from dairy and beef cattle. One hundred genomically distinct assemblies were added to the NCBI database, doubling the available genomic sequences for these four species. Computer-based methods identified 11 predicted antimicrobial resistance genes in three species, with none being detected in M. bovis While computer-based analysis can identify antibiotic resistance genes within whole-genome sequences (genotype), it may not predict the actual antimicrobial resistance observed in a living organism (phenotype). Antimicrobial susceptibility testing on 64 H. somni , M. haemolytica , and P. multocida isolates had an overall concordance rate between genotype and phenotypic resistance to the associated class of antimicrobials of 72.7% ( P < 0.001), showing substantial discordance. Concordance rates varied greatly among different antimicrobial, antibiotic resistance gene, and bacterial species combinations. This suggests that antimicrobial susceptibility phenotypes are needed to complement genomically predicted antibiotic resistance gene genotypes to better understand how the presence of antibiotic resistance genes within a given bacterial species could potentially impact optimal bovine respiratory disease

The impact of sampling, PCR, and sequencing replication on discerning changes in drinking water bacterial community over diurnal time-scales.

PubMed

Bautista-de Los Santos, Quyen Melina; Schroeder, Joanna L; Blakemore, Oliver; Moses, Jonathan; Haffey, Mark; Sloan, William; Pinto, Ameet J

2016-03-01

High-throughput and deep DNA sequencing, particularly amplicon sequencing, is being increasingly utilized to reveal spatial and temporal dynamics of bacterial communities in drinking water systems. Whilst the sampling and methodological biases associated with PCR and sequencing have been studied in other environments, they have not been quantified for drinking water. These biases are likely to have the greatest effect on the ability to characterize subtle spatio-temporal patterns influenced by process/environmental conditions. In such cases, intra-sample variability may swamp any underlying small, systematic variation. To evaluate this, we undertook a study with replication at multiple levels including sampling sites, sample collection, PCR amplification, and high throughput sequencing of 16S rRNA amplicons. The variability inherent to the PCR amplification and sequencing steps is significant enough to mask differences between bacterial communities from replicate samples. This was largely driven by greater variability in detection of rare bacteria (relative abundance <0.01%) across PCR/sequencing replicates as compared to replicate samples. Despite this, we captured significant changes in bacterial community over diurnal time-scales and find that the extent and pattern of diurnal changes is specific to each sampling location. Further, we find diurnal changes in bacterial community arise due to differences in the presence/absence of the low abundance bacteria and changes in the relative abundance of dominant bacteria. Finally, we show that bacterial community composition is significantly different across sampling sites for time-periods during which there are typically rapid changes in water use. This suggests hydraulic changes (driven by changes in water demand) contribute to shaping the bacterial community in bulk drinking water over diurnal time-scales. Copyright © 2015 Elsevier Ltd. All rights reserved.

Using high throughput sequencing to explore the biodiversity in oral bacterial communities.

PubMed

Diaz, P I; Dupuy, A K; Abusleme, L; Reese, B; Obergfell, C; Choquette, L; Dongari-Bagtzoglou, A; Peterson, D E; Terzi, E; Strausbaugh, L D

2012-06-01

High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns. © 2012 John Wiley & Sons A/S.

Soil Parameters Drive the Structure, Diversity and Metabolic Potentials of the Bacterial Communities Across Temperate Beech Forest Soil Sequences.

PubMed

Jeanbille, M; Buée, M; Bach, C; Cébron, A; Frey-Klett, P; Turpault, M P; Uroz, S

2016-02-01

Soil and climatic conditions as well as land cover and land management have been shown to strongly impact the structure and diversity of the soil bacterial communities. Here, we addressed under a same land cover the potential effect of the edaphic parameters on the soil bacterial communities, excluding potential confounding factors as climate. To do this, we characterized two natural soil sequences occurring in the Montiers experimental site. Spatially distant soil samples were collected below Fagus sylvatica tree stands to assess the effect of soil sequences on the edaphic parameters, as well as the structure and diversity of the bacterial communities. Soil analyses revealed that the two soil sequences were characterized by higher pH and calcium and magnesium contents in the lower plots. Metabolic assays based on Biolog Ecoplates highlighted higher intensity and richness in usable carbon substrates in the lower plots than in the middle and upper plots, although no significant differences occurred in the abundance of bacterial and fungal communities along the soil sequences as assessed using quantitative PCR. Pyrosequencing analysis of 16S ribosomal RNA (rRNA) gene amplicons revealed that Proteobacteria, Acidobacteria and Bacteroidetes were the most abundantly represented phyla. Acidobacteria, Proteobacteria and Chlamydiae were significantly enriched in the most acidic and nutrient-poor soils compared to the Bacteroidetes, which were significantly enriched in the soils presenting the higher pH and nutrient contents. Interestingly, aluminium, nitrogen, calcium, nutrient availability and pH appeared to be the best predictors of the bacterial community structures along the soil sequences.

[Bacterial diversity in sequencing batch biofilm reactor (SBBR) for landfill leachate treatment using PCR-DGGE].

PubMed

Xiao, Yong; Yang, Zhao-hui; Zeng, Guang-ming; Ma, Yan-he; Liu, You-sheng; Wang, Rong-juan; Xu, Zheng-yong

2007-05-01

For studying the bacterial diversity and the mechanism of denitrification in sequencing bath biofilm reactor (SBBR) treating landfill leachate to provide microbial evidence for technique improvements, total microbial DNA was extracted from samples which were collected from natural landfill leachate and biofilm of a SBBR that could efficiently remove NH4+ -N and COD of high concentration. 16S rDNA fragments were amplified from the total DNA successfully using a pair of universal bacterial 16S rDNA primer, GC341F and 907R, and then were used for denaturing gradient gel electrophoresis (DGGE) analysis. The bands in the gel were analyzed by statistical methods and excided from the gel for sequencing, and the sequences were used for homology analysis and then two phylogenetic trees were constructed using DNAStar software. Results indicated that the bacterial diversity of the biofilm in SBBR and the landfill leachate was abundant, and no obvious change of community structure happened during running in the biofilm, in which most bacteria came from the landfill leachate. There may be three different modes of denitrification in the reactor because several different nitrifying bacteria, denitrifying bacteria and anaerobic ammonia oxidation bacteria coexisted in it. The results provided some valuable references for studying microbiological mechanism of denitrification in SBBR.

Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

PubMed

Christen, Matthias; Deutsch, Samuel; Christen, Beat

2015-08-21

Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

A Year of Infection in the Intensive Care Unit: Prospective Whole Genome Sequencing of Bacterial Clinical Isolates Reveals Cryptic Transmissions and Novel Microbiota

PubMed Central

Roach, David J.; Burton, Joshua N.; Lee, Choli; Stackhouse, Bethany; Butler-Wu, Susan M.; Cookson, Brad T.

2015-01-01

Bacterial whole genome sequencing holds promise as a disruptive technology in clinical microbiology, but it has not yet been applied systematically or comprehensively within a clinical context. Here, over the course of one year, we performed prospective collection and whole genome sequencing of nearly all bacterial isolates obtained from a tertiary care hospital’s intensive care units (ICUs). This unbiased collection of 1,229 bacterial genomes from 391 patients enables detailed exploration of several features of clinical pathogens. A sizable fraction of isolates identified as clinically relevant corresponded to previously undescribed species: 12% of isolates assigned a species-level classification by conventional methods actually qualified as distinct, novel genomospecies on the basis of genomic similarity. Pan-genome analysis of the most frequently encountered pathogens in the collection revealed substantial variation in pan-genome size (1,420 to 20,432 genes) and the rate of gene discovery (1 to 152 genes per isolate sequenced). Surprisingly, although potential nosocomial transmission of actively surveilled pathogens was rare, 8.7% of isolates belonged to genomically related clonal lineages that were present among multiple patients, usually with overlapping hospital admissions, and were associated with clinically significant infection in 62% of patients from which they were recovered. Multi-patient clonal lineages were particularly evident in the neonatal care unit, where seven separate Staphylococcus epidermidis clonal lineages were identified, including one lineage associated with bacteremia in 5/9 neonates. Our study highlights key differences in the information made available by conventional microbiological practices versus whole genome sequencing, and motivates the further integration of microbial genome sequencing into routine clinical care. PMID:26230489

Identification of the bacterial community of maple sap by using amplified ribosomal DNA (rDNA) restriction analysis and rDNA sequencing.

PubMed

Lagacé, L; Pitre, M; Jacques, M; Roy, D

2004-04-01

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the gamma- and beta-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). gamma-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control.

Identification of the Bacterial Community of Maple Sap by Using Amplified Ribosomal DNA (rDNA) Restriction Analysis and rDNA Sequencing

PubMed Central

Lagacé, L.; Pitre, M.; Jacques, M.; Roy, D.

2004-01-01

The bacterial community of maple sap was characterized by analysis of samples obtained at the taphole of maple trees for the 2001 and 2002 seasons. Among the 190 bacterial isolates, 32 groups were formed according to the similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). A subset of representative isolates for each ARDRA group was identified by 16S rRNA gene fragment sequencing. Results showed a wide variety of organisms, with 22 different genera encountered. Pseudomonas and Ralstonia, of the γ- and β-Proteobacteria, respectively, were the most frequently encountered genera. Gram-positive bacteria were also observed, and Staphylococcus, Plantibacter, and Bacillus were the most highly represented genera. The sampling period corresponding to 50% of the cumulative sap flow percentage presented the greatest bacterial diversity according to its Shannon diversity index value (1.1). γ-Proteobacteria were found to be dominant almost from the beginning of the season to the end. These results are providing interesting insights on maple sap microflora that will be useful for further investigation related to microbial contamination and quality of maple products and also for guiding new strategies on taphole contamination control. PMID:15066796

A comprehensive insight into bacterial virulence in drinking water using 454 pyrosequencing and Illumina high-throughput sequencing.

PubMed

Huang, Kailong; Zhang, Xu-Xiang; Shi, Peng; Wu, Bing; Ren, Hongqiang

2014-11-01

In order to comprehensively investigate bacterial virulence in drinking water, 454 pyrosequencing and Illumina high-throughput sequencing were used to detect potential pathogenic bacteria and virulence factors (VFs) in a full-scale drinking water treatment and distribution system. 16S rRNA gene pyrosequencing revealed high bacterial diversity in the drinking water (441-586 operational taxonomic units). Bacterial diversity decreased after chlorine disinfection, but increased after pipeline distribution. α-Proteobacteria was the most dominant taxonomic class. Alignment against the established pathogen database showed that several types of putative pathogens were present in the drinking water and Pseudomonas aeruginosa had the highest abundance (over 11‰ of total sequencing reads). Many pathogens disappeared after chlorine disinfection, but P. aeruginosa and Leptospira interrogans were still detected in the tap water. High-throughput sequencing revealed prevalence of various pathogenicity islands and virulence proteins in the drinking water, and translocases, transposons, Clp proteases and flagellar motor switch proteins were the predominant VFs. Both diversity and abundance of the detectable VFs increased after the chlorination, and decreased after the pipeline distribution. This study indicates that joint use of 454 pyrosequencing and Illumina sequencing can comprehensively characterize environmental pathogenesis, and several types of putative pathogens and various VFs are prevalent in drinking water. Copyright © 2014 Elsevier Inc. All rights reserved.

SIMBA: a web tool for managing bacterial genome assembly generated by Ion PGM sequencing technology.

PubMed

Mariano, Diego C B; Pereira, Felipe L; Aguiar, Edgar L; Oliveira, Letícia C; Benevides, Leandro; Guimarães, Luís C; Folador, Edson L; Sousa, Thiago J; Ghosh, Preetam; Barh, Debmalya; Figueiredo, Henrique C P; Silva, Artur; Ramos, Rommel T J; Azevedo, Vasco A C

2016-12-15

The evolution of Next-Generation Sequencing (NGS) has considerably reduced the cost per sequenced-base, allowing a significant rise of sequencing projects, mainly in prokaryotes. However, the range of available NGS platforms requires different strategies and software to correctly assemble genomes. Different strategies are necessary to properly complete an assembly project, in addition to the installation or modification of various software. This requires users to have significant expertise in these software and command line scripting experience on Unix platforms, besides possessing the basic expertise on methodologies and techniques for genome assembly. These difficulties often delay the complete genome assembly projects. In order to overcome this, we developed SIMBA (SImple Manager for Bacterial Assemblies), a freely available web tool that integrates several component tools for assembling and finishing bacterial genomes. SIMBA provides a friendly and intuitive user interface so bioinformaticians, even with low computational expertise, can work under a centralized administrative control system of assemblies managed by the assembly center head. SIMBA guides the users to execute assembly process through simple and interactive pages. SIMBA workflow was divided in three modules: (i) projects: allows a general vision of genome sequencing projects, in addition to data quality analysis and data format conversions; (ii) assemblies: allows de novo assemblies with the software Mira, Minia, Newbler and SPAdes, also assembly quality validations using QUAST software; and (iii) curation: presents methods to finishing assemblies through tools for scaffolding contigs and close gaps. We also presented a case study that validated the efficacy of SIMBA to manage bacterial assemblies projects sequenced using Ion Torrent PGM. Besides to be a web tool for genome assembly, SIMBA is a complete genome assemblies project management system, which can be useful for managing of several

AgdbNet – antigen sequence database software for bacterial typing

PubMed Central

Jolley, Keith A; Maiden, Martin CJ

2006-01-01

Background Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences. Results Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script. Conclusion The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated. PMID:16790057

Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR.

PubMed

Bulgari, Daniela; Casati, Paola; Brusetti, Lorenzo; Quaglino, Fabio; Brasca, Milena; Daffonchio, Daniele; Bianco, Piero Attilio

2009-08-01

Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

Detection of Mixed Infection from Bacterial Whole Genome Sequence Data Allows Assessment of Its Role in Clostridium difficile Transmission

PubMed Central

Eyre, David W.; Cule, Madeleine L.; Griffiths, David; Crook, Derrick W.; Peto, Tim E. A.

2013-01-01

Bacterial whole genome sequencing offers the prospect of rapid and high precision investigation of infectious disease outbreaks. Close genetic relationships between microorganisms isolated from different infected cases suggest transmission is a strong possibility, whereas transmission between cases with genetically distinct bacterial isolates can be excluded. However, undetected mixed infections—infection with ≥2 unrelated strains of the same species where only one is sequenced—potentially impairs exclusion of transmission with certainty, and may therefore limit the utility of this technique. We investigated the problem by developing a computationally efficient method for detecting mixed infection without the need for resource-intensive independent sequencing of multiple bacterial colonies. Given the relatively low density of single nucleotide polymorphisms within bacterial sequence data, direct reconstruction of mixed infection haplotypes from current short-read sequence data is not consistently possible. We therefore use a two-step maximum likelihood-based approach, assuming each sample contains up to two infecting strains. We jointly estimate the proportion of the infection arising from the dominant and minor strains, and the sequence divergence between these strains. In cases where mixed infection is confirmed, the dominant and minor haplotypes are then matched to a database of previously sequenced local isolates. We demonstrate the performance of our algorithm with in silico and in vitro mixed infection experiments, and apply it to transmission of an important healthcare-associated pathogen, Clostridium difficile. Using hospital ward movement data in a previously described stochastic transmission model, 15 pairs of cases enriched for likely transmission events associated with mixed infection were selected. Our method identified four previously undetected mixed infections, and a previously undetected transmission event, but no direct transmission between

Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability.

PubMed

Goldenzweig, Adi; Goldsmith, Moshe; Hill, Shannon E; Gertman, Or; Laurino, Paola; Ashani, Yacov; Dym, Orly; Unger, Tamar; Albeck, Shira; Prilusky, Jaime; Lieberman, Raquel L; Aharoni, Amir; Silman, Israel; Sussman, Joel L; Tawfik, Dan S; Fleishman, Sarel J

2016-07-21

Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Metagenomic study of bacterial microbiota in persistent endodontic infections using Next-generation sequencing.

PubMed

Sánchez-Sanhueza, G; Bello-Toledo, H; González-Rocha, G; Gonçalves, A T; Valenzuela, V; Gallardo-Escárate, C

2018-05-22

To determine the bacterial microbiota in root canals associated with persistent apical periodontitis and their relationship with the clinical characteristics of patients using next-generation sequencing (NGS). Bacterial samples from root canals associated with teeth having persistent apical periodontitis were taken from 24 patients undergoing root canal retreatment. Bacterial DNA was extracted, and V3-V4 variable regions of the 16S rRNA gene were amplified. The amplification was deep sequenced by Illumina technology to establish the metagenetic relationships among the bacterial species identified. The composition and diversity of microbial communities in the root canal and their relationships with clinical features were analysed. Parametric and nonparametric tests were used to analyse differences between patient characteristics and microbial data. A total of 86 different operational taxonomic units (OTUs) were identified and Good's nonparametric coverage estimator method indicated that 99.9 ± 0.00001% diversity was recovered per sample. The largest number of bacteria belonged to the phylum Proteobacteria. According to the medical history from the American Society of Anesthesiologists (ASA) Classification System, ASA II-III had higher richness estimates and distinct phylogenetic relationships compared to ASA I individuals (P < 0.05). Periapical index (PAI) score 5 was associated with increased microbiota diversity in comparison to PAI score 4, and this index was reduced in symptomatic patients. Based on the findings of this study, it is possible to suggest a close relationship between several clinical features and greater microbiota diversity with persistent endodontic infections. This work provides a better understanding on how microbial communities interact with their host and vice versa. © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Complete Genome Sequence of a Putative New Bacterial Strain, I507, Isolated from the Indian Ocean

PubMed Central

Wang, Shu-yan; Wei, Jia-qiang

2018-01-01

ABSTRACT Bacterial strain I507 was isolated from the central Indian Ocean and may be a potential novel species, according to the 16S rRNA gene sequence. Here, we present its complete genome sequence and expect that it will provide researchers with valuable information to further understand its classification and function in the future. PMID:29674539

Insights into the Emergent Bacterial Pathogen Cronobacter spp., Generated by Multilocus Sequence Typing and Analysis

PubMed Central

Joseph, Susan; Forsythe, Stephen J.

2012-01-01

Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognized route of infection being the consumption of contaminated infant formula. As a recently recognized bacterial pathogen of considerable importance and regulatory control, appropriate detection, and identification schemes are required. The application of multilocus sequence typing (MLST) and analysis (MLSA) of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB, and ppsA (concatenated length 3036 base pairs) has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognized genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti) and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health. PMID:23189075

Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

PubMed

Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

2016-12-01

We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Using DGGE and 16S rRNA gene sequence analysis to evaluate changes in oral bacterial composition.

PubMed

Chen, Zhou; Trivedi, Harsh M; Chhun, Nok; Barnes, Virginia M; Saxena, Deepak; Xu, Tao; Li, Yihong

2011-01-01

To investigate whether a standard dental prophylaxis followed by tooth brushing with an antibacterial dentifrice will affect the oral bacterial community, as determined by denaturing gradient gel electrophoresis (DGGE) combined with 16S rRNA gene sequence analysis. Twenty-four healthy adults were instructed to brush their teeth using commercial dentifrice for 1 week during a washout period. An initial set of pooled supragingival plaque samples was collected from each participant at baseline (0 h) before prophylaxis treatment. The subjects were given a clinical examination and dental prophylaxis and asked to brush for 1 min with a dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride (Colgate Total). On the following day, a second set of pooled supragingival plaque samples (24 h) was collected. Total bacterial genomic DNA was isolated from the samples. Differences in the microbial composition before and after the prophylactic procedure and tooth brushing were assessed by comparing the DGGE profiles and 16S rRNA gene segments sequence analysis. Two distinct clusters of DGGE profiles were found, suggesting that a shift in the microbial composition had occurred 24 h after the prophylaxis and brushing. A detailed sequencing analysis of 16S rRNA gene segments further identified 6 phyla and 29 genera, including known and unknown bacterial species. Importantly, an increase in bacterial diversity was observed after 24 h, including members of the Streptococcaceae family, Prevotella, Corynebacterium, TM7 and other commensal bacteria. The results suggest that the use of a standard prophylaxis followed by the use of the dentifrice containing 0.3% triclosan, 2.0% PVM/MA copolymer and 0.243% sodium fluoride may promote a healthier composition within the oral bacterial community.

BG7: A New Approach for Bacterial Genome Annotation Designed for Next Generation Sequencing Data

PubMed Central

Pareja-Tobes, Pablo; Manrique, Marina; Pareja-Tobes, Eduardo; Pareja, Eduardo; Tobes, Raquel

2012-01-01

BG7 is a new system for de novo bacterial, archaeal and viral genome annotation based on a new approach specifically designed for annotating genomes sequenced with next generation sequencing technologies. The system is versatile and able to annotate genes even in the step of preliminary assembly of the genome. It is especially efficient detecting unexpected genes horizontally acquired from bacterial or archaeal distant genomes, phages, plasmids, and mobile elements. From the initial phases of the gene annotation process, BG7 exploits the massive availability of annotated protein sequences in databases. BG7 predicts ORFs and infers their function based on protein similarity with a wide set of reference proteins, integrating ORF prediction and functional annotation phases in just one step. BG7 is especially tolerant to sequencing errors in start and stop codons, to frameshifts, and to assembly or scaffolding errors. The system is also tolerant to the high level of gene fragmentation which is frequently found in not fully assembled genomes. BG7 current version – which is developed in Java, takes advantage of Amazon Web Services (AWS) cloud computing features, but it can also be run locally in any operating system. BG7 is a fast, automated and scalable system that can cope with the challenge of analyzing the huge amount of genomes that are being sequenced with NGS technologies. Its capabilities and efficiency were demonstrated in the 2011 EHEC Germany outbreak in which BG7 was used to get the first annotations right the next day after the first entero-hemorrhagic E. coli genome sequences were made publicly available. The suitability of BG7 for genome annotation has been proved for Illumina, 454, Ion Torrent, and PacBio sequencing technologies. Besides, thanks to its plasticity, our system could be very easily adapted to work with new technologies in the future. PMID:23185310

Solving the problem of comparing whole bacterial genomes across different sequencing platforms.

PubMed

Kaas, Rolf S; Leekitcharoenphon, Pimlapas; Aarestrup, Frank M; Lund, Ole

2014-01-01

Whole genome sequencing (WGS) shows great potential for real-time monitoring and identification of infectious disease outbreaks. However, rapid and reliable comparison of data generated in multiple laboratories and using multiple technologies is essential. So far studies have focused on using one technology because each technology has a systematic bias making integration of data generated from different platforms difficult. We developed two different procedures for identifying variable sites and inferring phylogenies in WGS data across multiple platforms. The methods were evaluated on three bacterial data sets and sequenced on three different platforms (Illumina, 454, Ion Torrent). We show that the methods are able to overcome the systematic biases caused by the sequencers and infer the expected phylogenies. It is concluded that the cause of the success of these new procedures is due to a validation of all informative sites that are included in the analysis. The procedures are available as web tools.

Complete Genome Sequence of Lactobacillus rhamnosus Strain BPL5 (CECT 8800), a Probiotic for Treatment of Bacterial Vaginosis.

PubMed

Chenoll, Empar; Codoñer, Francisco M; Martinez-Blanch, Juan F; Ramón, Daniel; Genovés, Salvador; Menabrito, Marco

2016-04-21

ITALIC! Lactobacillus rhamnosusBPL5 (CECT 8800), is a probiotic strain suitable for the treatment of bacterial vaginosis. Here, we report its complete genome sequence deciphered by PacBio single-molecule real-time (SMRT) technology. Analysis of the sequence may provide insight into its functional activity. Copyright © 2016 Chenoll et al.

Comparison of ZP3 protein sequences among vertebrate species: to obtain a consensus sequence for immunocontraception.

PubMed

Zhu, X; Naz, R K

1999-03-01

The deduced ZP3 amino acid (aa) sequences of 13 vertebrate species namely mouse, hamster, rabbit, pig, porcine, cow, dog, cat, human, bonnet, marmoset, carp, and frog were compared using the PILEUP and PRETTY alignment programs (GCG, Wisconsin, USA). The published aa sequences obtained from 13 vertebrate species indicated the overall evolutionarily conservation in the N-terminus, central region, and C-terminus of the ZP3 polypeptide. More variations of ZP3 polypeptide sequences were seen in the alignments of carp and frog from the 11 mammalian species making the leader sequence more prominent. The canonical furin proteolytic processing signal at the C-terminus was found in all the ZP3 polypeptide sequences except of carp and frog. In the central region, the ZP3 deduced aa sequences of all the 13 vertebrate species aligned well, and six relatively conserved sequences were found. There are 11 conserved cysteine residues in the central region across all species including carp and frog, indicating that these residues have longer evolutionary history. The ZP3 aa sequence similarities were examined using the GAP program (GCG). The highest aa similarities are observed between the members of the same order within the class mammalia, and also (95.4%) between pig (ungulata) and rabbit (lagomorpha). The deduced ZP3 aa sequences per se may not be enough to build a phylogenetic tree.

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

PubMed Central

Reuter, Sandra; Ellington, Matthew J.; Cartwright, Edward J. P.; Köser, Claudio U.; Török, M. Estée; Gouliouris, Theodore; Harris, Simon R.; Brown, Nicholas M.; Holden, Matthew T. G.; Quail, Mike; Parkhill, Julian; Smith, Geoffrey P.; Bentley, Stephen D.; Peacock, Sharon J.

2014-01-01

IMPORTANCE The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of

Bacterial Diversity in Human Subgingival Plaque

PubMed Central

Paster, Bruce J.; Boches, Susan K.; Galvin, Jamie L.; Ericson, Rebecca E.; Lau, Carol N.; Levanos, Valerie A.; Sahasrabudhe, Ashish; Dewhirst, Floyd E.

2001-01-01

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our

Bacterial taxa–area and distance–decay relationships in marine environments

PubMed Central

Zinger, L; Boetius, A; Ramette, A

2014-01-01

The taxa–area relationship (TAR) and the distance–decay relationship (DDR) both describe spatial turnover of taxa and are central patterns of biodiversity. Here, we compared TAR and DDR of bacterial communities across different marine realms and ecosystems at the global scale. To obtain reliable global estimates for both relationships, we quantified the poorly assessed effects of sequencing depth, rare taxa removal and number of sampling sites. Slope coefficients of bacterial TARs were within the range of those of plants and animals, whereas slope coefficients of bacterial DDR were much lower. Slope coefficients were mostly affected by removing rare taxa and by the number of sampling sites considered in the calculations. TAR and DDR slope coefficients were overestimated at sequencing depth <4000 sequences per sample. Noticeably, bacterial TAR and DDR patterns did not correlate with each other both within and across ecosystem types, suggesting that (i) TAR cannot be directly derived from DDR and (ii) TAR and DDR may be influenced by different ecological factors. Nevertheless, we found marine bacterial TAR and DDR to be steeper in ecosystems associated with high environmental heterogeneity or spatial isolation, namely marine sediments and coastal environments compared with pelagic ecosystems. Hence, our study provides information on macroecological patterns of marine bacteria, as well as methodological and conceptual insights, at a time when biodiversity surveys increasingly make use of high-throughput sequencing technologies. PMID:24460915

Diversity and Variation of Bacterial Community Revealed by MiSeq Sequencing in Chinese Dark Teas

PubMed Central

Fu, Jianyu; Lv, Haipeng; Chen, Feng

2016-01-01

Chinese dark teas (CDTs) are now among the popular tea beverages worldwide due to their unique health benefits. Because the production of CDTs involves fermentation that is characterized by the effect of microbes, microorganisms are believed to play critical roles in the determination of the chemical characteristics of CDTs. Some dominant fungi have been identified from CDTs. In contrast, little, if anything, is known about the composition of bacterial community in CDTs. This study was set to investigate the diversity and variation of bacterial community in four major types of CDTs from China. First, the composition of the bacterial community of CDTs was determined using MiSeq sequencing. From the four typical CDTs, a total of 238 genera that belong to 128 families of bacteria were detected, including most of the families of beneficial bacteria known to be associated with fermented food. While different types of CDTs had generally distinct bacterial structures, the two types of brick teas produced from adjacent regions displayed strong similarity in bacterial composition, suggesting that the producing environment and processing condition perhaps together influence bacterial succession in CDTs. The global characterization of bacterial communities in CDTs is an essential first step for us to understand their function in fermentation and their potential impact on human health. Such knowledge will be important guidance for improving the production of CDTs with higher quality and elevated health benefits. PMID:27690376

Characterization of bovine ruminal epithelial bacterial communities using 16S rRNA sequencing, PCR-DGGE, and qRT-PCR analysis.

PubMed

Li, Meiju; Zhou, Mi; Adamowicz, Elizabeth; Basarab, John A; Guan, Le Luo

2012-02-24

Currently, knowledge regarding the ecology and function of bacteria attached to the epithelial tissue of the rumen wall is limited. In this study, the diversity of the bacterial community attached to the rumen epithelial tissue was compared to the rumen content bacterial community using 16S rRNA gene sequencing, PCR-DGGE, and qRT-PCR analysis. Sequence analysis of 2785 randomly selected clones from six 16S rDNA (∼1.4kb) libraries showed that the community structures of three rumen content libraries clustered together and were separated from the rumen tissue libraries. The diversity index of each library revealed that ruminal content bacterial communities (4.12/4.42/4.88) were higher than ruminal tissue communities (2.90/2.73/3.23), based on 97% similarity. The phylum Firmicutes was predominant in the ruminal tissue communities, while the phylum Bacteroidetes was predominant in the ruminal content communities. The phyla Fibrobacteres, Planctomycetes, and Verrucomicrobia were only detected in the ruminal content communities. PCR-DGGE analysis of the bacterial profiles of the rumen content and ruminal epithelial tissue samples from 22 steers further confirmed that there is a distinct bacterial community that inhibits the rumen epithelium. The distinctive epimural bacterial communities suggest that Firmicutes, together with other epithelial-specific species, may have additional functions other than food digestion. Copyright © 2011 Elsevier B.V. All rights reserved.

Short- and Long-term Evolutionary Dynamics of Bacterial Insertion Sequences: Insights from Wolbachia Endosymbionts

PubMed Central

Cerveau, Nicolas; Leclercq, Sébastien; Leroy, Elodie; Bouchon, Didier; Cordaux, Richard

2011-01-01

Transposable elements (TE) are one of the major driving forces of genome evolution, raising the question of the long-term dynamics underlying their evolutionary success. Long-term TE evolution can readily be reconstructed in eukaryotes, thanks to many degraded copies constituting genomic fossil records of past TE proliferations. By contrast, bacterial genomes usually experience high sequence turnover and short TE retention times, thereby obscuring ancient TE evolutionary patterns. We found that Wolbachia bacterial genomes contain 52–171 insertion sequence (IS) TEs. IS account for 11% of Wolbachia wRi, which is one of the highest IS genomic coverage reported in prokaryotes to date. We show that many IS groups are currently expanding in various Wolbachia genomes and that IS horizontal transfers are frequent among strains, which can explain the apparent synchronicity of these IS proliferations. Remarkably, >70% of Wolbachia IS are nonfunctional. They constitute an unusual bacterial IS genomic fossil record providing direct empirical evidence for a long-term IS evolutionary dynamics following successive periods of intense transpositional activity. Our results show that comprehensive IS annotations have the potential to provide new insights into prokaryote TE evolution and, more generally, prokaryote genome evolution. Indeed, the identification of an important IS genomic fossil record in Wolbachia demonstrates that IS elements are not always of recent origin, contrary to the conventional view of TE evolution in prokaryote genomes. Our results also raise the question whether the abundance of IS fossils is specific to Wolbachia or it may be a general, albeit overlooked, feature of prokaryote genomes. PMID:21940637

Short- and long-term evolutionary dynamics of bacterial insertion sequences: insights from Wolbachia endosymbionts.

PubMed

Cerveau, Nicolas; Leclercq, Sébastien; Leroy, Elodie; Bouchon, Didier; Cordaux, Richard

2011-01-01

Transposable elements (TE) are one of the major driving forces of genome evolution, raising the question of the long-term dynamics underlying their evolutionary success. Long-term TE evolution can readily be reconstructed in eukaryotes, thanks to many degraded copies constituting genomic fossil records of past TE proliferations. By contrast, bacterial genomes usually experience high sequence turnover and short TE retention times, thereby obscuring ancient TE evolutionary patterns. We found that Wolbachia bacterial genomes contain 52-171 insertion sequence (IS) TEs. IS account for 11% of Wolbachia wRi, which is one of the highest IS genomic coverage reported in prokaryotes to date. We show that many IS groups are currently expanding in various Wolbachia genomes and that IS horizontal transfers are frequent among strains, which can explain the apparent synchronicity of these IS proliferations. Remarkably, >70% of Wolbachia IS are nonfunctional. They constitute an unusual bacterial IS genomic fossil record providing direct empirical evidence for a long-term IS evolutionary dynamics following successive periods of intense transpositional activity. Our results show that comprehensive IS annotations have the potential to provide new insights into prokaryote TE evolution and, more generally, prokaryote genome evolution. Indeed, the identification of an important IS genomic fossil record in Wolbachia demonstrates that IS elements are not always of recent origin, contrary to the conventional view of TE evolution in prokaryote genomes. Our results also raise the question whether the abundance of IS fossils is specific to Wolbachia or it may be a general, albeit overlooked, feature of prokaryote genomes.

Exploring bacterial epigenomics in the next-generation sequencing era: a new approach for an emerging frontier.

PubMed

Chen, Poyin; Jeannotte, Richard; Weimer, Bart C

2014-05-01

Epigenetics has an important role for the success of foodborne pathogen persistence in diverse host niches. Substantial challenges exist in determining DNA methylation to situation-specific phenotypic traits. DNA modification, mediated by restriction-modification systems, functions as an immune response against antagonistic external DNA, and bacteriophage-acquired methyltransferases (MTase) and orphan MTases - those lacking the cognate restriction endonuclease - facilitate evolution of new phenotypes via gene expression modulation via DNA and RNA modifications, including methylation and phosphorothioation. Recent establishment of large-scale genome sequencing projects will result in a significant increase in genome availability that will lead to new demands for data analysis including new predictive bioinformatics approaches that can be verified with traditional scientific rigor. Sequencing technologies that detect modification coupled with mass spectrometry to discover new adducts is a powerful tactic to study bacterial epigenetics, which is poised to make novel and far-reaching discoveries that link biological significance and the bacterial epigenome. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bacterial Communities Associated with Host-Adapted Populations of Pea Aphids Revealed by Deep Sequencing of 16S Ribosomal DNA

PubMed Central

Gauthier, Jean-Pierre; Outreman, Yannick; Mieuzet, Lucie; Simon, Jean-Christophe

2015-01-01

Associations between microbes and animals are ubiquitous and hosts may benefit from harbouring microbial communities through improved resource exploitation or resistance to environmental stress. The pea aphid, Acyrthosiphon pisum, is the host of heritable bacterial symbionts, including the obligate endosymbiont Buchnera aphidicola and several facultative symbionts. While obligate symbionts supply aphids with key nutrients, facultative symbionts influence their hosts in many ways such as protection against natural enemies, heat tolerance, color change and reproduction alteration. The pea aphid also encompasses multiple plant-specialized biotypes, each adapted to one or a few legume species. Facultative symbiont communities differ strongly between biotypes, although bacterial involvement in plant specialization is uncertain. Here, we analyse the diversity of bacterial communities associated with nine biotypes of the pea aphid complex using amplicon pyrosequencing of 16S rRNA genes. Combined clustering and phylogenetic analyses of 16S sequences allowed identifying 21 bacterial OTUs (Operational Taxonomic Unit). More than 98% of the sequencing reads were assigned to known pea aphid symbionts. The presence of Wolbachia was confirmed in A. pisum while Erwinia and Pantoea, two gut associates, were detected in multiple samples. The diversity of bacterial communities harboured by pea aphid biotypes was very low, ranging from 3 to 11 OTUs across samples. Bacterial communities differed more between than within biotypes but this difference did not correlate with the genetic divergence between biotypes. Altogether, these results confirm that the aphid microbiota is dominated by a few heritable symbionts and that plant specialization is an important structuring factor of bacterial communities associated with the pea aphid complex. However, since we examined the microbiota of aphid samples kept a few generations in controlled conditions, it may be that bacterial diversity was

Investigation of bacterial and archaeal communities: novel protocols using modern sequencing by Illumina MiSeq and traditional DGGE-cloning.

PubMed

Kraková, Lucia; Šoltys, Katarína; Budiš, Jaroslav; Grivalský, Tomáš; Ďuriš, František; Pangallo, Domenico; Szemes, Tomáš

2016-09-01

Different protocols based on Illumina high-throughput DNA sequencing and denaturing gradient gel electrophoresis (DGGE)-cloning were developed and applied for investigating hot spring related samples. The study was focused on three target genes: archaeal and bacterial 16S rRNA and mcrA of methanogenic microflora. Shorter read lengths of the currently most popular technology of sequencing by Illumina do not allow analysis of the complete 16S rRNA region, or of longer gene fragments, as was the case of Sanger sequencing. Here, we demonstrate that there is no need for special indexed or tailed primer sets dedicated to short variable regions of 16S rRNA since the presented approach allows the analysis of complete bacterial 16S rRNA amplicons (V1-V9) and longer archaeal 16S rRNA and mcrA sequences. Sample augmented with transposon is represented by a set of approximately 300 bp long fragments that can be easily sequenced by Illumina MiSeq. Furthermore, a low proportion of chimeric sequences was observed. DGGE-cloning based strategies were performed combining semi-nested PCR, DGGE and clone library construction. Comparing both investigation methods, a certain degree of complementarity was observed confirming that the DGGE-cloning approach is not obsolete. Novel protocols were created for several types of laboratories, utilizing the traditional DGGE technique or using the most modern Illumina sequencing.

Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags

PubMed Central

2010-01-01

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80°C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method. PMID:20673359

Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags.

PubMed

Wu, Gary D; Lewis, James D; Hoffmann, Christian; Chen, Ying-Yu; Knight, Rob; Bittinger, Kyle; Hwang, Jennifer; Chen, Jun; Berkowsky, Ronald; Nessel, Lisa; Li, Hongzhe; Bushman, Frederic D

2010-07-30

Intense interest centers on the role of the human gut microbiome in health and disease, but optimal methods for analysis are still under development. Here we present a study of methods for surveying bacterial communities in human feces using 454/Roche pyrosequencing of 16S rRNA gene tags. We analyzed fecal samples from 10 individuals and compared methods for storage, DNA purification and sequence acquisition. To assess reproducibility, we compared samples one cm apart on a single stool specimen for each individual. To analyze storage methods, we compared 1) immediate freezing at -80 degrees C, 2) storage on ice for 24 or 3) 48 hours. For DNA purification methods, we tested three commercial kits and bead beating in hot phenol. Variations due to the different methodologies were compared to variation among individuals using two approaches--one based on presence-absence information for bacterial taxa (unweighted UniFrac) and the other taking into account their relative abundance (weighted UniFrac). In the unweighted analysis relatively little variation was associated with the different analytical procedures, and variation between individuals predominated. In the weighted analysis considerable variation was associated with the purification methods. Particularly notable was improved recovery of Firmicutes sequences using the hot phenol method. We also carried out surveys of the effects of different 454 sequencing methods (FLX versus Titanium) and amplification of different 16S rRNA variable gene segments. Based on our findings we present recommendations for protocols to collect, process and sequence bacterial 16S rDNA from fecal samples--some major points are 1) if feasible, bead-beating in hot phenol or use of the PSP kit improves recovery; 2) storage methods can be adjusted based on experimental convenience; 3) unweighted (presence-absence) comparisons are less affected by lysis method.

Draft Genome Sequence of Xanthomonas arboricola pv. pruni Strain Xap33, Causal Agent of Bacterial Spot Disease on Almond

PubMed Central

Garita-Cambronero, J.; Sena-Vélez, M.; Palacio-Bielsa, A.

2014-01-01

We report the annotated genome sequence of Xanthomonas arboricola pv. pruni strain Xap33, isolated from almond leaves showing bacterial spot disease symptoms in Spain. The availability of this genome sequence will aid our understanding of the infection mechanism of this bacterium as well as its relationship to other species of the same genus. PMID:24903863

Next-generation sequencing showing potential leachate influence on bacterial communities around a landfill in China.

PubMed

Rajasekar, Adharsh; Sekar, Raju; Medina-Roldán, Eduardo; Bridge, Jonathan; Moy, Charles K S; Wilkinson, Stephen

2018-04-10

The impact of contaminated leachate on groundwater from landfills is well known, but the specific effects on bacterial consortia are less well-studied. Bacterial communities in a landfill and an urban site located in Suzhou, China, were studied using Illumina high-throughput sequencing. A total of 153 944 good-quality reads were produced and sequences assigned to 6388 operational taxonomic units. Bacterial consortia consisted of up to 16 phyla, including Proteobacteria (31.9%-94.9% at landfill, 25.1%-43.3% at urban sites), Actinobacteria (0%-28.7% at landfill, 9.9%-34.3% at urban sites), Bacteroidetes (1.4%-25.6% at landfill, 5.6%-7.8% at urban sites), Chloroflexi (0.4%-26.5% at urban sites only), and unclassified bacteria. Pseudomonas was the dominant (67%-93%) genus in landfill leachate. Arsenic concentrations in landfill raw leachate (RL) (1.11 × 10 3 μg/L) and fresh leachate (FL2) (1.78 × 10 3 μg/L) and mercury concentrations in RL (10.9 μg/L) and FL2 (7.37 μg/L) exceeded Chinese State Environmental Protection Administration standards for leachate in landfills. The Shannon diversity index and Chao1 richness estimate showed RL and FL2 lacked richness and diversity when compared with other samples. This is consistent with stresses imposed by elevated arsenic and mercury and has implications for ecological site remediation by bioremediation or natural attenuation.

Phyllosphere Bacterial Community of Floating Macrophytes in Paddy Soil Environments as Revealed by Illumina High-Throughput Sequencing

PubMed Central

Xie, Wan-Ying

2014-01-01

The phyllosphere of floating macrophytes in paddy soil ecosystems, a unique habitat, may support large microbial communities but remains largely unknown. We took Wolffia australiana as a representative floating plant and investigated its phyllosphere bacterial community and the underlying driving forces of community modulation in paddy soil ecosystems using Illumina HiSeq 2000 platform-based 16S rRNA gene sequence analysis. The results showed that the phyllosphere of W. australiana harbored considerably rich communities of bacteria, with Proteobacteria and Bacteroidetes as the predominant phyla. The core microbiome in the phyllosphere contained genera such as Acidovorax, Asticcacaulis, Methylibium, and Methylophilus. Complexity of the phyllosphere bacterial communities in terms of class number and α-diversity was reduced compared to those in corresponding water and soil. Furthermore, the bacterial communities exhibited structures significantly different from those in water and soil. These findings and the following redundancy analysis (RDA) suggest that species sorting played an important role in the recruitment of bacterial species in the phyllosphere. The compositional structures of the phyllosphere bacterial communities were modulated predominantly by water physicochemical properties, while the initial soil bacterial communities had limited impact. Taken together, the findings from this study reveal the diversity and uniqueness of the phyllosphere bacterial communities associated with the floating macrophytes in paddy soil environments. PMID:25362067

Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.

PubMed

Woo, P C Y; Lau, S K P; Teng, J L L; Tse, H; Yuen, K-Y

2008-10-01

In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rDNA sequencing has played a pivotal role in the accurate identification of bacterial isolates and the discovery of novel bacteria in clinical microbiology laboratories. For bacterial identification, 16S rDNA sequencing is particularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, uncultivable bacteria and culture-negative infections. Not only has it provided insights into aetiologies of infectious disease, but it also helps clinicians in choosing antibiotics and in determining the duration of treatment and infection control procedures. With the use of 16S rDNA sequencing, 215 novel bacterial species, 29 of which belong to novel genera, have been discovered from human specimens in the past 7 years of the 21st century (2001-2007). One hundred of the 215 novel species, 15 belonging to novel genera, have been found in four or more subjects. The largest number of novel species discovered were of the genera Mycobacterium (n = 12) and Nocardia (n = 6). The oral cavity/dental-related specimens (n = 19) and the gastrointestinal tract (n = 26) were the most important sites for discovery and/or reservoirs of novel species. Among the 100 novel species, Streptococcus sinensis, Laribacter hongkongensis, Clostridium hathewayi and Borrelia spielmanii have been most thoroughly characterized, with the reservoirs and routes of transmission documented, and S. sinensis, L. hongkongensis and C. hathewayi have been found globally. One of the greatest hurdles in putting 16S rDNA sequencing into routine use in clinical microbiology laboratories is automation of the technology. The only step that can be automated at the moment is input of the 16S rDNA sequence of the bacterial isolate for identification into one of the software packages that will generate the result of the identity of the isolate on the basis of its sequence database. However

Complete Genome Sequence and Immunoproteomic Analyses of the Bacterial Fish Pathogen Streptococcus parauberis▿†

PubMed Central

Nho, Seong Won; Hikima, Jun-ichi; Cha, In Seok; Park, Seong Bin; Jang, Ho Bin; del Castillo, Carmelo S.; Kondo, Hidehiro; Hirono, Ikuo; Aoki, Takashi; Jung, Tae Sung

2011-01-01

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae. PMID:21531805

Calculating the quality of public high-throughput sequencing data to obtain a suitable subset for reanalysis from the Sequence Read Archive.

PubMed

Ohta, Tazro; Nakazato, Takeru; Bono, Hidemasa

2017-06-01

It is important for public data repositories to promote the reuse of archived data. In the growing field of omics science, however, the increasing number of submissions of high-throughput sequencing (HTSeq) data to public repositories prevents users from choosing a suitable data set from among the large number of search results. Repository users need to be able to set a threshold to reduce the number of results to obtain a suitable subset of high-quality data for reanalysis. We calculated the quality of sequencing data archived in a public data repository, the Sequence Read Archive (SRA), by using the quality control software FastQC. We obtained quality values for 1 171 313 experiments, which can be used to evaluate the suitability of data for reuse. We also visualized the data distribution in SRA by integrating the quality information and metadata of experiments and samples. We provide quality information of all of the archived sequencing data, which enable users to obtain sufficient quality sequencing data for reanalyses. The calculated quality data are available to the public in various formats. Our data also provide an example of enhancing the reuse of public data by adding metadata to published research data by a third party. © The Authors 2017. Published by Oxford University Press.

Calculating the quality of public high-throughput sequencing data to obtain a suitable subset for reanalysis from the Sequence Read Archive

PubMed Central

Nakazato, Takeru; Bono, Hidemasa

2017-01-01

Abstract It is important for public data repositories to promote the reuse of archived data. In the growing field of omics science, however, the increasing number of submissions of high-throughput sequencing (HTSeq) data to public repositories prevents users from choosing a suitable data set from among the large number of search results. Repository users need to be able to set a threshold to reduce the number of results to obtain a suitable subset of high-quality data for reanalysis. We calculated the quality of sequencing data archived in a public data repository, the Sequence Read Archive (SRA), by using the quality control software FastQC. We obtained quality values for 1 171 313 experiments, which can be used to evaluate the suitability of data for reuse. We also visualized the data distribution in SRA by integrating the quality information and metadata of experiments and samples. We provide quality information of all of the archived sequencing data, which enable users to obtain sufficient quality sequencing data for reanalyses. The calculated quality data are available to the public in various formats. Our data also provide an example of enhancing the reuse of public data by adding metadata to published research data by a third party. PMID:28449062

A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes

PubMed Central

2010-01-01

Background Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT). Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins. Results Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the repeat may be disseminated by

Phyllosphere bacterial community of floating macrophytes in paddy soil environments as revealed by illumina high-throughput sequencing.

PubMed

Xie, Wan-Ying; Su, Jian-Qiang; Zhu, Yong-Guan

2015-01-01

The phyllosphere of floating macrophytes in paddy soil ecosystems, a unique habitat, may support large microbial communities but remains largely unknown. We took Wolffia australiana as a representative floating plant and investigated its phyllosphere bacterial community and the underlying driving forces of community modulation in paddy soil ecosystems using Illumina HiSeq 2000 platform-based 16S rRNA gene sequence analysis. The results showed that the phyllosphere of W. australiana harbored considerably rich communities of bacteria, with Proteobacteria and Bacteroidetes as the predominant phyla. The core microbiome in the phyllosphere contained genera such as Acidovorax, Asticcacaulis, Methylibium, and Methylophilus. Complexity of the phyllosphere bacterial communities in terms of class number and α-diversity was reduced compared to those in corresponding water and soil. Furthermore, the bacterial communities exhibited structures significantly different from those in water and soil. These findings and the following redundancy analysis (RDA) suggest that species sorting played an important role in the recruitment of bacterial species in the phyllosphere. The compositional structures of the phyllosphere bacterial communities were modulated predominantly by water physicochemical properties, while the initial soil bacterial communities had limited impact. Taken together, the findings from this study reveal the diversity and uniqueness of the phyllosphere bacterial communities associated with the floating macrophytes in paddy soil environments. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Comprehensive phylogenetic analysis of bacterial reverse transcriptases.

PubMed

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology.

Comprehensive Phylogenetic Analysis of Bacterial Reverse Transcriptases

PubMed Central

Toro, Nicolás; Nisa-Martínez, Rafael

2014-01-01

Much less is known about reverse transcriptases (RTs) in prokaryotes than in eukaryotes, with most prokaryotic enzymes still uncharacterized. Two surveys involving BLAST searches for RT genes in prokaryotic genomes revealed the presence of large numbers of diverse, uncharacterized RTs and RT-like sequences. Here, using consistent annotation across all sequenced bacterial species from GenBank and other sources via RAST, available from the PATRIC (Pathogenic Resource Integration Center) platform, we have compiled the data for currently annotated reverse transcriptases from completely sequenced bacterial genomes. RT sequences are broadly distributed across bacterial phyla, but green sulfur bacteria and cyanobacteria have the highest levels of RT sequence diversity (≤85% identity) per genome. By contrast, phylum Actinobacteria, for which a large number of genomes have been sequenced, was found to have a low RT sequence diversity. Phylogenetic analyses revealed that bacterial RTs could be classified into 17 main groups: group II introns, retrons/retron-like RTs, diversity-generating retroelements (DGRs), Abi-like RTs, CRISPR-Cas-associated RTs, group II-like RTs (G2L), and 11 other groups of RTs of unknown function. Proteobacteria had the highest potential functional diversity, as they possessed most of the RT groups. Group II introns and DGRs were the most widely distributed RTs in bacterial phyla. Our results provide insights into bacterial RT phylogeny and the basis for an update of annotation systems based on sequence/domain homology. PMID:25423096

Assessing the Robustness of Complete Bacterial Genome Segmentations

NASA Astrophysics Data System (ADS)

Devillers, Hugo; Chiapello, Hélène; Schbath, Sophie; El Karoui, Meriem

Comparison of closely related bacterial genomes has revealed the presence of highly conserved sequences forming a "backbone" that is interrupted by numerous, less conserved, DNA fragments. Segmentation of bacterial genomes into backbone and variable regions is particularly useful to investigate bacterial genome evolution. Several software tools have been designed to compare complete bacterial chromosomes and a few online databases store pre-computed genome comparisons. However, very few statistical methods are available to evaluate the reliability of these software tools and to compare the results obtained with them. To fill this gap, we have developed two local scores to measure the robustness of bacterial genome segmentations. Our method uses a simulation procedure based on random perturbations of the compared genomes. The scores presented in this paper are simple to implement and our results show that they allow to discriminate easily between robust and non-robust bacterial genome segmentations when using aligners such as MAUVE and MGA.

Towards rationally redesigning bacterial signaling systems using information encoded in abundant sequence data

NASA Astrophysics Data System (ADS)

Cheng, Ryan; Morcos, Faruck; Levine, Herbert; Onuchic, Jose

2014-03-01

An important challenge in biology is to distinguish the subset of residues that allow bacterial two-component signaling (TCS) proteins to preferentially interact with their correct TCS partner such that they can bind and transfer signal. Detailed knowledge of this information would allow one to search sequence-space for mutations that can systematically tune the signal transmission between TCS partners as well as re-encode a TCS protein to preferentially transfer signals to a non-partner. Motivated by the notion that this detailed information is found in sequence data, we explore the mutual sequence co-evolution between signaling partners to infer how mutations can positively or negatively alter their interaction. Using Direct Coupling Analysis (DCA) for determining evolutionarily conserved interprotein interactions, we apply a DCA-based metric to quantify mutational changes in the interaction between TCS proteins and demonstrate that it accurately correlates with experimental mutagenesis studies probing the mutational change in the in vitro phosphotransfer. Our methodology serves as a potential framework for the rational design of TCS systems as well as a framework for the system-level study of protein-protein interactions in sequence-rich systems. This research has been supported by the NSF INSPIRE award MCB-1241332 and by the CTBP sponsored by the NSF (Grant PHY-1308264).

Next-generation sequencing identification of pathogenic bacterial genes and their relationship with fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal.

PubMed

Ghaju Shrestha, Rajani; Tanaka, Yasuhiro; Malla, Bikash; Bhandari, Dinesh; Tandukar, Sarmila; Inoue, Daisuke; Sei, Kazunari; Sherchand, Jeevan B; Haramoto, Eiji

2017-12-01

Bacteriological analysis of drinking water leads to detection of only conventional fecal indicator bacteria. This study aimed to explore and characterize bacterial diversity, to understand the extent of pathogenic bacterial contamination, and to examine the relationship between pathogenic bacteria and fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal. Sixteen water samples were collected from shallow dug wells (n=12), a deep tube well (n=1), a spring (n=1), and rivers (n=2) in September 2014 for 16S rRNA gene next-generation sequencing. A total of 525 genera were identified, of which 81 genera were classified as possible pathogenic bacteria. Acinetobacter, Arcobacter, and Clostridium were detected with a relatively higher abundance (>0.1% of total bacterial genes) in 16, 13, and 5 of the 16 samples, respectively, and the highest abundance ratio of Acinetobacter (85.14%) was obtained in the deep tube well sample. Furthermore, the bla OXA23-like genes of Acinetobacter were detected using SYBR Green-based quantitative PCR in 13 (35%) of 37 water samples, including the 16 samples that were analyzed for next-generation sequencing, with concentrations ranging 5.3-7.5logcopies/100mL. There was no sufficient correlation found between fecal indicator bacteria, such as Escherichia coli and total coliforms, and potential pathogenic bacteria, as well as the bla OXA23-like gene of Acinetobacter. These results suggest the limitation of using conventional fecal indicator bacteria in evaluating the pathogenic bacteria contamination of different water sources in the Kathmandu Valley. Copyright © 2017 Elsevier B.V. All rights reserved.

PathogenFinder--distinguishing friend from foe using bacterial whole genome sequence data.

PubMed

Cosentino, Salvatore; Voldby Larsen, Mette; Møller Aarestrup, Frank; Lund, Ole

2013-01-01

Although the majority of bacteria are harmless or even beneficial to their host, others are highly virulent and can cause serious diseases, and even death. Due to the constantly decreasing cost of high-throughput sequencing there are now many completely sequenced genomes available from both human pathogenic and innocuous strains. The data can be used to identify gene families that correlate with pathogenicity and to develop tools to predict the pathogenicity of newly sequenced strains, investigations that previously were mainly done by means of more expensive and time consuming experimental approaches. We describe PathogenFinder (http://cge.cbs.dtu.dk/services/PathogenFinder/), a web-server for the prediction of bacterial pathogenicity by analysing the input proteome, genome, or raw reads provided by the user. The method relies on groups of proteins, created without regard to their annotated function or known involvement in pathogenicity. The method has been built to work with all taxonomic groups of bacteria and using the entire training-set, achieved an accuracy of 88.6% on an independent test-set, by correctly classifying 398 out of 449 completely sequenced bacteria. The approach here proposed is not biased on sets of genes known to be associated with pathogenicity, thus the approach could aid the discovery of novel pathogenicity factors. Furthermore the pathogenicity prediction web-server could be used to isolate the potential pathogenic features of both known and unknown strains.

Amplification and sequence analysis of partial bacterial 16S ribosomal RNA gene in gallbladder bile from patients with primary biliary cirrhosis.

PubMed

Hiramatsu, K; Harada, K; Tsuneyama, K; Sasaki, M; Fujita, S; Hashimoto, T; Kaneko, S; Kobayashi, K; Nakanuma, Y

2000-07-01

The etiopathogenesis of bile duct lesion in primary biliary cirrhosis is unknown, though the participation of bacteria and/or their components and products is suspected. In this study, we tried to detect and identify bacteria in the bile of patients with primary biliary cirrhosis by polymerase chain reaction using universal bacterial primers of the 16S ribosomal RNA gene. Gallbladder bile samples from 15 patients with primary biliary cirrhosis, 5 with primary sclerosing cholangitis, 5 with hepatitis C virus-related liver cirrhosis, 11 with cholecystolithiasis, and from 12 normal adult gallbladders were used. In addition to the culture study, partial bacterial 16S ribosomal RNA gene was amplified by polymerase chain reaction (PCR) taking advantage of universal primers that can amplify the gene of almost all bacterial species, and the amplicons were cloned and sequenced. Sequence homology with specific bacterial species was analyzed by database research. Bacterial contamination at every step of the bile sampling, DNA extraction and PCR study was avoided. Furthermore, to confirm whether bacterial DNA is detectable in liver explants, the same analysis was performed using 10 liver explants of patients with primary biliary cirrhosis. In primary biliary cirrhosis, 75% (p<0.0001) of 100 clones were identified as so-called gram-positive cocci while these cocci were positive in only 5% in cholecystolithiasis (p<0.0001). In cholecystolithiasis gram-negative rods were predominant instead. One bacterial species detected in a normal adult was not related to those detected in primary biliary cirrhosis and cholecystolithiasis patients. No bacterial DNA was detected by PCR amplification in 10 liver explants of patients with primary biliary cirrhosis. The present results raise several possible roles of gram-positive bacteria in bile in the etiopathogenesis of primary biliary cirrhosis. However, these results could also reflect an epiphenomenon due to decreased bile flow in the

Obtaining a more resolute teleost growth hormone phylogeny by the introduction of gaps in sequence alignment.

PubMed

Rubin, D A; Dores, R M

1995-06-01

In order to obtain a more resolute phylogeny of teleosts based on growth hormone (GH) sequences, phylogenetic analyses were performed in which deletions (gaps), which appear to be order specific, were upheld to maintain GH's structural information. Sequences were analyzed at 194 amino acid positions. In addition, the two closest genealogically related groups to the teleosts, Amia calva and Acipenser guldenstadti, were used as outgroups. Modified sequence alignments were also analyzed to determine clade stability. Analyses indicated, in the most parsimonious cladogram, that molecular and morphological relationships for the orders of fishes are congruent. With GH molecular sequence data it was possible to resolve all clades at the familial level. Analyses of the primary sequence data indicate that: (a) the halecomorphean and chondrostean GH sequences are the appropriate outgroups for generating the most parsimonious cladogram for teleosts; (b) proper alignment of teleost GH sequence by the inclusion of gaps is necessary for resolution of the Percomorpha; and (c) removal of sequence information by deleting improperly aligned sequence decreases the phylogenetic signal obtained.

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

Response of soil bacterial communities to lead and zinc pollution revealed by Illumina MiSeq sequencing investigation.

PubMed

Xu, Xihui; Zhang, Zhou; Hu, Shunli; Ruan, Zhepu; Jiang, Jiandong; Chen, Chen; Shen, Zhenguo

2017-01-01

Soil provides a critical environment for microbial community development. However, microorganisms may be sensitive to substances such as heavy metals (HMs), which are common soil contaminants. This study investigated bacterial communities using 16S ribosomal RNA (rRNA) gene fragment sequencing in geographic regions with and without HM pollution to elucidate the effects of soil properties and HMs on bacterial communities. No obvious changes in the richness or diversity of bacterial communities were observed between samples from mining and control areas. Significant differences in bacterial richness and diversity were detected between samples from different geographic regions, indicating that the basic soil characteristics were the most important factors affecting bacterial communities other than HMs. However, the abundances of several phyla and genera differed significantly between mining and control samples, suggesting that Zn and Pb pollution may impact the soil bacterial community composition. Moreover, regression analyses showed that the relative abundances of these phyla and genera were correlated significantly with the soil-available Zn and Pb contents. Redundancy analysis indicated that the soil K, ammoniacal nitrogen (NH 4 + -N), total Cu, and available Zn and Cu contents were the most important factors. Our results not only suggested that the soil bacteria were sensitive to HM stresses but also indicated that other soil properties may affect soil microorganisms to a greater extent.

LLNL Genomic Assessment: Viral and Bacterial Sequencing Needs for TMTI, Task 1.4.2 Report

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

Slezak, T; Borucki, M; Lam, M

Good progress has been made on both bacterial and viral sequencing by the TMTI centers. While access to appropriate samples is a limiting factor to throughput, excellent progress has been made with respect to getting agreements in place with key sources of relevant materials. Sharing of sequenced genomes funded by TMTI has been extremely limited to date. The April 2010 exercise should force a resolution to this, but additional managerial pressures may be needed to ensure that rapid sharing of TMTI-funded sequencing occurs, regardless of collaborator constraints concerning ultimate publication(s). Policies to permit TMTI-internal rapid sharing of sequenced genomes shouldmore » be written into all TMTI agreements with collaborators now being negotiated. TMTI needs to establish a Web-based system for tracking samples destined for sequencing. This includes metadata on sample origins and contributor, information on sample shipment/receipt, prioritization by TMTI, assignment to one or more sequencing centers (including possible TMTI-sponsored sequencing at a contributor site), and status history of the sample sequencing effort. While this system could be a component of the AFRL system, it is not part of any current development effort. Policy and standardized procedures are needed to ensure appropriate verification of all TMTI samples prior to the investment in sequencing. PCR, arrays, and classical biochemical tests are examples of potential verification methods. Verification is needed to detect miss-labeled, degraded, mixed or contaminated samples. Regular QC exercises are needed to ensure that the TMTI-funded centers are meeting all standards for producing quality genomic sequence data.« less

High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain.

PubMed

Dobson, Alleson; O'Sullivan, Orla; Cotter, Paul D; Ross, Paul; Hill, Colin

2011-07-01

Lacticin 3147 is a two-peptide broad spectrum lantibiotic produced by Lactococcus lactis DPC3147 shown to inhibit a number of clinically relevant Gram-positive pathogens. Initially isolated from an Irish kefir grain, lacticin 3147 is one of the most extensively studied lantibiotics to date. In this study, the bacterial diversity of the Irish kefir grain from which L. lactis DPC3147 was originally isolated was for the first time investigated using a high-throughput parallel sequencing strategy. A total of 17 416 unique V4 variable regions of the 16S rRNA gene were analysed from both the kefir starter grain and its derivative kefir-fermented milk. Firmicutes (which includes the lactic acid bacteria) was the dominant phylum accounting for > 92% of sequences. Within the Firmicutes, dramatic differences in abundance were observed when the starter grain and kefir milk fermentate were compared. The kefir grain-associated bacterial community was largely composed of the Lactobacillaceae family while Streptococcaceae (primarily Lactococcus spp.) was the dominant family within the kefir milk fermentate. Sequencing data confirmed previous findings that the microbiota of kefir milk and the starter grain are quite different while at the same time, establishing that the microbial diversity of the starter grain is not uniform with a greater level of diversity associated with the interior kefir starter grain compared with the exterior. © 2011 Teagasc Food Research Centre, Moorepark. FEMS Microbiology Letters © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

Bacterial diversity characterization in petroleum samples from Brazilian reservoirs

PubMed Central

de Oliveira, Valéria Maia; Sette, Lara Durães; Simioni, Karen Christina Marques; dos Santos Neto, Eugênio Vaz

2008-01-01

This study aimed at evaluating potential differences among the bacterial communities from formation water and oil samples originated from biodegraded and non-biodegraded Brazilian petroleum reservoirs by using a PCR-DGGE based approach. Environmental DNA was isolated and used in PCR reactions with bacterial primers, followed by separation of 16S rDNA fragments in the DGGE. PCR products were also cloned and sequenced, aiming at the taxonomic affiliation of the community members. The fingerprints obtained allowed the direct comparison among the bacterial communities from oil samples presenting distinct degrees of biodegradation, as well as between the communities of formation water and oil sample from the non-biodegraded reservoir. Very similar DGGE band profiles were observed for all samples, and the diversity of the predominant bacterial phylotypes was shown to be low. Cloning and sequencing results revealed major differences between formation water and oil samples from the non-biodegraded reservoir. Bacillus sp. and Halanaerobium sp. were shown to be the predominant components of the bacterial community from the formation water sample, whereas the oil sample also included Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. and Acidithiobacillus ferrooxidans. The PCR-DGGE technique, combined with cloning and sequencing of PCR products, revealed the presence of taxonomic groups not found previously in these samples when using cultivation-based methods and 16S rRNA gene library assembly, confirming the need of a polyphasic study in order to improve the knowledge of the extent of microbial diversity in such extreme environments. PMID:24031244

Assembled sequence contigs by SOAPdenova and Volvet algorithms from metagenomic short reads of a new bacterial isolate of gut origin

USDA-ARS?s Scientific Manuscript database

Assembled sequence contigs by SOAPdenova and Volvet algorithms from metagenomic short reads of a new bacterial isolate of gut origin. This study included 2 submissions with a total of 9.8 million bp of assembled contigs....

Comparison of the diagnostic performance of bacterial culture of nasopharyngeal swab and bronchoalveolar lavage fluid samples obtained from calves with bovine respiratory disease.

PubMed

Capik, Sarah F; White, Brad J; Lubbers, Brian V; Apley, Michael D; DeDonder, Keith D; Larson, Robert L; Harhay, Greg P; Chitko-McKown, Carol G; Harhay, Dayna M; Kalbfleisch, Ted S; Schuller, Gennie; Clawson, Michael L

2017-03-01

OBJECTIVE To compare predictive values, extent of agreement, and gamithromycin susceptibility between bacterial culture results of nasopharyngeal swab (NPS) and bronchoalveolar lavage fluid (BALF) samples obtained from calves with bovine respiratory disease (BRD). ANIMALS 28 beef calves with clinical BRD. PROCEDURES Pooled bilateral NPS samples and BALF samples were obtained for bacterial culture from calves immediately before and at various times during the 5 days after gamithromycin (6 mg/kg, SC, once) administration. For each culture-positive sample, up to 12 Mannheimia haemolytica, 6 Pasteurella multocida, and 6 Histophilus somni colonies underwent gamithromycin susceptibility testing. Whole-genome sequencing was performed on all M haemolytica isolates. For paired NPS and BALF samples collected 5 days after gamithromycin administration, the positive and negative predictive values for culture results of NPS samples relative to those of BALF samples and the extent of agreement between the sampling methods were determined. RESULTS Positive and negative predictive values of NPS samples were 67% and 100% for M haemolytica, 75% and 100% for P multocida, and 100% and 96% for H somni. Extent of agreement between results for NPS and BALF samples was substantial for M haemolytica (κ, 0.71) and H somni (κ, 0.78) and almost perfect for P multocida (κ, 0.81). Gamithromycin susceptibility varied within the same sample and between paired NPS and BALF samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated culture results of NPS and BALF samples from calves with BRD should be interpreted cautiously considering disease prevalence within the population, sample collection relative to antimicrobial administration, and limitations of diagnostic testing methods.

High-Throughput rRNA Gene Sequencing Reveals High
and Complex Bacterial Diversity Associated with
Brazilian Coffee Bean Fermentation

PubMed Central

Vinícius de Melo, Gilberto

2018-01-01

Summary Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97% of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing.

Necessary Sequencing Depth and Clustering Method to Obtain Relatively Stable Diversity Patterns in Studying Fish Gut Microbiota.

PubMed

Xiao, Fanshu; Yu, Yuhe; Li, Jinjin; Juneau, Philippe; Yan, Qingyun

2018-05-25

The 16S rRNA gene is one of the most commonly used molecular markers for estimating bacterial diversity during the past decades. However, there is no consistency about the sequencing depth (from thousand to millions of sequences per sample), and the clustering methods used to generate OTUs may also be different among studies. These inconsistent premises make effective comparisons among studies difficult or unreliable. This study aims to examine the necessary sequencing depth and clustering method that would be needed to ensure a stable diversity patterns for studying fish gut microbiota. A total number of 42 samples dataset of Siniperca chuatsi (carnivorous fish) gut microbiota were used to test how the sequencing depth and clustering may affect the alpha and beta diversity patterns of fish intestinal microbiota. Interestingly, we found that the sequencing depth (resampling 1000-11,000 per sample) and the clustering methods (UPARSE and UCLUST) did not bias the estimates of the diversity patterns during the fish development from larva to adult. Although we should acknowledge that a suitable sequencing depth may differ case by case, our finding indicates that a shallow sequencing such as 1000 sequences per sample may be also enough to reflect the general diversity patterns of fish gut microbiota. However, we have shown in the present study that strict pre-processing of the original sequences is required to ensure reliable results. This study provides evidences to help making a strong scientific choice of the sequencing depth and clustering method for future studies on fish gut microbiota patterns, but at the same time reducing as much as possible the costs related to the analysis.

Responses of Baltic Sea Ice and Open-Water Natural Bacterial Communities to Salinity Change

PubMed Central

Kaartokallio, Hermanni; Laamanen, Maria; Sivonen, Kaarina

2005-01-01

To investigate the responses of Baltic Sea wintertime bacterial communities to changing salinity (5 to 26 practical salinity units), an experimental study was conducted. Bacterial communities of Baltic seawater and sea ice from a coastal site in southwest Finland were used in two batch culture experiments run for 17 or 18 days at 0°C. Bacterial abundance, cell volume, and leucine and thymidine incorporation were measured during the experiments. The bacterial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified partial 16S rRNA genes with sequencing of DGGE bands from initial communities and communities of day 10 or 13 of the experiment. The sea ice-derived bacterial community was metabolically more active than the open-water community at the start of the experiment. Ice-derived bacterial communities were able to adapt to salinity change with smaller effects on physiology and community structure, whereas in the open-water bacterial communities, the bacterial cell volume evolution, bacterial abundance, and community structure responses indicated the presence of salinity stress. The closest relatives for all eight partial 16S rRNA gene sequences obtained were either organisms found in polar sea ice and other cold habitats or those found in summertime Baltic seawater. All sequences except one were associated with the α- and γ-proteobacteria or the Cytophaga-Flavobacterium-Bacteroides group. The overall physiological and community structure responses were parallel in ice-derived and open-water bacterial assemblages, which points to a linkage between community structure and physiology. These results support previous assumptions of the role of salinity fluctuation as a major selective factor shaping the sea ice bacterial community structure. PMID:16085826

Genome sequence and plasmid transformation of the model high-yield bacterial cellulose producer Gluconacetobacter hansenii ATCC 53582

NASA Astrophysics Data System (ADS)

Florea, Michael; Reeve, Benjamin; Abbott, James; Freemont, Paul S.; Ellis, Tom

2016-03-01

Bacterial cellulose is a strong, highly pure form of cellulose that is used in a range of applications in industry, consumer goods and medicine. Gluconacetobacter hansenii ATCC 53582 is one of the highest reported bacterial cellulose producing strains and has been used as a model organism in numerous studies of bacterial cellulose production and studies aiming to increased cellulose productivity. Here we present a high-quality draft genome sequence for G. hansenii ATCC 53582 and find that in addition to the previously described cellulose synthase operon, ATCC 53582 contains two additional cellulose synthase operons and several previously undescribed genes associated with cellulose production. In parallel, we also develop optimized protocols and identify plasmid backbones suitable for transformation of ATCC 53582, albeit with low efficiencies. Together, these results provide important information for further studies into cellulose synthesis and for future studies aiming to genetically engineer G. hansenii ATCC 53582 for increased cellulose productivity.

Characterization of bacterial diversity associated with deep sea ferromanganese nodules from the South China Sea.

PubMed

Zhang, De-Chao; Liu, Yan-Xia; Li, Xin-Zheng

2015-09-01

Deep sea ferromanganese (FeMn) nodules contain metallic mineral resources and have great economic potential. In this study, a combination of culture-dependent and culture-independent (16S rRNA genes clone library and pyrosequencing) methods was used to investigate the bacterial diversity in FeMn nodules from Jiaolong Seamount, the South China Sea. Eleven bacterial strains including some moderate thermophiles were isolated. The majority of strains belonged to the phylum Proteobacteria; one isolate belonged to the phylum Firmicutes. A total of 259 near full-length bacterial 16S rRNA gene sequences in a clone library and 67,079 valid reads obtained using pyrosequencing indicated that members of the Gammaproteobacteria dominated, with the most abundant bacterial genera being Pseudomonas and Alteromonas. Sequence analysis indicated the presence of many organisms whose closest relatives are known manganese oxidizers, iron reducers, hydrogen-oxidizing bacteria and methylotrophs. This is the first reported investigation of bacterial diversity associated with deep sea FeMn nodules from the South China Sea.

Bacterial diversity in three distinct sub-habitats within the pitchers of the northern pitcher plant, Sarracenia purpurea.

PubMed

Krieger, Joseph R; Kourtev, Peter S

2012-03-01

Pitcher plants have been widely used in ecological studies of food webs; however, their bacterial communities are poorly characterized. Pitchers of Sarracenia purpurea contain several distinct sub-habitats, namely the bottom sediment, the liquid, and the internal pitcher wall. We hypothesized that those three sub-habitats within pitcher plants are inhabited by distinct bacterial populations. We used denaturing gradient gel electrophoresis and 16S rRNA gene sequencing to characterize bacterial populations in pitchers from three bogs. DGGE and sequencing revealed that in any given pitcher, the three sub-habitats contain significantly different bacterial populations. However, there was significant variability between bacterial populations inhabiting the same type of habitat in different pitchers, even at the same site. Therefore, no consistent set of bacterial populations was enriched in any of the three sub-habitats. All sub-habitats appeared to be dominated by alpha- and betaproteobacteria in differing proportions. In addition, sequences from the Bacteroidetes and Firmicutes were obtained from all three sub-habitats. We conclude that container aquatic habitats such as the pitchers of S. purpurea possess a very high bacterial diversity, with many unique bacterial populations enriched in individual pitchers. Within an individual pitcher, populations of certain bacterial families may be enriched in one of the three studied sub-habitats. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Next-Generation Sequencing Analyses of Bacterial Community Structures in Soybean Pastes Produced in Northeast China.

PubMed

Lee, Mi-Hwa; Li, Fan-Zhu; Lee, Jiyeon; Kang, Jisu; Lim, Seong-Il; Nam, Young-Do

2017-04-01

Fermented soybean foods contain nutritional components including easily digestible peptides, cholesterol-free oils, minerals, and vitamins. Various fermented soybean foods have been developed and are consumed as flavoring condiments in Asian regions. While the quality of fermented soybean foods is largely affected by microorganisms that participate in the fermentation process, our knowledge about the microorganisms in soybean pastes manufactured in Northeast China is limited. The current study used a culture-independent barcoded pyrosequencing method targeting hypervariable V1/V2 regions of the 16S rRNA gene to evaluate Korean doenjang and soybean pastes prepared by the Hun Chinese (SPHC) and Korean minority (SPKM) populations in Northeast China. In total, 63399 high-quality sequences were derived from 16 soybean paste samples collected in Northeast China. Each bacterial species-level taxon of SPHC, SPKM, and Korean doenjang was clustered separately. Each paste contained representative bacterial species that could be distinguished from each other: Bacillus subtilis in SPKM, Tetragenococcus halophilus in SPHC, and Enterococcus durans in Korean doenjang. This is the 1st massive sequencing-based study analyzing microbial communities in soybean pastes manufactured in Northeast China, compared to Korean doenjang. Our results clearly showed that each soybean paste contained unique microbial communities that varied depending on the manufacturing process and location. © 2017 Institute of Food Technologists®.

Bacterial community composition of chronic periodontitis and novel oral sampling sites for detecting disease indicators

PubMed Central

2014-01-01

Background Periodontitis is an infectious and inflammatory disease of polymicrobial etiology that can lead to the destruction of bones and tissues that support the teeth. The management of chronic periodontitis (CP) relies heavily on elimination or at least control of known pathogenic consortia associated with the disease. Until now, microbial plaque obtained from the subgingival (SubG) sites has been the primary focus for bacterial community analysis using deep sequencing. In addition to the use of SubG plaque, here, we investigated whether plaque obtained from supragingival (SupG) and tongue dorsum sites can serve as alternatives for monitoring CP-associated bacterial biomarkers. Results Using SubG, SupG, and tongue plaque DNA from 11 healthy and 13 diseased subjects, we sequenced V3 regions (approximately 200 bases) of the 16S rRNA gene using Illumina sequencing. After quality filtering, approximately 4.1 million sequences were collapsed into operational taxonomic units (OTUs; sequence identity cutoff of >97%) that were classified to a total of 19 phyla spanning 114 genera. Bacterial community diversity and overall composition was not affected by health or disease, and multiresponse permutation procedure (MRPP) on Bray-Curtis distance measures only supported weakly distinct bacterial communities in SubG and tongue plaque depending on health or disease status (P < 0.05). Nonetheless, in SubG and tongue sites, the relative abundance of Firmicutes was increased significantly from health to disease and members of Synergistetes were found in higher abundance across all sites in disease. Taxa indicative of CP were identified in all three locations (for example, Treponema denticola, Porphyromonas gingivalis, Synergistes oral taxa 362 and 363). Conclusions For the first time, this study demonstrates that SupG and tongue dorsum plaque can serve as alternative sources for detecting and enumerating known and novel bacterial biomarkers of CP. This finding is clinically

Whole Genome Sequence Analysis of Pig Respiratory Bacterial Pathogens with Elevated Minimum Inhibitory Concentrations for Macrolides.

PubMed

Dayao, Denise Ann Estarez; Seddon, Jennifer M; Gibson, Justine S; Blackall, Patrick J; Turni, Conny

2016-10-01

Macrolides are often used to treat and control bacterial pathogens causing respiratory disease in pigs. This study analyzed the whole genome sequences of one clinical isolate of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, and Bordetella bronchiseptica, all isolated from Australian pigs to identify the mechanism underlying the elevated minimum inhibitory concentrations (MICs) for erythromycin, tilmicosin, or tulathromycin. The H. parasuis assembled genome had a nucleotide transition at position 2059 (A to G) in the six copies of the 23S rRNA gene. This mutation has previously been associated with macrolide resistance but this is the first reported mechanism associated with elevated macrolide MICs in H. parasuis. There was no known macrolide resistance mechanism identified in the other three bacterial genomes. However, strA and sul2, aminoglycoside and sulfonamide resistance genes, respectively, were detected in one contiguous sequence (contig 1) of A. pleuropneumoniae assembled genome. This contig was identical to plasmids previously identified in Pasteurellaceae. This study has provided one possible explanation of elevated MICs to macrolides in H. parasuis. Further studies are necessary to clarify the mechanism causing the unexplained macrolide resistance in other Australian pig respiratory pathogens including the role of efflux systems, which were detected in all analyzed genomes.

Maize Endophytic Bacterial Diversity as Affected by Soil Cultivation History.

PubMed

Correa-Galeote, David; Bedmar, Eulogio J; Arone, Gregorio J

2018-01-01

The bacterial endophytic communities residing within roots of maize ( Zea mays L.) plants cultivated by a sustainable management in soils from the Quechua maize belt (Peruvian Andes) were examined using tags pyrosequencing spanning the V4 and V5 hypervariable regions of the 16S rRNA. Across four replicate libraries, two corresponding to sequences of endophytic bacteria from long time maize-cultivated soils and the other two obtained from fallow soils, 793 bacterial sequences were found that grouped into 188 bacterial operational taxonomic units (OTUs, 97% genetic similarity). The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from fallow soils. A mean of 30 genera were found in the fallow soil libraries and 47 were in those from the maize-cultivated soils. Both alpha and beta diversity indexes showed clear differences between bacterial endophytic populations from plants with different soil cultivation history and that the soils cultivated for long time requires a higher diversity of endophytes. The number of sequences corresponding to main genera Sphingomonas, Herbaspirillum, Bradyrhizobium and Methylophilus in the maize-cultivated libraries were statistically more abundant than those from the fallow soils. Sequences of genera Dyella and Sreptococcus were significantly more abundant in the libraries from the fallow soils. Relative abundance of genera Burkholderia, candidatus Glomeribacter, Staphylococcus, Variovorax, Bacillus and Chitinophaga were similar among libraries. A canonical correspondence analysis of the relative abundance of the main genera showed that the four libraries distributed in two clearly separated groups. Our results suggest that cultivation history is an important driver of endophytic colonization of maize and that after a long time of cultivation of the soil the maize plants need to increase the richness of the bacterial endophytes communities.

Maize Endophytic Bacterial Diversity as Affected by Soil Cultivation History

PubMed Central

Correa-Galeote, David; Bedmar, Eulogio J.; Arone, Gregorio J.

2018-01-01

The bacterial endophytic communities residing within roots of maize (Zea mays L.) plants cultivated by a sustainable management in soils from the Quechua maize belt (Peruvian Andes) were examined using tags pyrosequencing spanning the V4 and V5 hypervariable regions of the 16S rRNA. Across four replicate libraries, two corresponding to sequences of endophytic bacteria from long time maize-cultivated soils and the other two obtained from fallow soils, 793 bacterial sequences were found that grouped into 188 bacterial operational taxonomic units (OTUs, 97% genetic similarity). The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from fallow soils. A mean of 30 genera were found in the fallow soil libraries and 47 were in those from the maize-cultivated soils. Both alpha and beta diversity indexes showed clear differences between bacterial endophytic populations from plants with different soil cultivation history and that the soils cultivated for long time requires a higher diversity of endophytes. The number of sequences corresponding to main genera Sphingomonas, Herbaspirillum, Bradyrhizobium and Methylophilus in the maize-cultivated libraries were statistically more abundant than those from the fallow soils. Sequences of genera Dyella and Sreptococcus were significantly more abundant in the libraries from the fallow soils. Relative abundance of genera Burkholderia, candidatus Glomeribacter, Staphylococcus, Variovorax, Bacillus and Chitinophaga were similar among libraries. A canonical correspondence analysis of the relative abundance of the main genera showed that the four libraries distributed in two clearly separated groups. Our results suggest that cultivation history is an important driver of endophytic colonization of maize and that after a long time of cultivation of the soil the maize plants need to increase the richness of the bacterial endophytes communities. PMID:29662471

Bacterial diversity in a glacier foreland of the high Arctic.

PubMed

Schütte, Ursel M E; Abdo, Zaid; Foster, James; Ravel, Jacques; Bunge, John; Solheim, Bjørn; Forney, Larry J

2010-03-01

Over the past 100 years, Arctic temperatures have increased at almost twice the global average rate. One consequence is the acceleration of glacier retreat, exposing new habitats that are colonized by microorganisms whose diversity and function are unknown. Here, we characterized bacterial diversity along two approximately parallel chronosequences in an Arctic glacier forefield that span six time points following glacier retreat. We assessed changes in phylotype richness, evenness and turnover rate through the analysis of 16S rRNA gene sequences recovered from 52 samples taken from surface layers along the chronosequences. An average of 4500 sequences was obtained from each sample by 454 pyrosequencing. Using parametric methods, it was estimated that bacterial phylotype richness was high, and that it increased significantly from an average of 4000 (at a threshold of 97% sequence similarity) at locations exposed for 5 years to an average of 7050 phylotypes per 0.5 g of soil at sites that had been exposed for 150 years. Phylotype evenness also increased over time, with an evenness of 0.74 for 150 years since glacier retreat reflecting large proportions of rare phylotypes. The bacterial species turnover rate was especially high between sites exposed for 5 and 19 years. The level of bacterial diversity present in this High Arctic glacier foreland was comparable with that found in temperate and tropical soils, raising the question whether global patterns of bacterial species diversity parallel that of plants and animals, which have been found to form a latitudinal gradient and be lower in polar regions compared with the tropics.

Illumina sequencing-based analyses of bacterial communities during short-chain fatty-acid production from food waste and sewage sludge fermentation at different pH values.

PubMed

Cheng, Weixiao; Chen, Hong; Yan, ShuHai; Su, Jianqiang

2014-09-01

Short-chain fatty acids (SCFAs) can be produced by primary and waste activated sludge anaerobic fermentation. The yield and product spectrum distribution of SCFAs can be significantly affected by different initial pH values. However, most studies have focused on the physical and chemical aspects of SCFA production by waste activated sludge fermentation at different pH values. Information on the bacterial community structures during acidogenic fermentation is limited. In this study, comparisons of the bacterial communities during the co-substrate fermentation of food wastes and sewage sludge at different pH values were performed using the barcoded Illumina paired-end sequencing method. The results showed that different pH environments harbored a characteristic bacterial community, including sequences related to Lactobacillus, Prevotella, Mitsuokella, Treponema, Clostridium, and Ureibacillus. The most abundant bacterial operational taxonomic units in the different pH environments were those related to carbohydrate-degrading bacteria, which are associated with constituents of co-substrate fermentation. Further analyses showed that during organic matter fermentation, a core microbiota composed of Firmicutes, Proteobacteria, and Bacteroidetes existed. Comparison analyses revealed that the bacterial community during fermentation was significantly affected by the pH, and that the diverse product distribution was related to the shift in bacterial communities.

Bacterial diversity in permanently cold and alkaline ikaite columns from Greenland.

PubMed

Schmidt, Mariane; Priemé, Anders; Stougaard, Peter

2006-12-01

Bacterial diversity in alkaline (pH 10.4) and permanently cold (4 degrees C) ikaite tufa columns from the Ikka Fjord, SW Greenland, was investigated using growth characterization of cultured bacterial isolates with Terminal-restriction fragment length polymorphism (T-RFLP) and sequence analysis of bacterial 16S rRNA gene fragments. More than 200 bacterial isolates were characterized with respect to pH and temperature tolerance, and it was shown that the majority were cold-active alkaliphiles. T-RFLP analysis revealed distinct bacterial communities in different fractions of three ikaite columns, and, along with sequence analysis, it showed the presence of rich and diverse bacterial communities. Rarefaction analysis showed that the 109 sequenced clones in the 16S rRNA gene library represented between 25 and 65% of the predicted species richness in the three ikaite columns investigated. Phylogenetic analysis of the 16S rRNA gene sequences revealed many sequences with similarity to alkaliphilic or psychrophilic bacteria, and showed that 33% of the cloned sequences and 33% of the cultured bacteria showed less than 97% sequence identity to known sequences in databases, and may therefore represent yet unknown species.

Dynamic succession of substrate-associated bacterial composition and function during Ganoderma lucidum growth

PubMed Central

Li, Qiang; Zou, Jie; Tan, Hao; Tan, Wei; Peng, Weihong

2018-01-01

Background Ganoderma lucidum, a valuable medicinal fungus, is widely distributed in China. It grows alongside with a complex microbial ecosystem in the substrate. As sequencing technology advances, it is possible to reveal the composition and functions of substrate-associated bacterial communities. Methods We analyzed the bacterial community dynamics in the substrate during the four typical growth stages of G. lucidum using next-generation sequencing. Results The physicochemical properties of the substrate (e.g. acidity, moisture, total nitrogen, total phosphorus and total potassium) changed between different growth stages. A total of 598,771 sequences from 12 samples were obtained and assigned to 22 bacterial phyla. Proteobacteria and Firmicutes were the dominant phyla. Bacterial community composition and diversity significantly differed between the elongation stage and the other three growth stages. LEfSe analysis revealed a large number of bacterial taxa (e.g. Bacteroidetes, Acidobacteria and Nitrospirae) with significantly higher abundance at the elongation stage. Functional pathway prediction uncovered significant abundance changes of a number of bacterial functional pathways between the elongation stage and other growth stages. At the elongation stage, the abundance of the environmental information processing pathway (mainly membrane transport) decreased, whereas that of the metabolism-related pathways increased. Discussion The changes in bacterial community composition, diversity and predicted functions were most likely related to the changes in the moisture and nutrient conditions in the substrate with the growth of G. lucidum, particularly at the elongation stage. Our findings shed light on the G. lucidum-bacteria-substrate relationships, which should facilitate the industrial cultivation of G. lucidum. PMID:29915697

Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and “Nonbacterial” Prostatitis

PubMed Central

Tanner, Michael A.; Shoskes, Daniel; Shahed, Asha; Pace, Norman R.

1999-01-01

The etiology of chronic prostatitis syndromes in men is controversial, particularly when positive cultures for established uropathogens are lacking. Although identification of bacteria in prostatic fluid has relied on cultivation and microscopy, most microorganisms in the environment, including some human pathogens, are resistant to cultivation. We report here on an rRNA-based molecular phylogenetic approach to the identification of bacteria in prostate fluid from prostatitis patients. Positive bacterial signals were seen for 65% of patients with chronic prostatitis overall. Seven of 11 patients with bacterial signals but none of 6 patients without bacterial signals were cured with antibiotic-based therapy. Results indicate the occurrence in the prostate fluid of a wide spectrum of bacterial species representing several genera. Most rRNA genes were closely related to those of species belonging to the genera Corynebacterium, Staphylococcus, Peptostreptococcus, Streptococcus, and Escherichia. Unexpectedly, a wide diversity of Corynebacterium species was found in high proportion compared to the proportions of other bacterial species found. A subset of these 16S rRNA sequences represent those of undescribed species on the basis of their positions in phylogenetic trees. These uncharacterized organisms were not detected in control samples, suggesting that the organisms have a role in the disease or are the consequence of the disease. These studies show that microorganisms associated with prostatitis generally occur as complex microbial communities that differ between patients. The results also indicate that microbial communities distinct from those associated with prostatitis may occur at low levels in normal prostatic fluid. PMID:10325338

Bacterial community changes in an industrial algae production system.

PubMed

Fulbright, Scott P; Robbins-Pianka, Adam; Berg-Lyons, Donna; Knight, Rob; Reardon, Kenneth F; Chisholm, Stephen T

2018-04-01

While microalgae are a promising feedstock for production of fuels and other chemicals, a challenge for the algal bioproducts industry is obtaining consistent, robust algae growth. Algal cultures include complex bacterial communities and can be difficult to manage because specific bacteria can promote or reduce algae growth. To overcome bacterial contamination, algae growers may use closed photobioreactors designed to reduce the number of contaminant organisms. Even with closed systems, bacteria are known to enter and cohabitate, but little is known about these communities. Therefore, the richness, structure, and composition of bacterial communities were characterized in closed photobioreactor cultivations of Nannochloropsis salina in F/2 medium at different scales, across nine months spanning late summer-early spring, and during a sequence of serially inoculated cultivations. Using 16S rRNA sequence data from 275 samples, bacterial communities in small, medium, and large cultures were shown to be significantly different. Larger systems contained richer bacterial communities compared to smaller systems. Relationships between bacterial communities and algae growth were complex. On one hand, blooms of a specific bacterial type were observed in three abnormal, poorly performing replicate cultivations, while on the other, notable changes in the bacterial community structures were observed in a series of serial large-scale batch cultivations that had similar growth rates. Bacteria common to the majority of samples were identified, including a single OTU within the class Saprospirae that was found in all samples. This study contributes important information for crop protection in algae systems, and demonstrates the complex ecosystems that need to be understood for consistent, successful industrial algae cultivation. This is the first study to profile bacterial communities during the scale-up process of industrial algae systems.

Estimating Bacterial Diversity for Ecological Studies: Methods, Metrics, and Assumptions

PubMed Central

Birtel, Julia; Walser, Jean-Claude; Pichon, Samuel; Bürgmann, Helmut; Matthews, Blake

2015-01-01

Methods to estimate microbial diversity have developed rapidly in an effort to understand the distribution and diversity of microorganisms in natural environments. For bacterial communities, the 16S rRNA gene is the phylogenetic marker gene of choice, but most studies select only a specific region of the 16S rRNA to estimate bacterial diversity. Whereas biases derived from from DNA extraction, primer choice and PCR amplification are well documented, we here address how the choice of variable region can influence a wide range of standard ecological metrics, such as species richness, phylogenetic diversity, β-diversity and rank-abundance distributions. We have used Illumina paired-end sequencing to estimate the bacterial diversity of 20 natural lakes across Switzerland derived from three trimmed variable 16S rRNA regions (V3, V4, V5). Species richness, phylogenetic diversity, community composition, β-diversity, and rank-abundance distributions differed significantly between 16S rRNA regions. Overall, patterns of diversity quantified by the V3 and V5 regions were more similar to one another than those assessed by the V4 region. Similar results were obtained when analyzing the datasets with different sequence similarity thresholds used during sequences clustering and when the same analysis was used on a reference dataset of sequences from the Greengenes database. In addition we also measured species richness from the same lake samples using ARISA Fingerprinting, but did not find a strong relationship between species richness estimated by Illumina and ARISA. We conclude that the selection of 16S rRNA region significantly influences the estimation of bacterial diversity and species distributions and that caution is warranted when comparing data from different variable regions as well as when using different sequencing techniques. PMID:25915756

Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

DOE PAGES

Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian; ...

2016-06-02

We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungalmore » community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.« less

Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

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

Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian

We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungalmore » community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation.« less

Size Matters: Assessing Optimum Soil Sample Size for Fungal and Bacterial Community Structure Analyses Using High Throughput Sequencing of rRNA Gene Amplicons

PubMed Central

Penton, C. Ryan; Gupta, Vadakattu V. S. R.; Yu, Julian; Tiedje, James M.

2016-01-01

We examined the effect of different soil sample sizes obtained from an agricultural field, under a single cropping system uniform in soil properties and aboveground crop responses, on bacterial and fungal community structure and microbial diversity indices. DNA extracted from soil sample sizes of 0.25, 1, 5, and 10 g using MoBIO kits and from 10 and 100 g sizes using a bead-beating method (SARDI) were used as templates for high-throughput sequencing of 16S and 28S rRNA gene amplicons for bacteria and fungi, respectively, on the Illumina MiSeq and Roche 454 platforms. Sample size significantly affected overall bacterial and fungal community structure, replicate dispersion and the number of operational taxonomic units (OTUs) retrieved. Richness, evenness and diversity were also significantly affected. The largest diversity estimates were always associated with the 10 g MoBIO extractions with a corresponding reduction in replicate dispersion. For the fungal data, smaller MoBIO extractions identified more unclassified Eukaryota incertae sedis and unclassified glomeromycota while the SARDI method retrieved more abundant OTUs containing unclassified Pleosporales and the fungal genera Alternaria and Cercophora. Overall, these findings indicate that a 10 g soil DNA extraction is most suitable for both soil bacterial and fungal communities for retrieving optimal diversity while still capturing rarer taxa in concert with decreasing replicate variation. PMID:27313569

Whole-genome sequencing in bacteriology: state of the art

PubMed Central

Dark, Michael J

2013-01-01

Over the last ten years, genome sequencing capabilities have expanded exponentially. There have been tremendous advances in sequencing technology, DNA sample preparation, genome assembly, and data analysis. This has led to advances in a number of facets of bacterial genomics, including metagenomics, clinical medicine, bacterial archaeology, and bacterial evolution. This review examines the strengths and weaknesses of techniques in bacterial genome sequencing, upcoming technologies, and assembly techniques, as well as highlighting recent studies that highlight new applications for bacterial genomics. PMID:24143115

The impact of persistent bacterial bronchitis on the pulmonary microbiome of children

PubMed Central

Bingle, Lynne; Cookson, William O. C. M.; Everard, Mark L.; Moffatt, Miriam F.

2017-01-01

Introduction Persistent bacterial bronchitis (PBB) is a leading cause of chronic wet cough in young children. This study aimed to characterise the respiratory bacterial microbiota of healthy children and to assess the impact of the changes associated with the development of PBB. Blind, protected brushings were obtained from 20 healthy controls and 24 children with PBB, with an additional directed sample obtained from PBB patients. DNA was extracted, quantified using a 16S rRNA gene quantitative PCR assay prior to microbial community analysis by 16S rRNA gene sequencing. Results No significant difference in bacterial diversity or community composition (R2 = 0.01, P = 0.36) was observed between paired blind and non-blind brushes, showing that blind brushings are a valid means of accessing the airway microbiota. This has important implications for collecting lower respiratory samples from healthy children. A significant decrease in bacterial diversity (P < 0.001) and change in community composition (R2 = 0.08, P = 0.004) was observed among controls, in comparison with patients. Bacterial communities within patients with PBB were dominated by Proteobacteria, and indicator species analysis showed that Haemophilus and Neisseria were significantly associated with the patient group. In 15 (52.9%) cases the dominant organism by sequencing was not identified by standard routine clinical culture. Conclusion The bacteria present in the lungs of patients with PBB were less diverse in terms of richness and evenness. The results validate the clinical diagnosis, and suggest that more attention to bacterial communities in children with chronic cough may lead to more rapid recognition of this condition with earlier treatment and reduction in disease burden. PMID:29281698

A framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates.

PubMed

Helbling, Damian E; Johnson, David R; Lee, Tae Kwon; Scheidegger, Andreas; Fenner, Kathrin

2015-03-01

The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific

Low bacterial community diversity in two introduced aphid pests revealed with 16S rRNA amplicon sequencing

PubMed Central

Ortiz-Martínez, Sebastían; Silva, Andrea X.; Lavandero, Blas

2018-01-01

Bacterial endosymbionts that produce important phenotypic effects on their hosts are common among plant sap-sucking insects. Aphids have become a model system of insect-symbiont interactions. However, endosymbiont research has focused on a few aphid species, making it necessary to make greater efforts to other aphid species through different regions, in order to have a better understanding of the role of endosymbionts in aphids as a group. Aphid endosymbionts have frequently been studied by PCR-based techniques, using species-specific primers, nevertheless this approach may omit other non-target bacteria cohabiting a particular host species. Advances in high-throughput sequencing technologies are complementing our knowledge of microbial communities by allowing us the study of whole microbiome of different organisms. We used a 16S rRNA amplicon sequencing approach to study the microbiome of aphids in order to describe the bacterial community diversity in introduced populations of the cereal aphids, Sitobion avenae and Rhopalosiphum padi in Chile (South America). An absence of secondary endosymbionts and two common secondary endosymbionts of aphids were found in the aphids R. padi and S. avenae, respectively. Of those endosymbionts, Regiella insecticola was the dominant secondary endosymbiont among the aphid samples. In addition, the presence of a previously unidentified bacterial species closely related to a phytopathogenic Pseudomonad species was detected. We discuss these results in relation to the bacterial endosymbiont diversity found in other regions of the native and introduced range of S. avenae and R. padi. A similar endosymbiont diversity has been reported for both aphid species in their native range. However, variation in the secondary endosymbiont infection could be observed among the introduced and native populations of the aphid S. avenae, indicating that aphid-endosymbiont associations can vary across the geographic range of an aphid species. In

MIPS bacterial genomes functional annotation benchmark dataset.

PubMed

Tetko, Igor V; Brauner, Barbara; Dunger-Kaltenbach, Irmtraud; Frishman, Goar; Montrone, Corinna; Fobo, Gisela; Ruepp, Andreas; Antonov, Alexey V; Surmeli, Dimitrij; Mewes, Hans-Wernen

2005-05-15

Any development of new methods for automatic functional annotation of proteins according to their sequences requires high-quality data (as benchmark) as well as tedious preparatory work to generate sequence parameters required as input data for the machine learning methods. Different program settings and incompatible protocols make a comparison of the analyzed methods difficult. The MIPS Bacterial Functional Annotation Benchmark dataset (MIPS-BFAB) is a new, high-quality resource comprising four bacterial genomes manually annotated according to the MIPS functional catalogue (FunCat). These resources include precalculated sequence parameters, such as sequence similarity scores, InterPro domain composition and other parameters that could be used to develop and benchmark methods for functional annotation of bacterial protein sequences. These data are provided in XML format and can be used by scientists who are not necessarily experts in genome annotation. BFAB is available at http://mips.gsf.de/proj/bfab

BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads.

PubMed

Hong, Lewis Z; Hong, Shuzhen; Wong, Han Teng; Aw, Pauline P K; Cheng, Yan; Wilm, Andreas; de Sessions, Paola F; Lim, Seng Gee; Nagarajan, Niranjan; Hibberd, Martin L; Quake, Stephen R; Burkholder, William F

2014-01-01

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.

FoodMicrobionet: A database for the visualisation and exploration of food bacterial communities based on network analysis.

PubMed

Parente, Eugenio; Cocolin, Luca; De Filippis, Francesca; Zotta, Teresa; Ferrocino, Ilario; O'Sullivan, Orla; Neviani, Erasmo; De Angelis, Maria; Cotter, Paul D; Ercolini, Danilo

2016-02-16

Amplicon targeted high-throughput sequencing has become a popular tool for the culture-independent analysis of microbial communities. Although the data obtained with this approach are portable and the number of sequences available in public databases is increasing, no tool has been developed yet for the analysis and presentation of data obtained in different studies. This work describes an approach for the development of a database for the rapid exploration and analysis of data on food microbial communities. Data from seventeen studies investigating the structure of bacterial communities in dairy, meat, sourdough and fermented vegetable products, obtained by 16S rRNA gene targeted high-throughput sequencing, were collated and analysed using Gephi, a network analysis software. The resulting database, which we named FoodMicrobionet, was used to analyse nodes and network properties and to build an interactive web-based visualisation. The latter allows the visual exploration of the relationships between Operational Taxonomic Units (OTUs) and samples and the identification of core- and sample-specific bacterial communities. It also provides additional search tools and hyperlinks for the rapid selection of food groups and OTUs and for rapid access to external resources (NCBI taxonomy, digital versions of the original articles). Microbial interaction network analysis was carried out using CoNet on datasets extracted from FoodMicrobionet: the complexity of interaction networks was much lower than that found for other bacterial communities (human microbiome, soil and other environments). This may reflect both a bias in the dataset (which was dominated by fermented foods and starter cultures) and the lower complexity of food bacterial communities. Although some technical challenges exist, and are discussed here, the net result is a valuable tool for the exploration of food bacterial communities by the scientific community and food industry. Copyright © 2015. Published by

Bacterial and archaeal phylogenetic diversity of a cold sulfur-rich spring on the shoreline of Lake Erie, Michigan

USGS Publications Warehouse

Chaudhary, A.; Haack, S.K.; Duris, J.W.; Marsh, T.L.

2009-01-01

Studies of sulfidic springs have provided new insights into microbial metabolism, groundwater biogeochemistry, and geologic processes. We investigated Great Sulphur Spring on the western shore of Lake Erie and evaluated the phylogenetic affiliations of 189 bacterial and 77 archaeal 16S rRNA gene sequences from three habitats: the spring origin (11-m depth), bacterial-algal mats on the spring pond surface, and whitish filamentous materials from the spring drain. Water from the spring origin water was cold, pH 6.3, and anoxic (H2, 5.4 nM; CH4, 2.70 ??M) with concentrations of S2- (0.03 mM), SO42- (14.8 mM), Ca2+ (15.7 mM), and HCO3- (4.1 mM) similar to those in groundwater from the local aquifer. No archaeal and few bacterial sequences were >95% similar to sequences of cultivated organisms. Bacterial sequences were largely affiliated with sulfur-metabolizing or chemolithotrophic taxa in Beta-, Gamma-, Delta-, and Epsilonproteobacteria. Epsilonproteobacteria sequences similar to those obtained from other sulfidic environments and a new clade of Cyanobacteria sequences were particularly abundant (16% and 40%, respectively) in the spring origin clone library. Crenarchaeota sequences associated with archaeal-bacterial consortia in whitish filaments at a German sulfidic spring were detected only in a similar habitat at Great Sulphur Spring. This study expands the geographic distribution of many uncultured Archaea and Bacteria sequences to the Laurentian Great Lakes, indicates possible roles for epsilonproteobacteria in local aquifer chemistry and karst formation, documents new oscillatorioid Cyanobacteria lineages, and shows that uncultured, cold-adapted Crenarchaeota sequences may comprise a significant part of the microbial community of some sulfidic environments. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China

PubMed Central

Shao, Ming-Wei; Lu, Yi-Hui; Miao, Shuang; Zhang, Yun; Chen, Ting-Ting; Zhang, Ying-Lao

2015-01-01

The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds. PMID:26221957

Diversity, Bacterial Symbionts and Antibacterial Potential of Gut-Associated Fungi Isolated from the Pantala flavescens Larvae in China.

PubMed

Shao, Ming-Wei; Lu, Yi-Hui; Miao, Shuang; Zhang, Yun; Chen, Ting-Ting; Zhang, Ying-Lao

2015-01-01

The diversity of fungi associated with the gut of Pantala flavescens larvae was investigated using a culture-dependent method and molecular identification based on an analysis of the internally transcribed spacer sequence. In total, 48 fungal isolates were obtained from P. flavescens larvae. Based on phylogenetic analyses, the fungal isolates were grouped in 5 classes and 12 different genera. Fourteen bacterial 16S rDNA sequences derived from total genomic DNA extractions of fungal mycelia were obtained. The majority of the sequences were associated with Proteobacteria (13/14), and one Bacillaceae (1/14) was included. Leclercia sp., Oceanobacillus oncorhynchi and Methylobacterium extorquens, were reported for the first time as bacterial endosymbionts in fungi. High-performance liquid chromatography (HPLC) analysis indicated that bacterial symbionts produced specific metabolites and also exerted an inhibitory effect on fungal metabolites. The biological activity of the fungal culture extracts against the pathogenic bacteria Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 8739) was investigated, and 20 extracts (42%) exhibited antibacterial activity against at least one of the tested bacterial strains. This study is the first report on the diversity and antibacterial activity of symbiotic fungi residing in the gut of P. flavescens larvae, and the results show that these fungi are highly diverse and could be exploited as a potential source of bioactive compounds.

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.

Bacterial diversity in different regions of gastrointestinal tract of Giant African Snail (Achatina fulica)

PubMed Central

Pawar, Kiran D; Banskar, Sunil; Rane, Shailendra D; Charan, Shakti S; Kulkarni, Girish J; Sawant, Shailesh S; Ghate, Hemant V; Patole, Milind S; Shouche, Yogesh S

2012-01-01

The gastrointestinal (GI) tract of invasive land snail Achatina fulica is known to harbor metabolically active bacterial communities. In this study, we assessed the bacterial diversity in the different regions of GI tract of Giant African snail, A. fulica by culture-independent and culture-dependent methods. Five 16S rRNA gene libraries from different regions of GI tract of active snails indicated that sequences affiliated to phylum γ-Proteobacteria dominated the esophagus, crop, intestine, and rectum libraries, whereas sequences affiliated to Tenericutes dominated the stomach library. On phylogenetic analysis, 30, 27, 9, 27, and 25 operational taxonomic units (OTUs) from esophagus, crop, stomach, intestine, and rectum libraries were identified, respectively. Estimations of the total bacterial diversity covered along with environmental cluster analysis showed highest bacterial diversity in the esophagus and lowest in the stomach. Thirty-three distinct bacterial isolates were obtained, which belonged to 12 genera of two major bacterial phyla namely γ-Proteobacteria and Firmicutes. Among these, Lactococcus lactis and Kurthia gibsonii were the dominant bacteria present in all GI tract regions. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated significant differences in bacterial load in different GI tract regions of active and estivating snails. The difference in the bacterial load between the intestines of active and estivating snail was maximum. Principal component analysis (PCA) of terminal restriction fragment length polymorphism suggested that bacterial community structure changes only in intestine when snail enters estivation state. PMID:23233413

Soil bacterial diversity patterns and drivers along an elevational gradient on Shennongjia Mountain, China

PubMed Central

Zhang, Yuguang; Cong, Jing; Lu, Hui; Li, Guangliang; Xue, Yadong; Deng, Ye; Li, Hui; Zhou, Jizhong; Li, Diqiang

2015-01-01

Understanding biological diversity elevational pattern and the driver factors are indispensable to develop the ecological theories. Elevational gradient may minimize the impact of environmental factors and is the ideal places to study soil microbial elevational patterns. In this study, we selected four typical vegetation types from 1000 to 2800 m above the sea level on the northern slope of Shennongjia Mountain in central China, and analysed the soil bacterial community composition, elevational patterns and the relationship between soil bacterial diversity and environmental factors by using the 16S rRNA Illumina sequencing and multivariate statistical analysis. The results revealed that the dominant bacterial phyla were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Verrucomicrobia, which accounted for over 75% of the bacterial sequences obtained from tested samples, and the soil bacterial operational taxonomic unit (OTU) richness was a significant monotonous decreasing (P < 0.01) trend with the elevational increasing. The similarity of soil bacterial population composition decreased significantly (P < 0.01) with elevational distance increased as measured by the Jaccard and Bray–Curtis index. Canonical correspondence analysis and Mantel test analysis indicated that plant diversity and soil pH were significantly correlated (P < 0.01) with the soil bacterial community. Therefore, the soil bacterial diversity on Shennongjia Mountain had a significant and different elevational pattern, and plant diversity and soil pH may be the key factors in shaping the soil bacterial spatial pattern. PMID:26032124

Sequence analyses and evolutionary relationships among the energy-coupling proteins Enzyme I and HPr of the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

PubMed Central

Reizer, J.; Hoischen, C.; Reizer, A.; Pham, T. N.; Saier, M. H.

1993-01-01

We have previously reported the overexpression, purification, and biochemical properties of the Bacillus subtilis Enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) (Reizer, J., et al., 1992, J. Biol. Chem. 267, 9158-9169). We now report the sequencing of the ptsI gene of B. subtilis encoding Enzyme I (570 amino acids and 63,076 Da). Putative transcriptional regulatory signals are identified, and the pts operon is shown to be subject to carbon source-dependent regulation. Multiple alignments of the B. subtilis Enzyme I with (1) six other sequenced Enzymes I of the PTS from various bacterial species, (2) phosphoenolpyruvate synthase of Escherichia coli, and (3) bacterial and plant pyruvate: phosphate dikinases (PPDKs) revealed regions of sequence similarity as well as divergence. Statistical analyses revealed that these three types of proteins comprise a homologous family, and the phylogenetic tree of the 11 sequenced protein members of this family was constructed. This tree was compared with that of the 12 sequence HPr proteins or protein domains. Antibodies raised against the B. subtilis and E. coli Enzymes I exhibited immunological cross-reactivity with each other as well as with PPDK of Bacteroides symbiosus, providing support for the evolutionary relationships of these proteins suggested from the sequence comparisons. Putative flexible linkers tethering the N-terminal and the C-terminal domains of protein members of the Enzyme I family were identified, and their potential significance with regard to Enzyme I function is discussed. The codon choice pattern of the B. subtilis and E. coli ptsI and ptsH genes was found to exhibit a bias toward optimal codons in these organisms.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7686067

Bacterial community dynamics in a cooling tower with emphasis on pathogenic bacteria and Legionella species using universal and genus-specific deep sequencing.

PubMed

Pereira, Rui P A; Peplies, Jörg; Höfle, Manfred G; Brettar, Ingrid

2017-10-01

Cooling towers are the major source of outbreaks of legionellosis in Europe and worldwide. These outbreaks are mostly associated with Legionella species, primarily L. pneumophila, and its surveillance in cooling tower environments is of high relevance to public health. In this study, a combined NGS-based approach was used to study the whole bacterial community, specific waterborne and water-based bacterial pathogens, especially Legionella species, targeting the 16S rRNA gene. This approach was applied to water from a cooling tower obtained by monthly sampling during two years. The studied cooling tower was an open circuit cooling tower with lamellar cooling situated in Braunschweig, Germany. A highly diverse bacterial community was observed with 808 genera including 25 potentially pathogenic taxa using universal 16S rRNA primers. Sphingomonas and Legionella were the most abundant pathogenic genera. By applying genus-specific primers for Legionella, a diverse community with 85 phylotypes, and a representative core community with substantial temporal heterogeneity was observed. A high percentage of sequences (65%) could not be affiliated to an acknowledged species. L. pneumophila was part of the core community and the most abundant Legionella species reinforcing the importance of cooling towers as its environmental reservoir. Major temperature shifts (>10 °C) were the key environmental factor triggering the reduction or dominance of the Legionella species in the Legionella community dynamics. In addition, interventions by chlorine dioxide had a strong impact on the Legionella community composition but not on the whole bacterial community. Overall, the presented results demonstrated the value of a combined NGS approach for the molecular monitoring and surveillance of health related pathogens in man-made freshwater systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

Using PacBio sequencing to investigate the bacterial microbiota of traditional Buryatian cottage cheese and comparison with Italian and Kazakhstan artisanal cheeses.

PubMed

Jin, Hao; Mo, Lanxin; Pan, Lin; Hou, Qaingchaun; Li, Chuanjuan; Darima, Iaptueva; Yu, Jie

2018-05-09

Traditional fermented dairy foods including cottage cheese have been major components of the Buryatia diet for centuries. Buryatian cheeses have maintained not only their unique taste and flavor but also their rich natural lactic acid bacteria (LAB) content. However, relatively few studies have described their microbial communities or explored their potential to serve as LAB resources. In this study, the bacterial microbiota community of 7 traditional artisan cheeses produced by local Buryatian families was investigated using single-molecule, real-time sequencing. In addition, we compared the bacterial microbiota of the Buryatian cheese samples with data sets of cheeses from Kazakhstan and Italy. Furthermore, we isolated and preserved several LAB samples from Buryatian cheese. A total of 62 LAB strains (belonging to 6 genera and 14 species or subspecies) were isolated from 7 samples of Buryatian cheese. Full-length 16S rRNA sequencing of the microbiota revealed 145 species of 82 bacterial genera, belonging to 7 phyla. The most dominant species was Lactococcus lactis (43.89%). Data sets of cheeses from Italy and Kazakhstan were retrieved from public databases. Principal component analysis and multivariate ANOVA showed marked differences in the structure of the microbiota communities in the cheese data sets from the 3 regions. Linear discriminant analyses of the effect size identified 48 discriminant bacterial clades among the 3 groups, which might have contributed to the observed structural differences. Our results indicate that the bacterial communities of traditional artisan cheeses vary depending on geographic origin. In addition, we isolated novel and valuable LAB resources for the improvement of cottage cheese production. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

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.

New perspectives on bacterial ferredoxin evolution

NASA Technical Reports Server (NTRS)

George, D. G.; Hunt, L. T.; Yeh, L.-S. L.; Barker, W. C.

1985-01-01

Ferredoxins are low-molecular-weight, nonheme, iron proteins which function as electron carriers in a wide variety of electron transport chains. Howard et al. (1983) have suggested that the amino end of Azotobacter vinelandii ferredoxin shows a greater similarity to the carboxyl end of ferredoxin from Chromatium vinosum and that their half-chain sequences are homologous when the half-chains of either species are considered in inverse order. Examination of this proposition has made it necessary to reevaluate previous conclusions concerning the evolution of bacterial ferredoxin. Attention is given to the properties of the bacterial ferredoxin sequences, and the evolution of the bacterial ferredoxins.

Consequences of organ choice in describing bacterial pathogen assemblages in a rodent population.

PubMed

Villette, P; Afonso, E; Couval, G; Levret, A; Galan, M; Tatard, C; Cosson, J F; Giraudoux, P

2017-10-01

High-throughput sequencing technologies now allow for rapid cost-effective surveys of multiple pathogens in many host species including rodents, but it is currently unclear if the organ chosen for screening influences the number and identity of bacteria detected. We used 16S rRNA amplicon sequencing to identify bacterial pathogens in the heart, liver, lungs, kidneys and spleen of 13 water voles (Arvicola terrestris) collected in Franche-Comté, France. We asked if bacterial pathogen assemblages within organs are similar and if all five organs are necessary to detect all of the bacteria present in an individual animal. We identified 24 bacteria representing 17 genera; average bacterial richness for each organ ranged from 1·5 ± 0·4 (mean ± standard error) to 2·5 ± 0·4 bacteria/organ and did not differ significantly between organs. The average bacterial richness when organ assemblages were pooled within animals was 4·7 ± 0·6 bacteria/animal; Operational Taxonomic Unit accumulation analysis indicates that all five organs are required to obtain this. Organ type influences bacterial assemblage composition in a systematic way (PERMANOVA, 999 permutations, pseudo-F 4,51 = 1·37, P = 0·001). Our results demonstrate that the number of organs sampled influences the ability to detect bacterial pathogens, which can inform sampling decisions in public health and wildlife ecology.

Protocol to obtain targeted transcript sequence data from snake venom samples collected in the Colombian field.

PubMed

Fonseca, Alejandra; Renjifo-Ibáñez, Camila; Renjifo, Juan Manuel; Cabrera, Rodrigo

2018-03-21

Snake venoms are a mixture of different molecules that can be used in the design of drugs for various diseases. The study of these venoms has relied on strategies that use complete venom extracted from animals in captivity or from venom glands that require the sacrifice of the animals. Colombia, a country with political and geographical conflicts has difficult access to certain regions. A strategy that can prevent the sacrifice of animals and could allow the study of samples collected in the field is necessary. We report the use of lyophilized venom from Crotalus durissus cumanensis as a model to test, for the first time, a protocol for the amplification of complete toxins from Colombian venom samples collected in the field. In this protocol, primers were designed from conserved region from Crotalus sp. mRNA and EST regions to maximize the likelihood of coding sequence amplification. We obtained the sequences of Metalloproteinases II, Disintegrins, Disintegrin-Like, Phospholipases A 2, C-type Lectins and Serine proteinases from Crotalus durissus cumanensis and compared them to different Crotalus sp sequences available on databases obtaining concordance between the toxins amplified and those reported. Our strategy allows the use of lyophilized venom to obtain complete toxin sequences from samples collected in the field and the study of poorly characterized venoms in challenging environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Genome sequence determination and metagenomic characterization of a Dehalococcoides mixed culture grown on cis-1,2-dichloroethene.

PubMed

Yohda, Masafumi; Yagi, Osami; Takechi, Ayane; Kitajima, Mizuki; Matsuda, Hisashi; Miyamura, Naoaki; Aizawa, Tomoko; Nakajima, Mutsuyasu; Sunairi, Michio; Daiba, Akito; Miyajima, Takashi; Teruya, Morimi; Teruya, Kuniko; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Juan, Ayaka; Nakano, Kazuma; Aoyama, Misako; Terabayashi, Yasunobu; Satou, Kazuhito; Hirano, Takashi

2015-07-01

A Dehalococcoides-containing bacterial consortium that performed dechlorination of 0.20 mM cis-1,2-dichloroethene to ethene in 14 days was obtained from the sediment mud of the lotus field. To obtain detailed information of the consortium, the metagenome was analyzed using the short-read next-generation sequencer SOLiD 3. Matching the obtained sequence tags with the reference genome sequences indicated that the Dehalococcoides sp. in the consortium was highly homologous to Dehalococcoides mccartyi CBDB1 and BAV1. Sequence comparison with the reference sequence constructed from 16S rRNA gene sequences in a public database showed the presence of Sedimentibacter, Sulfurospirillum, Clostridium, Desulfovibrio, Parabacteroides, Alistipes, Eubacterium, Peptostreptococcus and Proteocatella in addition to Dehalococcoides sp. After further enrichment, the members of the consortium were narrowed down to almost three species. Finally, the full-length circular genome sequence of the Dehalococcoides sp. in the consortium, D. mccartyi IBARAKI, was determined by analyzing the metagenome with the single-molecule DNA sequencer PacBio RS. The accuracy of the sequence was confirmed by matching it to the tag sequences obtained by SOLiD 3. The genome is 1,451,062 nt and the number of CDS is 1566, which includes 3 rRNA genes and 47 tRNA genes. There exist twenty-eight RDase genes that are accompanied by the genes for anchor proteins. The genome exhibits significant sequence identity with other Dehalococcoides spp. throughout the genome, but there exists significant difference in the distribution RDase genes. The combination of a short-read next-generation DNA sequencer and a long-read single-molecule DNA sequencer gives detailed information of a bacterial consortium. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Analysis of bacterial populations in the environment using two-dimensional gel electrophoresis of genomic DNA and complementary DNA.

PubMed

Liu, Guo-Hua; Nakamura, Tatsuo; Amemiya, Takashi; Rajendran, Narasimmalu; Itoh, Kiminori

2011-01-01

Two-dimensional gel electrophoresis (2-DGE) mapping of genomic DNA and complementary DNA (cDNA) amplicons was attempted to analyze total and active bacterial populations within soil and activated sludge samples. Distinct differences in the number and species of bacterial populations and those that were metabolically active at the time of sampling were visually observed especially for the soil community. Statistical analyses and sequencing based on the 2-DGE data further revealed the relationships between total and active bacterial populations within each community. This high-resolution technique would be useful for obtaining a better understanding of bacterial population structures in the environment.

Temporal-spatial variation of bacterial diversity in estuary sediments in the south of Zhejiang Province, China.

PubMed

Lu, Xiao-Ming; Chen, Chang; Zheng, Tian-Ling; Chen, Jian-Jun

2016-03-01

The winter and summer microbial community structure in sediment samples obtained from the estuaries of the wastewater-polluted River Ou (DO and XO), River Feiyun (DF and XF), and River Ao (DA and XA) in the south of Zhejiang Province in China was determined using 454 pyrosequencing. Sediment samples (DD and XD) were also correspondingly collected near the shore far from the estuaries for comparison. For the above sediments, 294,870 effective sequences were obtained to do the bacterial diversity and abundance determination. In total, 1924, 1517, 2071, 1956, 1995, 1800, 2261, and 2097 operational taxonomic units were obtained at 3 % distance cutoff in the DO, XO, DF, XF, DA, XA, DD, and XD sediments, respectively. Bacterial phylotype richness in DD was higher than the other sediments, and XO had the least richness. The most dominant class in the DA, DD, DF, DO, and XA sediments is Gammaproteobacteria. Deltaproteobacteria is the most dominant one in XD, XO, and XF. Circa 14.4 % sequences in XD were found to be affiliated with the Flavobacteriales order. Characterization of the estuarine sediment bacterial communities indicated that chemical pollution has the potential to decrease the natural variability that exists among estuary ecosystems. However, chemical pollutants did not cause clear bio-homogenization in these estuaries.

Bacterial discrimination by means of a universal array approach mediated by LDR (ligase detection reaction)

PubMed Central

Busti, Elena; Bordoni, Roberta; Castiglioni, Bianca; Monciardini, Paolo; Sosio, Margherita; Donadio, Stefano; Consolandi, Clarissa; Rossi Bernardi, Luigi; Battaglia, Cristina; De Bellis, Gianluca

2002-01-01

Background PCR amplification of bacterial 16S rRNA genes provides the most comprehensive and flexible means of sampling bacterial communities. Sequence analysis of these cloned fragments can provide a qualitative and quantitative insight of the microbial population under scrutiny although this approach is not suited to large-scale screenings. Other methods, such as denaturing gradient gel electrophoresis, heteroduplex or terminal restriction fragment analysis are rapid and therefore amenable to field-scale experiments. A very recent addition to these analytical tools is represented by microarray technology. Results Here we present our results using a Universal DNA Microarray approach as an analytical tool for bacterial discrimination. The proposed procedure is based on the properties of the DNA ligation reaction and requires the design of two probes specific for each target sequence. One oligo carries a fluorescent label and the other a unique sequence (cZipCode or complementary ZipCode) which identifies a ligation product. Ligated fragments, obtained in presence of a proper template (a PCR amplified fragment of the 16s rRNA gene) contain either the fluorescent label or the unique sequence and therefore are addressed to the location on the microarray where the ZipCode sequence has been spotted. Such an array is therefore "Universal" being unrelated to a specific molecular analysis. Here we present the design of probes specific for some groups of bacteria and their application to bacterial diagnostics. Conclusions The combined use of selective probes, ligation reaction and the Universal Array approach yielded an analytical procedure with a good power of discrimination among bacteria. PMID:12243651

Contamination of sequence databases with adaptor sequences

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

Yoshikawa, Takeo; Sanders, A.R.; Detera-Wadleigh, S.D.

Because of the exponential increase in the amount of DNA sequences being added to the public databases on a daily basis, it has become imperative to identify sources of contamination rapidly. Previously, contaminations of sequence databases have been reported to alert the scientific community to the problem. These contaminations can be divided into two categories. The first category comprises host sequences that have been difficult for submitters to manage or control. Examples include anomalous sequences derived from Escherichia coli, which are inserted into the chromosomes (and plasmids) of the bacterial hosts. Insertion sequences are highly mobile and are capable ofmore » transposing themselves into plasmids during cloning manipulation. Another example of the first category is the infection with yeast genomic DNA or with bacterial DNA of some commercially available cDNA libraries from Clontech. The second category of database contamination is due to the inadvertent inclusion of nonhost sequences. This category includes incorporation of cloning-vector sequences and multicloning sites in the database submission. M13-derived artifacts have been common, since M13-based vectors have been widely used for subcloning DNA fragments. Recognizing this problem, the National Center for Biotechnology Information (NCBI) started to screen, in April 1994, all sequences directly submitted to GenBank, against a set of vector data retrieved from GenBank by use of key-word searches, such as {open_quotes}vector.{close_quotes} In this report, we present evidence for another sequence artifact that is widespread but that, to our knowledge, has not yet been reported. 11 refs., 1 tab.« less

Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

PubMed Central

Wong, Michael; Daly, Seth M.; Greenberg, David E.; Toprak, Erdal

2016-01-01

The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities. PMID:27631336

Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing

PubMed Central

Marino, Marilena; Innocente, Nadia; Maifreni, Michela; Mounier, Jérôme; Cobo-Díaz, José F.; Coton, Emmanuel; Carraro, Lisa; Cardazzo, Barbara

2017-01-01

This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines. PMID:29163411

Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables.

PubMed

Jackson, Colin R; Randolph, Kevin C; Osborn, Shelly L; Tyler, Heather L

2013-12-01

Plants harbor a diverse bacterial community, both as epiphytes on the plant surface and as endophytes within plant tissue. While some plant-associated bacteria act as plant pathogens or promote plant growth, others may be human pathogens. The aim of the current study was to determine the bacterial community composition of organic and conventionally grown leafy salad vegetables at the point of consumption using both culture-dependent and culture-independent methods. Total culturable bacteria on salad vegetables ranged from 8.0 × 10(3) to 5.5 × 10(8) CFU g(-1). The number of culturable endophytic bacteria from surface sterilized plants was significantly lower, ranging from 2.2 × 10(3) to 5.8 × 10(5) CFU g(-1). Cultured isolates belonged to six major bacterial phyla, and included representatives of Pseudomonas, Pantoea, Chryseobacterium, and Flavobacterium. Eleven different phyla and subphyla were identified by culture-independent pyrosequencing, with Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes being the most dominant lineages. Other bacterial lineages identified (e.g. Firmicutes, Alphaproteobacteria, Acidobacteria, and Actinobacteria) typically represented less than 1% of sequences obtained. At the genus level, sequences classified as Pseudomonas were identified in all samples and this was often the most prevalent genus. Ralstonia sequences made up a greater portion of the community in surface sterilized than non-surface sterilized samples, indicating that it was largely endophytic, while Acinetobacter sequences appeared to be primarily associated with the leaf surface. Analysis of molecular variance indicated there were no significant differences in bacterial community composition between organic versus conventionally grown, or surface-sterilized versus non-sterilized leaf vegetables. While culture-independent pyrosequencing identified significantly more bacterial taxa, the dominant taxa from pyrosequence data were also detected by traditional

Culture dependent and independent analysis of bacterial communities associated with commercial salad leaf vegetables

PubMed Central

2013-01-01

Background Plants harbor a diverse bacterial community, both as epiphytes on the plant surface and as endophytes within plant tissue. While some plant-associated bacteria act as plant pathogens or promote plant growth, others may be human pathogens. The aim of the current study was to determine the bacterial community composition of organic and conventionally grown leafy salad vegetables at the point of consumption using both culture-dependent and culture-independent methods. Results Total culturable bacteria on salad vegetables ranged from 8.0 × 103 to 5.5 × 108 CFU g-1. The number of culturable endophytic bacteria from surface sterilized plants was significantly lower, ranging from 2.2 × 103 to 5.8 × 105 CFU g-1. Cultured isolates belonged to six major bacterial phyla, and included representatives of Pseudomonas, Pantoea, Chryseobacterium, and Flavobacterium. Eleven different phyla and subphyla were identified by culture-independent pyrosequencing, with Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes being the most dominant lineages. Other bacterial lineages identified (e.g. Firmicutes, Alphaproteobacteria, Acidobacteria, and Actinobacteria) typically represented less than 1% of sequences obtained. At the genus level, sequences classified as Pseudomonas were identified in all samples and this was often the most prevalent genus. Ralstonia sequences made up a greater portion of the community in surface sterilized than non-surface sterilized samples, indicating that it was largely endophytic, while Acinetobacter sequences appeared to be primarily associated with the leaf surface. Analysis of molecular variance indicated there were no significant differences in bacterial community composition between organic versus conventionally grown, or surface-sterilized versus non-sterilized leaf vegetables. While culture-independent pyrosequencing identified significantly more bacterial taxa, the dominant taxa from pyrosequence data were also detected by

Evaluating bacterial pathogen DNA preservation in museum osteological collections

PubMed Central

Barnes, Ian; Thomas, Mark G

2005-01-01

Reports of bacterial pathogen DNA sequences obtained from archaeological bone specimens raise the possibility of greatly improving our understanding of the history of infectious diseases. However, the survival of pathogen DNA over long time periods is poorly characterized, and scepticism remains about the reliability of these data. In order to explore the survival of bacterial pathogen DNA in bone specimens, we analysed samples from 59 eighteenth and twentieth century individuals known to have been infected with either Mycobacterium tuberculosis or Treponema pallidum. No reproducible evidence of surviving pathogen DNA was obtained, despite the use of extraction and PCR-amplification methods determined to be highly sensitive. These data suggest that previous studies need to be interpreted with caution, and we propose that a much greater emphasis is placed on understanding how pathogen DNA survives in archaeological material, and how its presence can be properly verified and used. PMID:16608682

Whole genome sequencing options for bacterial strain typing and epidemiologic analysis based on single nucleotide polymorphism versus gene-by-gene-based approaches.

PubMed

Schürch, A C; Arredondo-Alonso, S; Willems, R J L; Goering, R V

2018-04-01

Whole genome sequence (WGS)-based strain typing finds increasing use in the epidemiologic analysis of bacterial pathogens in both public health as well as more localized infection control settings. This minireview describes methodologic approaches that have been explored for WGS-based epidemiologic analysis and considers the challenges and pitfalls of data interpretation. Personal collection of relevant publications. When applying WGS to study the molecular epidemiology of bacterial pathogens, genomic variability between strains is translated into measures of distance by determining single nucleotide polymorphisms in core genome alignments or by indexing allelic variation in hundreds to thousands of core genes, assigning types to unique allelic profiles. Interpreting isolate relatedness from these distances is highly organism specific, and attempts to establish species-specific cutoffs are unlikely to be generally applicable. In cases where single nucleotide polymorphism or core gene typing do not provide the resolution necessary for accurate assessment of the epidemiology of bacterial pathogens, inclusion of accessory gene or plasmid sequences may provide the additional required discrimination. As with all epidemiologic analysis, realizing the full potential of the revolutionary advances in WGS-based approaches requires understanding and dealing with issues related to the fundamental steps of data generation and interpretation. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

oriTfinder: a web-based tool for the identification of origin of transfers in DNA sequences of bacterial mobile genetic elements.

PubMed

Li, Xiaobin; Xie, Yingzhou; Liu, Meng; Tai, Cui; Sun, Jingyong; Deng, Zixin; Ou, Hong-Yu

2018-05-04

oriTfinder is a web server that facilitates the rapid identification of the origin of transfer site (oriT) of a conjugative plasmid or chromosome-borne integrative and conjugative element. The utilized back-end database oriTDB was built upon more than one thousand known oriT regions of bacterial mobile genetic elements (MGEs) as well as the known MGE-encoding relaxases and type IV coupling proteins (T4CP). With a combination of similarity searches for the oriTDB-archived oriT nucleotide sequences and the co-localization of the flanking relaxase homologous genes, the oriTfinder can predict the oriT region with high accuracy in the DNA sequence of a bacterial plasmid or chromosome in minutes. The server also detects the other transfer-related modules, including the potential relaxase gene, T4CP gene and the type IV secretion system gene cluster, and the putative genes coding for virulence factors and acquired antibiotic resistance determinants. oriTfinder may contribute to meeting the increasing demands of re-annotations for bacterial conjugative, mobilizable or non-transferable elements and aid in the rapid risk accession of disease-relevant trait dissemination in pathogenic bacteria of interest. oriTfinder is freely available to all users without any login requirement at http://bioinfo-mml.sjtu.edu.cn/oriTfinder.

Bacterial diversity in different regions of gastrointestinal tract of Giant African snail (Achatina fulica).

PubMed

Pawar, Kiran D; Banskar, Sunil; Rane, Shailendra D; Charan, Shakti S; Kulkarni, Girish J; Sawant, Shailesh S; Ghate, Hemant V; Patole, Milind S; Shouche, Yogesh S

2012-12-01

The gastrointestinal (GI) tract of invasive land snail Achatina fulica is known to harbor metabolically active bacterial communities. In this study, we assessed the bacterial diversity in the different regions of GI tract of Giant African snail, A. fulica by culture-independent and culture-dependent methods. Five 16S rRNA gene libraries from different regions of GI tract of active snails indicated that sequences affiliated to phylum γ-Proteobacteria dominated the esophagus, crop, intestine, and rectum libraries, whereas sequences affiliated to Tenericutes dominated the stomach library. On phylogenetic analysis, 30, 27, 9, 27, and 25 operational taxonomic units (OTUs) from esophagus, crop, stomach, intestine, and rectum libraries were identified, respectively. Estimations of the total bacterial diversity covered along with environmental cluster analysis showed highest bacterial diversity in the esophagus and lowest in the stomach. Thirty-three distinct bacterial isolates were obtained, which belonged to 12 genera of two major bacterial phyla namely γ-Proteobacteria and Firmicutes. Among these, Lactococcus lactis and Kurthia gibsonii were the dominant bacteria present in all GI tract regions. Quantitative real-time polymerase chain reaction (qPCR) analysis indicated significant differences in bacterial load in different GI tract regions of active and estivating snails. The difference in the bacterial load between the intestines of active and estivating snail was maximum. Principal component analysis (PCA) of terminal restriction fragment length polymorphism suggested that bacterial community structure changes only in intestine when snail enters estivation state. © 2012 The Authors. Published by Blackwell Publishing Ltd.

StrainSeeker: fast identification of bacterial strains from raw sequencing reads using user-provided guide trees.

PubMed

Roosaare, Märt; Vaher, Mihkel; Kaplinski, Lauris; Möls, Märt; Andreson, Reidar; Lepamets, Maarja; Kõressaar, Triinu; Naaber, Paul; Kõljalg, Siiri; Remm, Maido

2017-01-01

Fast, accurate and high-throughput identification of bacterial isolates is in great demand. The present work was conducted to investigate the possibility of identifying isolates from unassembled next-generation sequencing reads using custom-made guide trees. A tool named StrainSeeker was developed that constructs a list of specific k -mers for each node of any given Newick-format tree and enables the identification of bacterial isolates in 1-2 min. It uses a novel algorithm, which analyses the observed and expected fractions of node-specific k -mers to test the presence of each node in the sample. This allows StrainSeeker to determine where the isolate branches off the guide tree and assign it to a clade whereas other tools assign each read to a reference genome. Using a dataset of 100 Escherichia coli isolates, we demonstrate that StrainSeeker can predict the clades of E. coli with 92% accuracy and correct tree branch assignment with 98% accuracy. Twenty-five thousand Illumina HiSeq reads are sufficient for identification of the strain. StrainSeeker is a software program that identifies bacterial isolates by assigning them to nodes or leaves of a custom-made guide tree. StrainSeeker's web interface and pre-computed guide trees are available at http://bioinfo.ut.ee/strainseeker. Source code is stored at GitHub: https://github.com/bioinfo-ut/StrainSeeker.

Novel division level bacterial diversity in a Yellowstone hot spring.

PubMed

Hugenholtz, P; Pitulle, C; Hershberger, K L; Pace, N R

1998-01-01

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These

Spatial distribution of marine airborne bacterial communities

PubMed Central

Seifried, Jasmin S; Wichels, Antje; Gerdts, Gunnar

2015-01-01

The spatial distribution of bacterial populations in marine bioaerosol samples was investigated during a cruise from the North Sea to the Baltic Sea via Skagerrak and Kattegat. The analysis of the sampled bacterial communities with a pyrosequencing approach revealed that the most abundant phyla were represented by the Proteobacteria (49.3%), Bacteroidetes (22.9%), Actinobacteria (16.3%), and Firmicutes (8.3%). Cyanobacteria were assigned to 1.5% of all bacterial reads. A core of 37 bacterial OTUs made up more than 75% of all bacterial sequences. The most abundant OTU was Sphingomonas sp. which comprised 17% of all bacterial sequences. The most abundant bacterial genera were attributed to distinctly different areas of origin, suggesting highly heterogeneous sources for bioaerosols of marine and coastal environments. Furthermore, the bacterial community was clearly affected by two environmental parameters – temperature as a function of wind direction and the sampling location itself. However, a comparison of the wind directions during the sampling and calculated backward trajectories underlined the need for more detailed information on environmental parameters for bioaerosol investigations. The current findings support the assumption of a bacterial core community in the atmosphere. They may be emitted from strong aerosolizing sources, probably being mixed and dispersed over long distances. PMID:25800495

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

Independent studies using deep sequencing resolve the same set of core bacterial species dominating gut communities of honey bees.

PubMed

Sabree, Zakee L; Hansen, Allison K; Moran, Nancy A

2012-01-01

Starting in 2003, numerous studies using culture-independent methodologies to characterize the gut microbiota of honey bees have retrieved a consistent and distinctive set of eight bacterial species, based on near identity of the 16S rRNA gene sequences. A recent study [Mattila HR, Rios D, Walker-Sperling VE, Roeselers G, Newton ILG (2012) Characterization of the active microbiotas associated with honey bees reveals healthier and broader communities when colonies are genetically diverse. PLoS ONE 7(3): e32962], using pyrosequencing of the V1-V2 hypervariable region of the 16S rRNA gene, reported finding entirely novel bacterial species in honey bee guts, and used taxonomic assignments from these reads to predict metabolic activities based on known metabolisms of cultivable species. To better understand this discrepancy, we analyzed the Mattila et al. pyrotag dataset. In contrast to the conclusions of Mattila et al., we found that the large majority of pyrotag sequences belonged to clusters for which representative sequences were identical to sequences from previously identified core species of the bee microbiota. On average, they represent 95% of the bacteria in each worker bee in the Mattila et al. dataset, a slightly lower value than that found in other studies. Some colonies contain small proportions of other bacteria, mostly species of Enterobacteriaceae. Reanalysis of the Mattila et al. dataset also did not support a relationship between abundances of Bifidobacterium and of putative pathogens or a significant difference in gut communities between colonies from queens that were singly or multiply mated. Additionally, consistent with previous studies, the dataset supports the occurrence of considerable strain variation within core species, even within single colonies. The roles of these bacteria within bees, or the implications of the strain variation, are not yet clear.

Sequence and phylogenetic analysis of chicken anaemia virus obtained from backyard and commercial chickens in Nigeria.

PubMed

Oluwayelu, D O; Todd, D; Olaleye, O D

2008-12-01

This work reports the first molecular analysis study of chicken anaemia virus (CAV) in backyard chickens in Africa using molecular cloning and sequence analysis to characterize CAV strains obtained from commercial chickens and Nigerian backyard chickens. Partial VP1 gene sequences were determined for three CAVs from commercial chickens and for six CAV variants present in samples from a backyard chicken. Multiple alignment analysis revealed that the 6% and 4% nucleotide diversity obtained respectively for the commercial and backyard chicken strains translated to only 2% amino acid diversity for each breed. Overall, the amino acid composition of Nigerian CAVs was found to be highly conserved. Since the partial VP1 gene sequence of two backyard chicken cloned CAV strains (NGR/CI-8 and NGR/CI-9) were almost identical and evolutionarily closely related to the commercial chicken strains NGR-1, and NGR-4 and NGR-5, respectively, we concluded that CAV infections had crossed the farm boundary.

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

NASA Technical Reports Server (NTRS)

Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

2003-01-01

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

A DNA sequence obtained by replacement of the dopamine RNA aptamer bases is not an aptamer.

PubMed

Álvarez-Martos, Isabel; Ferapontova, Elena E

2017-08-05

A unique specificity of the aptamer-ligand biorecognition and binding facilitates bioanalysis and biosensor development, contributing to discrimination of structurally related molecules, such as dopamine and other catecholamine neurotransmitters. The aptamer sequence capable of specific binding of dopamine is a 57 nucleotides long RNA sequence reported in 1997 (Biochemistry, 1997, 36, 9726). Later, it was suggested that the DNA homologue of the RNA aptamer retains the specificity of dopamine binding (Biochem. Biophys. Res. Commun., 2009, 388, 732). Here, we show that the DNA sequence obtained by the replacement of the RNA aptamer bases for their DNA analogues is not able of specific biorecognition of dopamine, in contrast to the original RNA aptamer sequence. This DNA sequence binds dopamine and structurally related catecholamine neurotransmitters non-specifically, as any DNA sequence, and, thus, is not an aptamer and cannot be used neither for in vivo nor in situ analysis of dopamine in the presence of structurally related neurotransmitters. Copyright © 2017 Elsevier Inc. All rights reserved.

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Wang, S S; Lechner, R L; Karawya, E; Hesse, J; Martin, R G

1985-07-01

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.

Differential signatures of bacterial and mammalian IMP dehydrogenase enzymes.

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

Zhang, R.; Evans, G.; Rotella, F.

1999-06-01

IMP dehydrogenase (IMPDH) is an essential enzyme of de novo guanine nucleotide synthesis. IMPDH inhibitors have clinical utility as antiviral, anticancer or immunosuppressive agents. The essential nature of this enzyme suggests its therapeutic applications may be extended to the development of antimicrobial agents. Bacterial IMPDH enzymes show bio- chemical and kinetic characteristics that are different than the mammalian IMPDH enzymes, suggesting IMPDH may be an attractive target for the development of antimicrobial agents. We suggest that the biochemical and kinetic differences between bacterial and mammalian enzymes are a consequence of the variance of specific, identifiable amino acid residues. Identification ofmore » these residues or combination of residues that impart this mammalian or bacterial enzyme signature is a prerequisite for the rational identification of agents that specifically target the bacterial enzyme. We used sequence alignments of IMPDH proteins to identify sequence signatures associated with bacterial or eukaryotic IMPDH enzymes. These selections were further refined to discern those likely to have a role in catalysis using information derived from the bacterial and mammalian IMPDH crystal structures and site-specific mutagenesis. Candidate bacterial sequence signatures identified by this process include regions involved in subunit interactions, the active site flap and the NAD binding region. Analysis of sequence alignments in these regions indicates a pattern of catalytic residues conserved in all enzymes and a secondary pattern of amino acid conservation associated with the major phylogenetic groups. Elucidation of the basis for this mammalian/bacterial IMPDH signature will provide insight into the catalytic mechanism of this enzyme and the foundation for the development of highly specific inhibitors.« less

Statistical analysis of the Bacterial Carbohydrate Structure Data Base (BCSDB): Characteristics and diversity of bacterial carbohydrates in comparison with mammalian glycans

PubMed Central

Herget, Stephan; Toukach, Philip V; Ranzinger, René; Hull, William E; Knirel, Yuriy A; von der Lieth, Claus-Wilhelm

2008-01-01

Background There are considerable differences between bacterial and mammalian glycans. In contrast to most eukaryotic carbohydrates, bacterial glycans are often composed of repeating units with diverse functions ranging from structural reinforcement to adhesion, colonization and camouflage. Since bacterial glycans are typically displayed at the cell surface, they can interact with the environment and, therefore, have significant biomedical importance. Results The sequence characteristics of glycans (monosaccharide composition, modifications, and linkage patterns) for the higher bacterial taxonomic classes have been examined and compared with the data for mammals, with both similarities and unique features becoming evident. Compared to mammalian glycans, the bacterial glycans deposited in the current databases have a more than ten-fold greater diversity at the monosaccharide level, and the disaccharide pattern space is approximately nine times larger. Specific bacterial subclasses exhibit characteristic glycans which can be distinguished on the basis of distinctive structural features or sequence properties. Conclusion For the first time a systematic database analysis of the bacterial glycome has been performed. This study summarizes the current knowledge of bacterial glycan architecture and diversity and reveals putative targets for the rational design and development of therapeutic intervention strategies by comparing bacterial and mammalian glycans. PMID:18694500

Analysis of the distal gut bacterial community by 454-pyrosequencing in captive giraffes (Giraffa camelopardalis).

PubMed

AlZahal, Ousama; Valdes, Eduardo V; McBride, Brian W

2016-01-01

The objective of this study was to characterize the structure of the fecal bacterial community of five giraffes (Giraffa camelopardalis) at Disney's Animal Kingdom, FL. Fecal genomic DNA was extracted and variable regions 1-3 of the 16S rRNA gene was PCR-amplified and then sequenced. The MOTHUR software-program was used for sequence processing, diversity analysis, and classification. A total of 181,689 non-chimeric bacterial sequences were obtained, and average number of sequences per sample was 36,338 -± 8,818. Sequences were assigned to 8,284 operational taxonomic units (OTU) with 95% of genetic similarity, which included 2,942 singletons (36%). Number of OTUs per sample was 2,554 ± 264. Samples were normalized and alpha (intra-sample) diversity indices; Chao1, Inverse Simpson, Shannon, and coverage were estimated as 3,712 ± 430, 116 -± 70, 6.1 ± 0.4, and 96 ± 1%, respectively. Thirteen phyla were detected and Firmicutes, Bacteroidetes, and Spirochaetes were the most dominant phyla (more than 2% of total sequences), and constituted 92% of the classified sequences, 66% of total sequences, and 43% of total OTUs. Our computation predicted that three OTUs were likely to be present in at least three of the five samples at greater than 1% dominance rate. These OTUs were Treponema, an unidentified OTU belonging to the order Bacteroidales, and Ruminococcus. This report was the first to characterize the bacterial community of the distal gut in giraffes utilizing fecal samples, and it demonstrated that the distal gut of giraffes is likely a potential reservoir for a number of undocumented species of bacteria. © 2015 Wiley Periodicals, Inc.

Molecular diversity of bacterial endosymbionts associated with dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) reveals a high degree of phylogenetic congruence with their host.

PubMed

Palomares-Rius, Juan E; Archidona-Yuste, Antonio; Cantalapiedra-Navarrete, Carolina; Prieto, Pilar; Castillo, Pablo

2016-12-01

Bacterial endosymbionts have been detected in some groups of plant-parasitic nematodes, but few cases have been reported compared to other groups in the phylum Nematoda, such as animal-parasitic or free-living nematodes. This study was performed on a wide variety of plant-parasitic nematode families and species from different host plants and nematode populations. A total of 124 nematode populations (previously identified morphologically and molecularly) were screened for the presence of potential bacterial endosymbionts using the partial 16S rRNA gene and fluorescence in situ hybridization (FISH) and confocal microscopy. Potential bacterial endosymbionts were only detected in nematode species belonging to the genus Xiphinema and specifically in the X. americanum group. Fifty-seven partial 16S rRNA sequences were obtained from bacterial endosymbionts in this study. One group of sequences was closely related to the genus 'Candidatus Xiphinematobacter' (19 bacterial endosymbiont sequences were associated with seven nematode host species, including two that have already been described and three unknown bacterial endosymbionts). The second bacterial endosymbiont group (38 bacterial endosymbiont sequences associated with six nematode species) was related to the family Burkholderiaceae, which includes fungal and soil-plant bacterial endosymbionts. These endosymbionts were reported for the first time in the phylum Nematoda. Our findings suggest that there is a highly specific symbiotic relationship between nematode host and bacterial endosymbionts. Overall, these results were corroborated by a phylogeny of nematode host and bacterial endosymbionts that suggested that there was a high degree of phylogenetic congruence and long-term evolutionary persistence between hosts and endosymbionts. © 2016 John Wiley & Sons Ltd.

Phylogenetic mapping of bacterial morphology

NASA Technical Reports Server (NTRS)

Siefert, J. L.; Fox, G. E.

1998-01-01

The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

Serratia marcescens outbreak in a neonatal intensive care unit (NICU): new insights from next-generation sequencing applications.

PubMed

Martineau, Christine; Li, Xuejing; Lalancette, Cindy; Perreault, Thérèse; Fournier, Eric; Tremblay, Julien; Gonzales, Milagros; Yergeau, Étienne; Quach, Caroline

2018-06-13

Serratia marcescens is an environmental bacterium commonly associated with outbreaks in neonatal intensive care units (NICU). Investigation of S. marcescens outbreaks requires efficient recovery and typing of clinical and environmental isolates. In this study, we described how the use of next-generation sequencing applications, such as bacterial whole-genome sequencing (WGS) and bacterial community profiling, could improve S. marcescens outbreak investigation. Phylogenomic links and potential antibiotic resistance genes and plasmids in S. marcescens isolates were investigated using WGS, while bacterial communities and relative abundances of Serratia in environmental samples were assessed using sequencing of bacterial phylogenetic marker genes (16S rRNA and gyrB genes). Typing results obtained using WGS for the ten S. marcescens isolates recovered during a NICU outbreak investigation were highly consistent with those from pulse-field gel electrophoresis (PFGE), the current gold standard typing method for this bacterium. WGS also allowed for the identification of genes associated with antibiotic resistance in all isolates, while no plasmid was detected. Sequencing of the 16S rRNA and gyrB genes both showed higher relative abundances of Serratia in environmental sampling sites that were in close contact with infected babies. Much lower relative abundances of Serratia were observed following disinfection of a room, indicating that the protocol used was efficient. Variations in the bacterial community composition and structure following room disinfection and between sampling sites were also identified through 16S rRNA gene sequencing. Globally, results from this study highlight the potential for next-generation sequencing tools to improve and facilitate outbreak investigation. Copyright © 2018 American Society for Microbiology.

Evaluation of the Bacterial Diversity in the Human Tongue Coating Based on Genus-Specific Primers for 16S rRNA Sequencing.

PubMed

Sun, Beili; Zhou, Dongrui; Tu, Jing; Lu, Zuhong

2017-01-01

The characteristics of tongue coating are very important symbols for disease diagnosis in traditional Chinese medicine (TCM) theory. As a habitat of oral microbiota, bacteria on the tongue dorsum have been proved to be the cause of many oral diseases. The high-throughput next-generation sequencing (NGS) platforms have been widely applied in the analysis of bacterial 16S rRNA gene. We developed a methodology based on genus-specific multiprimer amplification and ligation-based sequencing for microbiota analysis. In order to validate the efficiency of the approach, we thoroughly analyzed six tongue coating samples from lung cancer patients with different TCM types, and more than 600 genera of bacteria were detected by this platform. The results showed that ligation-based parallel sequencing combined with enzyme digestion and multiamplification could expand the effective length of sequencing reads and could be applied in the microbiota analysis.

Draft genome sequences of 9 LA-MRSA ST5 isolates obtained from humans after short term swine contact

USDA-ARS?s Scientific Manuscript database

Livestock associated methicillin resistant Staphylococcus aureus (LA-MRSA) sequence type 5 have raised concerns surrounding the potential for these isolates to colonize or cause disease in humans with swine contact. Here, we report draft genome sequences for 9 LA-MRSA ST5 isolates obtained from huma...

THE ANAEROBIC BIODEGRADATION OF O-,M- AND P-CRESOL BY SULFATE-REDUCING BACTERIAL ENRICHMENT CULTURES OBTAINED FROM A SHALLOW ANOXIC AQUIFER

EPA Science Inventory

Sulfate-reducing bacterial enrichments were obtained from a shallow anoxic aquifer for their ability to metabolize either o-, m-, orp-cresol. GC/MS and simultaneous adaptation experiments suggested that the anaerobic decomposition of p-cresol proceeds ...

Complete Genome Sequences of Two Geographically Distinct Legionella micdadei Clinical Isolates

PubMed Central

Jose, Bethany R.; Perry, Jasper; Smeele, Zoe; Aitken, Jack; Gardner, Paul P.

2017-01-01

ABSTRACT Legionella is a highly diverse genus of intracellular bacterial pathogens that cause Legionnaire’s disease (LD), an often severe form of pneumonia. Two L. micdadei sp. clinical isolates, obtained from patients hospitalized with LD from geographically distinct areas, were sequenced using PacBio SMRT cell technology, identifying incomplete phage regions, which may impact virulence. PMID:28572318

Impact of cultivation on characterisation of species composition of soil bacterial communities.

PubMed

McCaig, A E.; Grayston, S J.; Prosser, J I.; Glover, L A.

2001-03-01

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated

Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

PubMed Central

Sanz, Yolanda

2017-01-01

Abstract The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems. PMID:28605506

Crossroads between Bacterial and Mammalian Glycosyltransferases

PubMed Central

Brockhausen, Inka

2014-01-01

Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures. PMID:25368613

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

PubMed Central

Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

2003-01-01

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 107 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years. PMID:12676695

Analysis of bacterial and archaeal diversity in coastal microbial mats using massive parallel 16S rRNA gene tag sequencing.

PubMed

Bolhuis, Henk; Stal, Lucas J

2011-11-01

Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity.

Sulfamethoxazole and COD increase abundance of sulfonamide resistance genes and change bacterial community structures within sequencing batch reactors.

PubMed

Guo, Xueping; Pang, Weihai; Dou, Chunling; Yin, Daqiang

2017-05-01

The abundant microbial community in biological treatment processes in wastewater treatment plants (WWTPs) may potentially enhance the horizontal gene transfer of antibiotic resistance genes with the presence of antibiotics. A lab-scale sequencing batch reactor was designed to investigate response of sulfonamide resistance genes (sulI, sulII) and bacterial communities to various concentrations of sulfamethoxazole (SMX) and chemical oxygen demand (COD) of wastewater. The SMX concentrations (0.001 mg/L, 0.1 mg/L and 10 mg/L) decreased with treatment time and higher SMX level was more difficult to remove. The presence of SMX also significantly reduced the removal efficiency of ammonia nitrogen, affecting the normal function of WWTPs. All three concentrations of SMX raised both sulI and sulII genes with higher concentrations exhibiting greater increases. The abundance of sul genes was positive correlated with treatment time and followed the second-order reaction kinetic model. Interestingly, these two genes have rather similar activity. SulI and sulII gene abundance also performed similar response to COD. Simpson index and Shannon-Weiner index did not show changes in the microbial community diversity. However, the 16S rRNA gene cloning and sequencing results showed the bacterial community structures varied during different stages. The results demonstrated that influent antibiotics into WWTPs may facilitate selection of ARGs and affect the wastewater conventional treatment as well as the bacteria community structures. Copyright © 2017 Elsevier Ltd. All rights reserved.

Draft Sequences of the Radish (Raphanus sativus L.) Genome

PubMed Central

Kitashiba, Hiroyasu; Li, Feng; Hirakawa, Hideki; Kawanabe, Takahiro; Zou, Zhongwei; Hasegawa, Yoichi; Tonosaki, Kaoru; Shirasawa, Sachiko; Fukushima, Aki; Yokoi, Shuji; Takahata, Yoshihito; Kakizaki, Tomohiro; Ishida, Masahiko; Okamoto, Shunsuke; Sakamoto, Koji; Shirasawa, Kenta; Tabata, Satoshi; Nishio, Takeshi

2014-01-01

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified. PMID:24848699

Illumina Amplicon Sequencing of 16S rRNA Tag Reveals Bacterial Community Development in the Rhizosphere of Apple Nurseries at a Replant Disease Site and a New Planting Site

PubMed Central

Sun, Jian; Zhang, Qiang; Zhou, Jia; Wei, Qinping

2014-01-01

We used a next-generation, Illumina-based sequencing approach to characterize the bacterial community development of apple rhizosphere soil in a replant site (RePlant) and a new planting site (NewPlant) in Beijing. Dwarfing apple nurseries of ‘Fuji’/SH6/Pingyitiancha trees were planted in the spring of 2013. Before planting, soil from the apple rhizosphere of the replant site (ReSoil) and from the new planting site (NewSoil) was sampled for analysis on the Illumina MiSeq platform. In late September, the rhizosphere soil from both sites was resampled (RePlant and NewPlant). More than 16,000 valid reads were obtained for each replicate, and the community was composed of five dominant groups (Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes and Actinobacteria). The bacterial diversity decreased after apple planting. Principal component analyses revealed that the rhizosphere samples were significantly different among treatments. Apple nursery planting showed a large impact on the soil bacterial community, and the community development was significantly different between the replanted and newly planted soils. Verrucomicrobia were less abundant in RePlant soil, while Pseudomonas and Lysobacter were increased in RePlant compared with ReSoil and NewPlant. Both RePlant and ReSoil showed relatively higher invertase and cellulase activities than NewPlant and NewSoil, but only NewPlant soil showed higher urease activity, and this soil also had the higher plant growth. Our experimental results suggest that planting apple nurseries has a significant impact on soil bacterial community development at both replant and new planting sites, and planting on new site resulted in significantly higher soil urease activity and a different bacterial community composition. PMID:25360786

Cronobacter, the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis.

PubMed

Forsythe, Stephen J; Dickins, Benjamin; Jolley, Keith A

2014-12-16

Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database (http://pubmlst.org/cronobacter/) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infections Whole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains. The Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes 'on the fly', and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range

Bacterial collagen-like proteins that form triple-helical structures

PubMed Central

Yu, Zhuoxin; An, Bo; Ramshaw, John A.M.; Brodsky, Barbara

2014-01-01

A large number of collagen-like proteins have been identified in bacteria during the past ten years, principally from analysis of genome databases. These bacterial collagens share the distinctive Gly-Xaa-Yaa repeating amino acid sequence of animal collagens which underlies their unique triple-helical structure. A number of the bacterial collagens have been expressed in E. coli, and they all adopt a triple-helix conformation. Unlike animal collagens, these bacterial proteins do not contain the post-translationally modified amino acid, hydroxyproline, which is known to stabilize the triple-helix structure and may promote self-assembly. Despite the absence of collagen hydroxylation, the triple-helix structures of the bacterial collagens studied exhibit a high thermal stability of 35–39 °C, close to that seen for mammalian collagens. These bacterial collagens are readily produced in large quantities by recombinant methods, either in the original amino acid sequence or in genetically manipulated sequences. This new family of recombinant, easy to modify collagens could provide a novel system for investigating structural and functional motifs in animal collagens and could also form the basis of new biomedical materials with designed structural properties and functions. PMID:24434612

Whole-Genome Sequences of Cronobacter sakazakii Isolates Obtained from Foods of Plant Origin and Dried-Food Manufacturing Environments.

PubMed

Jang, Hyein; Addy, Nicole; Ewing, Laura; Jean-Gilles Beaubrun, Junia; Lee, YouYoung; Woo, JungHa; Negrete, Flavia; Finkelstein, Samantha; Tall, Ben D; Lehner, Angelika; Eshwar, Athmanya; Gopinath, Gopal R

2018-04-12

Here, we present draft genome sequences of 29 Cronobacter sakazakii isolates obtained from foods of plant origin and dried-food manufacturing facilities. Assemblies and annotations resulted in genome sizes ranging from 4.3 to 4.5 Mb and 3,977 to 4,256 gene-coding sequences with G+C contents of ∼57.0%.

Characterization of bacterial community associated with phytoplankton bloom in a eutrophic lake in South Norway using 16S rRNA gene amplicon sequence analysis.

PubMed

Parulekar, Niranjan Nitin; Kolekar, Pandurang; Jenkins, Andrew; Kleiven, Synne; Utkilen, Hans; Johansen, Anette; Sawant, Sangeeta; Kulkarni-Kale, Urmila; Kale, Mohan; Sæbø, Mona

2017-01-01

Interactions between different phytoplankton taxa and heterotrophic bacterial communities within aquatic environments can differentially support growth of various heterotrophic bacterial species. In this study, phytoplankton diversity was studied using traditional microscopic techniques and the bacterial communities associated with phytoplankton bloom were studied using High Throughput Sequencing (HTS) analysis of 16S rRNA gene amplicons from the V1-V3 and V3-V4 hypervariable regions. Samples were collected from Lake Akersvannet, a eutrophic lake in South Norway, during the growth season from June to August 2013. Microscopic examination revealed that the phytoplankton community was mostly represented by Cyanobacteria and the dinoflagellate Ceratium hirundinella. The HTS results revealed that Proteobacteria (Alpha, Beta, and Gamma), Bacteriodetes, Cyanobacteria, Actinobacteria and Verrucomicrobia dominated the bacterial community, with varying relative abundances throughout the sampling season. Species level identification of Cyanobacteria showed a mixed population of Aphanizomenon flos-aquae, Microcystis aeruginosa and Woronichinia naegeliana. A significant proportion of the microbial community was composed of unclassified taxa which might represent locally adapted freshwater bacterial groups. Comparison of cyanobacterial species composition from HTS and microscopy revealed quantitative discrepancies, indicating a need for cross validation of results. To our knowledge, this is the first study that uses HTS methods for studying the bacterial community associated with phytoplankton blooms in a Norwegian lake. The study demonstrates the value of considering results from multiple methods when studying bacterial communities.

Insect Gut Bacterial Diversity Determined by Environmental Habitat, Diet, Developmental Stage, and Phylogeny of Host

PubMed Central

Yun, Ji-Hyun; Roh, Seong Woon; Whon, Tae Woong; Jung, Mi-Ja; Kim, Min-Soo; Park, Doo-Sang; Yoon, Changmann; Nam, Young-Do; Kim, Yun-Ji; Choi, Jung-Hye; Kim, Joon-Yong; Shin, Na-Ri; Kim, Sung-Hee; Lee, Won-Jae

2014-01-01

Insects are the most abundant animals on Earth, and the microbiota within their guts play important roles by engaging in beneficial and pathological interactions with these hosts. In this study, we comprehensively characterized insect-associated gut bacteria of 305 individuals belonging to 218 species in 21 taxonomic orders, using 454 pyrosequencing of 16S rRNA genes. In total, 174,374 sequence reads were obtained, identifying 9,301 bacterial operational taxonomic units (OTUs) at the 3% distance level from all samples, with an average of 84.3 (±97.7) OTUs per sample. The insect gut microbiota were dominated by Proteobacteria (62.1% of the total reads, including 14.1% Wolbachia sequences) and Firmicutes (20.7%). Significant differences were found in the relative abundances of anaerobes in insects and were classified according to the criteria of host environmental habitat, diet, developmental stage, and phylogeny. Gut bacterial diversity was significantly higher in omnivorous insects than in stenophagous (carnivorous and herbivorous) insects. This insect-order-spanning investigation of the gut microbiota provides insights into the relationships between insects and their gut bacterial communities. PMID:24928884

An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences.

PubMed

Gerritsen, K E H; Olieslagers, T I; Groeneweg, M; Voorter, C E M; Tilanus, M G J

2014-11-01

The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Whole-Genome Sequences of Cronobacter sakazakii Isolates Obtained from Foods of Plant Origin and Dried-Food Manufacturing Environments

PubMed Central

Addy, Nicole; Ewing, Laura; Jean-Gilles Beaubrun, Junia; Lee, YouYoung; Woo, JungHa; Negrete, Flavia; Finkelstein, Samantha; Tall, Ben D.; Lehner, Angelika; Eshwar, Athmanya; Gopinath, Gopal R.

2018-01-01

ABSTRACT Here, we present draft genome sequences of 29 Cronobacter sakazakii isolates obtained from foods of plant origin and dried-food manufacturing facilities. Assemblies and annotations resulted in genome sizes ranging from 4.3 to 4.5 Mb and 3,977 to 4,256 gene-coding sequences with G+C contents of ∼57.0%. PMID:29650569

Response of Intestinal Bacterial Flora to the Long-term Feeding of Aflatoxin B1 (AFB1) in Mice.

PubMed

Yang, Xiai; Liu, Liangliang; Chen, Jing; Xiao, Aiping

2017-10-12

In order to investigate the influence of aflatoxin B1 (AFB1) on intestinal bacterial flora, 24 Kunming mice (KM mice) were randomly placed into four groups, which were labeled as control, low-dose, medium-dose, and high-dose groups. They were fed intragastrically with 0.4 mL of 0 mg/L, 2.5 mg/L, 4 mg/L, or 10 mg/L of AFB1 solutions, twice a day for 2 months. The hypervariable region V3 + V4 on 16S rDNA of intestinal bacterial flora was sequenced by the use of a high-flux sequencing system on a Miseq Illumina platform; then, the obtained sequences were analyzed. The results showed that, when compared with the control group, both genera and phyla of intestinal bacteria in the three treatment groups decreased. About one third of the total genera and one half of the total phyla remained in the high-dose group. The dominant flora were Lactobacillus and Bacteroides in all groups. There were significant differences in the relative abundance of intestinal bacterial flora among groups. Most bacteria decreased as a whole from the control to the high-dose groups, but several beneficial and pathogenic bacterial species increased significantly with increasing dose of AFB1. Thus, the conclusion was that intragastric feeding with 2.5~10 mg/mL AFB1 for 2 months could decrease the majority of intestinal bacterial flora and induce the proliferation of some intestinal bacteria flora.

Microbial Culturomics Broadens Human Vaginal Flora Diversity: Genome Sequence and Description of Prevotella lascolaii sp. nov. Isolated from a Patient with Bacterial Vaginosis.

PubMed

Diop, Khoudia; Diop, Awa; Levasseur, Anthony; Mediannikov, Oleg; Robert, Catherine; Armstrong, Nicholas; Couderc, Carine; Bretelle, Florence; Raoult, Didier; Fournier, Pierre-Edouard; Fenollar, Florence

2018-03-01

Microbial culturomics is a new subfield of postgenomic medicine and omics biotechnology application that has broadened our awareness on bacterial diversity of the human microbiome, including the human vaginal flora bacterial diversity. Using culturomics, a new obligate anaerobic Gram-stain-negative rod-shaped bacterium designated strain khD1 T was isolated in the vagina of a patient with bacterial vaginosis and characterized using taxonogenomics. The most abundant cellular fatty acids were C 15:0 anteiso (36%), C 16:0 (19%), and C 15:0 iso (10%). Based on an analysis of the full-length 16S rRNA gene sequences, phylogenetic analysis showed that the strain khD1 T exhibited 90% sequence similarity with Prevotella loescheii, the phylogenetically closest validated Prevotella species. With 3,763,057 bp length, the genome of strain khD1 T contained (mol%) 48.7 G + C and 3248 predicted genes, including 3194 protein-coding and 54 RNA genes. Given the phenotypical and biochemical characteristic results as well as genome sequencing, strain khD1 T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella lascolaii sp. nov. is proposed. The type strain is khD1 T ( = CSUR P0109, = DSM 101754). These results show that microbial culturomics greatly improves the characterization of the human microbiome repertoire by isolating potential putative new species. Further studies will certainly clarify the microbial mechanisms of pathogenesis of these new microbes and their role in health and disease. Microbial culturomics is an important new addition to the diagnostic medicine toolbox and warrants attention in future medical, global health, and integrative biology postgraduate teaching curricula.

Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

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

Jansson, Janet; Edlund, Anna; Hardeman, Fredrik

Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179 mV, -64 mV and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labeled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities weremore » most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labeled DNA and DNA from reverse transcription PCR (rt-PCR) showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.« less

Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial, archaeal and eukaryotic microorganisms in rumen microbial communities.

PubMed

Kittelmann, Sandra; Seedorf, Henning; Walters, William A; Clemente, Jose C; Knight, Rob; Gordon, Jeffrey I; Janssen, Peter H

2013-01-01

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.

Determining the culturability of the rumen bacterial microbiome

PubMed Central

Creevey, Christopher J; Kelly, William J; Henderson, Gemma; Leahy, Sinead C

2014-01-01

The goal of the Hungate1000 project is to generate a reference set of rumen microbial genome sequences. Toward this goal we have carried out a meta-analysis using information from culture collections, scientific literature, and the NCBI and RDP databases and linked this with a comparative study of several rumen 16S rRNA gene-based surveys. In this way we have attempted to capture a snapshot of rumen bacterial diversity to examine the culturable fraction of the rumen bacterial microbiome. Our analyses have revealed that for cultured rumen bacteria, there are many genera without a reference genome sequence. Our examination of culture-independent studies highlights that there are few novel but many uncultured taxa within the rumen bacterial microbiome. Taken together these results have allowed us to compile a list of cultured rumen isolates that are representative of abundant, novel and core bacterial species in the rumen. In addition, we have identified taxa, particularly within the phylum Bacteroidetes, where further cultivation efforts are clearly required. This information is being used to guide the isolation efforts and selection of bacteria from the rumen microbiota for sequencing through the Hungate1000. PMID:24986151

Dynamic Succession of Soil Bacterial Community during Continuous Cropping of Peanut (Arachis hypogaea L.)

PubMed Central

Chen, Mingna; Li, Xiao; Yang, Qingli; Chi, Xiaoyuan; Pan, Lijuan; Chen, Na; Yang, Zhen; Wang, Tong; Wang, Mian; Yu, Shanlin

2014-01-01

Plant health and soil fertility are affected by plant–microbial interactions in soils. Peanut is an important oil crop worldwide and shows considerable adaptability, but growth and yield are negatively affected by continuous cropping. In this study, 16S rRNA gene clone library analyses were used to study the succession of soil bacterial communities under continuous peanut cultivation. Six libraries were constructed for peanut over three continuous cropping cycles and during its seedling and pod-maturing growth stages. Cluster analyses indicated that soil bacterial assemblages obtained from the same peanut cropping cycle were similar, regardless of growth period. The diversity of bacterial sequences identified in each growth stage library of the three peanut cropping cycles was high and these sequences were affiliated with 21 bacterial groups. Eight phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, Proteobacteria and Verrucomicrobia were dominant. The related bacterial phylotypes dynamic changed during continuous cropping progress of peanut. This study demonstrated that the bacterial populations especially the beneficial populations were positively selected. The simplification of the beneficial microbial communities such as the phylotypes of Alteromonadales, Burkholderiales, Flavobacteriales, Pseudomonadales, Rhizobiales and Rhodospirillales could be important factors contributing to the decline in peanut yield under continuous cropping. The microbial phylotypes that did not successively changed with continuous cropping, such as populations related to Rhizobiales and Rhodospirillales, could potentially resist stress due to continuous cropping and deserve attention. In addition, some phylotypes, such as Acidobacteriales, Chromatiales and Gemmatimonadales, showed a contrary tendency, their abundance or diversity increased with continuous peanut cropping progress. Some bacterial phylotypes including Acidobacteriales

Bacterial population dynamics during the ensiling of Medicago sativa (alfalfa) and subsequent exposure to air.

PubMed

McGarvey, J A; Franco, R B; Palumbo, J D; Hnasko, R; Stanker, L; Mitloehner, F M

2013-06-01

To describe, at high resolution, the bacterial population dynamics and chemical transformations during the ensiling of alfalfa and subsequent exposure to air. Samples of alfalfa, ensiled alfalfa and silage exposed to air were collected and their bacterial population structures compared using 16S rRNA gene libraries containing approximately 1900 sequences each. Cultural and chemical analyses were also performed to complement the 16S gene sequence data. Sequence analysis revealed significant differences (P < 0·05) in the bacterial populations at each time point. The alfalfa-derived library contained mostly sequences associated with the Gammaproteobacteria (including the genera: Enterobacter, Erwinia and Pantoea); the ensiled material contained mostly sequences associated with the lactic acid bacteria (LAB) (including the genera: Lactobacillus, Pediococcus and Lactococcus). Exposure to air resulted in even greater percentages of LAB, especially among the genus Lactobacillus, and a significant drop in bacterial diversity. In-depth 16S rRNA gene sequence analysis revealed significant bacterial population structure changes during ensiling and again during exposure to air. This in-depth description of the bacterial population dynamics that occurred during ensiling and simulated feed out expands our knowledge of these processes. © 2013 The Society for Applied Microbiology No claim to US Government works.

Bacterial microbiota of Kazakhstan cheese revealed by single molecule real time (SMRT) sequencing and its comparison with Belgian, Kalmykian and Italian artisanal cheeses.

PubMed

Li, Jing; Zheng, Yi; Xu, Haiyan; Xi, Xiaoxia; Hou, Qiangchuan; Feng, Shuzhen; Wuri, Laga; Bian, Yanfei; Yu, Zhongjie; Kwok, Lai-Yu; Sun, Zhihong; Sun, Tiansong

2017-01-09

In Kazakhstan, traditional artisanal cheeses have a long history and are widely consumed. The unique characteristics of local artisanal cheeses are almost completely preserved. However, their microbial communities have rarely been reported. The current study firstly generated the Single Molecule, Real-Time (SMRT) sequencing bacterial diversity profiles of 6 traditional artisanal cheese samples of Kazakhstan origin, followed by comparatively analyzed the microbiota composition between the current dataset and those from cheeses originated from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy. Across the Kazakhstan cheese samples, a total of 238 bacterial species belonging to 14 phyla and 140 genera were identified. Lactococcus lactis (28.93%), Lactobacillus helveticus (26.43%), Streptococcus thermophilus (12.18%) and Lactobacillus delbrueckii (12.15%) were the dominant bacterial species for these samples. To further evaluate the cheese bacterial diversity of Kazakhstan cheeses in comparison with those from other geographic origins, 16S rRNA datasets of 36 artisanal cheeses from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy were retrieved from public databases. The cheese bacterial microbiota communities were largely different across sample origins. By principal coordinate analysis (PCoA) and multivariate analysis of variance (MANOVA), the structure of the Kazakhstan artisanal cheese samples was found to be different from those of the other geographic origins. Furthermore, the redundancy analysis (RDA) identified 16 bacterial OTUs as the key variables responsible for such microbiota structural difference. Our results together suggest that the diversity of bacterial communities in different groups is stratified by geographic region. This study does not only provide novel information on the bacterial microbiota of traditional artisanal cheese of Kazakhstan at species level, but also interesting insights into the bacterial diversity of artisanal

Arginine-lysine positional swap of the LL-37 peptides reveals evolutional advantages of the native sequence and leads to bacterial probes.

PubMed

Wang, Xiuqing; Junior, José Carlos Bozelli; Mishra, Biswajit; Lushnikova, Tamara; Epand, Richard M; Wang, Guangshun

2017-08-01

Antimicrobial peptides are essential components of the innate immune system of multicellular organisms. Although cationic and hydrophobic amino acids are known determinants of these amphipathic molecules for bacterial killing, it is not clear how lysine-arginine (K-R) positional swaps influence peptide structure and activity. This study addresses this question by investigating two groups of peptides (GF-17 and 17BIPHE2) derived from human cathelicidin LL-37. K-R positional swap showed little effect on minimal inhibitory concentrations of the peptides. However, there are clear differences in bacterial killing kinetics. The membrane permeation patterns vary with peptide and bacterial types, but not changes in fluorescent dyes, salts or pH. In general, the original peptide is more efficient in bacterial killing, but less toxic to human cells, than the K-R swapped peptides, revealing the evolutionary significance of the native sequence for host defense. The characteristic membrane permeation patterns for different bacteria suggest a possible application of these K-R positional-swapped peptides as molecular probes for the type of bacteria. Such differences are related to bacterial membrane compositions: minimal for Gram-positive Staphylococcus aureus with essentially all anionic lipids (cardiolipin and phosphatidylglycerol), but evident for Gram-negative Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli with a mixture of phosphatidylethanolamine and phosphatidylglycerol. Biophysical characterization found similar structures and binding affinities for these peptides in vesicle systems mimicking E. coli and S. aureus. It seems that interfacial arginines of GF-17 are preferred over lysines in bacterial membrane permeation. Our study sheds new light on the design of cationic amphipathic peptides. Copyright © 2017 Elsevier B.V. All rights reserved.

Bacterial flora analysis of coliforms in sewage, river water, and ground water using MALDI-TOF mass spectrometry.

PubMed

Suzuki, Yoshihiro; Niina, Kouki; Matsuwaki, Tomonori; Nukazawa, Kei; Iguchi, Atsushi

2018-01-28

The aim of this study was to rapidly and effectively analyze coliforms, which are the most fundamental indicators of water quality for fecal pollution, using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Coliform bacteria were isolated from municipal sewage, river water, and groundwater. For each sample, 100 isolates were determined by MALDI-TOF MS. In addition, these same 100 isolates were also identified via 16S rRNA gene sequence analysis. Obtained MALDI-TOF MS data were compared with the 16S rRNA sequencing analysis, and the validity of MALDI-TOF MS for classification of coliform bacteria was examined. The concordance rate of bacterial identification for the 100 isolates obtained by MALDI-TOF MS analysis and 16S rRNA gene sequence analysis for sewage, river water, and ground water were 96%, 74%, and 62% at the genus level, respectively. Among the sewage, river water, and ground water samples, the coliform bacterial flora were distinct. The dominant genus of coliforms in sewage, river water, and groundwater were Klebsiella spp., Enterobacter spp., and Serratia spp., respectively. We determined that MALDI-TOF MS is a rapid and accurate tool that can be used to identify coliforms. Therefore, without using conventional 16S rRNA sequencing, it is possible to rapidly and effectively classify coliforms in water using MALDI-TOF MS.

Bacterial community composition characterization of a lead-contaminated Microcoleus sp. consortium.

PubMed

Giloteaux, Ludovic; Solé, Antoni; Esteve, Isabel; Duran, Robert

2011-08-01

A Microcoleus sp. consortium, obtained from the Ebro delta microbial mat, was maintained under different conditions including uncontaminated, lead-contaminated, and acidic conditions. Terminal restriction fragment length polymorphism and 16S rRNA gene library analyses were performed in order to determine the effect of lead and culture conditions on the Microcoleus sp. consortium. The bacterial composition inside the consortium revealed low diversity and the presence of specific terminal-restriction fragments under lead conditions. 16S rRNA gene library analyses showed that members of the consortium were affiliated to the Alpha, Beta, and Gammaproteobacteria and Cyanobacteria. Sequences closely related to Achromobacter spp., Alcaligenes faecalis, and Thiobacillus species were exclusively found under lead conditions while sequences related to Geitlerinema sp., a cyanobacterium belonging to the Oscillatoriales, were not found in presence of lead. This result showed a strong lead selection of the bacterial members present in the Microcoleus sp. consortium. Several of the 16S rRNA sequences were affiliated to nitrogen-fixing microorganisms including members of the Rhizobiaceae and the Sphingomonadaceae. Additionally, confocal laser scanning microscopy and scanning and transmission electron microscopy showed that under lead-contaminated condition Microcoleus sp. cells were grouped and the number of electrodense intracytoplasmic inclusions was increased.

Diagnosis of Meningococcal Meningitis by Broad-Range Bacterial PCR with Cerebrospinal Fluid

PubMed Central

Kotilainen, Pirkko; Jalava, Jari; Meurman, Olli; Lehtonen, Olli-Pekka; Rintala, Esa; Seppälä, Olli-Pekka; Eerola, Erkki; Nikkari, Simo

1998-01-01

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the Neisseria meningitidis sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded Listeria monocytogenes. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis. PMID:9665992

Prevalent bacterial species and novel phylotypes in advanced noma lesions.

PubMed

Paster, B J; Falkler Jr, W A; Enwonwu, C O; Idigbe, E O; Savage, K O; Levanos, V A; Tamer, M A; Ericson, R L; Lau, C N; Dewhirst, F E

2002-06-01

The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods. 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned into Escherichia coli. Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or closest relatives by comparison with sequences of known species or phylotypes. Nearly complete sequences of approximately 1,500 bases were obtained for most of the potentially novel species. A total of 67 bacterial species or phylotypes were detected, 25 of which have not yet been grown in vitro. Nineteen of the species or phylotypes, including Propionibacterium acnes, Staphylococcus spp., and the opportunistic pathogens Stenotrophomonas maltophilia and Ochrobactrum anthropi were detected in more than one subject. Other known species that were detected included Achromobacter spp., Afipia spp., Brevundimonas diminuta, Capnocytophaga spp., Cardiobacterium sp., Eikenella corrodens, Fusobacterium spp., Gemella haemoylsans, and Neisseria spp. Phylotypes that were unique to noma infections included those in the genera Eubacterium, Flavobacterium, Kocuria, Microbacterium, and Porphyromonas and the related Streptococcus salivarius and genera Sphingomonas and TREPONEMA: Since advanced noma lesions are infections open to the environment, it was not surprising to detect species not commonly associated with the oral cavity, e.g., from soil. Several species previously implicated as putative pathogens of noma, such as spirochetes and Fusobacterium spp., were detected in at least one subject. However, due to the limited number of available noma subjects, it was not possible at this time to associate specific species with the disease.

Bacterial DNA Detected in Japanese Rice Wines and the Fermentation Starters.

PubMed

Terasaki, Momoka; Fukuyama, Akari; Takahashi, Yurika; Yamada, Masato; Nishida, Hiromi

2017-12-01

As Japanese rice wine (sake) brewing is not done aseptically, bacterial contamination is conceivable during the process of sake production. There are two types of the fermentation starter, sokujo-moto and yamahai-moto (kimoto). We identified bacterial DNA found in various sakes, the sokujo-moto and the yamahai-moto making just after sake yeast addition. Each sake has a unique variety of bacterial DNA not observed in other sakes. Although most bacterial DNA sequences detected in the sokujo-moto were found in sakes of different sake breweries, most bacterial DNA sequences detected in the yamahai-moto at the early stage of the starter fermentation were not detected in any sakes. Our findings demonstrate that various bacteria grow and then die during the process of sake brewing, as indicated by the presence of trace levels of bacterial DNA.

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters

PubMed Central

Bao, Weichen; Mi, Zhihui; Xu, Haiyan; Zheng, Yi; Kwok, Lai Yu; Zhang, Heping; Zhang, Wenyi

2016-01-01

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, γ-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the organic acids content. By SMRT analysis of the microbial composition in eight silage samples, we found that the bacterial species number and relative abundances shifted apparently after fermentation. Such changes were specific to the LAB species in the additives. Particularly, Bacillus megaterium was the initial dominant species in the raw materials; and after the fermentation process, Pediococcus acidilactici and Lactobacillus plantarum became the most prevalent species, both of which were intrinsically present in the LAB additives. Our data have demonstrated that the SMRT sequencing platform is applicable in assessing the quality of silage. PMID:27340760

Assessing quality of Medicago sativa silage by monitoring bacterial composition with single molecule, real-time sequencing technology and various physiological parameters.

PubMed

Bao, Weichen; Mi, Zhihui; Xu, Haiyan; Zheng, Yi; Kwok, Lai Yu; Zhang, Heping; Zhang, Wenyi

2016-06-24

The present study applied the PacBio single molecule, real-time sequencing technology (SMRT) in evaluating the quality of silage production. Specifically, we produced four types of Medicago sativa silages by using four different lactic acid bacteria-based additives (AD-I, AD-II, AD-III and AD-IV). We monitored the changes in pH, organic acids (including butyric acid, the ratio of acetic acid/lactic acid, γ-aminobutyric acid, 4-hyroxy benzoic acid and phenyl lactic acid), mycotoxins, and bacterial microbiota during silage fermentation. Our results showed that the use of the additives was beneficial to the silage fermentation by enhancing a general pH and mycotoxin reduction, while increasing the organic acids content. By SMRT analysis of the microbial composition in eight silage samples, we found that the bacterial species number and relative abundances shifted apparently after fermentation. Such changes were specific to the LAB species in the additives. Particularly, Bacillus megaterium was the initial dominant species in the raw materials; and after the fermentation process, Pediococcus acidilactici and Lactobacillus plantarum became the most prevalent species, both of which were intrinsically present in the LAB additives. Our data have demonstrated that the SMRT sequencing platform is applicable in assessing the quality of silage.

Comparative phylobiomic analysis of the bacterial community of water kefir by 16S rRNA gene amplicon sequencing and ARDRA analysis.

PubMed

Gulitz, A; Stadie, J; Ehrmann, M A; Ludwig, W; Vogel, R F

2013-04-01

The aim of this study was to analyse the bacterial microbiota of water kefir using culture-independent methods. We compared four water kefirs of different origins using 16S rDNA amplicon sequencing and ARDRA. The microbiota consisted of different proportions of the genera Lactobacillus (Lact.), Leuconostoc (Leuc.), Acetobacter (Acet.) and Gluconobacter. Surprisingly, varying but consistently high numbers of sequences representing members of the genus Bifidobacterium (Bif.) were found in all kefirs. Whereas part of the bifidobacterial sequences could be assigned to Bifidobacterium psychraerophilum, a majority of sequences identical to each other could not be assigned to any known species. A nearly full-length sequence of the latter exhibited a beyond-species similarity (96.4%) with the sequence from the closest relative species Bif. psychraerophilum. A Bifidobacterium-specific ARDRA analysis reflected the abundance of the novel Bifidobacterium species by revealing its unique MboI restriction profile. Attempts to isolate the bifidobacteria were successful for Bif. psychraerophilum only. The complexity of the water kefir microbiota has been underestimated in previously studies. The occurrence of bifidobacteria as part of the consortium is novel. These data give new insights into the understanding of the complexity of food fermentations and underline the need for approaches detecting noncultivable organisms. © 2013 The Society for Applied Microbiology.

Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy

PubMed Central

Ulrich, Nikea; Rosenberger, Abigail; Brislawn, Colin; Wright, Justin; Kessler, Collin; Toole, David; Solomon, Caroline; Strutt, Steven; McClure, Erin

2016-01-01

ABSTRACT Bacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in which Betaproteobacteria and Gammaproteobacteria decreased in 16S rRNA gene relative abundance, while the relative abundance of members of the Firmicutes increased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains of Legionella, Campylobacter, Arcobacter, and Helicobacter, as well as bacteria of fecal origin (e.g., Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event. IMPORTANCE In order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE PAGES

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.; ...

2018-02-09

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

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

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Primer development to obtain complete coding sequence of HA and NA genes of influenza A/H3N2 virus.

PubMed

Agustiningsih, Agustiningsih; Trimarsanto, Hidayat; Setiawaty, Vivi; Artika, I Made; Muljono, David Handojo

2016-08-30

Influenza is an acute respiratory illness and has become a serious public health problem worldwide. The need to study the HA and NA genes in influenza A virus is essential since these genes frequently undergo mutations. This study describes the development of primer sets for RT-PCR to obtain complete coding sequence of Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 virus from Indonesia. The primers were developed based on influenza A/H3N2 sequence worldwide from Global Initiative on Sharing All Influenza Data (GISAID) and further tested using Indonesian influenza A/H3N2 archived samples of influenza-like illness (ILI) surveillance from 2008 to 2009. An optimum RT-PCR condition was acquired for all HA and NA fragments designed to cover complete coding sequence of HA and NA genes. A total of 71 samples were successfully sequenced for complete coding sequence both of HA and NA genes out of 145 samples of influenza A/H3N2 tested. The developed primer sets were suitable for obtaining complete coding sequences of HA and NA genes of Indonesian samples from 2008 to 2009.

Changes in rhizosphere bacterial gene expression following glyphosate treatment.

PubMed

Newman, Molli M; Lorenz, Nicola; Hoilett, Nigel; Lee, Nathan R; Dick, Richard P; Liles, Mark R; Ramsier, Cliff; Kloepper, Joseph W

2016-05-15

In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria. Copyright © 2016 The Authors. Published by Elsevier B.V. All

Ericoid Roots and Mycospheres Govern Plant-Specific Bacterial Communities in Boreal Forest Humus.

PubMed

Timonen, Sari; Sinkko, Hanna; Sun, Hui; Sietiö, Outi-Maaria; Rinta-Kanto, Johanna M; Kiheri, Heikki; Heinonsalo, Jussi

2017-05-01

In this study, the bacterial populations of roots and mycospheres of the boreal pine forest ericoid plants, heather (Calluna vulgaris), bilberry (Vaccinium myrtillus), and lingonberry (Vaccinium vitis-idaea), were studied by qPCR and next-generation sequencing (NGS). All bacterial communities of mycosphere soils differed from soils uncolonized by mycorrhizal mycelia. Colonization by mycorrhizal hyphae increased the total number of bacterial 16S ribosomal DNA (rDNA) gene copies in the humus but decreased the number of different bacterial operational taxonomic units (OTUs). Nevertheless, ericoid roots and mycospheres supported numerous OTUs not present in uncolonized humus. Bacterial communities in bilberry mycospheres were surprisingly similar to those in pine mycospheres but not to bacterial communities in heather and lingonberry mycospheres. In contrast, bacterial communities of ericoid roots were more similar to each other than to those of pine roots. In all sample types, the relative abundances of bacterial sequences belonging to Alphaproteobacteria and Acidobacteria were higher than the sequences belonging to other classes. Soil samples contained more Actinobacteria, Deltaproteobacteria, Opitutae, and Planctomycetia, whereas Armatimonadia, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia were more common to roots. All mycosphere soils and roots harbored bacteria unique to that particular habitat. Our study suggests that the habitation by ericoid plants increases the overall bacterial diversity of boreal forest soils.

A Primer on Infectious Disease Bacterial Genomics

PubMed Central

Petkau, Aaron; Knox, Natalie; Graham, Morag; Van Domselaar, Gary

2016-01-01

SUMMARY The number of large-scale genomics projects is increasing due to the availability of affordable high-throughput sequencing (HTS) technologies. The use of HTS for bacterial infectious disease research is attractive because one whole-genome sequencing (WGS) run can replace multiple assays for bacterial typing, molecular epidemiology investigations, and more in-depth pathogenomic studies. The computational resources and bioinformatics expertise required to accommodate and analyze the large amounts of data pose new challenges for researchers embarking on genomics projects for the first time. Here, we present a comprehensive overview of a bacterial genomics projects from beginning to end, with a particular focus on the planning and computational requirements for HTS data, and provide a general understanding of the analytical concepts to develop a workflow that will meet the objectives and goals of HTS projects. PMID:28590251

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

PubMed

Hosokawa, Masahito; Nishikawa, Yohei; Kogawa, Masato; Takeyama, Haruko

2017-07-12

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Anchoring 9,371 Maize Expressed Sequence Tagged Unigenes to the Bacterial Artificial Chromosome Contig Map by Two-Dimensional Overgo Hybridization1

PubMed Central

Gardiner, Jack; Schroeder, Steven; Polacco, Mary L.; Sanchez-Villeda, Hector; Fang, Zhiwei; Morgante, Michele; Landewe, Tim; Fengler, Kevin; Useche, Francisco; Hanafey, Michael; Tingey, Scott; Chou, Hugh; Wing, Rod; Soderlund, Carol; Coe, Edward H.

2004-01-01

Our goal is to construct a robust physical map for maize (Zea mays) comprehensively integrated with the genetic map. We have used a two-dimensional 24 × 24 overgo pooling strategy to anchor maize expressed sequence tagged (EST) unigenes to 165,888 bacterial artificial chromosomes (BACs) on high-density filters. A set of 70,716 public maize ESTs seeded derivation of 10,723 EST unigene assemblies. From these assemblies, 10,642 overgo sequences of 40 bp were applied as hybridization probes. BAC addresses were obtained for 9,371 overgo probes, representing an 88% success rate. More than 96% of the successful overgo probes identified two or more BACs, while 5% identified more than 50 BACs. The majority of BACs identified (79%) were hybridized with one or two overgos. A small number of BACs hybridized with eight or more overgos, suggesting that these BACs must be gene rich. Approximately 5,670 overgos identified BACs assembled within one contig, indicating that these probes are highly locus specific. A total of 1,795 megabases (Mb; 87%) of the total 2,050 Mb in BAC contigs were associated with one or more overgos, which are serving as sequence-tagged sites for single nucleotide polymorphism development. Overgo density ranged from less than one overgo per megabase to greater than 20 overgos per megabase. The majority of contigs (52%) hit by overgos contained three to nine overgos per megabase. Analysis of approximately 1,022 Mb of genetically anchored BAC contigs indicates that 9,003 of the total 13,900 overgo-contig sites are genetically anchored. Our results indicate overgos are a powerful approach for generating gene-specific hybridization probes that are facilitating the assembly of an integrated genetic and physical map for maize. PMID:15020742

Gene finding in metatranscriptomic sequences.

PubMed

Ismail, Wazim Mohammed; Ye, Yuzhen; Tang, Haixu

2014-01-01

Metatranscriptomic sequencing is a highly sensitive bioassay of functional activity in a microbial community, providing complementary information to the metagenomic sequencing of the community. The acquisition of the metatranscriptomic sequences will enable us to refine the annotations of the metagenomes, and to study the gene activities and their regulation in complex microbial communities and their dynamics. In this paper, we present TransGeneScan, a software tool for finding genes in assembled transcripts from metatranscriptomic sequences. By incorporating several features of metatranscriptomic sequencing, including strand-specificity, short intergenic regions, and putative antisense transcripts into a Hidden Markov Model, TranGeneScan can predict a sense transcript containing one or multiple genes (in an operon) or an antisense transcript. We tested TransGeneScan on a mock metatranscriptomic data set containing three known bacterial genomes. The results showed that TranGeneScan performs better than metagenomic gene finders (MetaGeneMark and FragGeneScan) on predicting protein coding genes in assembled transcripts, and achieves comparable or even higher accuracy than gene finders for microbial genomes (Glimmer and GeneMark). These results imply, with the assistance of metatranscriptomic sequencing, we can obtain a broad and precise picture about the genes (and their functions) in a microbial community. TransGeneScan is available as open-source software on SourceForge at https://sourceforge.net/projects/transgenescan/.

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1

PubMed Central

2011-01-01

Background The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. Results Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. Conclusion Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans. PMID:21477375

Production, purification, sequencing and activity spectra of mutacins D-123.1 and F-59.1.

PubMed

Nicolas, Guillaume G; LaPointe, Gisèle; Lavoie, Marc C

2011-04-10

The increase in bacterial resistance to antibiotics impels the development of new anti-bacterial substances. Mutacins (bacteriocins) are small antibacterial peptides produced by Streptococcus mutans showing activity against bacterial pathogens. The objective of the study was to produce and characterise additional mutacins in order to find new useful antibacterial substances. Mutacin F-59.1 was produced in liquid media by S. mutans 59.1 while production of mutacin D-123.1 by S. mutans 123.1 was obtained in semi-solid media. Mutacins were purified by hydrophobic chromatography. The amino acid sequences of the mutacins were obtained by Edman degradation and their molecular mass was determined by mass spectrometry. Mutacin F-59.1 consists of 25 amino acids, containing the YGNGV consensus sequence of pediocin-like bacteriocins with a molecular mass calculated at 2719 Da. Mutacin D-123.1 has an identical molecular mass (2364 Da) with the same first 9 amino acids as mutacin I. Mutacins D-123.1 and F-59.1 have wide activity spectra inhibiting human and food-borne pathogens. The lantibiotic mutacin D-123.1 possesses a broader activity spectrum than mutacin F-59.1 against the bacterial strains tested. Mutacin F-59.1 is the first pediocin-like bacteriocin identified and characterised that is produced by Streptococcus mutans. Mutacin D-123.1 appears to be identical to mutacin I previously identified in different strains of S. mutans.

Flexible taxonomic assignment of ambiguous sequencing reads

PubMed Central

2011-01-01

Background To characterize the diversity of bacterial populations in metagenomic studies, sequencing reads need to be accurately assigned to taxonomic units in a given reference taxonomy. Reads that cannot be reliably assigned to a unique leaf in the taxonomy (ambiguous reads) are typically assigned to the lowest common ancestor of the set of species that match it. This introduces a potentially severe error in the estimation of bacteria present in the sample due to false positives, since all species in the subtree rooted at the ancestor are implicitly assigned to the read even though many of them may not match it. Results We present a method that maps each read to a node in the taxonomy that minimizes a penalty score while balancing the relevance of precision and recall in the assignment through a parameter q. This mapping can be obtained in time linear in the number of matching sequences, because LCA queries to the reference taxonomy take constant time. When applied to six different metagenomic datasets, our algorithm produces different taxonomic distributions depending on whether coverage or precision is maximized. Including information on the quality of the reads reduces the number of unassigned reads but increases the number of ambiguous reads, stressing the relevance of our method. Finally, two measures of performance are described and results with a set of artificially generated datasets are discussed. Conclusions The assignment strategy of sequencing reads introduced in this paper is a versatile and a quick method to study bacterial communities. The bacterial composition of the analyzed samples can vary significantly depending on how ambiguous reads are assigned depending on the value of the q parameter. Validation of our results in an artificial dataset confirm that a combination of values of q produces the most accurate results. PMID:21211059

Bacterial Community Diversity of Oil-Contaminated Soils Assessed by High Throughput Sequencing of 16S rRNA Genes.

PubMed

Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

2015-09-24

Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future.

Simultaneous Amplicon Sequencing to Explore Co-Occurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities

PubMed Central

Kittelmann, Sandra; Seedorf, Henning; Walters, William A.; Clemente, Jose C.; Knight, Rob; Gordon, Jeffrey I.; Janssen, Peter H.

2013-01-01

Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats. PMID:23408926

Bacterial diversity in the oral cavity of ten healthy individuals

PubMed Central

Bik, Elisabeth M.; Long, Clara Davis; Armitage, Gary C.; Loomer, Peter; Emerson, Joanne; Mongodin, Emmanuel F.; Nelson, Karen E.; Gill, Steven R.; Fraser-Liggett, Claire M.; Relman, David A.

2010-01-01

The composition of the oral microbiota from 10 individuals with healthy oral tissues was determined using culture-independent techniques. From each individual, 26 specimens, each from different oral sites at a single point in time, were collected and pooled. An eleventh pool was constructed using portions of the subgingival specimens from all 10 individuals. The 16S rRNA gene was amplified using broad-range bacterial primers, and clone libraries from the individual and subgingival pools were constructed. From a total of 11 368 high-quality, non-chimeric, near full-length sequences, 247 species-level phylotypes (using a 99% sequence identity threshold) and 9 bacteria phyla were identified. At least 15 bacterial genera were conserved among all 10 individuals, with significant interindividual differences at the species and strain level. Comparisons of these oral bacterial sequences to near full-length sequences found previously in the large intestines and feces of other healthy individuals suggest that the mouth and intestinal tract harbor distinct sets of bacteria. Co-occurrence analysis demonstrated significant segregation of taxa when community membership was examined at the level of genus, but not at the level of species, suggesting that ecologically-significant, competitive interactions are more apparent at a broader taxonomic level than species. This study is one of the more comprehensive, high-resolution analyses of bacterial diversity within the healthy human mouth to date, and highlights the value of tools from macroecology for enhancing our understanding of bacterial ecology in human health. PMID:20336157

Detecting macroecological patterns in bacterial communities across independent studies of global soils.

PubMed

Ramirez, Kelly S; Knight, Christopher G; de Hollander, Mattias; Brearley, Francis Q; Constantinides, Bede; Cotton, Anne; Creer, Si; Crowther, Thomas W; Davison, John; Delgado-Baquerizo, Manuel; Dorrepaal, Ellen; Elliott, David R; Fox, Graeme; Griffiths, Robert I; Hale, Chris; Hartman, Kyle; Houlden, Ashley; Jones, David L; Krab, Eveline J; Maestre, Fernando T; McGuire, Krista L; Monteux, Sylvain; Orr, Caroline H; van der Putten, Wim H; Roberts, Ian S; Robinson, David A; Rocca, Jennifer D; Rowntree, Jennifer; Schlaeppi, Klaus; Shepherd, Matthew; Singh, Brajesh K; Straathof, Angela L; Bhatnagar, Jennifer M; Thion, Cécile; van der Heijden, Marcel G A; de Vries, Franciska T

2018-02-01

The emergence of high-throughput DNA sequencing methods provides unprecedented opportunities to further unravel bacterial biodiversity and its worldwide role from human health to ecosystem functioning. However, despite the abundance of sequencing studies, combining data from multiple individual studies to address macroecological questions of bacterial diversity remains methodically challenging and plagued with biases. Here, using a machine-learning approach that accounts for differences among studies and complex interactions among taxa, we merge 30 independent bacterial data sets comprising 1,998 soil samples from 21 countries. Whereas previous meta-analysis efforts have focused on bacterial diversity measures or abundances of major taxa, we show that disparate amplicon sequence data can be combined at the taxonomy-based level to assess bacterial community structure. We find that rarer taxa are more important for structuring soil communities than abundant taxa, and that these rarer taxa are better predictors of community structure than environmental factors, which are often confounded across studies. We conclude that combining data from independent studies can be used to explore bacterial community dynamics, identify potential 'indicator' taxa with an important role in structuring communities, and propose hypotheses on the factors that shape bacterial biogeography that have been overlooked in the past.

Identification of a novel calcium binding motif based on the detection of sequence insertions in the animal peroxidase domain of bacterial proteins.

PubMed

Santamaría-Hernando, Saray; Krell, Tino; Ramos-González, María-Isabel

2012-01-01

Proteins of the animal heme peroxidase (ANP) superfamily differ greatly in size since they have either one or two catalytic domains that match profile PS50292. The orf PP_2561 of Pseudomonas putida KT2440 that we have called PepA encodes a two-domain ANP. The alignment of these domains with those of PepA homologues revealed a variable number of insertions with the consensus G-x-D-G-x-x-[GN]-[TN]-x-D-D. This motif has also been detected in the structure of pseudopilin (pdb 3G20), where it was found to be involved in Ca(2+) coordination although a sequence analysis did not reveal the presence of any known calcium binding motifs in this protein. Isothermal titration calorimetry revealed that a peptide containing this consensus motif bound specifically calcium ions with affinities ranging between 33-79 µM depending on the pH. Microcalorimetric titrations of the purified N-terminal ANP-like domain of PepA revealed Ca(2+) binding with a K(D) of 12 µM and stoichiometry of 1.25 calcium ions per protein monomer. This domain exhibited peroxidase activity after its reconstitution with heme. These data led to the definition of a novel calcium binding motif that we have termed PERCAL and which was abundantly present in animal peroxidase-like domains of bacterial proteins. Bacterial heme peroxidases thus possess two different types of calcium binding motifs, namely PERCAL and the related hemolysin type calcium binding motif, with the latter being located outside the catalytic domains and in their C-terminal end. A phylogenetic tree of ANP-like catalytic domains of bacterial proteins with PERCAL motifs, including single domain peroxidases, was divided into two major clusters, representing domains with and without PERCAL motif containing insertions. We have verified that the recently reported classification of bacterial heme peroxidases in two families (cd09819 and cd09821) is unrelated to these insertions. Sequences matching PERCAL were detected in all kingdoms of life.

Benthic bacterial diversity in submerged sinkhole ecosystems.

PubMed

Nold, Stephen C; Pangborn, Joseph B; Zajack, Heidi A; Kendall, Scott T; Rediske, Richard R; Biddanda, Bopaiah A

2010-01-01

Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities.

Analysis of Bacterial Community Structure in Sulfurous-Oil-Containing Soils and Detection of Species Carrying Dibenzothiophene Desulfurization (dsz) Genes

PubMed Central

Duarte, Gabriela Frois; Rosado, Alexandre Soares; Seldin, Lucy; de Araujo, Welington; van Elsas, Jan Dirk

2001-01-01

The selective effects of sulfur-containing hydrocarbons, with respect to changes in bacterial community structure and selection of desulfurizing organisms and genes, were studied in soil. Samples taken from a polluted field soil (A) along a concentration gradient of sulfurous oil and from soil microcosms treated with dibenzothiophene (DBT)-containing petroleum (FSL soil) were analyzed. Analyses included plate counts of total bacteria and of DBT utilizers, molecular community profiling via soil DNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and detection of genes that encode enzymes involved in the desulfurization of hydrocarbons, i.e., dszA, dszB, and dszC.Data obtained from the A soil showed no discriminating effects of oil levels on the culturable bacterial numbers on either medium used. Generally, counts of DBT degraders were 10- to 100-fold lower than the total culturable counts. However, PCR-DGGE showed that the numbers of bands detected in the molecular community profiles decreased with increasing oil content of the soil. Analysis of the sequences of three prominent bands of the profiles generated with the highly polluted soil samples suggested that the underlying organisms were related to Actinomyces sp., Arthrobacter sp., and a bacterium of uncertain affiliation. dszA, dszB, and dszC genes were present in all A soil samples, whereas a range of unpolluted soils gave negative results in this analysis. Results from the study of FSL soil revealed minor effects of the petroleum-DBT treatment on culturable bacterial numbers and clear effects on the DBT-utilizing communities. The molecular community profiles were largely stable over time in the untreated soil, whereas they showed a progressive change over time following treatment with DBT-containing petroleum. Direct PCR assessment revealed the presence of dszB-related signals in the untreated FSL soil and the apparent selection of dszA- and dszC-related sequences by the petroleum-DBT treatment

Modeling the integration of bacterial rRNA fragments into the human cancer genome.

PubMed

Sieber, Karsten B; Gajer, Pawel; Dunning Hotopp, Julie C

2016-03-21

Cancer is a disease driven by the accumulation of genomic alterations, including the integration of exogenous DNA into the human somatic genome. We previously identified in silico evidence of DNA fragments from a Pseudomonas-like bacteria integrating into the 5'-UTR of four proto-oncogenes in stomach cancer sequencing data. The functional and biological consequences of these bacterial DNA integrations remain unknown. Modeling of these integrations suggests that the previously identified sequences cover most of the sequence flanking the junction between the bacterial and human DNA. Further examination of these reads reveals that these integrations are rich in guanine nucleotides and the integrated bacterial DNA may have complex transcript secondary structures. The models presented here lay the foundation for future experiments to test if bacterial DNA integrations alter the transcription of the human genes.

Base-Calling Algorithm with Vocabulary (BCV) Method for Analyzing Population Sequencing Chromatograms

PubMed Central

Fantin, Yuri S.; Neverov, Alexey D.; Favorov, Alexander V.; Alvarez-Figueroa, Maria V.; Braslavskaya, Svetlana I.; Gordukova, Maria A.; Karandashova, Inga V.; Kuleshov, Konstantin V.; Myznikova, Anna I.; Polishchuk, Maya S.; Reshetov, Denis A.; Voiciehovskaya, Yana A.; Mironov, Andrei A.; Chulanov, Vladimir P.

2013-01-01

Sanger sequencing is a common method of reading DNA sequences. It is less expensive than high-throughput methods, and it is appropriate for numerous applications including molecular diagnostics. However, sequencing mixtures of similar DNA of pathogens with this method is challenging. This is important because most clinical samples contain such mixtures, rather than pure single strains. The traditional solution is to sequence selected clones of PCR products, a complicated, time-consuming, and expensive procedure. Here, we propose the base-calling with vocabulary (BCV) method that computationally deciphers Sanger chromatograms obtained from mixed DNA samples. The inputs to the BCV algorithm are a chromatogram and a dictionary of sequences that are similar to those we expect to obtain. We apply the base-calling function on a test dataset of chromatograms without ambiguous positions, as well as one with 3–14% sequence degeneracy. Furthermore, we use BCV to assemble a consensus sequence for an HIV genome fragment in a sample containing a mixture of viral DNA variants and to determine the positions of the indels. Finally, we detect drug-resistant Mycobacterium tuberculosis strains carrying frameshift mutations mixed with wild-type bacteria in the pncA gene, and roughly characterize bacterial communities in clinical samples by direct 16S rRNA sequencing. PMID:23382983

Bacterial diversity at different stages of the composting process

PubMed Central

2010-01-01

Background Composting is an aerobic microbiological process that is facilitated by bacteria and fungi. Composting is also a method to produce fertilizer or soil conditioner. Tightened EU legislation now requires treatment of the continuously growing quantities of organic municipal waste before final disposal. However, some full-scale composting plants experience difficulties with the efficiency of biowaste degradation and with the emission of noxious odours. In this study we examine the bacterial species richness and community structure of an optimally working pilot-scale compost plant, as well as a full-scale composting plant experiencing typical problems. Bacterial species composition was determined by isolating total DNA followed by amplifying and sequencing the gene encoding the 16S ribosomal RNA. Results Over 1500 almost full-length 16S rRNA gene sequences were analysed and of these, over 500 were present only as singletons. Most of the sequences observed in either one or both of the composting processes studied here were similar to the bacterial species reported earlier in composts, including bacteria from the phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Deinococcus-Thermus. In addition, a number of previously undetected bacterial phylotypes were observed. Statistical calculations estimated a total bacterial diversity of over 2000 different phylotypes in the studied composts. Conclusions Interestingly, locally enriched or evolved bacterial variants of familiar compost species were observed in both composts. A detailed comparison of the bacterial diversity revealed a large difference in composts at the species and strain level from the different composting plants. However, at the genus level, the difference was much smaller and illustrated a delay of the composting process in the full-scale, sub-optimally performing plants. PMID:20350306

Towards Spectral Library-free MALDI-TOF MS Bacterial Identification.

PubMed

Cheng, Ding; Qiao, Liang; Horvatovich, Péter

2018-05-11

Bacterial identification is of great importance in clinical diagnosis, environmental monitoring and food safety control. Among various strategies, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has drawn significant interests, and has been clinically used. Nevertheless, current bioinformatics solutions use spectral libraries for the identification of bacterial strains. Spectral library generation requires acquisition of MALDI-TOF spectra from monoculture bacterial colonies, which is time-consuming and not possible for many species and strains. We propose a strategy for bacterial typing by MALDI-TOF using protein sequences from public database, i.e. UniProt. Ten genes were identified to encode proteins most often observed by MALD-TOF from bacteria through 500 times repeated a 10-fold double cross-validation procedure, using 403 MALDI-TOF spectra corresponding to 14 genera, 81 species and 403 strains, and the protein sequences of 1276 species in UniProt. The 10 genes were then used to annotate peaks on MALDI-TOF spectra of bacteria for bacterial identification. With the approach, bacteria can be identified at the genus level by searching against a database containing the protein sequences of 42 genera of bacteria from UniProt. Our approach identified 84.1% of the 403 spectra correctly at the genus level. Source code of the algorithm is available at https://github.com/dipcarbon/BacteriaMSLF.

Molecular Diagnosis of Orthopedic-Device-Related Infection Directly from Sonication Fluid by Metagenomic Sequencing

PubMed Central

Sanderson, Nicholas D.; Atkins, Bridget L.; Brent, Andrew J.; Cole, Kevin; Foster, Dona; McNally, Martin A.; Oakley, Sarah; Peto, Leon; Taylor, Adrian; Peto, Tim E. A.; Crook, Derrick W.; Eyre, David W.

2017-01-01

ABSTRACT Culture of multiple periprosthetic tissue samples is the current gold standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through culture of sonication fluid from explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of total DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 derivation samples, we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and confirmed our findings in 47 independent validation samples. Compared to results from sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69 (88%; 95% confidence interval [CI], 77 to 94%; for derivation samples 35/38 [92%; 95% CI, 79 to 98%]; for validation samples, 26/31 [84%; 95% CI, 66 to 95%]), and genus-level sensitivity was 64/69 (93%; 95% CI, 84 to 98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97 (88%; 95% CI, 79 to 93%; for derivation samples, 43/50 [86%; 95% CI, 73 to 94%]; for validation samples, 42/47 [89%; 95% CI, 77 to 96%]). High levels of human DNA contamination were seen despite the use of laboratory methods to remove it. Rigorous laboratory good practice was required to minimize bacterial DNA contamination. We demonstrate that metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings

Complete Genome Sequence of a New Firmicutes Species Isolated from Anaerobic Biomass Hydrolysis.

PubMed

Abendroth, Christian; Hahnke, Sarah; Codoñer, Francisco M; Klocke, Michael; Luschnig, Olaf; Porcar, Manuel

2017-10-05

A new Firmicutes isolate, strain HV4-6-A5C, was obtained from the hydrolysis stage of a mesophilic and anaerobic two-stage lab-scale leach-bed system for biomethanation of fresh grass. It is assumed that the bacterial isolate contributes to plant biomass degradation. Here, we report a draft annotated genome sequence of this organism. Copyright © 2017 Abendroth et al.

A Diverse Soil Microbiome Degrades More Crude Oil than Specialized Bacterial Assemblages Obtained in Culture.

PubMed

Bell, Terrence H; Stefani, Franck O P; Abram, Katrina; Champagne, Julie; Yergeau, Etienne; Hijri, Mohamed; St-Arnaud, Marc

2016-09-15

Soil microbiome modification may alter system function, which may enhance processes like bioremediation. In this study, we filled microcosms with gamma-irradiated soil that was reinoculated with the initial soil or cultivated bacterial subsets obtained on regular media (REG-M) or media containing crude oil (CO-M). We allowed 8 weeks for microbiome stabilization, added crude oil and monoammonium phosphate, incubated the microcosms for another 6 weeks, and then measured the biodegradation of crude oil components, bacterial taxonomy, and functional gene composition. We hypothesized that the biodegradation of targeted crude oil components would be enhanced by limiting the microbial taxa competing for resources and by specifically selecting bacteria involved in crude oil biodegradation (i.e., CO-M). Postincubation, large differences in taxonomy and functional gene composition between the three microbiome types remained, indicating that purposeful soil microbiome structuring is feasible. Although phylum-level bacterial taxonomy was constrained, operational taxonomic unit composition varied between microbiome types. Contrary to our hypothesis, the biodegradation of C10 to C50 hydrocarbons was highest when the original microbiome was reinoculated, despite a higher relative abundance of alkane hydroxylase genes in the CO-M microbiomes and of carbon-processing genes in the REG-M microbiomes. Despite increases in the relative abundances of genes potentially linked to hydrocarbon processing in cultivated subsets of the microbiome, reinoculation of the initial microbiome led to maximum biodegradation. In this study, we show that it is possible to sustainably modify microbial assemblages in soil. This has implications for biotechnology, as modification of gut microbial assemblages has led to improved treatments for diseases like Clostridium difficile infection. Although the soil environment determined which major phylogenetic groups of bacteria would dominate the assemblage, we

Use of 16S rRNA sequencing and quantitative PCR to correlate venous leg ulcer bacterial bioburden dynamics with wound expansion, antibiotic therapy, and healing

PubMed Central

Sprockett, Daniel D.; Ammons, Christine G.; Tuttle, Marie S.

2016-01-01

Clinical diagnosis of infection in chronic wounds is currently limited to subjective clinical signs and culture-based methods that underestimate the complexity of wound microbial bioburden as revealed by DNA-based microbial identification methods. Here, we use 16S rRNA next generation sequencing and quantitative polymerase chain reaction to characterize weekly changes in bacterial load, community structure, and diversity associated with a chronic venous leg ulcer over the 15-week course of treatment and healing. Our DNA-based methods and detailed sampling scheme reveal that the bacterial bioburden of the wound is unexpectedly dynamic, including changes in the bacterial load and community structure that correlate with wound expansion, antibiotic therapy, and healing. We demonstrate that these multidimensional changes in bacterial bioburden can be summarized using swabs taken prior to debridement, and therefore, can be more easily collected serially than debridement or biopsy samples. Overall, this case illustrates the importance of detailed clinical indicators and longitudinal sampling to determine the pathogenic significance of chronic wound microbial dynamics and guide best use of antimicrobials for improvement of healing outcomes. PMID:25902876

Isolation and identification of biocellulose-producing bacterial strains from Malaysian acidic fruits.

PubMed

Voon, W W Y; Rukayadi, Y; Meor Hussin, A S

2016-05-01

Biocellulose (BC) is pure extracellular cellulose produced by several species of micro-organisms that has numerous applications in the food, biomedical and paper industries. However, the existing biocellulose-producing bacterial strain with high yield was limited. The aim of this study was to isolate and identify the potential biocellulose-producing bacterial isolates from Malaysian acidic fruits. One hundred and ninety-three bacterial isolates were obtained from 19 local acidic fruits collected in Malaysia and screened for their ability to produce BC. A total of 15 potential bacterial isolates were then cultured in standard Hestrin-Schramm (HS) medium statically at 30°C for 2 weeks to determine the BC production. The most potent bacterial isolates were identified using 16S rRNA gene sequence analysis, morphological and biochemical characteristics. Three new and potent biocellulose-producing bacterial strains were isolated from soursop fruit and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. Stenotrophomonas maltophilia WAUPM42 was the most potent biocellulose-producing bacterial strain that produced the highest amount of BC 0·58 g l(-1) in standard HS medium. Whereas, the isolates P. vagans WAUPM45 and B. fluminensis WAUPM53 showed 0·50 and 0·52 g l(-1) of BC production, respectively. Biocellulose (BC) is pure extracellular cellulose that is formed by many micro-organisms in the presence of carbon source and acidic condition. It can replace plant-based cellulose in multifarious applications due to its unique characteristics. In this study, three potential biocellulose-producing bacterial strains were obtained from Malaysian acidic fruits and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. This study reports for the first time the new biocellulose-producing bacterial strains isolated from Malaysian acidic fruits. © 2016 The

Cryptic species revealed by molecular phylogenetic analysis of sequences obtained from basidiomata of Tulasnella.

PubMed

Cruz, Darío; Suárez, Juan Pablo; Kottke, Ingrid; Piepenbring, Meike

2014-01-01

Delimitation of species and the search for a proper threshold for defining phylogenetic species in fungi are under discussion. In this study, morphological and molecular data are correlated to delimit species of Tulasnella, the most important mycobionts of Orchidaceae, which suffer from poor taxonomy. Resupinate basidiomata of Tulasnella species were collected in Ecuador and Germany, and 11 specimens (seven from Ecuador, four from Germany) were assigned to traditional species concepts by use of morphological keys. The specimens were compared by micro-anatomical examination with 75 specimens of Tulasnella borrowed from fungaria to obtain better insights on variation of characters. Sequences of the ITS region (127) were obtained after cloning from the fresh basidiomata and from pure cultures. Proportional variability of ITS sequences was analyzed within and among the cultures and the specimens designated to different morphospecies. Results suggested an intragenomic variation of less than 2%, an intraspecific variation of up to 4% and an interspecific divergence of more than 9% in Tulasnella. Cryptic species in Tulasnella, mostly from Ecuador, were revealed by phylogenetic analyses with 4% intraspecific divergence as a minimum threshold for delimiting species. Conventional diagnostic morphological characters appeared insufficient for species characterization. Arguments are presented for molecular delimitation of the established species Tulasnella albida, T. asymmetrica, T. eichleriana, T. cf. pinicola, T. tomaculum and T. violea. © 2014 by The Mycological Society of America.

Gamma-irradiated bacterial preparation having anti-tumor activity

DOEpatents

Vass, Arpad A.; Tyndall, Richard L.; Terzaghi-Howe, Peggy

1999-01-01

A bacterial preparation from Pseudomonas species isolated #15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

Bacterial meningitis.

PubMed

Heckenberg, Sebastiaan G B; Brouwer, Matthijs C; van de Beek, Diederik

2014-01-01

Bacterial meningitis is a neurologic emergency. Vaccination against common pathogens has decreased the burden of disease. Early diagnosis and rapid initiation of empiric antimicrobial and adjunctive therapy are vital. Therapy should be initiated as soon as blood cultures have been obtained, preceding any imaging studies. Clinical signs suggestive of bacterial meningitis include fever, headache, meningismus, and an altered level of consciousness but signs may be scarce in children, in the elderly, and in meningococcal disease. Host genetic factors are major determinants of susceptibility to meningococcal and pneumococcal disease. Dexamethasone therapy has been implemented as adjunctive treatment of adults with pneumococcal meningitis. Adequate and prompt treatment of bacterial meningitis is critical to outcome. In this chapter we review the epidemiology, pathophysiology, and management of bacterial meningitis. © 2014 Elsevier B.V. All rights reserved.

Bacterial community analysis of drinking water biofilms in southern Sweden.

PubMed

Lührig, Katharina; Canbäck, Björn; Paul, Catherine J; Johansson, Tomas; Persson, Kenneth M; Rådström, Peter

2015-01-01

Next-generation sequencing of the V1-V2 and V3 variable regions of the 16S rRNA gene generated a total of 674,116 reads that described six distinct bacterial biofilm communities from both water meters and pipes. A high degree of reproducibility was demonstrated for the experimental and analytical work-flow by analyzing the communities present in parallel water meters, the rare occurrence of biological replicates within a working drinking water distribution system. The communities observed in water meters from households that did not complain about their drinking water were defined by sequences representing Proteobacteria (82-87%), with 22-40% of all sequences being classified as Sphingomonadaceae. However, a water meter biofilm community from a household with consumer reports of red water and flowing water containing elevated levels of iron and manganese had fewer sequences representing Proteobacteria (44%); only 0.6% of all sequences were classified as Sphingomonadaceae; and, in contrast to the other water meter communities, markedly more sequences represented Nitrospira and Pedomicrobium. The biofilm communities in pipes were distinct from those in water meters, and contained sequences that were identified as Mycobacterium, Nocardia, Desulfovibrio, and Sulfuricurvum. The approach employed in the present study resolved the bacterial diversity present in these biofilm communities as well as the differences that occurred in biofilms within a single distribution system, and suggests that next-generation sequencing of 16S rRNA amplicons can show changes in bacterial biofilm communities associated with different water qualities.

Short-Sequence DNA Repeats in Prokaryotic Genomes

PubMed Central

van Belkum, Alex; Scherer, Stewart; van Alphen, Loek; Verbrugh, Henri

1998-01-01

Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10−4 per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant. PMID:9618442

DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure

DOE PAGES

None

2014-12-01

The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illuminamore » 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.« less

Accurate multiplex polony sequencing of an evolved bacterial genome.

PubMed

Shendure, Jay; Porreca, Gregory J; Reppas, Nikos B; Lin, Xiaoxia; McCutcheon, John P; Rosenbaum, Abraham M; Wang, Michael D; Zhang, Kun; Mitra, Robi D; Church, George M

2005-09-09

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

Transposon facilitated DNA sequencing

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

Berg, D.E.; Berg, C.M.; Huang, H.V.

1990-01-01

The purpose of this research is to investigate and develop methods that exploit the power of bacterial transposable elements for large scale DNA sequencing: Our premise is that the use of transposons to put primer binding sites randomly in target DNAs should provide access to all portions of large DNA fragments, without the inefficiencies of methods involving random subcloning and attendant repetitive sequencing, or of sequential synthesis of many oligonucleotide primers that are used to match systematically along a DNA molecule. Two unrelated bacterial transposons, Tn5 and {gamma}{delta}, are being used because they have both proven useful for molecular analyses,more » and because they differ sufficiently in mechanism and specificity of transposition to merit parallel development.« less

Bacterial Genome Engineering and Synthetic Biology: Combating Pathogens

DTIC Science & Technology

2016-11-04

engineering and SB methods such as recombineering, clustered regularly interspaced short palindromic repeats ( CRISPR ), and bacterial cell-cell...Cholera# Yersinia pseudotuberculosis# Staphylococcus aureus* Phage Engineering CRISPR /Cas9 Delivery of CRISPR genes and RNA guides for sequence...bear very close sequence alignment to the harmless strains via the use of the CRISPR /Cas9 system. The CRISPR system specifically targets a DNA sequence

An integrated semiconductor device enabling non-optical genome sequencing.

PubMed

Rothberg, Jonathan M; Hinz, Wolfgang; Rearick, Todd M; Schultz, Jonathan; Mileski, William; Davey, Mel; Leamon, John H; Johnson, Kim; Milgrew, Mark J; Edwards, Matthew; Hoon, Jeremy; Simons, Jan F; Marran, David; Myers, Jason W; Davidson, John F; Branting, Annika; Nobile, John R; Puc, Bernard P; Light, David; Clark, Travis A; Huber, Martin; Branciforte, Jeffrey T; Stoner, Isaac B; Cawley, Simon E; Lyons, Michael; Fu, Yutao; Homer, Nils; Sedova, Marina; Miao, Xin; Reed, Brian; Sabina, Jeffrey; Feierstein, Erika; Schorn, Michelle; Alanjary, Mohammad; Dimalanta, Eileen; Dressman, Devin; Kasinskas, Rachel; Sokolsky, Tanya; Fidanza, Jacqueline A; Namsaraev, Eugeni; McKernan, Kevin J; Williams, Alan; Roth, G Thomas; Bustillo, James

2011-07-20

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

Streptococcus massiliensis in the human mouth: a phylogenetic approach for the inference of bacterial habitats.

PubMed

Póntigo, F; Silva, C; Moraga, M; Flores, S V

2015-12-29

Streptococcus is a diverse bacterial lineage. Species of this genus occupy a myriad of environments inside humans and other animals. Despite the elucidation of several of these habitats, many remain to be identified. Here, we explore a methodological approach to reveal unknown bacterial environments. Specifically, we inferred the phylogeny of the Mitis group by analyzing the sequences of eight genes. In addition, information regarding habitat use of species belonging to this group was obtained from the scientific literature. The oral cavity emerged as a potential, previously unknown, environment of Streptococcus massiliensis. This phylogeny-based prediction was confirmed by species-specific polymerase chain reaction (PCR) amplification. We propose employing a similar approach, i.e., use of bibliographic data and molecular phylogenetics as predictive methods, and species-specific PCR as confirmation, in order to reveal other unknown habitats in further bacterial taxa.

Gamma-irradiated bacterial preparation having anti-tumor activity

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

Vass, A.A.; Tyndall, R.L.; Terzaghi-Howe, P.

1999-11-16

This application describes a bacterial preparation from Pseudomonas species isolated {number{underscore}sign}15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

Bacterial expression of self-assembling peptide hydrogelators

NASA Astrophysics Data System (ADS)

Sonmez, Cem

For tissue regeneration and drug delivery applications, various architectures are explored to serve as biomaterial tools. Via de novo design, functional peptide hydrogel materials have been developed as scaffolds for biomedical applications. The objective of this study is to investigate bacterial expression as an alternative method to chemical synthesis for the recombinant production of self-assembling peptides that can form rigid hydrogels under physiological conditions. The Schneider and Pochan Labs have designed and characterized a 20 amino acid beta-hairpin forming amphiphilic peptide containing a D-residue in its turn region (MAX1). As a result, this peptide must be prepared chemically. Peptide engineering, using the sequence of MAX1 as a template, afforded a small family of peptides for expression (EX peptides) that have different turn sequences consisting of natural amino acids and amenable to bacterial expression. Each sequence was initially chemically synthesized to quickly assess the material properties of its corresponding gel. One model peptide EX1, was chosen to start the bacterial expression studies. DNA constructs facilitating the expression of EX1 were designed in such that the peptide could be expressed with different fusion partners and subsequently cleaved by enzymatic or chemical means to afford the free peptide. Optimization studies were performed to increase the yield of pure peptide that ultimately allowed 50 mg of pure peptide to be harvested from one liter of culture, providing an alternate means to produce this hydrogel-forming peptide. Recombinant production of other self-assembling hairpins with different turn sequences was also successful using this optimized protocol. The studies demonstrate that new beta-hairpin self-assembling peptides that are amenable to bacterial production and form rigid hydrogels at physiological conditions can be designed and produced by fermentation in good yield at significantly reduced cost when compared to

Bacterial communities associated with the ectoparasitic mites Varroa destructor and Tropilaelaps mercedesae of the honey bee (Apis mellifera).

PubMed

Pakwan, Chonthicha; Kaltenpoth, Martin; Weiss, Benjamin; Chantawannakul, Panuwan; Jun, Guo; Disayathanoowat, Terd

2017-12-01

Varroa and Tropilaelaps mites have been reported as serious ectoparasites of the honey bee (Apis mellifera). In this study, bacterial communities associated with Varroa destructor and Tropilaelaps mercedesae from northern Thailand were determined, using both culture-dependent and culture-independent approaches. Adult female mites were collected from apiaries in Chiang Mai and Lampang provinces. Culturable bacteria were isolated from individual mites. On average, we observed approximately 1340 and 1140 CFU/mite in Varroa and Tropilaelaps, respectively. All isolates were assigned to the genus Enterococcus. Six samples of genomic DNA from 30-50 mites were extracted and subjected to pyrosequencing of bacterial 16S rRNA amplicons. The resulting 81 717 sequences obtained from Varroa were grouped into 429 operational taxonomic units. The most abundant bacteria in Varroa mites belonged to the family Enterobacteriaceae, especially the genera Arsenophonus, Enterobacter and Proteus. For Tropilaelaps mites, 84 075 sequences were obtained and clustered into 166 operational taxonomic units, within which the family Enterococcaceae (particularly the genus Enterococcus) was predominant. Localization of bacteria in the mites using fluorescence in situ hybridization with two universal bacterial probes revealed that these bacteria were in the cecum of the mites. Taxon-specific Enterobacteriaceae and Arsenophonus probes also confirmed their localization in the cecum of Varroa. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence typing primers.

PubMed

Adamiak, Paul; Vanderkooi, Otto G; Kellner, James D; Schryvers, Anthony B; Bettinger, Julie A; Alcantara, Joenel

2014-06-03

Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate's sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles. The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years. The inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative

Draft Genome Sequence of Two Strains of Xanthomonas arboricola Isolated from Prunus persica Which Are Dissimilar to Strains That Cause Bacterial Spot Disease on Prunus spp.

PubMed Central

Garita-Cambronero, Jerson; Palacio-Bielsa, Ana; López, María M.

2016-01-01

The draft genome sequences of two strains of Xanthomonas arboricola, isolated from asymptomatic peach trees in Spain, are reported here. These strains are avirulent and do not belong to the same phylogroup as X. arboricola pv. pruni, a causal agent of bacterial spot disease of stone fruits and almonds. PMID:27609931

Amylase production potentials of bacterial isolates obtained from the gut of Oryctes rhinoceros larvae

NASA Astrophysics Data System (ADS)

Aryati, P. C.; Pangastuti, A.; Sari, S. L. A.

2017-04-01

Amylase is one of the main enzymes used in industry, such as food, detergent, textile, and pharmaceutical industry. Amylase can be produced by plants, animals, and microorganisms. However, bacterial and fungal amylases have dominated application in industries. This research was aimed to determine amylolytic activity of bacteria isolated from the gut of Oryctes rhinoceros larvae. Based on clear zone formation, 9 from 11 isolates showed amylolytic activity. Isolates with the widest clear zone, i.e Bacillus subtilis GOR1, Bacillus cereus GOR3, and Bacillus pumilus GOR2, were screened for amylolytic activity based on reduction sugar production. The result showed that Bacillus subtilis GOR1 was the most potential as amylase producer, showed by the widest clear zone 5.224 cm2 and highest reduction sugar production 0.0235 mg/ml. Highest amylase specific activity (0.1447 U/mg protein) was obtained at 60°C and pH 7. Amylase activity was stable for 3 hours at 60°C with residual activity respectively was 59.7%.

Correlation between genome reduction and bacterial growth.

PubMed

Kurokawa, Masaomi; Seno, Shigeto; Matsuda, Hideo; Ying, Bei-Wen

2016-12-01

Genome reduction by removing dispensable genomic sequences in bacteria is commonly used in both fundamental and applied studies to determine the minimal genetic requirements for a living system or to develop highly efficient bioreactors. Nevertheless, whether and how the accumulative loss of dispensable genomic sequences disturbs bacterial growth remains unclear. To investigate the relationship between genome reduction and growth, a series of Escherichia coli strains carrying genomes reduced in a stepwise manner were used. Intensive growth analyses revealed that the accumulation of multiple genomic deletions caused decreases in the exponential growth rate and the saturated cell density in a deletion-length-dependent manner as well as gradual changes in the patterns of growth dynamics, regardless of the growth media. Accordingly, a perspective growth model linking genome evolution to genome engineering was proposed. This study provides the first demonstration of a quantitative connection between genomic sequence and bacterial growth, indicating that growth rate is potentially associated with dispensable genomic sequences. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Identification and Characterization of Domesticated Bacterial Transposases

PubMed Central

Gallie, Jenna; Rainey, Paul B.

2017-01-01

Abstract Selfish genetic elements, such as insertion sequences and transposons are found in most genomes. Transposons are usually identifiable by their high copy number within genomes. In contrast, REP-associated tyrosine transposases (RAYTs), a recently described class of bacterial transposase, are typically present at just one copy per genome. This suggests that RAYTs no longer copy themselves and thus they no longer function as a typical transposase. Motivated by this possibility we interrogated thousands of fully sequenced bacterial genomes in order to determine patterns of RAYT diversity, their distribution across chromosomes and accessory elements, and rate of duplication. RAYTs encompass exceptional diversity and are divisible into at least five distinct groups. They possess features more similar to housekeeping genes than insertion sequences, are predominantly vertically transmitted and have persisted through evolutionary time to the point where they are now found in 24% of all species for which at least one fully sequenced genome is available. Overall, the genomic distribution of RAYTs suggests that they have been coopted by host genomes to perform a function that benefits the host cell. PMID:28910967

Site-Specific Integration of Foreign DNA into Minimal Bacterial and Human Target Sequences Mediated by a Conjugative Relaxase

PubMed Central

Agúndez, Leticia; González-Prieto, Coral; Machón, Cristina; Llosa, Matxalen

2012-01-01

Background Bacterial conjugation is a mechanism for horizontal DNA transfer between bacteria which requires cell to cell contact, usually mediated by self-transmissible plasmids. A protein known as relaxase is responsible for the processing of DNA during bacterial conjugation. TrwC, the relaxase of conjugative plasmid R388, is also able to catalyze site-specific integration of the transferred DNA into a copy of its target, the origin of transfer (oriT), present in a recipient plasmid. This reaction confers TrwC a high biotechnological potential as a tool for genomic engineering. Methodology/Principal Findings We have characterized this reaction by conjugal mobilization of a suicide plasmid to a recipient cell with an oriT-containing plasmid, selecting for the cointegrates. Proteins TrwA and IHF enhanced integration frequency. TrwC could also catalyze integration when it is expressed from the recipient cell. Both Y18 and Y26 catalytic tyrosil residues were essential to perform the reaction, while TrwC DNA helicase activity was dispensable. The target DNA could be reduced to 17 bp encompassing TrwC nicking and binding sites. Two human genomic sequences resembling the 17 bp segment were accepted as targets for TrwC-mediated site-specific integration. TrwC could also integrate the incoming DNA molecule into an oriT copy present in the recipient chromosome. Conclusions/Significance The results support a model for TrwC-mediated site-specific integration. This reaction may allow R388 to integrate into the genome of non-permissive hosts upon conjugative transfer. Also, the ability to act on target sequences present in the human genome underscores the biotechnological potential of conjugative relaxase TrwC as a site-specific integrase for genomic modification of human cells. PMID:22292089

Transforming microbial genotyping: a robotic pipeline for genotyping bacterial strains.

PubMed

O'Farrell, Brian; Haase, Jana K; Velayudhan, Vimalkumar; Murphy, Ronan A; Achtman, Mark

2012-01-01

Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost.

Transforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial Strains

PubMed Central

Velayudhan, Vimalkumar; Murphy, Ronan A.; Achtman, Mark

2012-01-01

Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spread-sheets. The efficiency, reliability and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. We developed a medium- throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline was implemented through a combination of four automated liquid handling systems, a Laboratory Information Management System (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a Sample Management System, plus numerous Python scripts. All tubes and microwell racks were bar-coded and their locations and status were recorded in the LIMS. We also created a hierarchical set of items that could be used to represent bacterial species, their products and experiments. The LIMS allowed reliable, semi-automated, traceable bacterial genotyping from initial single colony isolation and sub-cultivation through DNA extraction and normalization to PCRs, sequencing and MLST sequence trace evaluation. We also describe robotic sequencing to facilitate cherrypicking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of < €25 per strain. Since developing this pipeline, >200,000 items were processed by two to three people. Our sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support, and provides a general framework for semi-automated bacterial genotyping of large numbers of samples at low cost. PMID:23144721

Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples.

PubMed

Marsh, Alan J; O'Sullivan, Orla; Hill, Colin; Ross, R Paul; Cotter, Paul D

2014-04-01

Kombucha is a sweetened tea beverage that, as a consequence of fermentation, contains ethanol, carbon dioxide, a high concentration of acid (gluconic, acetic and lactic) as well as a number of other metabolites and is thought to contain a number of health-promoting components. The sucrose-tea solution is fermented by a symbiosis of bacteria and yeast embedded within a cellulosic pellicle, which forms a floating mat in the tea, and generates a new layer with each successful fermentation. The specific identity of the microbial populations present has been the focus of attention but, to date, the majority of studies have relied on culture-based analyses. To gain a more comprehensive insight into the kombucha microbiota we have carried out the first culture-independent, high-throughput sequencing analysis of the bacterial and fungal populations of 5 distinct pellicles as well as the resultant fermented kombucha at two time points. Following the analysis it was established that the major bacterial genus present was Gluconacetobacter, present at >85% in most samples, with only trace populations of Acetobacter detected (<2%). A prominent Lactobacillus population was also identified (up to 30%), with a number of sub-dominant genera, not previously associated with kombucha, also being revealed. The yeast populations were found to be dominated by Zygosaccharomyces at >95% in the fermented beverage, with a greater fungal diversity present in the cellulosic pellicle, including numerous species not identified in kombucha previously. Ultimately, this study represents the most accurate description of the microbiology of kombucha to date. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dynamics of indigenous bacterial communities associated with crude oil degradation in soil microcosms during nutrient-enhanced bioremediation.

PubMed

Chikere, Chioma B; Surridge, Karen; Okpokwasili, Gideon C; Cloete, Thomas E

2012-03-01

Bacterial population dynamics were examined during bioremediation of an African soil contaminated with Arabian light crude oil and nutrient enrichment (biostimulation). Polymerase chain reaction followed by denaturing gradient gel electrophoresis (DGGE) were used to generate bacterial community fingerprints of the different treatments employing the 16S ribosomal ribonucleic acid (rRNA) gene as molecular marker. The DGGE patterns of the nutrient-amended soils indicated the presence of distinguishable bands corresponding to the oil-contaminated-nutrient-enriched soils, which were not present in the oil-contaminated and pristine control soils. Further characterization of the dominant DGGE bands after excision, reamplification and sequencing revealed that Corynebacterium spp., Dietzia spp., Rhodococcus erythropolis sp., Nocardioides sp., Low G+C (guanine plus cytosine) Gram positive bacterial clones and several uncultured bacterial clones were the dominant bacterial groups after biostimulation. Prominent Corynebacterium sp. IC10 sequence was detected across all nutrient-amended soils but not in oil-contaminated control soil. Total heterotrophic and hydrocarbon utilizing bacterial counts increased significantly in the nutrient-amended soils 2 weeks post contamination whereas oil-contaminated and pristine control soils remained fairly stable throughout the experimental period. Gas chromatographic analysis of residual hydrocarbons in biostimulated soils showed marked attenuation of contaminants starting from the second to the sixth week after contamination whereas no significant reduction in hydrocarbon peaks were seen in the oil-contaminated control soil throughout the 6-week experimental period. Results obtained indicated that nutrient amendment of oil-contaminated soil selected and enriched the bacterial communities mainly of the Actinobacteria phylogenetic group capable of surviving in toxic contamination with concomitant biodegradation of the hydrocarbons. The

CRISPR-like sequences in Helicobacter pylori and application in genotyping.

PubMed

Bangpanwimon, Khotchawan; Sottisuporn, Jaksin; Mittraparp-Arthorn, Pimonsri; Ueaphatthanaphanich, Warattaya; Rattanasupar, Attapon; Pourcel, Christine; Vuddhakul, Varaporn

2017-01-01

Many bacteria and archaea possess a defense system called clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (CRISPR-Cas system) against invaders such as phages or plasmids. This system has not been demonstrated in Helicobacter pylori . The numbers of spacer in CRISPR array differ among bacterial strains and can be used as a genetic marker for bacterial typing. A total of 36 H. pylori isolates were collected from patients in three hospitals located in the central (PBH) and southern (SKH) regions of Thailand. It is of interest that CRISPR-like sequences of this bacterium were detected in vlpC encoded for VacA-like protein C. Virulence genes were investigated and the most pathogenic genotype ( cagA vacA s1m1) was detected in 17 out of 29 (58.6%) isolates from PBH and 5 out of 7 (71.4%) from SKH. vapD gene was identified in each one isolate from PBH and SKH. CRISPR-like sequences and virulence genes of 20 isolates of H. pylori obtained in this study were analyzed and CRISPR-virulence typing was constructed and compared to profiles obtained by the random amplification of polymorphic DNA (RAPD) technique. The discriminatory power (DI) of CRISPR-virulence typing was not different from RAPD typing. CRISPR-virulence typing in H. pylori is easy and reliable for epidemiology and can be used for inter-laboratory interpretation.

Bacterial Community Analysis of Drinking Water Biofilms in Southern Sweden

PubMed Central

Lührig, Katharina; Canbäck, Björn; Paul, Catherine J.; Johansson, Tomas; Persson, Kenneth M.; Rådström, Peter

2015-01-01

Next-generation sequencing of the V1–V2 and V3 variable regions of the 16S rRNA gene generated a total of 674,116 reads that described six distinct bacterial biofilm communities from both water meters and pipes. A high degree of reproducibility was demonstrated for the experimental and analytical work-flow by analyzing the communities present in parallel water meters, the rare occurrence of biological replicates within a working drinking water distribution system. The communities observed in water meters from households that did not complain about their drinking water were defined by sequences representing Proteobacteria (82–87%), with 22–40% of all sequences being classified as Sphingomonadaceae. However, a water meter biofilm community from a household with consumer reports of red water and flowing water containing elevated levels of iron and manganese had fewer sequences representing Proteobacteria (44%); only 0.6% of all sequences were classified as Sphingomonadaceae; and, in contrast to the other water meter communities, markedly more sequences represented Nitrospira and Pedomicrobium. The biofilm communities in pipes were distinct from those in water meters, and contained sequences that were identified as Mycobacterium, Nocardia, Desulfovibrio, and Sulfuricurvum. The approach employed in the present study resolved the bacterial diversity present in these biofilm communities as well as the differences that occurred in biofilms within a single distribution system, and suggests that next-generation sequencing of 16S rRNA amplicons can show changes in bacterial biofilm communities associated with different water qualities. PMID:25739379

Analysis of the Bacterial Diversity in Liver Abscess: Differences between Pyogenic and Amebic Abscesses

PubMed Central

Reyna-Fabián, Miriam E.; Zermeño, Valeria; Ximénez, Cecilia; Flores, Janin; Romero, Miguel F.; Diaz, Daniel; Argueta, Jesús; Moran, Patricia; Valadez, Alicia; Cerritos, René

2016-01-01

Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba. PMID:26572872

Techniques for Large-Scale Bacterial Genome Manipulation and Characterization of the Mutants with Respect to In Silico Metabolic Reconstructions.

PubMed

diCenzo, George C; Finan, Turlough M

2018-01-01

The rate at which all genes within a bacterial genome can be identified far exceeds the ability to characterize these genes. To assist in associating genes with cellular functions, a large-scale bacterial genome deletion approach can be employed to rapidly screen tens to thousands of genes for desired phenotypes. Here, we provide a detailed protocol for the generation of deletions of large segments of bacterial genomes that relies on the activity of a site-specific recombinase. In this procedure, two recombinase recognition target sequences are introduced into known positions of a bacterial genome through single cross-over plasmid integration. Subsequent expression of the site-specific recombinase mediates recombination between the two target sequences, resulting in the excision of the intervening region and its loss from the genome. We further illustrate how this deletion system can be readily adapted to function as a large-scale in vivo cloning procedure, in which the region excised from the genome is captured as a replicative plasmid. We next provide a procedure for the metabolic analysis of bacterial large-scale genome deletion mutants using the Biolog Phenotype MicroArray™ system. Finally, a pipeline is described, and a sample Matlab script is provided, for the integration of the obtained data with a draft metabolic reconstruction for the refinement of the reactions and gene-protein-reaction relationships in a metabolic reconstruction.

[Congenital skull base defect causing recurrent bacterial meningitis].

PubMed

Berliner, Elihay; Bar Meir, Maskit; Megged, Orli

2012-08-01

Bacterial meningitis is a life threatening disease. Most patients will experience only one episode throughout life. Children who experience bacterial meningitis more than once, require further immunologic or anatomic evaluation. We report a 9 year old child with five episodes of bacterial meningitis due to a congenital defect of the skull base. A two and a half year old boy first presented to our medical center with pneumococcal meningitis. He was treated with antibiotics and fully recovered. Two months later he presented again with a similar clinical picture. Streptococcus pneumoniae grew in cerebrospinal fluid (CSF) culture. CT scan and later MRI of the brain revealed a defect in the anterior middle fossa floor, with protrusion of brain tissue into the sphenoidal sinus. Corrective surgery was recommended but the parents refused. Three months later, a third episode of pneumococcal meningitis occurred. The child again recovered with antibiotics and this time corrective surgery was performed. Five years later, the boy presented once again with clinical signs and symptoms consistent with bacterial meningitis. CSF culture was positive, but the final identification of the bacteria was conducted by broad spectrum 16S ribosomal RNA PCR (16S rRNA PCR) which revealed a sequence of Neisseria lactamica. CT and MRI showed recurrence of the skull base defect with encephalocele in the sphenoid sinus. The parents again refused neurosurgical intervention. A year later the patient presented with bacterial meningitis. CSF culture obtained after initiation of antibiotics was negative, but actinobacillus was identified in the CSF by 16S rRNA PCR. The patient is scheduled for neurosurgical intervention. In patients with recurrent bacterial meningitis caused by organisms colonizing the oropharynx or nasopharynx, an anatomical defect should be carefully sought and surgically repaired.

Bacterial community diversity of the deep-sea octocoral Paramuricea placomus.

PubMed

Kellogg, Christina A; Ross, Steve W; Brooke, Sandra D

2016-01-01

Compared to tropical corals, much less is known about deep-sea coral biology and ecology. Although the microbial communities of some deep-sea corals have been described, this is the first study to characterize the bacterial community associated with the deep-sea octocoral, Paramuricea placomus . Samples from five colonies of P. placomus were collected from Baltimore Canyon (379-382 m depth) in the Atlantic Ocean off the east coast of the United States of America. DNA was extracted from the coral samples and 16S rRNA gene amplicons were pyrosequenced using V4-V5 primers. Three samples sequenced deeply (>4,000 sequences each) and were further analyzed. The dominant microbial phylum was Proteobacteria, but other major phyla included Firmicutes and Planctomycetes. A conserved community of bacterial taxa held in common across the three P. placomus colonies was identified, comprising 68-90% of the total bacterial community depending on the coral individual. The bacterial community of P. placomus does not appear to include the genus Endozoicomonas , which has been found previously to be the dominant bacterial associate in several temperate and tropical gorgonians. Inferred functionality suggests the possibility of nitrogen cycling by the core bacterial community.

Bacterial community diversity of the deep-sea octocoral Paramuricea placomus

USGS Publications Warehouse

Kellogg, Christina A.; Ross, Steve W.; Brooke, Sandra D.

2016-01-01

Compared to tropical corals, much less is known about deep-sea coral biology and ecology. Although the microbial communities of some deep-sea corals have been described, this is the first study to characterize the bacterial community associated with the deep-sea octocoral, Paramuricea placomus. Samples from five colonies of P. placomus were collected from Baltimore Canyon (379–382 m depth) in the Atlantic Ocean off the east coast of the United States of America. DNA was extracted from the coral samples and 16S rRNA gene amplicons were pyrosequenced using V4-V5 primers. Three samples sequenced deeply (>4,000 sequences each) and were further analyzed. The dominant microbial phylum was Proteobacteria, but other major phyla included Firmicutes and Planctomycetes. A conserved community of bacterial taxa held in common across the three P. placomuscolonies was identified, comprising 68–90% of the total bacterial community depending on the coral individual. The bacterial community of P. placomusdoes not appear to include the genus Endozoicomonas, which has been found previously to be the dominant bacterial associate in several temperate and tropical gorgonians. Inferred functionality suggests the possibility of nitrogen cycling by the core bacterial community.

Molecular Survey of Bacterial Communities Associated with Bacterial Chondronecrosis with Osteomyelitis (BCO) in Broilers

PubMed Central

Jiang, Tieshan; Mandal, Rabindra K.; Wideman, Robert F.; Khatiwara, Anita; Pevzner, Igal; Min Kwon, Young

2015-01-01

Bacterial chondronecrosis with osteomyelitis (BCO) is recognized as an important cause of lameness in commercial broiler chickens (meat-type chickens). Relatively little is known about the microbial communities associated with BCO. This study was conducted to increase our understanding of the microbial factors associated with BCO using a culture-independent approach. Using Illumina sequencing of the hyper-variable region V6 in the 16S rRNA gene, we characterized the bacterial communities in 97 femoral or tibial heads from normal and lame broilers carefully selected to represent diverse variations in age, line, lesion type, floor type, clinical status and bone type. Our in-depth survey based on 14 million assembled sequence reads revealed that complex bacterial communities exist in all samples, including macroscopically normal bones from clinically healthy birds. Overall, Proteobacteria (mean 90.9%) comprised the most common phylum, followed by Firmicutes (6.1%) and Actinobacteria (2.6%), accounting for more than 99% of all reads. Statistical analyses demonstrated that there are differences in bacterial communities in different types of bones (femur vs. tibia), lesion types (macroscopically normal femora or tibiae vs. those with pathognomonic BCO lesions), and among individual birds. This analysis also showed that BCO samples overrepresented genera Staphylococcus, whose species have been frequently isolated in BCO samples in previous studies. Rarefaction analysis demonstrated the general tendency that increased severities of BCO lesions were associated with reduced species diversity in both femoral and tibial samples when compared to macroscopically normal samples. These observations suggest that certain bacterial subgroups are preferentially selected in association with the development of BCO lesions. Understanding the microbial species associated with BCO will identify opportunities for understanding and modulating the pathogenesis of this form of lameness in

Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study

PubMed Central

Jansen van Rensburg, Melissa J.; Swift, Craig; Cody, Alison J.; Jenkins, Claire

2016-01-01

The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli. Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE. The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software. PMID:27733632

Identification of Microbial Profile of Koji Using Single Molecule, Real-Time Sequencing Technology.

PubMed

Hui, Wenyan; Hou, Qiangchuan; Cao, Chenxia; Xu, Haiyan; Zhen, Yi; Kwok, Lai-Yu; Sun, Tiansong; Zhang, Heping; Zhang, Wenyi

2017-05-01

Koji is a kind of Japanese traditional fermented starter that has been used for centuries. Many fermented foods are made from koji, such as sake, miso, and soy sauce. This study used the single molecule real-time sequencing technology (SMRT) to investigate the bacterial and fungal microbiota of 3 Japanese koji samples. After SMRT analysis, a total of 39121 high-quality sequences were generated, including 14354 bacterial and 24767 fungal sequence reads. The high-quality gene sequences were assigned to 5 bacterial and 2 fungal plyla, dominated by Proteobacteria and Ascomycota, respectively. At the genus level, Ochrobactrum and Wickerhamomyces were the most abundant bacterial and fungal genera, respectively. The predominant bacterial and fungal species were Ochrobactrum lupini and Wickerhamomyces anomalus, respectively. Our study profiled the microbiota composition of 3 Japanese koji samples to the species level precision. The results may be useful for further development of traditional fermented products, especially optimization of koji preparation. Meanwhile, this study has demonstrated that SMRT is a robust tool for analyzing the microbial composition in food samples. © 2017 Institute of Food Technologists®.

Bacterial analysis of combined periodontal-endodontic lesions by polymerase chain reaction-denaturing gradient gel electrophoresis.

PubMed

Xia, Minghui; Qi, Qingguo

2013-01-01

We used denaturing gradient gel electrophoresis (DGGE) to compare bacterial profiles in periodontium and root canals of teeth with combined periodontal-endodontic lesions. Samples of dental plaque and necrotic pulp were collected from thirteen extracted teeth with advanced periodontitis. Genomic DNA was extracted for polymerase chain reaction (PCR) analysis using universal bacterial primers. The PCR products were then loaded onto DGGE gels to obtain fractionated bands. Characteristic DGGE bands were excised and DNA was cloned and sequenced. The number of bands, which indicates the number of bacterial species, was compared between dental plaques and necrotic pulp tissues from the same tooth. Although the difference was statistically significant (P < 0.01), there was no positive correlation; similarity (Dice coefficient) was 13.1% to 62.5%. Some bacteria species were present in both the periodontal pockets and root canals of the same tooth; however, periodontal bacteria did not always invade the root canals, and some bacteria in root canals were not present in periodontal pockets of the same tooth. In some teeth, unique bacteria in root canals had not passed from periodontal pockets. A basic local alignment search tool (BLAST) sequence search in Genbank indicated that new bacteria species were present in periodontal pockets and root canals. Their characteristics must thus be further analyzed.

Analysis of five complete genome sequences for members of the class Peribacteria in the recently recognized Peregrinibacteria bacterial phylum

DOE PAGES

Anantharaman, Karthik; Brown, Christopher T.; Burstein, David; ...

2016-01-28

Five closely related populations of bacteria from the Candidate Phylum (CP) Peregrinibacteria, part of the bacterial Candidate Phyla Radiation (CPR), were sampled from filtered groundwater obtained from an aquifer adjacent to the Colorado River near the town of Rifle, CO, USA. Here, we present the first complete genome sequences for organisms from this phylum. These bacteria have small genomes and, unlike most organisms from other lineages in the CPR, have the capacity for nucleotide synthesis. They invest significantly in biosynthesis of cell wall and cell envelope components, including peptidoglycan, isoprenoids via the mevalonate pathway, and a variety of amino sugarsmore » including perosamine and rhamnose. The genomes encode an intriguing set of large extracellular proteins, some of which are very cysteine-rich and may function in attachment, possibly to other cells. Strain variation in these proteins is an important source of genotypic variety. Overall, the cell envelope features, combined with the lack of biosynthesis capacities for many required cofactors, fatty acids, and most amino acids point to a symbiotic lifestyle. Furthermore, phylogenetic analyses indicate that these bacteria likely represent a new class within the Peregrinibacteria phylum, although they ultimately may be recognized as members of a separate phylum. In conclusion, we propose the provisional taxonomic assignment as ‘ Candidatus Peribacter riflensis’, Genus Peribacter, Family Peribacteraceae, Order Peribacterales, Class Peribacteria in the phylum Peregrinibacteria.« less

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

PubMed

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Benthic Bacterial Diversity in Submerged Sinkhole Ecosystems▿ †

PubMed Central

Nold, Stephen C.; Pangborn, Joseph B.; Zajack, Heidi A.; Kendall, Scott T.; Rediske, Richard R.; Biddanda, Bopaiah A.

2010-01-01

Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities. PMID:19880643

Spatial and vertical distribution of bacterial community in the northern South China Sea.

PubMed

Sun, Fu-Lin; Wang, You-Shao; Wu, Mei-Lin; Sun, Cui-Ci; Cheng, Hao

2015-10-01

Microbial communities are highly diverse in coastal oceans and response rapidly with changing environments. Learning about this will help us understand the ecology of microbial populations in marine ecosystems. This study aimed to assess the spatial and vertical distributions of the bacterial community in the northern South China Sea. Multi-dimensional scaling analyses revealed structural differences of the bacterial community among sampling sites and vertical depth. Result also indicated that bacterial community in most sites had higher diversity in 0-75 m depths than those in 100-200 m depths. Bacterial community of samples was positively correlation with salinity and depth, whereas was negatively correlation with temperature. Proteobacteria and Cyanobacteria were the dominant groups, which accounted for the majority of sequences. The α-Proteobacteria was highly diverse, and sequences belonged to Rhodobacterales bacteria were dominant in all characterized sequences. The current data indicate that the Rhodobacterales bacteria, especially Roseobacter clade are the diverse group in the tropical waters.

Complete Genome Sequence of Mycobacterium marinum ATCC 927T, Obtained Using Nanopore and Illumina Sequencing Technologies.

PubMed

Yoshida, Mitsunori; Fukano, Hanako; Miyamoto, Yuji; Shibayama, Keigo; Suzuki, Masato; Hoshino, Yoshihiko

2018-05-17

Mycobacterium marinum is a slowly growing, broad-host-range mycobacterial species. Here, we report the complete genome sequence of a Mycobacterium marinum type strain that was isolated from tubercles of diseased fish. This sequence will provide essential information for future taxonomic and comparative genome studies of its relatives. Copyright © 2018 Yoshida et al.

Assessing the impact of fungicide enostroburin application on bacterial community in wheat phyllosphere.

PubMed

Gu, Likun; Bai, Zhihui; Jin, Bo; Hu, Qing; Wang, Huili; Zhuang, Guoqiang; Zhang, Hongxun

2010-01-01

Fungicides have been used extensively for controlling fungal pathogens of plants. However, little is known regarding the effects that fungicides upon the indigenous bacterial communities within the plant phyllosphere. The aims of this study were to assess the impact of fungicide enostroburin upon bacterial communities in wheat phyllosphere. Culture-independent methodologies of 16S rDNA clone library and 16S rDNA directed polymerase chain reaction with denaturing gradient gel electrophoresis (PCR-DGGE) were used for monitoring the change of bacterial community. The 16S rDNA clone library and PCR-DGGE analysis both confirmed the microbial community of wheat plant phyllosphere were predominantly of the gamma-Proteobacteria phyla. Results from PCR-DGGE analysis indicated a significant change in bacterial community structure within the phyllosphere following fungicide enostroburin application. Bands sequenced within control cultures were predominantly of Pseudomonas genus, but those bands sequenced in the treated samples were predominantly strains of Pantoea genus and Pseudomonas genus. Of interest was the appearance of two DGGE bands following fungicide treatment, one of which had sequence similarities (98%) to Pantoea sp. which might be a competitor of plant pathogens. This study revealed the wheat phyllosphere bacterial community composition and a shift in the bacterial community following fungicide enostroburin application.

Diversity of Bacterial Communities in Container Habitats of Mosquitoes

PubMed Central

Ponnusamy, Loganathan; Xu, Ning; Stav, Gil; Wesson, Dawn M.; Schal, Coby

2010-01-01

We investigated the bacterial diversity of microbial communities in water-filled, human-made and natural container habitats of the mosquitoes Aedes aegypti and Aedes albopictus in suburban landscapes of New Orleans, Louisiana in 2003. We collected water samples from three classes of containers, including tires (n=12), cemetery urns (n=23), and miscellaneous containers that included two tree holes (n=19). Total genomic DNA was extracted from water samples, and 16S ribosomal DNA fragments (operational taxonomic units, OTUs) were amplified by PCR and separated by denaturing gradient gel electrophoresis (DGGE). The bacterial communities in containers represented diverse DGGE-DNA banding patterns that were not related to the class of container or to the local spatial distribution of containers. Mean richness and evenness of OTUs were highest in water samples from tires. Bacterial phylotypes were identified by comparative sequence analysis of 90 16S rDNA DGGE band amplicons. The majority of sequences were placed in five major taxa: Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and an unclassified group; Proteobacteria and Bacteroidetes were the predominant heterotrophic bacteria in containers. The bacterial communities in human-made containers consisted mainly of undescribed species, and a phylogenetic analysis based on 16S rRNA sequences suggested that species composition was independent of both container type and the spatial distribution of containers. Comparative PCR-based, cultivation-independent rRNA surveys of microbial communities associated with mosquito habitats can provide significant insight into community organization and dynamics of bacterial species. PMID:18373113

Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste

PubMed Central

Arregui, Lucía; Arroyo, Miguel; Mendoza, José Antonio; Álvarez, Cristina; García-Vera, Cristina; Marquina, Domingo; Santos, Antonio; Serrano, Susana

2018-01-01

Quorum sensing (QS) is a mechanism dependent on bacterial density. This coordinated process is mediated by the synthesis and the secretion of signal molecules, called autoinducers (AIs). N-acyl-homoserine lactones (AHLs) are the most common AIs that are used by Gram-negative bacteria and are involved in biofilm formation. Quorum Quenching (QQ) is the interference of QS by producing hydrolyzing enzymes, among other strategies. The main objective of the present study was to identify QS and QQ strains from MBR wastewater treatment plants. A total of 99 strains were isolated from two Spanish plants that were intended to treat leachate from municipal solid waste. Five AHL producers were detected using AHL biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1). Fifteen strains of seventy-one Gram-positive were capable of eliminating or reducing at least one AHL activity. The analysis of 16S rRNA gene sequence showed the importance of the Pseudomonas genus in the production of biofilms and the relevance of the genus Bacillus in the disruption of the QS mechanism, in which the potential activity of lactonase or acylase enzymes was investigated with the aim to contribute to solve biofouling problems and to increase the useful lifespan of membranes. PMID:29783658

Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste.

PubMed

Soler, Albert; Arregui, Lucía; Arroyo, Miguel; Mendoza, José Antonio; Muras, Andrea; Álvarez, Cristina; García-Vera, Cristina; Marquina, Domingo; Santos, Antonio; Serrano, Susana

2018-05-18

Quorum sensing (QS) is a mechanism dependent on bacterial density. This coordinated process is mediated by the synthesis and the secretion of signal molecules, called autoinducers (AIs). N -acyl-homoserine lactones (AHLs) are the most common AIs that are used by Gram-negative bacteria and are involved in biofilm formation. Quorum Quenching (QQ) is the interference of QS by producing hydrolyzing enzymes, among other strategies. The main objective of the present study was to identify QS and QQ strains from MBR wastewater treatment plants. A total of 99 strains were isolated from two Spanish plants that were intended to treat leachate from municipal solid waste. Five AHL producers were detected using AHL biosensor strains ( Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1). Fifteen strains of seventy-one Gram-positive were capable of eliminating or reducing at least one AHL activity. The analysis of 16S rRNA gene sequence showed the importance of the Pseudomonas genus in the production of biofilms and the relevance of the genus Bacillus in the disruption of the QS mechanism, in which the potential activity of lactonase or acylase enzymes was investigated with the aim to contribute to solve biofouling problems and to increase the useful lifespan of membranes.

Detection of Low-Copy-Number Genomic DNA Sequences in Individual Bacterial Cells by Using Peptide Nucleic Acid-Assisted Rolling-Circle Amplification and Fluorescence In Situ Hybridization▿ †

PubMed Central

Smolina, Irina; Lee, Charles; Frank-Kamenetskii, Maxim

2007-01-01

An approach is proposed for in situ detection of short signature DNA sequences present in single copies per bacterial genome. The site is locally opened by peptide nucleic acids, and a circular oligonucleotide is assembled. The amplicon generated by rolling circle amplification is detected by hybridization with fluorescently labeled decorator probes. PMID:17293504

A Bioinformatic Strategy for the Detection, Classification and Analysis of Bacterial Autotransporters

PubMed Central

Celik, Nermin; Webb, Chaille T.; Leyton, Denisse L.; Holt, Kathryn E.; Heinz, Eva; Gorrell, Rebecca; Kwok, Terry; Naderer, Thomas; Strugnell, Richard A.; Speed, Terence P.; Teasdale, Rohan D.; Likić, Vladimir A.; Lithgow, Trevor

2012-01-01

Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters. PMID:22905239

Isolation and identification of efficient Egyptian malathion-degrading bacterial isolates.

PubMed

Hamouda, S A; Marzouk, M A; Abbassy, M A; Abd-El-Haleem, D A; Shamseldin, Abdelaal

2015-03-01

Bacterial isolates degrading malathion were isolated from the soil and agricultural waste water due to their ability to grow on minimal salt media amended with malathion as a sole carbon source. Efficiencies of native Egyptian bacterial malathion-degrading isolates were investigated and the study generated nine highly effective malathion-degrading bacterial strains among 40. Strains were identified by partial sequencing of 16S rDNA analysis. Comparative analysis of 16S rDNA sequences revealed that these bacteria are similar with the genus Acinetobacter and Bacillus spp. and RFLP based PCR of 16S rDNA gave four different RFLP patterns among strains with enzyme HinfI while with enzyme HaeI they gave two RFLP profiles. The degradation rate of malathion in liquid culture was estimated using gas chromatography. Bacterial strains could degrade more than 90% of the initial malathion concentration (1000 ppm) within 4 days. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Neptune: a bioinformatics tool for rapid discovery of genomic variation in bacterial populations

PubMed Central

Marinier, Eric; Zaheer, Rahat; Berry, Chrystal; Weedmark, Kelly A.; Domaratzki, Michael; Mabon, Philip; Knox, Natalie C.; Reimer, Aleisha R.; Graham, Morag R.; Chui, Linda; Patterson-Fortin, Laura; Zhang, Jian; Pagotto, Franco; Farber, Jeff; Mahony, Jim; Seyer, Karine; Bekal, Sadjia; Tremblay, Cécile; Isaac-Renton, Judy; Prystajecky, Natalie; Chen, Jessica; Slade, Peter

2017-01-01

Abstract The ready availability of vast amounts of genomic sequence data has created the need to rethink comparative genomics algorithms using ‘big data’ approaches. Neptune is an efficient system for rapidly locating differentially abundant genomic content in bacterial populations using an exact k-mer matching strategy, while accommodating k-mer mismatches. Neptune’s loci discovery process identifies sequences that are sufficiently common to a group of target sequences and sufficiently absent from non-targets using probabilistic models. Neptune uses parallel computing to efficiently identify and extract these loci from draft genome assemblies without requiring multiple sequence alignments or other computationally expensive comparative sequence analyses. Tests on simulated and real datasets showed that Neptune rapidly identifies regions that are both sensitive and specific. We demonstrate that this system can identify trait-specific loci from different bacterial lineages. Neptune is broadly applicable for comparative bacterial analyses, yet will particularly benefit pathogenomic applications, owing to efficient and sensitive discovery of differentially abundant genomic loci. The software is available for download at: http://github.com/phac-nml/neptune. PMID:29048594

Analysis of Bacterial Community Composition of Corroded Steel Immersed in Sanya and Xiamen Seawaters in China via Method of Illumina MiSeq Sequencing

PubMed Central

Li, Xiaohong; Duan, Jizhou; Xiao, Hui; Li, Yongqian; Liu, Haixia; Guan, Fang; Zhai, Xiaofan

2017-01-01

Metal corrosion is of worldwide concern because it is the cause of major economic losses, and because it creates significant safety issues. The mechanism of the corrosion process, as influenced by bacteria, has been studied extensively. However, the bacterial communities that create the biofilms that form on metals are complicated, and have not been well studied. This is why we sought to analyze the composition of bacterial communities living on steel structures, together with the influence of ecological factors on these communities. The corrosion samples were collected from rust layers on steel plates that were immersed in seawater for two different periods at Sanya and Xiamen, China. We analyzed the bacterial communities on the samples by targeted 16S rRNA gene (V3–V4 region) sequencing using the Illumina MiSeq. Phylogenetic analysis revealed that the bacteria fell into 13 phylotypes (similarity level = 97%). Proteobacteria, Firmicutes and Bacteroidetes were the dominant phyla, accounting for 88.84% of the total. Deltaproteobacteria, Clostridia and Gammaproteobacteria were the dominant classes, and accounted for 70.90% of the total. Desulfovibrio spp., Desulfobacter spp. and Desulfotomaculum spp. were the dominant genera and accounted for 45.87% of the total. These genera are sulfate-reducing bacteria that are known to corrode steel. Bacterial diversity on the 6 months immersion samples was much higher than that of the samples that had been immersed for 8 years (P < 0.001, Student’s t-test). The average complexity of the biofilms from the 8-years immersion samples from Sanya was greater than those from Xiamen, but not significantly so (P > 0.05, Student’s t-test). Overall, the data showed that the rust layers on the steel plates carried many bacterial species. The bacterial community composition was influenced by the immersion time. The results of our study will be of benefit to the further studies of bacterial corrosion mechanisms and corrosion resistance

Characterization of the indigenous microflora in raw and pasteurized buffalo milk during storage at refrigeration temperature by high-throughput sequencing

USDA-ARS?s Scientific Manuscript database

The effect of refrigeration on bacterial communities within raw and pasteurized buffalo milk was studied using high-throughput sequencing. High quality samples of raw buffalo milk were obtained from five dairy farms in the Guangxi province of China. A sample of each milk was pasteurized, and both r...

BAC-end sequence-based SNP mining in Allotetraploid Cotton (Gossypium) utilizing re-sequencing data, phylogenetic inferences and perspectives for genetic mapping

USDA-ARS?s Scientific Manuscript database

A bacterial artificial chromosome (BAC) library and BAC-end sequences for Gossypium hirsutum L. have recently been developed. Here we report on genomic-based genome-wide SNP mining utilizing re-sequencing data with a BAC-end sequence reference for twelve G. hirsutum L. lines, one G. barbadense L. li...

Insertion Sequences

PubMed Central

Mahillon, Jacques; Chandler, Michael

1998-01-01

Insertion sequences (ISs) constitute an important component of most bacterial genomes. Over 500 individual ISs have been described in the literature to date, and many more are being discovered in the ongoing prokaryotic and eukaryotic genome-sequencing projects. The last 10 years have also seen some striking advances in our understanding of the transposition process itself. Not least of these has been the development of various in vitro transposition systems for both prokaryotic and eukaryotic elements and, for several of these, a detailed understanding of the transposition process at the chemical level. This review presents a general overview of the organization and function of insertion sequences of eubacterial, archaebacterial, and eukaryotic origins with particular emphasis on bacterial elements and on different aspects of the transposition mechanism. It also attempts to provide a framework for classification of these elements by assigning them to various families or groups. A total of 443 members of the collection have been grouped in 17 families based on combinations of the following criteria: (i) similarities in genetic organization (arrangement of open reading frames); (ii) marked identities or similarities in the enzymes which mediate the transposition reactions, the recombinases/transposases (Tpases); (iii) similar features of their ends (terminal IRs); and (iv) fate of the nucleotide sequence of their target sites (generation of a direct target duplication of determined length). A brief description of the mechanism(s) involved in the mobility of individual ISs in each family and of the structure-function relationships of the individual Tpases is included where available. PMID:9729608

Salivary bacterial fingerprints of established oral disease revealed by the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) technique

PubMed Central

Belstrøm, Daniel; Paster, Bruce J.; Fiehn, Nils-Erik; Bardow, Allan; Holmstrup, Palle

2016-01-01

Background and objective The composition of the salivary microbiota, as determined using various molecular methods, has been reported to differentiate oral health from diseases. Thus, the purpose of this study was to utilize the newly developed molecular technique HOMINGS (Human Oral Microbe Identification using Next Generation Sequencing) for comparison of the salivary microbiota in patients with periodontitis, patients with dental caries, and orally healthy individuals. The hypothesis was that this method could add on to the existing knowledge on salivary bacterial profiles in oral health and disease. Design Stimulated saliva samples (n=30) were collected from 10 patients with untreated periodontitis, 10 patients with untreated dental caries, and 10 orally healthy individuals. Salivary microbiota was analyzed using HOMINGS and statistical analysis was performed using Kruskal–Wallis test with Benjamini–Hochberg's correction. Results From a total of 30 saliva samples, a mean number of probe targets of 205 (range 120–353) were identified, and a statistically significant higher mean number of targets was registered in samples from patients with periodontitis (mean 220, range 143–306) and dental caries (mean 221, range 165–353) as compared to orally healthy individuals (mean 174, range 120–260) (p=0.04 and p=0.04). Nine probe targets were identified with a different relative abundance between groups (p<0.05). Conclusions Cross-sectional comparison of salivary bacterial profiles by means of HOMINGS analysis showed that different salivary bacterial profiles were associated with oral health and disease. Future large-scale prospective studies are needed to evaluate if saliva-based screening for disease-associated oral bacterial profiles may be used for identification of patients at risk of acquiring periodontitis and dental caries. PMID:26782357

dBBQs: dataBase of Bacterial Quality scores.

PubMed

Wanchai, Visanu; Patumcharoenpol, Preecha; Nookaew, Intawat; Ussery, David

2017-12-28

It is well-known that genome sequencing technologies are becoming significantly cheaper and faster. As a result of this, the exponential growth in sequencing data in public databases allows us to explore ever growing large collections of genome sequences. However, it is less known that the majority of available sequenced genome sequences in public databases are not complete, drafts of varying qualities. We have calculated quality scores for around 100,000 bacterial genomes from all major genome repositories and put them in a fast and easy-to-use database. Prokaryotic genomic data from all sources were collected and combined to make a non-redundant set of bacterial genomes. The genome quality score for each was calculated by four different measurements: assembly quality, number of rRNA and tRNA genes, and the occurrence of conserved functional domains. The dataBase of Bacterial Quality scores (dBBQs) was designed to store and retrieve quality scores. It offers fast searching and download features which the result can be used for further analysis. In addition, the search results are shown in interactive JavaScript chart framework using DC.js. The analysis of quality scores across major public genome databases find that around 68% of the genomes are of acceptable quality for many uses. dBBQs (available at http://arc-gem.uams.edu/dbbqs ) provides genome quality scores for all available prokaryotic genome sequences with a user-friendly Web-interface. These scores can be used as cut-offs to get a high-quality set of genomes for testing bioinformatics tools or improving the analysis. Moreover, all data of the four measurements that were combined to make the quality score for each genome, which can potentially be used for further analysis. dBBQs will be updated regularly and is freely use for non-commercial purpose.

A Novel Lactobacilli-Based Teat Disinfectant for Improving Bacterial Communities in the Milks of Cow Teats with Subclinical Mastitis

PubMed Central

Yu, Jie; Ren, Yan; Xi, XiaoXia; Huang, Weiqiang; Zhang, Heping

2017-01-01

Teat disinfection pre- and post-milking is important for the overall health and hygiene of dairy cows. The objective of this study was to evaluate the efficacy of a novel probiotic lactobacilli-based teat disinfectant based on changes in somatic cell count (SCC) and profiling of the bacterial community. A total of 69 raw milk samples were obtained from eleven Holstein-Friesian dairy cows over 12 days of teat dipping in China. Single molecule, real-time sequencing technology (SMRT) was employed to profile changes in the bacterial community during the cleaning protocol and to compare the efficacy of probiotic lactic acid bacteria (LAB) and commercial teat disinfectants. The SCC gradually decreased following the cleaning protocol and the SCC of the LAB group was slightly lower than that of the commercial disinfectant (CD) group. Our SMRT sequencing results indicate that raw milk from both the LAB and CD groups contained diverse microbial populations that changed over the course of the cleaning protocol. The relative abundances of some species were significantly changed during the cleaning process, which may explain the observed bacterial community differences. Collectively, these results suggest that the LAB disinfectant could reduce mastitis-associated bacteria and improve the microbial environment of the cow teat. It could be used as an alternative to chemical pre- and post-milking teat disinfectants to maintain healthy teats and udders. In addition, the Pacific Biosciences SMRT sequencing with the full-length 16S ribosomal RNA gene was shown to be a powerful tool for monitoring changes in the bacterial population during the cleaning protocol. PMID:29018412

Assessing the impact of water treatment on bacterial biofilms in drinking water distribution systems using high-throughput DNA sequencing.

PubMed

Shaw, Jennifer L A; Monis, Paul; Fabris, Rolando; Ho, Lionel; Braun, Kalan; Drikas, Mary; Cooper, Alan

2014-12-01

Biofilm control in drinking water distribution systems (DWDSs) is crucial, as biofilms are known to reduce flow efficiency, impair taste and quality of drinking water and have been implicated in the transmission of harmful pathogens. Microorganisms within biofilm communities are more resistant to disinfection compared to planktonic microorganisms, making them difficult to manage in DWDSs. This study evaluates the impact of four unique drinking water treatments on biofilm community structure using metagenomic DNA sequencing. Four experimental DWDSs were subjected to the following treatments: (1) conventional coagulation, (2) magnetic ion exchange contact (MIEX) plus conventional coagulation, (3) MIEX plus conventional coagulation plus granular activated carbon, and (4) membrane filtration (MF). Bacterial biofilms located inside the pipes of each system were sampled under sterile conditions both (a) immediately after treatment application ('inlet') and (b) at a 1 km distance from the treatment application ('outlet'). Bacterial 16S rRNA gene sequencing revealed that the outlet biofilms were more diverse than those sampled at the inlet for all treatments. The lowest number of unique operational taxonomic units (OTUs) and lowest diversity was observed in the MF inlet. However, the MF system revealed the greatest increase in diversity and OTU count from inlet to outlet. Further, the biofilm communities at the outlet of each system were more similar to one another than to their respective inlet, suggesting that biofilm communities converge towards a common established equilibrium as distance from treatment application increases. Based on the results, MF treatment is most effective at inhibiting biofilm growth, but a highly efficient post-treatment disinfection regime is also critical in order to prevent the high rates of post-treatment regrowth. Copyright © 2014 Elsevier Ltd. All rights reserved.

Exploiting Bacterial Whole-Genome Sequencing Data for Evaluation of Diagnostic Assays: Campylobacter Species Identification as a Case Study.

PubMed

Jansen van Rensburg, Melissa J; Swift, Craig; Cody, Alison J; Jenkins, Claire; Maiden, Martin C J

2016-12-01

The application of whole-genome sequencing (WGS) to problems in clinical microbiology has had a major impact on the field. Clinical laboratories are now using WGS for pathogen identification, antimicrobial susceptibility testing, and epidemiological typing. WGS data also represent a valuable resource for the development and evaluation of molecular diagnostic assays, which continue to play an important role in clinical microbiology. To demonstrate this application of WGS, this study used publicly available genomic data to evaluate a duplex real-time PCR (RT-PCR) assay that targets mapA and ceuE for the detection of Campylobacter jejuni and Campylobacter coli, leading global causes of bacterial gastroenteritis. In silico analyses of mapA and ceuE primer and probe sequences from 1,713 genetically diverse C. jejuni and C. coli genomes, supported by RT-PCR testing, indicated that the assay was robust, with 1,707 (99.7%) isolates correctly identified. The high specificity of the mapA-ceuE assay was the result of interspecies diversity and intraspecies conservation of the target genes in C. jejuni and C. coli Rare instances of a lack of specificity among C. coli isolates were due to introgression in mapA or sequence diversity in ceuE The results of this study illustrate how WGS can be exploited to evaluate molecular diagnostic assays by using publicly available data, online databases, and open-source software. Copyright © 2016 Jansen van Rensburg et al.

Bacterial diversity in faeces from polar bear (Ursus maritimus) in Arctic Svalbard.

PubMed

Glad, Trine; Bernhardsen, Pål; Nielsen, Kaare M; Brusetti, Lorenzo; Andersen, Magnus; Aars, Jon; Sundset, Monica A

2010-01-14

Polar bears (Ursus maritimus) are major predators in the Arctic marine ecosystem, feeding mainly on seals, and living closely associated with sea ice. Little is known of their gut microbial ecology and the main purpose of this study was to investigate the microbial diversity in faeces of polar bears in Svalbard, Norway (74-81 degrees N, 10-33 degrees E). In addition the level of blaTEM alleles, encoding ampicillin resistance (ampr) were determined. In total, ten samples were collected from ten individual bears, rectum swabs from five individuals in 2004 and faeces samples from five individuals in 2006. A 16S rRNA gene clone library was constructed, and all sequences obtained from 161 clones showed affiliation with the phylum Firmicutes, with 160 sequences identified as Clostridiales and one sequence identified as unclassified Firmicutes. The majority of the sequences (70%) were affiliated with the genus Clostridium. Aerobic heterotrophic cell counts on chocolate agar ranged between 5.0 x 10(4) to 1.6 x 10(6) colony forming units (cfu)/ml for the rectum swabs and 4.0 x 10(3) to 1.0 x 10(5) cfu/g for the faeces samples. The proportion of ampr bacteria ranged from 0% to 44%. All of 144 randomly selected ampr isolates tested positive for enzymatic beta-lactamase activity. Three % of the ampr isolates from the rectal samples yielded positive results when screened for the presence of blaTEM genes by PCR. BlaTEM alleles were also detected by PCR in two out of three total faecal DNA samples from polar bears. The bacterial diversity in faeces from polar bears in their natural environment in Svalbard is low compared to other animal species, with all obtained clones affiliating to Firmicutes. Furthermore, only low levels of blaTEM alleles were detected in contrast to their increasing prevalence in some clinical and commensal bacterial populations.

Bacterial diversity in faeces from polar bear (Ursus maritimus) in Arctic Svalbard

PubMed Central

2010-01-01

Background Polar bears (Ursus maritimus) are major predators in the Arctic marine ecosystem, feeding mainly on seals, and living closely associated with sea ice. Little is known of their gut microbial ecology and the main purpose of this study was to investigate the microbial diversity in faeces of polar bears in Svalbard, Norway (74-81°N, 10-33°E). In addition the level of blaTEM alleles, encoding ampicillin resistance (ampr) were determined. In total, ten samples were collected from ten individual bears, rectum swabs from five individuals in 2004 and faeces samples from five individuals in 2006. Results A 16S rRNA gene clone library was constructed, and all sequences obtained from 161 clones showed affiliation with the phylum Firmicutes, with 160 sequences identified as Clostridiales and one sequence identified as unclassified Firmicutes. The majority of the sequences (70%) were affiliated with the genus Clostridium. Aerobic heterotrophic cell counts on chocolate agar ranged between 5.0 × 104 to 1.6 × 106 colony forming units (cfu)/ml for the rectum swabs and 4.0 × 103 to 1.0 × 105 cfu/g for the faeces samples. The proportion of ampr bacteria ranged from 0% to 44%. All of 144 randomly selected ampr isolates tested positive for enzymatic β-lactamase activity. Three % of the ampr isolates from the rectal samples yielded positive results when screened for the presence of blaTEM genes by PCR. BlaTEM alleles were also detected by PCR in two out of three total faecal DNA samples from polar bears. Conclusion The bacterial diversity in faeces from polar bears in their natural environment in Svalbard is low compared to other animal species, with all obtained clones affiliating to Firmicutes. Furthermore, only low levels of blaTEM alleles were detected in contrast to their increasing prevalence in some clinical and commensal bacterial populations. PMID:20074323

Kullback Leibler divergence in complete bacterial and phage genomes

PubMed Central

Akhter, Sajia; Kashef, Mona T.; Ibrahim, Eslam S.; Bailey, Barbara

2017-01-01

The amino acid content of the proteins encoded by a genome may predict the coding potential of that genome and may reflect lifestyle restrictions of the organism. Here, we calculated the Kullback–Leibler divergence from the mean amino acid content as a metric to compare the amino acid composition for a large set of bacterial and phage genome sequences. Using these data, we demonstrate that (i) there is a significant difference between amino acid utilization in different phylogenetic groups of bacteria and phages; (ii) many of the bacteria with the most skewed amino acid utilization profiles, or the bacteria that host phages with the most skewed profiles, are endosymbionts or parasites; (iii) the skews in the distribution are not restricted to certain metabolic processes but are common across all bacterial genomic subsystems; (iv) amino acid utilization profiles strongly correlate with GC content in bacterial genomes but very weakly correlate with the G+C percent in phage genomes. These findings might be exploited to distinguish coding from non-coding sequences in large data sets, such as metagenomic sequence libraries, to help in prioritizing subsequent analyses. PMID:29204318

Kullback Leibler divergence in complete bacterial and phage genomes.

PubMed

Akhter, Sajia; Aziz, Ramy K; Kashef, Mona T; Ibrahim, Eslam S; Bailey, Barbara; Edwards, Robert A

2017-01-01

The amino acid content of the proteins encoded by a genome may predict the coding potential of that genome and may reflect lifestyle restrictions of the organism. Here, we calculated the Kullback-Leibler divergence from the mean amino acid content as a metric to compare the amino acid composition for a large set of bacterial and phage genome sequences. Using these data, we demonstrate that (i) there is a significant difference between amino acid utilization in different phylogenetic groups of bacteria and phages; (ii) many of the bacteria with the most skewed amino acid utilization profiles, or the bacteria that host phages with the most skewed profiles, are endosymbionts or parasites; (iii) the skews in the distribution are not restricted to certain metabolic processes but are common across all bacterial genomic subsystems; (iv) amino acid utilization profiles strongly correlate with GC content in bacterial genomes but very weakly correlate with the G+C percent in phage genomes. These findings might be exploited to distinguish coding from non-coding sequences in large data sets, such as metagenomic sequence libraries, to help in prioritizing subsequent analyses.

Bacterial mycophagy: definition and diagnosis of a unique bacterial-fungal interaction.

PubMed

Leveau, Johan H J; Preston, Gail M

2008-01-01

This review analyses the phenomenon of bacterial mycophagy, which we define as a set of phenotypic behaviours that enable bacteria to obtain nutrients from living fungi and thus allow the conversion of fungal into bacterial biomass. We recognize three types of bacterial strategies to derive nutrition from fungi: necrotrophy, extracellular biotrophy and endocellular biotrophy. Each is characterized by a set of uniquely sequential and differently overlapping interactions with the fungal target. We offer a detailed analysis of the nature of these interactions, as well as a comprehensive overview of methodologies for assessing and quantifying their individual contributions to the mycophagy phenotype. Furthermore, we discuss future prospects for the study and exploitation of bacterial mycophagy, including the need for appropriate tools to detect bacterial mycophagy in situ in order to be able to understand, predict and possibly manipulate the way in which mycophagous bacteria affect fungal activity, turnover, and community structure in soils and other ecosystems.

Histophagous ciliate Pseudocollinia brintoni and bacterial assemblage interaction with krill Nyctiphanes simplex. I. Transmission process.

PubMed

Gómez-Gutiérrez, Jaime; López-Cortés, Alejandro; Aguilar-Méndez, Mario J; Del Angel-Rodríguez, Jorge A; Tremblay, Nelly; Zenteno-Savín, Tania; Robinson, Carlos J

2015-10-27

Histophagous ciliates of the genus Pseudocollinia cause epizootic events that kill adult female krill (Euphausiacea), but their mode of transmission is unknown. We compared 16S rRNA sequences of bacterial strains isolated from stomachs of healthy krill Nyctiphanes simplex specimens with sequences of bacterial isolates and sequences of natural bacterial communities from the hemocoel of N. simplex specimens infected with P. brintoni to determine possible transmission pathways. All P. brintoni endoparasitic life stages and the transmission tomite stage (outside the host) were associated with bacterial assemblages. 16S rRNA sequences from isolated bacterial strains showed that Photobacterium spp. and Pseudoalteromonas spp. were dominant members of the bacterial assemblages during all life phases of P. brintoni and potential pathobionts. They were apparently unaffected by the krill's immune system or the histophagous activity of P. brintoni. However, other bacterial strains were found only in certain P. brintoni life phases, indicating that as the infection progressed, microhabitat conditions and microbial interactions may have become unfavorable for some strains of bacteria. Trophic infection is the most parsimonious explanation for how P. brintoni infects krill. We estimated N. simplex vulnerability to P. brintoni infection during more than three-fourths of their life span, infecting mostly adult females. The ciliates have relatively high prevalence levels (albeit at <10% of sampled stations) and a short life cycle (estimated <7 d). Histophagous ciliate-krill interactions may occur in other krill species, particularly those that form dense swarms and attain high population densities that potentially enhance trophic transmission and allow completion of the Pseudocollinia spp. life cycle.

Bacterial inosine 5'-monophosphate dehydrogenase ("IMPDH") DNA as a dominant selectable marker in mammals and other eukaryotes

DOEpatents

Huberman, Eliezer [Chicago, IL; Baccam, Mekhine J [Woodridge, IL

2007-02-27

The present invention relates to a nucleic acid sequence and its corresponding protein sequence useful as a dominant selectable marker in eukaryotes. More specifically the invention relates to a nucleic acid encoding a bacterial IMPDH gene that has been engineered into a eukaryotic expression vectors, thereby permitting bacterial IMPDH expression in mammalian cells. Bacterial IMPDH expression confers resistance to MPA which can be used as dominant selectable marker in eukaryotes including mammals. The invention also relates to expression vectors and cells that express the bacterial IMPDH gene as well as gene therapies and protein synthesis.

Soil bacterial community responses to revegetation of moving sand dune in semi-arid grassland.

PubMed

Cao, Chengyou; Zhang, Ying; Cui, Zhenbo; Feng, Shuwei; Wang, Tingting; Ren, Qing

2017-08-01

Grasslands in semi-arid Northern China are widely desertified, thus inducing the formation of a large area of moving sand lands. Revegetation of the sandy land is commonly adopted to restore degraded grasslands. The structure of the soil microbial community might dramatically change during degradation and recovery because microorganisms are one of the major drivers of ecological process through their interactions with plants and soil. Assuming that soil properties are the key determinants of the structure of soil bacterial community within the same soil type, whether the vegetation type causes the significant difference in the structure of soil bacterial community during revegetation and restoration of the degraded grasslands remains poorly understood. Our study aimed to (1) investigate the response of soil bacterial communities to the changes during vegetation degradation and recovery and (2) evaluate whether the soil bacterial communities under plantations return to their native state. We detected the shifts in diversities and compositions of the soil bacterial communities and the relative abundance of dominant bacterial taxa by using the high-throughput Illumina MiSeq sequencing technique in an area covered by 32-year-old Caragana microphylla, Artemisia halodendron, Hedysarum fruticosum, Pinus sylvestris var. mongolica, Populus simonii, and Salix gordejevii sand-fixing plantations and in the native community (NC) dominated by elm, and moving sandy dune (MS). We found that the obtained operational taxonomic units by 16S rRNA gene sequencing and diversity index in MS were all significantly lower than those in NC, and the number and composition of dominant genera were significantly different between NC and MS. Interestingly, the compositions of bacterial communities and the dominant genera in different sand-fixation plantations (C. microphylla, A. halodendron, H. fruticosum, P. sylvestris var. mongolica, P. simonii, and S. gordejevii) were all similar to those of

Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring.

PubMed

Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Joshi, Amit; Souza, Leila; Almeida, Paulo; Chauhan, Ashvini

2015-09-01

The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche.

Investigation of In vitro Mineral forming bacterial isolates from supragingival calculus.

PubMed

Baris, O; Demir, T; Gulluce, M

2017-12-01

Although it is known that bacterial mechanisms are involved in dental calculus formation, which is a predisposing factor in periodontal diseases, there have been few studies of such associations, and therefore, information available is limited. The purpose of this study was to isolate and identify aerobic bacteria responsible for direct calcification from supragingival calculus samples. The study was conducted using supragingival calculus samples from patients with periodontal disease, which was required as part of conventional treatment. Isolations were performed by sampling the supragingival calculus with buffer and inoculating the samples on media on which crystallization could be observed. The 16S recombinant DNA of the obtained pure cultures was then amplified and sequenced. A few bacterial species that have not previously been associated with mineralization or identified on bacterial plaque or calculus were detected. The bacteria that caused mineralization an aerobic environment are identified as Neisseria flava, Aggregatibacter segnis, Streptococcus tigurinus, and Morococcus cerebrosus. These findings proved that bacteria potentially play a role in the etiopathology of supragingival calculus. The association between the effects of the identified bacteria on periodontal diseases and calculus formation requires further studies.

The construction of recombinant industrial yeasts free of bacterial sequences by directed gene replacement into a nonessential region of the genome.

PubMed

Xiao, W; Rank, G H

1989-03-15

The yeast SMR1 gene was used as a dominant resistance-selectable marker for industrial yeast transformation and for targeting integration of an economically important gene at the homologous ILV2 locus. A MEL1 gene, which codes for alpha-galactosidase, was inserted into a dispensable upstream region of SMR1 in vitro; different treatments of the plasmid (pWX813) prior to transformation resulted in 3' end, 5' end and replacement integrations that exhibited distinct integrant structures. One-step replacement within a nonessential region of the host genome generated a stable integration of MEL1 devoid of bacterial plasmid DNA. Using this method, we have constructed several alpha-galactosidase positive industrial Saccharomyces strains. Our study provides a general method for stable gene transfer in most industrial Saccharomyces yeasts, including those used in the baking, brewing (ale and lager), distilling, wine and sake industries, with solely nucleotide sequences of interest. The absence of bacterial DNA in the integrant structure facilitates the commercial application of recombinant DNA technology in the food and beverage industry.

tRNADB-CE: tRNA gene database well-timed in the era of big sequence data.

PubMed

Abe, Takashi; Inokuchi, Hachiro; Yamada, Yuko; Muto, Akira; Iwasaki, Yuki; Ikemura, Toshimichi

2014-01-01

The tRNA gene data base curated by experts "tRNADB-CE" (http://trna.ie.niigata-u.ac.jp) was constructed by analyzing 1,966 complete and 5,272 draft genomes of prokaryotes, 171 viruses', 121 chloroplasts', and 12 eukaryotes' genomes plus fragment sequences obtained by metagenome studies of environmental samples. 595,115 tRNA genes in total, and thus two times of genes compiled previously, have been registered, for which sequence, clover-leaf structure, and results of sequence-similarity and oligonucleotide-pattern searches can be browsed. To provide collective knowledge with help from experts in tRNA researches, we added a column for enregistering comments to each tRNA. By grouping bacterial tRNAs with an identical sequence, we have found high phylogenetic preservation of tRNA sequences, especially at the phylum level. Since many species-unknown tRNAs from metagenomic sequences have sequences identical to those found in species-known prokaryotes, the identical sequence group (ISG) can provide phylogenetic markers to investigate the microbial community in an environmental ecosystem. This strategy can be applied to a huge amount of short sequences obtained from next-generation sequencers, as showing that tRNADB-CE is a well-timed database in the era of big sequence data. It is also discussed that batch-learning self-organizing-map with oligonucleotide composition is useful for efficient knowledge discovery from big sequence data.

Molecular comparison of the sampling efficiency of four types of airborne bacterial samplers.

PubMed

Li, Kejun

2011-11-15

In the present study, indoor and outdoor air samples were collected using four types of air samplers often used for airborne bacterial sampling. These air samplers included two solid impactors (BioStage and RCS), one liquid impinger (BioSampler), and one filter sampler with two kinds of filters (a gelatin and a cellulose acetate filter). The collected air samples were further processed to analyze the diversity and abundance of culturable bacteria and total bacteria through standard culture techniques, denaturing gradient gel electrophoresis (DGGE) fingerprinting and quantitative polymerase chain reaction (qPCR) analysis. The DGGE analysis indicated that the air samples collected using the BioStage and RCS samplers have higher culturable bacterial diversity, whereas the samples collected using the BioSampler and the cellulose acetate filter sampler have higher total bacterial diversity. To obtain more information on the sampled bacteria, some gel bands were excised and sequenced. In terms of sampling efficiency, results from the qPCR tests indicated that the collected total bacterial concentration was higher in samples collected using the BioSampler and the cellulose acetate filter sampler. In conclusion, the sampling bias and efficiency of four kinds of air sampling systems were compared in the present study and the two solid impactors were concluded to be comparatively efficient for culturable bacterial sampling, whereas the liquid impactor and the cellulose acetate filter sampler were efficient for total bacterial sampling. Copyright © 2011 Elsevier B.V. All rights reserved.

Bacterial Communities in Women with Bacterial Vaginosis: High Resolution Phylogenetic Analyses Reveal Relationships of Microbiota to Clinical Criteria

PubMed Central

Srinivasan, Sujatha; Hoffman, Noah G.; Morgan, Martin T.; Matsen, Frederick A.; Fiedler, Tina L.; Hall, Robert W.; Ross, Frederick J.; McCoy, Connor O.; Bumgarner, Roger; Marrazzo, Jeanne M.; Fredricks, David N.

2012-01-01

Background Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We investigated the associations between the presence of individual bacterial species and clinical diagnostic characteristics of BV. Methodology/Principal Findings Broad-range 16S rRNA gene PCR and pyrosequencing were performed on vaginal swabs from 220 women with and without BV. BV was assessed by Amsel’s clinical criteria and confirmed by Gram stain. Taxonomic classification was performed using phylogenetic placement tools that assigned 99% of query sequence reads to the species level. Women with BV had heterogeneous vaginal bacterial communities that were usually not dominated by a single taxon. In the absence of BV, vaginal bacterial communities were dominated by either Lactobacillus crispatus or Lactobacillus iners. Leptotrichia amnionii and Eggerthella sp. were the only two BV-associated bacteria (BVABs) significantly associated with each of the four Amsel’s criteria. Co-occurrence analysis revealed the presence of several sub-groups of BVABs suggesting metabolic co-dependencies. Greater abundance of several BVABs was observed in Black women without BV. Conclusions/Significance The human vaginal bacterial biota is heterogeneous and marked by greater species richness and diversity in women with BV; no species is universally present. Different bacterial species have different associations with the four clinical criteria, which may account for discrepancies often observed between Amsel and Nugent (Gram stain) diagnostic criteria. Several BVABs exhibited race-dependent prevalence when analyzed in separate groups by BV status which may contribute to increased incidence of BV in

Antarctic ice core samples: culturable bacterial diversity.

PubMed

Shivaji, Sisinthy; Begum, Zareena; Shiva Nageswara Rao, Singireesu Soma; Vishnu Vardhan Reddy, Puram V; Manasa, Poorna; Sailaja, Buddi; Prathiba, Mambatta S; Thamban, Meloth; Krishnan, Kottekkatu P; Singh, Shiv M; Srinivas, Tanuku N R

2013-01-01

Culturable bacterial abundance at 11 different depths of a 50.26 m ice core from the Tallaksenvarden Nunatak, Antarctica, varied from 0.02 to 5.8 × 10(3) CFU ml(-1) of the melt water. A total of 138 bacterial strains were recovered from the 11 different depths of the ice core. Based on 16S rRNA gene sequence analyses, the 138 isolates could be categorized into 25 phylotypes belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. All isolates had 16S rRNA sequences similar to previously determined sequences (97.2-100%). No correlation was observed in the distribution of the isolates at the various depths either at the phylum, genus or species level. The 25 phylotypes varied in growth temperature range, tolerance to NaCl, growth pH range and ability to produce eight different extracellular enzymes at either 4 or 18 °C. Iso-, anteiso-, unsaturated and saturated fatty acids together constituted a significant proportion of the total fatty acid composition. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Genetic Diversity of Bacterial Communities and Gene Transfer Agents in Northern South China Sea

PubMed Central

Sun, Fu-Lin; Wang, You-Shao; Wu, Mei-Lin; Jiang, Zhao-Yu; Sun, Cui-Ci; Cheng, Hao

2014-01-01

Pyrosequencing of the 16S ribosomal RNA gene (rDNA) amplicons was performed to investigate the unique distribution of bacterial communities in northern South China Sea (nSCS) and evaluate community structure and spatial differences of bacterial diversity. Cyanobacteria, Proteobacteria, Actinobacteria, and Bacteroidetes constitute the majority of bacteria. The taxonomic description of bacterial communities revealed that more Chroococcales, SAR11 clade, Acidimicrobiales, Rhodobacterales, and Flavobacteriales are present in the nSCS waters than other bacterial groups. Rhodobacterales were less abundant in tropical water (nSCS) than in temperate and cold waters. Furthermore, the diversity of Rhodobacterales based on the gene transfer agent (GTA) major capsid gene (g5) was investigated. Four g5 gene clone libraries were constructed from samples representing different regions and yielded diverse sequences. Fourteen g5 clusters could be identified among 197 nSCS clones. These clusters were also related to known g5 sequences derived from genome-sequenced Rhodobacterales. The composition of g5 sequences in surface water varied with the g5 sequences in the sampling sites; this result indicated that the Rhodobacterales population could be highly diverse in nSCS. Phylogenetic tree analysis result indicated distinguishable diversity patterns among tropical (nSCS), temperate, and cold waters, thereby supporting the niche adaptation of specific Rhodobacterales members in unique environments. PMID:25364820

Metagenomic analysis of bacterial and archaeal assemblages in the soil-mousse surrounding a geothermal spring

PubMed Central

Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Joshi, Amit; Souza, Leila; Almeida, Paulo; Chauhan, Ashvini

2015-01-01

The soil-mousse surrounding a geothermal spring was analyzed for bacterial and archaeal diversity using 16S rRNA gene amplicon metagenomic sequencing which revealed the presence of 18 bacterial phyla distributed across 109 families and 219 genera. Firmicutes, Actinobacteria, and the Deinococcus-Thermus group were the predominant bacterial assemblages with Crenarchaeota and Thaumarchaeota as the main archaeal assemblages in this largely understudied geothermal habitat. Several metagenome sequences remained taxonomically unassigned suggesting the presence of a repertoire of hitherto undescribed microbes in this geothermal soil-mousse econiche. PMID:26484255

Identifying the core seed bank of a complex boreal bacterial metacommunity.

PubMed

Ruiz-González, Clara; Niño-García, Juan Pablo; Kembel, Steven W; Del Giorgio, Paul A

2017-09-01

Seed banks are believed to contribute to compositional changes within and across microbial assemblages, but the application of this concept to natural communities remains challenging. Here we describe the core seed bank of a bacterial metacommunity from a boreal watershed, using the spatial distribution of bacterial operational taxonomic units (OTUs) across 223 heterogeneous terrestrial, aquatic and phyllosphere bacterial assemblages. Taxa were considered potential seeds if they transitioned from rare to abundant somewhere within the metacommunity and if they were ubiquitous and able to persist under unfavorable conditions, the latter assessed by checking their presence in three deeply sequenced samples (one soil, one river and one lake, 2.2-3 million reads per sample). We show that only a small fraction (13%) of all detected OTUs constitute a metacommunity seed bank that is shared between all terrestrial and aquatic communities, but not by phyllosphere assemblages, which seem to recruit from a different taxa pool. Our results suggest directional recruitment driven by the flow of water in the landscape, since most aquatic sequences were associated to OTUs found in a single deeply-sequenced soil sample, but only 45% of terrestrial sequences belonged to OTUs found in the two deeply-sequenced aquatic communities. Finally, we hypothesize that extreme rarity, and its interplay with water residence time and growth rates, may further constrain the size of the potential seed bank.

Bacterial membrane proteomics.

PubMed

Poetsch, Ansgar; Wolters, Dirk

2008-10-01

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

Characterization of the faecal bacterial community of wild young South American (Arctocephalus australis) and Subantarctic fur seals (Arctocephalus tropicalis).

PubMed

Medeiros, Aline Weber; Giongo, Adriana; Valdez, Fernanda P; Blaese de Amorin, Derek; Tavares, Maurício; d'Azevedo, Pedro A; Franco, Ana Claudia; Frazzon, Jeverson; Frazzon, Ana P G

2016-03-01

The microbiota of wild marine mammals is poorly understood, perhaps due to the migratory habits of some species and the difficulty in obtaining samples. Using high-throughput sequencing, the present study examines the faecal bacterial community of wild young South American (Arctocephalus australis) and Subantarctic fur seals (A. tropicalis). Faecal samples from South American (n = 6) and Subantarctic fur seals (n = 4) found dead along the south coast of Brazil were collected. Sequences were assigned to taxa using the Ribosomal Database Project-Bayesian classifier. Diversity of the microbiota was assessed by categorization of sequence reads into operational taxonomic units. Results indicate that Firmicutes (88.556%-84.016%) was the predominant phylum in South American and Subantarctic fur seals. The distribution of Actinobacteria and Proteobacteria varied according to the fur seal species. Fusobacteria and Bacteroidetes represented less than 1% of the sequences. The most abundant order in both fur seals was Clostridiales (88.64% and 87.49%). Individual variable incidences were observed in the composition of family among the fur seals, though the families Lachnospiraceae, Peptostreptococcaceae, Ruminococcaceae and Coriobacteriaceae were more prevalent. This study provides insight into the faecal bacterial community of wild young South American and Subantarctic fur seals. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bacterial microbiome of the nose of healthy dogs and dogs with nasal disease

PubMed Central

Dorn, Elisabeth S.; Suchodolski, Jan S.; Nisar, Tariq; Ravindran, Prajesh; Weber, Karin; Hartmann, Katrin; Schulz, Bianka S.

2017-01-01

The role of bacterial communities in canine nasal disease has not been studied so far using next generation sequencing methods. Sequencing of bacterial 16S rRNA genes has revealed that the canine upper respiratory tract harbors a diverse microbial community; however, changes in the composition of nasal bacterial communities in dogs with nasal disease have not been described so far. Aim of the study was to characterize the nasal microbiome of healthy dogs and compare it to that of dogs with histologically confirmed nasal neoplasia and chronic rhinitis. Nasal swabs were collected from healthy dogs (n = 23), dogs with malignant nasal neoplasia (n = 16), and dogs with chronic rhinitis (n = 8). Bacterial DNA was extracted and sequencing of the bacterial 16S rRNA gene was performed. Data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME). A total of 376 Operational Taxonomic Units out of 26 bacterial phyla were detected. In healthy dogs, Moraxella spp. was the most common species, followed by Phyllobacterium spp., Cardiobacteriaceae, and Staphylococcus spp. While Moraxella spp. were significantly decreased in diseased compared to healthy dogs (p = 0.005), Pasteurellaceae were significantly increased (p = 0.001). Analysis of similarities used on the unweighted UniFrac distance metric (p = 0.027) was significantly different when nasal microbial communities of healthy dogs were compared to those of dogs with nasal disease. The study showed that the canine nasal cavity is inhabited by a highly species-rich bacterial community, and suggests significant differences between the nasal microbiome of healthy dogs and dogs with nasal disease. PMID:28459886

Bacterial microbiome of the nose of healthy dogs and dogs with nasal disease.

PubMed

Tress, Barbara; Dorn, Elisabeth S; Suchodolski, Jan S; Nisar, Tariq; Ravindran, Prajesh; Weber, Karin; Hartmann, Katrin; Schulz, Bianka S

2017-01-01

The role of bacterial communities in canine nasal disease has not been studied so far using next generation sequencing methods. Sequencing of bacterial 16S rRNA genes has revealed that the canine upper respiratory tract harbors a diverse microbial community; however, changes in the composition of nasal bacterial communities in dogs with nasal disease have not been described so far. Aim of the study was to characterize the nasal microbiome of healthy dogs and compare it to that of dogs with histologically confirmed nasal neoplasia and chronic rhinitis. Nasal swabs were collected from healthy dogs (n = 23), dogs with malignant nasal neoplasia (n = 16), and dogs with chronic rhinitis (n = 8). Bacterial DNA was extracted and sequencing of the bacterial 16S rRNA gene was performed. Data were analyzed using Quantitative Insights Into Microbial Ecology (QIIME). A total of 376 Operational Taxonomic Units out of 26 bacterial phyla were detected. In healthy dogs, Moraxella spp. was the most common species, followed by Phyllobacterium spp., Cardiobacteriaceae, and Staphylococcus spp. While Moraxella spp. were significantly decreased in diseased compared to healthy dogs (p = 0.005), Pasteurellaceae were significantly increased (p = 0.001). Analysis of similarities used on the unweighted UniFrac distance metric (p = 0.027) was significantly different when nasal microbial communities of healthy dogs were compared to those of dogs with nasal disease. The study showed that the canine nasal cavity is inhabited by a highly species-rich bacterial community, and suggests significant differences between the nasal microbiome of healthy dogs and dogs with nasal disease.

Characterisation of the bacterial community structures in the intestine of Lampetra morii.

PubMed

Li, Yingying; Xie, Wenfang; Li, Qingwei

2016-07-01

The metagenomic analysis and 16S rDNA sequencing method were used to investigate the bacterial community in the intestines of Lampetra morii. The bacterial community structure in L. morii intestine was relatively simple. Eight different operational taxonomic units were observed. Chitinophagaceae_unclassified (26.5 %) and Aeromonas spp. (69.6 %) were detected as dominant members at the genus level. The non-dominant genera were as follows: Acinetobacter spp. (1.4 %), Candidatus Bacilloplasma (2.5 %), Enterobacteria spp. (1.5 %), Shewanella spp. (0.04 %), Vibrio spp. (0.09 %), and Yersinia spp. (1.8 %). The Shannon-Wiener (H) and Simpson (1-D) indexes were 0.782339 and 0.5546, respectively. The rarefaction curve representing the bacterial community richness and Shannon-Wiener curve representing the bacterial community diversity reached asymptote, which indicated that the sequence depth were sufficient to represent the majority of species richness and bacterial community diversity. The number of Aeromonas in lamprey intestine was two times higher after stimulation by lipopolysaccharide than PBS. This study provides data for understanding the bacterial community harboured in lamprey intestines and exploring potential key intestinal symbiotic bacteria essential for the L. morii immune response.

Insight into the effects of different cropping systems on soil bacterial community and tobacco bacterial wilt rate.

PubMed

Niu, Jiaojiao; Chao, Jin; Xiao, Yunhua; Chen, Wu; Zhang, Chao; Liu, Xueduan; Rang, Zhongwen; Yin, Huaqun; Dai, Linjian

2017-01-01

Rotation is an effective strategy to control crop disease and improve plant health. However, the effects of crop rotation on soil bacterial community composition and structure, and crop health remain unclear. In this study, using 16S rRNA gene sequencing, we explored the soil bacterial communities under four different cropping systems, continuous tobacco cropping (control group), tobacco-maize rotation, tobacco-lily rotation, and tobacco-turnip rotation. Results of detrended correspondence analysis and dissimilarity tests showed that soil bacterial community composition and structure changed significantly among the four groups, such that Acidobacteria and Actinobacteria were more abundant in the maize rotation group (16.6 and 11.5%, respectively) than in the control (8.5 and 7.1%, respectively). Compared with the control group (57.78%), maize and lily were effective rotation crops in controlling tobacco bacterial wilt (about 23.54 and 48.67%). On the other hand, tobacco bacterial wilt rate was increased in the turnip rotation (59.62%) relative to the control. Further study revealed that the abundances of several bacterial populations were directly correlated with tobacco bacterial wilt. For example, Acidobacteria and Actinobacteria were significantly negatively correlated to the tobacco bacterial wilt rate, so they may be probiotic bacteria. Canonical correspondence analysis showed that soil pH and calcium content were key factors in determining soil bacterial communities. In conclusion, our study revealed the composition and structure of bacterial communities under four different cropping systems and may unveil molecular mechanisms for the interactions between soil microorganisms and crop health. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Furnishing spaceship environment: evaluation of bacterial biofilms on different materials used inside International Space Station.

PubMed

Perrin, Elena; Bacci, Giovanni; Garrelly, Laurent; Canganella, Francesco; Bianconi, Giovanna; Fani, Renato; Mengoni, Alessio

2018-05-08

Performed inside International Space Station (ISS) from 2011 to 2016, VIABLE (eValuatIon And monitoring of microBiofiLms insidE International Space Station) ISS was a long-lasting experiment aimed at evaluating the bacterial contamination on different surface space materials subjected to different pre-treatment, to provide useful information for future space missions. In this work, surfaces samples of the VIABLE ISS experiment were analyzed to determine both the total bacterial load (ATP-metry, qPCR) and the composition of the microbial communities (16S rRNA genes amplicon sequencing). Data obtained showed a low bacterial contamination of all the surfaces, with values in agreement with those allowed inside ISS, and with a taxonomic composition similar to those found in previous studies (Enterobacteriales, Bacillales, Lactobacillales and Actinomycetales). No pre-treatment or material effect were observed on both the bacterial load and the composition of the communities, but for both a slight effect of the position (expose/not expose to air) was observed. In conclusion, under the conditions used for VIABLE ISS, no material or pre-treatment seems to be better than others in terms of quantity and type of bacterial contamination. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Shifts of live bacterial community in secondary effluent by chlorine disinfection revealed by Miseq high-throughput sequencing combined with propidium monoazide treatment.

PubMed

Pang, Yu-Chen; Xi, Jin-Ying; Xu, Yang; Huo, Zheng-Yang; Hu, Hong-Ying

2016-07-01

Chlorine disinfection is a commonly used disinfection process in wastewater treatment, but its effects on the indigenous bacterial community in treated wastewater have not been fully elucidated. In this study, secondary effluent samples collected in four wastewater treatment plants (WWTPs) were selected for chlorine disinfection. Shifts in the bacterial community compositions in secondary effluent samples upon chlorine disinfection, both immediately and after 24 h of storage, were investigated using Illumina MiSeq sequencing combined with propidium monoazide (PMA) treatment. The results showed that the phylum Proteobacteria was sensitive to chlorine, with the relative proportions of Proteobacteria decreased from 39.2 to 75.9 % in secondary effluent samples to 7.5 to 62.2 % immediately after chlorine disinfection. The phylogenetic analysis indicated that the most dominant genera belonging to Proteobacteria were sensitive to chlorine. In contrast, the phyla Firmicutes and Planctomycetes showed a certain resistance to chlorine, with their relative proportions increasing from 5.1 to 23.1 % and 0.8 to 9.3 % to 11.3 to 44.6 % and 1.5 to 13.3 %, respectively. Most dominant genera belonging to Firmicutes showed resistance to chlorine. A significant reduction in the richness and diversity of the bacterial community was observed after 24 h of storage of chlorinated secondary effluent. During the 24-h storage process, the relative proportions of most dominant phyla shifted in reverse from the changes induced by chlorine disinfection. Overall, chlorine disinfection not only changes the bacterial community compositions immediately after the disinfection process but also exerts further impacts over a longer period (24 h).

antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences.

PubMed

Medema, Marnix H; Blin, Kai; Cimermancic, Peter; de Jager, Victor; Zakrzewski, Piotr; Fischbach, Michael A; Weber, Tilmann; Takano, Eriko; Breitling, Rainer

2011-07-01

Bacterial and fungal secondary metabolism is a rich source of novel bioactive compounds with potential pharmaceutical applications as antibiotics, anti-tumor drugs or cholesterol-lowering drugs. To find new drug candidates, microbiologists are increasingly relying on sequencing genomes of a wide variety of microbes. However, rapidly and reliably pinpointing all the potential gene clusters for secondary metabolites in dozens of newly sequenced genomes has been extremely challenging, due to their biochemical heterogeneity, the presence of unknown enzymes and the dispersed nature of the necessary specialized bioinformatics tools and resources. Here, we present antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), the first comprehensive pipeline capable of identifying biosynthetic loci covering the whole range of known secondary metabolite compound classes (polyketides, non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides, beta-lactams, butyrolactones, siderophores, melanins and others). It aligns the identified regions at the gene cluster level to their nearest relatives from a database containing all other known gene clusters, and integrates or cross-links all previously available secondary-metabolite specific gene analysis methods in one interactive view. antiSMASH is available at http://antismash.secondarymetabolites.org.

Two ancient bacterial endosymbionts have coevolved with the planthoppers (Insecta: Hemiptera: Fulgoroidea).

PubMed

Urban, Julie M; Cryan, Jason R

2012-06-14

Members of the hemipteran suborder Auchenorrhyncha (commonly known as planthoppers, tree- and leafhoppers, spittlebugs, and cicadas) are unusual among insects known to harbor endosymbiotic bacteria in that they are associated with diverse assemblages of bacterial endosymbionts. Early light microscopic surveys of species representing the two major lineages of Auchenorrhyncha (the planthopper superfamily Fulgoroidea; and Cicadomorpha, comprising Membracoidea [tree- and leafhoppers], Cercopoidea [spittlebugs], and Cicadoidea [cicadas]), found that most examined species harbored at least two morphologically distinct bacterial endosymbionts, and some harbored as many as six. Recent investigations using molecular techniques have identified multiple obligate bacterial endosymbionts in Cicadomorpha; however, much less is known about endosymbionts of Fulgoroidea. In this study, we present the initial findings of an ongoing PCR-based survey (sequencing 16S rDNA) of planthopper-associated bacteria to document endosymbionts with a long-term history of codiversification with their fulgoroid hosts. Results of PCR surveys and phylogenetic analyses of 16S rDNA recovered a monophyletic clade of Betaproteobacteria associated with planthoppers; this clade included Vidania fulgoroideae, a recently described bacterium identified in exemplars of the planthopper family Cixiidae. We surveyed 77 planthopper species representing 18 fulgoroid families, and detected Vidania in 40 species (representing 13 families). Further, we detected the Sulcia endosymbiont (identified as an obligate endosymbiont of Auchenorrhyncha in previous studies) in 30 of the 40 species harboring Vidania. Concordance of the Vidania phylogeny with the phylogeny of the planthopper hosts (reconstructed based on sequence data from five genes generated from the same insect specimens from which the bacterial sequences were obtained) was supported by statistical tests of codiversification. Codiversification tests also

Two ancient bacterial endosymbionts have coevolved with the planthoppers (Insecta: Hemiptera: Fulgoroidea)

PubMed Central

2012-01-01

Background Members of the hemipteran suborder Auchenorrhyncha (commonly known as planthoppers, tree- and leafhoppers, spittlebugs, and cicadas) are unusual among insects known to harbor endosymbiotic bacteria in that they are associated with diverse assemblages of bacterial endosymbionts. Early light microscopic surveys of species representing the two major lineages of Auchenorrhyncha (the planthopper superfamily Fulgoroidea; and Cicadomorpha, comprising Membracoidea [tree- and leafhoppers], Cercopoidea [spittlebugs], and Cicadoidea [cicadas]), found that most examined species harbored at least two morphologically distinct bacterial endosymbionts, and some harbored as many as six. Recent investigations using molecular techniques have identified multiple obligate bacterial endosymbionts in Cicadomorpha; however, much less is known about endosymbionts of Fulgoroidea. In this study, we present the initial findings of an ongoing PCR-based survey (sequencing 16S rDNA) of planthopper-associated bacteria to document endosymbionts with a long-term history of codiversification with their fulgoroid hosts. Results Results of PCR surveys and phylogenetic analyses of 16S rDNA recovered a monophyletic clade of Betaproteobacteria associated with planthoppers; this clade included Vidania fulgoroideae, a recently described bacterium identified in exemplars of the planthopper family Cixiidae. We surveyed 77 planthopper species representing 18 fulgoroid families, and detected Vidania in 40 species (representing 13 families). Further, we detected the Sulcia endosymbiont (identified as an obligate endosymbiont of Auchenorrhyncha in previous studies) in 30 of the 40 species harboring Vidania. Concordance of the Vidania phylogeny with the phylogeny of the planthopper hosts (reconstructed based on sequence data from five genes generated from the same insect specimens from which the bacterial sequences were obtained) was supported by statistical tests of codiversification

Endosymbiont Dominated Bacterial Communities in a Dwarf Spider

PubMed Central

Vanthournout, Bram; Hendrickx, Frederik

2015-01-01

The microbial community of spiders is little known, with previous studies focussing primarily on the medical importance of spiders as vectors of pathogenic bacteria and on the screening of known cytoplasmic endosymbiont bacteria. These screening studies have been performed by means of specific primers that only amplify a selective set of endosymbionts, hampering the detection of unreported species in spiders. In order to have a more complete overview of the bacterial species that can be present in spiders, we applied a combination of a cloning assay, DGGE profiling and high-throughput sequencing on multiple individuals of the dwarf spider Oedothorax gibbosus. This revealed a co-infection of at least three known (Wolbachia, Rickettsia and Cardinium) and the detection of a previously unreported endosymbiont bacterium (Rhabdochlamydia) in spiders. 16S rRNA gene sequences of Rhabdochlamydia matched closely with those of Candidatus R. porcellionis, which is currently only reported as a pathogen from a woodlouse and with Candidatus R. crassificans reported from a cockroach. Remarkably, this bacterium appears to present in very high proportions in one of the two populations only, with all investigated females being infected. We also recovered Acinetobacter in high abundance in one individual. In total, more than 99% of approximately 4.5M high-throughput sequencing reads were restricted to these five bacterial species. In contrast to previously reported screening studies of terrestrial arthropods, our results suggest that the bacterial communities in this spider species are dominated by, or even restricted to endosymbiont bacteria. Given the high prevalence of endosymbiont species in spiders, this bacterial community pattern could be widespread in the Araneae order. PMID:25706947

Xylella genomics and bacterial pathogenicity to plants.

PubMed

Dow, J M; Daniels, M J

2000-12-01

Xylella fastidiosa, a pathogen of citrus, is the first plant pathogenic bacterium for which the complete genome sequence has been published. Inspection of the sequence reveals high relatedness to many genes of other pathogens, notably Xanthomonas campestris. Based on this, we suggest that Xylella possesses certain easily testable properties that contribute to pathogenicity. We also present some general considerations for deriving information on pathogenicity from bacterial genomics. Copyright 2000 John Wiley & Sons, Ltd.

The crystal structure of a bacterial Sufu-like protein defines a novel group of bacterial proteins that are similar to the N-terminal domain of human Sufu

PubMed Central

Das, Debanu; Finn, Robert D; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L; Bakolitsa, Constantina; Cai, Xiaohui; Carlton, Dennis; Chen, Connie; Chiu, Hsiu-Ju; Chiu, Michelle; Clayton, Thomas; Deller, Marc C; Duan, Lian; Ellrott, Kyle; Farr, Carol L; Feuerhelm, Julie; Grant, Joanna C; Grzechnik, Anna; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K; Klock, Heath E; Knuth, Mark W; Kozbial, Piotr; Sri Krishna, S; Kumar, Abhinav; Lam, Winnie W; Marciano, David; Miller, Mitchell D; Morse, Andrew T; Nigoghossian, Edward; Nopakun, Amanda; Okach, Linda; Puckett, Christina; Reyes, Ron; Tien, Henry J; Trame, Christine B; van den Bedem, Henry; Weekes, Dana; Wooten, Tiffany; Xu, Qingping; Yeh, Andrew; Zhou, Jiadong; Hodgson, Keith O; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M; Godzik, Adam; Lesley, Scott A; Wilson, Ian A

2010-01-01

Sufu (Suppressor of Fused), a two-domain protein, plays a critical role in regulating Hedgehog signaling and is conserved from flies to humans. A few bacterial Sufu-like proteins have previously been identified based on sequence similarity to the N-terminal domain of eukaryotic Sufu proteins, but none have been structurally or biochemically characterized and their function in bacteria is unknown. We have determined the crystal structure of a more distantly related Sufu-like homolog, NGO1391 from Neisseria gonorrhoeae, at 1.4 Å resolution, which provides the first biophysical characterization of a bacterial Sufu-like protein. The structure revealed a striking similarity to the N-terminal domain of human Sufu (r.m.s.d. of 2.6 Å over 93% of the NGO1391 protein), despite an extremely low sequence identity of ∼15%. Subsequent sequence analysis revealed that NGO1391 defines a new subset of smaller, Sufu-like proteins that are present in ∼200 bacterial species and has resulted in expansion of the SUFU (PF05076) family in Pfam. PMID:20836087

Bacterial spoilers of food: behavior, fitness and functional properties.

PubMed

Remenant, Benoît; Jaffrès, Emmanuel; Dousset, Xavier; Pilet, Marie-France; Zagorec, Monique

2015-02-01

Most food products are highly perishable as they constitute a rich nutrient source for microbial development. Among the microorganisms contaminating food, some present metabolic activities leading to spoilage. In addition to hygienic rules to reduce contamination, various treatments are applied during production and storage to avoid the growth of unwanted microbes. The nature and appearance of spoilage therefore depend on the physiological state of spoilers and on their ability to resist the processing/storage conditions and flourish on the food matrix. Spoilage also relies on the interactions between the microorganisms composing the ecosystems encountered in food. The recent rapid increase in publicly available bacterial genome sequences, as well as the access to high-throughput methods, should lead to a better understanding of spoiler behavior and to the possibility of decreasing food spoilage. This review lists the main bacterial species identified as food spoilers, their ability to develop during storage and/or processing, and the functions potentially involved in spoilage. We have also compiled an inventory of the available genome sequences of species encompassing spoilage strains. Combining in silico analysis of genome sequences with experimental data is proposed in order to understand and thus control the bacterial spoilage of food better. Copyright © 2014 Elsevier Ltd. All rights reserved.

Draft Genome Sequences of Endophytic Isolates of Klebsiella variicola and Klebsiella pneumoniae Obtained from the Same Sugarcane Plant

PubMed Central

2018-01-01

ABSTRACT Endophytic Klebsiella variicola KvMx2 and Klebsiella pneumoniae KpMx1 isolates obtained from the same sugarcane stem were used for whole-genome sequencing. The genomes revealed clear differences in essential genes for plant growth, development, and detoxification, as well as nitrogen fixation, catalases, cellulases, and shared virulence factors described in the K. pneumoniae pathogen. PMID:29567733

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.

Broad Surveys of DNA Viral Diversity Obtained through Viral Metagenomics of Mosquitoes

PubMed Central

Ng, Terry Fei Fan; Willner, Dana L.; Lim, Yan Wei; Schmieder, Robert; Chau, Betty; Nilsson, Christina; Anthony, Simon; Ruan, Yijun; Rohwer, Forest; Breitbart, Mya

2011-01-01

Viruses are the most abundant and diverse genetic entities on Earth; however, broad surveys of viral diversity are hindered by the lack of a universal assay for viruses and the inability to sample a sufficient number of individual hosts. This study utilized vector-enabled metagenomics (VEM) to provide a snapshot of the diversity of DNA viruses present in three mosquito samples from San Diego, California. The majority of the sequences were novel, suggesting that the viral community in mosquitoes, as well as the animal and plant hosts they feed on, is highly diverse and largely uncharacterized. Each mosquito sample contained a distinct viral community. The mosquito viromes contained sequences related to a broad range of animal, plant, insect and bacterial viruses. Animal viruses identified included anelloviruses, circoviruses, herpesviruses, poxviruses, and papillomaviruses, which mosquitoes may have obtained from vertebrate hosts during blood feeding. Notably, sequences related to human papillomaviruses were identified in one of the mosquito samples. Sequences similar to plant viruses were identified in all mosquito viromes, which were potentially acquired through feeding on plant nectar. Numerous bacteriophages and insect viruses were also detected, including a novel densovirus likely infecting Culex erythrothorax. Through sampling insect vectors, VEM enables broad survey of viral diversity and has significantly increased our knowledge of the DNA viruses present in mosquitoes. PMID:21674005

On the Search for Retrotransposons: Alternative Protocols to Obtain Sequences to Learn Profile Hidden Markov Models.

PubMed

Fischer, Carlos N; Campos, Victor De A; Barella, Victor H

2018-05-01

Profile hidden Markov models (pHMMs) have been used to search for transposable elements (TEs) in genomes. For the learning of pHMMs aimed to search for TEs of the retrotransposon class, the conventional protocol is to use the whole internal nucleotide portions of these elements as representative sequences. To further explore the potential of pHMMs in such a search, we propose five alternative ways to obtain the sets of representative sequences of TEs other than the conventional protocol. In this study, we are interested in Bel-PAO, Copia, Gypsy, and DIRS superfamilies from the retrotransposon class. We compared the pHMMs of all six protocols. The test results show that, for each TE superfamily, the pHMMs of at least two of the proposed protocols performed better than the conventional one and that the number of correct predictions provided by the latter can be improved by considering together the results of one or more of the alternative protocols.

SPARTA: Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis.

PubMed

Johnson, Benjamin K; Scholz, Matthew B; Teal, Tracy K; Abramovitch, Robert B

2016-02-04

Many tools exist in the analysis of bacterial RNA sequencing (RNA-seq) transcriptional profiling experiments to identify differentially expressed genes between experimental conditions. Generally, the workflow includes quality control of reads, mapping to a reference, counting transcript abundance, and statistical tests for differentially expressed genes. In spite of the numerous tools developed for each component of an RNA-seq analysis workflow, easy-to-use bacterially oriented workflow applications to combine multiple tools and automate the process are lacking. With many tools to choose from for each step, the task of identifying a specific tool, adapting the input/output options to the specific use-case, and integrating the tools into a coherent analysis pipeline is not a trivial endeavor, particularly for microbiologists with limited bioinformatics experience. To make bacterial RNA-seq data analysis more accessible, we developed a Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA). SPARTA is a reference-based bacterial RNA-seq analysis workflow application for single-end Illumina reads. SPARTA is turnkey software that simplifies the process of analyzing RNA-seq data sets, making bacterial RNA-seq analysis a routine process that can be undertaken on a personal computer or in the classroom. The easy-to-install, complete workflow processes whole transcriptome shotgun sequencing data files by trimming reads and removing adapters, mapping reads to a reference, counting gene features, calculating differential gene expression, and, importantly, checking for potential batch effects within the data set. SPARTA outputs quality analysis reports, gene feature counts and differential gene expression tables and scatterplots. SPARTA provides an easy-to-use bacterial RNA-seq transcriptional profiling workflow to identify differentially expressed genes between experimental conditions. This software will enable microbiologists with

A world without bacterial meningitis: how genomic epidemiology can inform vaccination strategy.

PubMed

Rodrigues, Charlene M C; Maiden, Martin C J

2018-01-01

Bacterial meningitis remains an important cause of global morbidity and mortality. Although effective vaccinations exist and are being increasingly used worldwide, bacterial diversity threatens their impact and the ultimate goal of eliminating the disease. Through genomic epidemiology, we can appreciate bacterial population structure and its consequences for transmission dynamics, virulence, antimicrobial resistance, and development of new vaccines. Here, we review what we have learned through genomic epidemiological studies, following the rapid implementation of whole genome sequencing that can help to optimise preventative strategies for bacterial meningitis.

Bacterial diversity and composition in the fluid of pitcher plants of the genus Nepenthes.

PubMed

Takeuchi, Yayoi; Chaffron, Samuel; Salcher, Michaela M; Shimizu-Inatsugi, Rie; Kobayashi, Masaki J; Diway, Bibian; von Mering, Christian; Pernthaler, Jakob; Shimizu, Kentaro K

2015-07-01

Pitchers are modified leaves used by carnivorous plants for trapping prey. Their fluids contain digestive enzymes from the plant and they harbor abundant microbes. In this study, the diversity of bacterial communities was assessed in Nepenthes pitcher fluids and the composition of the bacterial community was compared to that in other environments, including the phyllosphere of Arabidopsis, animal guts and another pitcher plant, Sarracenia. Diversity was measured by 454 pyrosequencing of 16S rRNA gene amplicons. A total of 232,823 sequences were obtained after chimera and singleton removal that clustered into 3260 distinct operational taxonomic units (OTUs) (3% dissimilarity), which were taxonomically distributed over 17 phyla, 25 classes, 45 orders, 100 families, and 195 genera. Pyrosequencing and fluorescence in situ hybridization yielded similar estimates of community composition. Most pitchers contained high proportions of unique OTUs, and only 22 OTUs (<0.6%) were shared by ≥14/16 samples, suggesting a unique bacterial assemblage in each pitcher at the OTU level. Diversity analysis at the class level revealed that the bacterial communities of both opened and unopened pitchers were most similar to that of Sarracenia and to that in the phyllosphere. Therefore, the bacterial community in pitchers may be formed by environmental filtering and/or by phyllosphere bacteria. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

Culturable endophytic bacterial communities associated with field-grown soybean.

PubMed

de Almeida Lopes, K B; Carpentieri-Pipolo, V; Oro, T H; Stefani Pagliosa, E; Degrassi, G

2016-03-01

Assess the diversity of the culturable endophytic bacterial population associated with transgenic and nontransgenic soybean grown in field trial sites in Brazil and characterize them phenotypically and genotypically focusing on characteristics related to plant growth promotion. Endophytic bacteria were isolated from roots, stems and leaves of soybean cultivars (nontransgenic (C) and glyphosate-resistant (GR) transgenic soybean), including the isogenic BRS133 and BRS245RR. Significant differences were observed in bacterial densities in relation to genotype and tissue from which the isolates were obtained. The highest number of bacteria was observed in roots and in GR soybean. Based on characteristics related to plant growth promotion, 54 strains were identified by partial 16S rRNA sequence analysis, with most of the isolates belonging to the species Enterobacter ludwigii and Variovorax paradoxus. Among the isolates, 44·4% were able to either produce indoleacetic acid (IAA) or solubilize phosphates, and 9·2% (all from GR soybean) presented both plant growth-promoting activities. The results from this study indicate that the abundance of endophytic bacterial communities of soybean differs between cultivars and in general it was higher in the transgenic cultivars than in nontransgenic cultivars. BRS 245 RR exhibited no significant difference in abundance compared to nontransgenic BRS133. This suggests that the impact of the management used in the GR soybean fields was comparable with the impacts of some enviromental factors. However, the bacterial endophytes associated to GR and nontransgenic soybean were different. The soybean-associated bacteria showing characteristics related to plant growth promotion were identified as belonging to the species Pantoea agglomerans and Variovorax paradoxus. Our study demonstrated differences concerning compostion of culturable endophytic bacterial population in nontransgenic and transgenic soybean. © 2016 The Society for Applied

Bacterial diversity of bacteriomes and organs of reproductive, digestive and excretory systems in two cicada species (Hemiptera: Cicadidae)

PubMed Central

Zheng, Zhou; Wang, Dandan; He, Hong

2017-01-01

Cicadas form intimate symbioses with bacteria to obtain nutrients that are scarce in the xylem fluid they feed on. The obligate symbionts in cicadas are purportedly confined to specialized bacteriomes, but knowledge of bacterial communities associated with cicadas is limited. Bacterial communities in the bacteriomes and organs of reproductive, digestive and excretory systems of two cicada species (Platypleura kaempferi and Meimuna mongolica) were investigated using different methods, and the bacterial diversity and distribution patterns of dominant bacteria in different tissues were compared. Within each species, the bacterial communities of testes are significantly different from those of bacteriomes and ovaries. The dominant endosymbiont Candidatus Sulcia muelleri is found not only in the bacteriomes and reproductive organs, but also in the “filter chamber + conical segment” of both species. The transmission mode of this endosymbiont in the alimentary canal and its effect on physiological processes merits further study. A novel bacterium of Rhizobiales, showing ~80% similarity to Candidatus Hodgkinia cicadicola, is dominant in the bacteriomes and ovaries of P. kaempferi. Given that the genome of H. cicadicola exhibits rapid sequence evolution, it is possible that this novel bacterium is a related endosymbiont with beneficial trophic functions similar to that of H. cicadicola in some other cicadas. Failure to detect H. cicadicola in M. mongolica suggests that it has been subsequently replaced by another bacterium, a yeast or gut microbiota which compensates for the loss of H. cicadicola. The distribution of this novel Rhizobiales species in other cicadas and its identification require further investigation to help establish the definition of the bacterial genus Candidatus Hodgkinia and to provide more information on sequence divergence of related endosymbionts of cicadas. Our results highlight the complex bacterial communities of cicadas, and are informative

Bacterial diversity of bacteriomes and organs of reproductive, digestive and excretory systems in two cicada species (Hemiptera: Cicadidae).

PubMed

Zheng, Zhou; Wang, Dandan; He, Hong; Wei, Cong

2017-01-01

Cicadas form intimate symbioses with bacteria to obtain nutrients that are scarce in the xylem fluid they feed on. The obligate symbionts in cicadas are purportedly confined to specialized bacteriomes, but knowledge of bacterial communities associated with cicadas is limited. Bacterial communities in the bacteriomes and organs of reproductive, digestive and excretory systems of two cicada species (Platypleura kaempferi and Meimuna mongolica) were investigated using different methods, and the bacterial diversity and distribution patterns of dominant bacteria in different tissues were compared. Within each species, the bacterial communities of testes are significantly different from those of bacteriomes and ovaries. The dominant endosymbiont Candidatus Sulcia muelleri is found not only in the bacteriomes and reproductive organs, but also in the "filter chamber + conical segment" of both species. The transmission mode of this endosymbiont in the alimentary canal and its effect on physiological processes merits further study. A novel bacterium of Rhizobiales, showing ~80% similarity to Candidatus Hodgkinia cicadicola, is dominant in the bacteriomes and ovaries of P. kaempferi. Given that the genome of H. cicadicola exhibits rapid sequence evolution, it is possible that this novel bacterium is a related endosymbiont with beneficial trophic functions similar to that of H. cicadicola in some other cicadas. Failure to detect H. cicadicola in M. mongolica suggests that it has been subsequently replaced by another bacterium, a yeast or gut microbiota which compensates for the loss of H. cicadicola. The distribution of this novel Rhizobiales species in other cicadas and its identification require further investigation to help establish the definition of the bacterial genus Candidatus Hodgkinia and to provide more information on sequence divergence of related endosymbionts of cicadas. Our results highlight the complex bacterial communities of cicadas, and are informative for

Use of Optical Mapping in Bacterial Genome Finishing

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

Kumar, Dibyendu

2010-06-03

Dibyendu Kumar from the University of Florida discusses whole-genome optical mapping to help validate bacterial genome assemblies on June 3, 2010 at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM.

Sequencing-Based Analysis of the Bacterial and Fungal Composition of Kefir Grains and Milks from Multiple Sources

PubMed Central

Marsh, Alan J.; O’Sullivan, Orla; Hill, Colin; Ross, R. Paul; Cotter, Paul D.

2013-01-01

Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists. PMID:23894461

Sequencing-based analysis of the bacterial and fungal composition of kefir grains and milks from multiple sources.

PubMed

Marsh, Alan J; O'Sullivan, Orla; Hill, Colin; Ross, R Paul; Cotter, Paul D

2013-01-01

Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists.

Complete Genome Sequences of Six Copper-Resistant Xanthomonas Strains Causing Bacterial Spot of Solaneous Plants, Belonging to X. gardneri, X. euvesicatoria, and X. vesicatoria, Using Long-Read Technology.

PubMed

Richard, Damien; Boyer, Claudine; Lefeuvre, Pierre; Canteros, Blanca I; Beni-Madhu, Shyam; Portier, Perrine; Pruvost, Olivier

2017-02-23

Xanthomonas vesicatoria , Xanthomonas euvesicatoria , and Xanthomonas gardneri cause bacterial spot disease. Copper has been applied since the 1920s as part of integrated management programs. The first copper-resistant strains were reported some decades later. Here, we fully sequenced six Xanthomonas strains pathogenic to tomato and/or pepper and having a copper-resistant phenotype. Copyright © 2017 Richard et al.

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

PubMed Central

Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Bælum, Jacob; Feld, Louise; Holben, William E.

2014-01-01

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition. PMID:24610853

From learning taxonomies to phylogenetic learning: integration of 16S rRNA gene data into FAME-based bacterial classification.

PubMed

Slabbinck, Bram; Waegeman, Willem; Dawyndt, Peter; De Vos, Paul; De Baets, Bernard

2010-01-30

Machine learning techniques have shown to improve bacterial species classification based on fatty acid methyl ester (FAME) data. Nonetheless, FAME analysis has a limited resolution for discrimination of bacteria at the species level. In this paper, we approach the species classification problem from a taxonomic point of view. Such a taxonomy or tree is typically obtained by applying clustering algorithms on FAME data or on 16S rRNA gene data. The knowledge gained from the tree can then be used to evaluate FAME-based classifiers, resulting in a novel framework for bacterial species classification. In view of learning in a taxonomic framework, we consider two types of trees. First, a FAME tree is constructed with a supervised divisive clustering algorithm. Subsequently, based on 16S rRNA gene sequence analysis, phylogenetic trees are inferred by the NJ and UPGMA methods. In this second approach, the species classification problem is based on the combination of two different types of data. Herein, 16S rRNA gene sequence data is used for phylogenetic tree inference and the corresponding binary tree splits are learned based on FAME data. We call this learning approach 'phylogenetic learning'. Supervised Random Forest models are developed to train the classification tasks in a stratified cross-validation setting. In this way, better classification results are obtained for species that are typically hard to distinguish by a single or flat multi-class classification model. FAME-based bacterial species classification is successfully evaluated in a taxonomic framework. Although the proposed approach does not improve the overall accuracy compared to flat multi-class classification, it has some distinct advantages. First, it has better capabilities for distinguishing species on which flat multi-class classification fails. Secondly, the hierarchical classification structure allows to easily evaluate and visualize the resolution of FAME data for the discrimination of bacterial

From learning taxonomies to phylogenetic learning: Integration of 16S rRNA gene data into FAME-based bacterial classification

PubMed Central

2010-01-01

Background Machine learning techniques have shown to improve bacterial species classification based on fatty acid methyl ester (FAME) data. Nonetheless, FAME analysis has a limited resolution for discrimination of bacteria at the species level. In this paper, we approach the species classification problem from a taxonomic point of view. Such a taxonomy or tree is typically obtained by applying clustering algorithms on FAME data or on 16S rRNA gene data. The knowledge gained from the tree can then be used to evaluate FAME-based classifiers, resulting in a novel framework for bacterial species classification. Results In view of learning in a taxonomic framework, we consider two types of trees. First, a FAME tree is constructed with a supervised divisive clustering algorithm. Subsequently, based on 16S rRNA gene sequence analysis, phylogenetic trees are inferred by the NJ and UPGMA methods. In this second approach, the species classification problem is based on the combination of two different types of data. Herein, 16S rRNA gene sequence data is used for phylogenetic tree inference and the corresponding binary tree splits are learned based on FAME data. We call this learning approach 'phylogenetic learning'. Supervised Random Forest models are developed to train the classification tasks in a stratified cross-validation setting. In this way, better classification results are obtained for species that are typically hard to distinguish by a single or flat multi-class classification model. Conclusions FAME-based bacterial species classification is successfully evaluated in a taxonomic framework. Although the proposed approach does not improve the overall accuracy compared to flat multi-class classification, it has some distinct advantages. First, it has better capabilities for distinguishing species on which flat multi-class classification fails. Secondly, the hierarchical classification structure allows to easily evaluate and visualize the resolution of FAME data for

Multilayer Approach for Characterization of Bacterial Diversity in a Marginal Sea: From Surface to Seabed

NASA Technical Reports Server (NTRS)

Ivana, Babic; Maja, Mucko; Ivica, Vilibic; Hrvoje, Mihanovic; Reffaella, Casotti; Zrinka, Ljubesic; Ivona, Cetinic; Cecilia, Balestra; Ines, Petric; Suncica, Bosak;

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

Draft Genome Sequences of Endophytic Isolates of Klebsiella variicola and Klebsiella pneumoniae Obtained from the Same Sugarcane Plant.

PubMed

Reyna-Flores, Fernando; Barrios-Camacho, Humberto; Dantán-González, Edgar; Ramírez-Trujillo, José Augusto; Lozano Aguirre Beltrán, Luis Fernando; Rodríguez-Medina, Nadia; Garza-Ramos, Ulises; Suárez-Rodríguez, Ramón

2018-03-22

Endophytic Klebsiella variicola KvMx2 and Klebsiella pneumoniae KpMx1 isolates obtained from the same sugarcane stem were used for whole-genome sequencing. The genomes revealed clear differences in essential genes for plant growth, development, and detoxification, as well as nitrogen fixation, catalases, cellulases, and shared virulence factors described in the K. pneumoniae pathogen. Copyright © 2018 Reyna-Flores et al.

Combining amplicon sequencing and metabolomics in cirrhotic patients highlights distinctive microbiota features involved in bacterial translocation, systemic inflammation and hepatic encephalopathy.

PubMed

Iebba, Valerio; Guerrieri, Francesca; Di Gregorio, Vincenza; Levrero, Massimo; Gagliardi, Antonella; Santangelo, Floriana; Sobolev, Anatoly P; Circi, Simone; Giannelli, Valerio; Mannina, Luisa; Schippa, Serena; Merli, Manuela

2018-05-29

In liver cirrhosis (LC), impaired intestinal functions lead to dysbiosis and possible bacterial translocation (BT). Bacteria or their byproducts within the bloodstream can thus play a role in systemic inflammation and hepatic encephalopathy (HE). We combined 16S sequencing, NMR metabolomics and network analysis to describe the interrelationships of members of the microbiota in LC biopsies, faeces, peripheral/portal blood and faecal metabolites with clinical parameters. LC faeces and biopsies showed marked dysbiosis with a heightened proportion of Enterobacteriaceae. Our approach showed impaired faecal bacterial metabolism of short-chain fatty acids (SCFAs) and carbon/methane sources in LC, along with an enhanced stress-related response. Sixteen species, mainly belonging to the Proteobacteria phylum, were shared between LC peripheral and portal blood and were functionally linked to iron metabolism. Faecal Enterobacteriaceae and trimethylamine were positively correlated with blood proinflammatory cytokines, while Ruminococcaceae and SCFAs played a protective role. Within the peripheral blood and faeces, certain species (Stenotrophomonas pavanii, Methylobacterium extorquens) and metabolites (methanol, threonine) were positively related to HE. Cirrhotic patients thus harbour a 'functional dysbiosis' in the faeces and peripheral/portal blood, with specific keystone species and metabolites related to clinical markers of systemic inflammation and HE.

Bacterial endophyte communities of three agricultural important grass species differ in their response towards management regimes

NASA Astrophysics Data System (ADS)

Wemheuer, Franziska; Kaiser, Kristin; Karlovsky, Petr; Daniel, Rolf; Vidal, Stefan; Wemheuer, Bernd

2017-01-01

Endophytic bacteria are critical for plant growth and health. However, compositional and functional responses of bacterial endophyte communities towards agricultural practices are still poorly understood. Hence, we analyzed the influence of fertilizer application and mowing frequency on bacterial endophytes in three agriculturally important grass species. For this purpose, we examined bacterial endophytic communities in aerial plant parts of Dactylis glomerata L., Festuca rubra L., and Lolium perenne L. by pyrotag sequencing of bacterial 16S rRNA genes over two consecutive years. Although management regimes influenced endophyte communities, observed responses were grass species-specific. This might be attributed to several bacteria specifically associated with a single grass species. We further predicted functional profiles from obtained 16S rRNA data. These profiles revealed that predicted abundances of genes involved in plant growth promotion or nitrogen metabolism differed between grass species and between management regimes. Moreover, structural and functional community patterns showed no correlation to each other indicating that plant species-specific selection of endophytes is driven by functional rather than phylogenetic traits. The unique combination of 16S rRNA data and functional profiles provided a holistic picture of compositional and functional responses of bacterial endophytes in agricultural relevant grass species towards management practices.

Insertion sequence diversity in archaea.

PubMed

Filée, J; Siguier, P; Chandler, M

2007-03-01

Insertion sequences (ISs) can constitute an important component of prokaryotic (bacterial and archaeal) genomes. Over 1,500 individual ISs are included at present in the ISfinder database (www-is.biotoul.fr), and these represent only a small portion of those in the available prokaryotic genome sequences and those that are being discovered in ongoing sequencing projects. In spite of this diversity, the transposition mechanisms of only a few of these ubiquitous mobile genetic elements are known, and these are all restricted to those present in bacteria. This review presents an overview of ISs within the archaeal kingdom. We first provide a general historical summary of the known properties and behaviors of archaeal ISs. We then consider how transposition might be regulated in some cases by small antisense RNAs and by termination codon readthrough. This is followed by an extensive analysis of the IS content in the sequenced archaeal genomes present in the public databases as of June 2006, which provides an overview of their distribution among the major archaeal classes and species. We show that the diversity of archaeal ISs is very great and comparable to that of bacteria. We compare archaeal ISs to known bacterial ISs and find that most are clearly members of families first described for bacteria. Several cases of lateral gene transfer between bacteria and archaea are clearly documented, notably for methanogenic archaea. However, several archaeal ISs do not have bacterial equivalents but can be grouped into Archaea-specific groups or families. In addition to ISs, we identify and list nonautonomous IS-derived elements, such as miniature inverted-repeat transposable elements. Finally, we present a possible scenario for the evolutionary history of ISs in the Archaea.

Assessing the Effect of Litter Species on the Dynamic of Bacterial and Fungal Communities during Leaf Decomposition in Microcosm by Molecular Techniques

PubMed Central

Xu, Wenjing; Shi, Lingling; Chan, Onchim; Li, Jiao; Casper, Peter; Zou, Xiaoming

2013-01-01

Although bacteria and fungi are well-known to be decomposers of leaf litter, few studies have examined their compositions and diversities during the decomposition process in tropical stream water. Xishuangbanna is a tropical region preserving one of the highest floristic diversity areas in China. In this study, leaf litter of four dominant plant species in Xishuangbanna was incubated in stream water for 42 days during which samples were taken regularly. Following DNA extraction, PCR-DGGE (denaturing gradient gel electrophoresis) and clone-sequencing analyses were performed using bacterial and fungal specific primers. Leaf species have slightly influences on bacterial community rather than fungal community. The richness and diversity of bacteria was higher than that of fungi, which increased towards the end of the 42-day-incubation. The bacterial community was initially more specific upon the type of leaves and gradually became similar at the later stage of decomposition with alpha-proteobacteria as major component. Sequences affiliated to methanotrophs were obtained that indicates potentially occurrence of methane oxidation and methanogenesis. For the fungal community, sequences affiliated to Aspergillus were predominant at the beginning and then shifted to Pleosporales. Our results suggest that the microorganisms colonizing leaf biofilm in tropical stream water were mostly generalists that could exploit the resources of leaves of various species equally well. PMID:24367682

Investigation of the bacterial retting community of kenaf (Hibiscus cannabinus) under different conditions using next-generation semiconductor sequencing.

PubMed

Visi, David K; D'Souza, Nandika; Ayre, Brian G; Webber Iii, Charles L; Allen, Michael S

2013-05-01

The microbial communities associated with kenaf (Hibiscus cannabinus) plant fibers during retting were determined in an effort to identify possible means of accelerating this process for industrial scale-up. Microbial communities were identified by semiconductor sequencing of 16S rRNA gene amplicons from DNA harvested from plant-surface associated samples and analyzed using an Ion Torrent PGM. The communities were sampled after 96 h from each of three different conditions, including amendments with pond water, sterilized pond water, or with a mixture of pectinolytic bacterial isolates. Additionally, plants from two different sources and having different pretreatment conditions were compared. We report that the best retting communities are dominated by members of the order Clostridiales. These bacteria appear to be naturally associated with the plant material, although slight variations between source materials were found. Additionally, heavy inoculations of pectinolytic bacteria established themselves and in addition their presence facilitated the rapid dominance of the original plant-associated Clostridiales. These data suggest that members of the order Clostridiales dominate the community and are most closely associated with efficient and effective retting. The results further suggest that establishment of the community structure is first driven by the switch to anaerobic conditions, and subsequently by possible competition for nitrogen. These findings reveal important bacterial groups involved in fiber retting, and suggest mechanisms for the manipulation of the community and retting efficiency by modifying nutrient availability.

Screening of bacterial strains isolated from uranium mill tailings porewaters for bioremediation purposes.

PubMed

Sánchez-Castro, Iván; Amador-García, Ahinara; Moreno-Romero, Cristina; López-Fernández, Margarita; Phrommavanh, Vannapha; Nos, Jeremy; Descostes, Michael; Merroun, Mohamed L

2017-01-01

The present work characterizes at different levels a number of bacterial strains isolated from porewaters sampled in the vicinity of two French uranium tailing repositories. The 16S rRNA gene from 33 bacterial isolates, corresponding to the different morphotypes recovered, was almost fully sequenced. The resulting sequences belonged to 13 bacterial genera comprised in the phyla Firmicutes, Actinobacteria and Proteobacteria. Further characterization at physiological level and metals/metalloid tolerance provided evidences for an appropriate selection of bacterial strains potentially useful for immobilization of uranium and other common contaminants. By using High Resolution Transmission Electron Microscope (HRTEM), this potential ability to immobilize uranium as U phosphate mineral phases was confirmed for the bacterial strains Br3 and Br5 corresponding to Arthrobacter sp. and Microbacterium oxydans, respectively. Scanning Transmission Electron Microscope- High-Angle Annular Dark-Field (STEM-HAADF) analysis showed U accumulates on the surface and within bacterial cytoplasm, in addition to the extracellular space. Energy Dispersive X-ray (EDX) element-distribution maps demonstrated the presence of U and P within these accumulates. These results indicate the potential of certain bacterial strains isolated from porewaters of U mill tailings for immobilizing uranium, likely as uranium phosphates. Some of these bacterial isolates might be considered as promising candidates in the design of uranium bioremediation strategies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Acute bacterial meningitis in adults.

PubMed

McGill, Fiona; Heyderman, Robert S; Panagiotou, Stavros; Tunkel, Allan R; Solomon, Tom

2016-12-17

Over the past several decades, the incidence of bacterial meningitis in children has decreased but there remains a significant burden of disease in adults, with a mortality of up to 30%. Although the pathogenesis of bacterial meningitis is not completely understood, knowledge of bacterial invasion and entry into the CNS is improving. Clinical features alone cannot determine whether meningitis is present and analysis of cerebrospinal fluid is essential for diagnosis. Newer technologies, such as multiplex PCR, and novel diagnostic platforms that incorporate proteomics and genetic sequencing, might help provide a quicker and more accurate diagnosis. Even with appropriate antimicrobial therapy, mortality is high and so attention has focused on adjunctive therapies; adjunctive corticosteroids are beneficial in certain circumstances. Any further improvements in outcome are likely to come from either modulation of the host response or novel approaches to therapy, rather than new antibiotics. Ultimately, the best hope to reduce the disease burden is with broadly protective vaccines. Copyright © 2016 Elsevier Ltd. All rights reserved.

Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

PubMed Central

Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

2001-01-01

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

Comprehensive Analysis of Bacterial Flora in Postoperative Maxillary Cyst Fluid by 16S rRNA Gene and Culture Methods

PubMed Central

Sano, Naoto; Yamashita, Yoshio; Fukuda, Kazumasa; Taniguchi, Hatsumi; Goto, Masaaki; Miyamoto, Hiroshi

2012-01-01

Intracystic fluid was aseptically collected from 11 patients with postoperative maxillary cyst (POMC), and DNA was extracted from the POMC fluid. Bacterial species were identified by sequencing after cloning of approximately 580 bp of the 16S rRNA gene. Identification of pathogenic bacteria was also performed by culture methods. The phylogenetic identity was determined by sequencing 517–596 bp in each of the 1139 16S rRNA gene clones. A total of 1114 clones were classified while the remaining 25 clones were unclassified. A total of 103 bacterial species belonging to 42 genera were identified in POMC fluid samples by 16S rRNA gene analysis. Species of Prevotella (91%), Neisseria (73%), Fusobacterium (73%), Porphyromonas (73%), and Propionibacterium (73%) were found to be highly prevalent in all patients. Streptococcus mitis (64%), Fusobacterium nucleatum (55%), Propionibacterium acnes (55%), Staphylococcus capitis (55%), and Streptococcus salivarius (55%) were detected in more than 6 of the 11 patients. The results obtained by the culture method were different from those obtained by 16S rRNA gene analysis, but both approaches may be necessary for the identification of pathogens, especially of bacteria that are difficult to detect by culture methods, and the development of rational treatments for patients with POMC. PMID:22685668

bcgTree: automatized phylogenetic tree building from bacterial core genomes.

PubMed

Ankenbrand, Markus J; Keller, Alexander

2016-10-01

The need for multi-gene analyses in scientific fields such as phylogenetics and DNA barcoding has increased in recent years. In particular, these approaches are increasingly important for differentiating bacterial species, where reliance on the standard 16S rDNA marker can result in poor resolution. Additionally, the assembly of bacterial genomes has become a standard task due to advances in next-generation sequencing technologies. We created a bioinformatic pipeline, bcgTree, which uses assembled bacterial genomes either from databases or own sequencing results from the user to reconstruct their phylogenetic history. The pipeline automatically extracts 107 essential single-copy core genes, found in a majority of bacteria, using hidden Markov models and performs a partitioned maximum-likelihood analysis. Here, we describe the workflow of bcgTree and, as a proof-of-concept, its usefulness in resolving the phylogeny of 293 publically available bacterial strains of the genus Lactobacillus. We also evaluate its performance in both low- and high-level taxonomy test sets. The tool is freely available at github ( https://github.com/iimog/bcgTree ) and our institutional homepage ( http://www.dna-analytics.biozentrum.uni-wuerzburg.de ).

Classifying the bacterial gut microbiota of termites and cockroaches: A curated phylogenetic reference database (DictDb).

PubMed

Mikaelyan, Aram; Köhler, Tim; Lampert, Niclas; Rohland, Jeffrey; Boga, Hamadi; Meuser, Katja; Brune, Andreas

2015-10-01

Recent developments in sequencing technology have given rise to a large number of studies that assess bacterial diversity and community structure in termite and cockroach guts based on large amplicon libraries of 16S rRNA genes. Although these studies have revealed important ecological and evolutionary patterns in the gut microbiota, classification of the short sequence reads is limited by the taxonomic depth and resolution of the reference databases used in the respective studies. Here, we present a curated reference database for accurate taxonomic analysis of the bacterial gut microbiota of dictyopteran insects. The Dictyopteran gut microbiota reference Database (DictDb) is based on the Silva database but was significantly expanded by the addition of clones from 11 mostly unexplored termite and cockroach groups, which increased the inventory of bacterial sequences from dictyopteran guts by 26%. The taxonomic depth and resolution of DictDb was significantly improved by a general revision of the taxonomic guide tree for all important lineages, including a detailed phylogenetic analysis of the Treponema and Alistipes complexes, the Fibrobacteres, and the TG3 phylum. The performance of this first documented version of DictDb (v. 3.0) using the revised taxonomic guide tree in the classification of short-read libraries obtained from termites and cockroaches was highly superior to that of the current Silva and RDP databases. DictDb uses an informative nomenclature that is consistent with the literature also for clades of uncultured bacteria and provides an invaluable tool for anyone exploring the gut community structure of termites and cockroaches. Copyright © 2015 Elsevier GmbH. All rights reserved.

Anti-Bacterial and Anti-Fungal Activity of Xanthones Obtained via Semi-Synthetic Modification of α-Mangostin from Garcinia mangostana.

PubMed

Narasimhan, Srinivasan; Maheshwaran, Shanmugam; Abu-Yousef, Imad A; Majdalawieh, Amin F; Rethavathi, Janarthanam; Das, Prince Edwin; Poltronieri, Palmiro

2017-02-12

The microbial contamination in food packaging has been a major concern that has paved the way to search for novel, natural anti-microbial agents, such as modified α-mangostin. In the present study, twelve synthetic analogs were obtained through semi-synthetic modification of α-mangostin by Ritter reaction, reduction by palladium-carbon (Pd-C), alkylation, and acetylation. The evaluation of the anti-microbial potential of the synthetic analogs showed higher bactericidal activity than the parent molecule. The anti-microbial studies proved that I E showed high anti-bacterial activity whereas I I showed the highest anti-fungal activity. Due to their microbicidal potential, modified α-mangostin derivatives could be utilized as active anti-microbial agents in materials for the biomedical and food industry.

Raw Cow Milk Bacterial Population Shifts Attributable to Refrigeration

PubMed Central

Lafarge, Véronique; Ogier, Jean-Claude; Girard, Victoria; Maladen, Véronique; Leveau, Jean-Yves; Gruss, Alexandra; Delacroix-Buchet, Agnès

2004-01-01

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4°C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database (∼150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4°C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4°C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated. PMID:15345453

Intestinal flora of FAP patients containing APC-like sequences.

PubMed

Hainova, K; Adamcikova, Z; Ciernikova, S; Stevurkova, V; Tyciakova, S; Zajac, V

2014-01-01

Colorectal cancer mortality is one of the most common cause of cancer-related mortality. A multiple risk factors are associated with colorectal cancer, including hereditary, enviromental and inflammatory syndromes affecting the gastrointestinal tract. Familial adenomatous polyposis (FAP) is characterized by the emergence of hundreds to thousands of colorectal adenomatous polyps and FAP syndrome is caused by mutations within the adenomatous polyposis coli (APC) tumor suppressor gene. We analyzed 21 rectal bacterial subclones isolated from FAP patient 41-1 with confirmed 5bp ACAAA deletion within codons 1060-1063 for the presence of APC-like sequences in longest exon 15. The studied section was defined by primers 15Efor-15Erev, what correlates with mutation cluster region (MCR) in which the 75% of all APC germline mutations were detected. More than 90% homology was showed by sequencing and subsequent software comparison. The expression of APC-like sequences was demostrated by Western blot analysis using monoclonal and polyclonal antibodies against APC protein. To study missing link between the DNA analysis (PCR, DNA sequencing) and protein expresion experiments (Western blotting) we analyzed bacterial transcripts containing the 15Efor-15Erev sequence of APC gene by reverse transcription-PCR, what indicated that an APC gene derived fragment may be produced. We observed 97-100 % homology after computer comparison of cDNA PCR products. Our results suggest that presence of APC-like sequences in intestinal/rectal bacteria is enrichment of bacterial genetic information in which horizontal gene transfer between humans and microflora play an important role.

Characterization of the Biodiversity of the Spoilage Microbiota in Chicken Meat Using Next Generation Sequencing and Culture Dependent Approach.

PubMed

Lee, Hee Soo; Kwon, Mirae; Heo, Sunhak; Kim, Min Gon; Kim, Geun-Bae

2017-01-01

This study investigated the psychrotrophic bacteria isolated from chicken meat to characterize their microbial composition during refrigerated storage. The bacterial community was identified by the Illumina MiSeq method based on bacterial DNA extracted from spoiled chicken meat. Molecular identification of the isolated psychrotrophic bacteria was carried out using 16S rDNA sequencing and their putrefactive potential was investigated by the growth at low temperature as well as their proteolytic activities in chicken meat. From the Illumina sequencing, a total of 187,671 reads were obtained from 12 chicken samples. Regardless of the type of chicken meat (i.e., whole meat and chicken breast) and storage temperatures (4°C and 10°C), Pseudomonas weihenstephanensis and Pseudomonas congelans were the most prominent bacterial species. Serratia spp. and Acinetobacter spp. were prominent in chicken breast and whole chicken meat, respectively. The 118 isolated strains of psychrotrophic bacteria comprised Pseudomonas spp. (58.48%), Serratia spp. (10.17%), and Morganella spp. (6.78%). All isolates grew well at 10°C and they induced different proteolytic activities depending on the species and strains. Parallel analysis of the next generation sequencing and culture dependent approach provides in-depth information on the biodiversity of the spoilage microbiota in chicken meat. Further study is needed to develop better preservation methods against these spoilage bacteria.

Office space bacterial abundance and diversity in three metropolitan areas.

PubMed

Hewitt, Krissi M; Gerba, Charles P; Maxwell, Sheri L; Kelley, Scott T

2012-01-01

People in developed countries spend approximately 90% of their lives indoors, yet we know little about the source and diversity of microbes in built environments. In this study, we combined culture-based cell counting and multiplexed pyrosequencing of environmental ribosomal RNA (rRNA) gene sequences to investigate office space bacterial diversity in three metropolitan areas. Five surfaces common to all offices were sampled using sterile double-tipped swabs, one tip for culturing and one for DNA extraction, in 30 different offices per city (90 offices, 450 total samples). 16S rRNA gene sequences were PCR amplified using bar-coded "universal" bacterial primers from 54 of the surfaces (18 per city) and pooled for pyrosequencing. A three-factorial Analysis of Variance (ANOVA) found significant differences in viable bacterial abundance between offices inhabited by men or women, among the various surface types, and among cities. Multiplex pyrosequencing identified more than 500 bacterial genera from 20 different bacterial divisions. The most abundant of these genera tended to be common inhabitants of human skin, nasal, oral or intestinal cavities. Other commonly occurring genera appeared to have environmental origins (e.g., soils). There were no significant differences in the bacterial diversity between offices inhabited by men or women or among surfaces, but the bacterial community diversity of the Tucson samples was clearly distinguishable from that of New York and San Francisco, which were indistinguishable. Overall, our comprehensive molecular analysis of office building microbial diversity shows the potential of these methods for studying patterns and origins of indoor bacterial contamination. "[H]umans move through a sea of microbial life that is seldom perceived except in the context of potential disease and decay." - Feazel et al. (2009).

Changes in Metabolically Active Bacterial Community during Rumen Development, and Their Alteration by Rhubarb Root Powder Revealed by 16S rRNA Amplicon Sequencing.

PubMed

Wang, Zuo; Elekwachi, Chijioke; Jiao, Jinzhen; Wang, Min; Tang, Shaoxun; Zhou, Chuanshe; Tan, Zhiliang; Forster, Robert J

2017-01-01

The objective of this present study was to explore the initial establishment of metabolically active bacteria and subsequent evolution in four fractions: rumen solid-phase (RS), liquid-phase (RL), protozoa-associated (RP), and epithelium-associated (RE) through early weaning and supplementing rhubarb root powder in 7 different age groups (1, 10, 20, 38, 41, 50, and 60 d) during rumen development. Results of the 16S rRNA sequencing based on RNA isolated from the four fractions revealed that the potentially active bacterial microbiota in four fractions were dominated by the phyla Proteobacteria, Firmicutes , and Bacteroidetes regardless of different ages. An age-dependent increment of Chao 1 richness was observed in the fractions of RL and RE. The principal coordinate analysis (PCoA) indicated that samples in four fractions all clustered based on different age groups, and the structure of the bacterial community in RE was distinct from those in other three fractions. The abundances of Proteobacteria decreased significantly ( P < 0.05) with age, while increases in the abundances of Firmicutes and Bacteroidetes were noted. At the genus level, the abundance of the predominant genus Mannheimia in the Proteobacteria phylum decreased significantly ( P < 0.05) after 1 d, while the genera Quinella, Prevotella, Fretibacterium, Ruminococcus, Lachnospiraceae NK3A20 group , and Atopobium underwent different manners of increases and dominated the bacterial microbiota across four fractions. Variations of the distributions of some specific bacterial genera across fractions were observed, and supplementation of rhubarb affected the relative abundance of various genera of bacteria.

Changes in Metabolically Active Bacterial Community during Rumen Development, and Their Alteration by Rhubarb Root Powder Revealed by 16S rRNA Amplicon Sequencing

PubMed Central

Wang, Zuo; Elekwachi, Chijioke; Jiao, Jinzhen; Wang, Min; Tang, Shaoxun; Zhou, Chuanshe; Tan, Zhiliang; Forster, Robert J.

2017-01-01

The objective of this present study was to explore the initial establishment of metabolically active bacteria and subsequent evolution in four fractions: rumen solid-phase (RS), liquid-phase (RL), protozoa-associated (RP), and epithelium-associated (RE) through early weaning and supplementing rhubarb root powder in 7 different age groups (1, 10, 20, 38, 41, 50, and 60 d) during rumen development. Results of the 16S rRNA sequencing based on RNA isolated from the four fractions revealed that the potentially active bacterial microbiota in four fractions were dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes regardless of different ages. An age-dependent increment of Chao 1 richness was observed in the fractions of RL and RE. The principal coordinate analysis (PCoA) indicated that samples in four fractions all clustered based on different age groups, and the structure of the bacterial community in RE was distinct from those in other three fractions. The abundances of Proteobacteria decreased significantly (P < 0.05) with age, while increases in the abundances of Firmicutes and Bacteroidetes were noted. At the genus level, the abundance of the predominant genus Mannheimia in the Proteobacteria phylum decreased significantly (P < 0.05) after 1 d, while the genera Quinella, Prevotella, Fretibacterium, Ruminococcus, Lachnospiraceae NK3A20 group, and Atopobium underwent different manners of increases and dominated the bacterial microbiota across four fractions. Variations of the distributions of some specific bacterial genera across fractions were observed, and supplementation of rhubarb affected the relative abundance of various genera of bacteria. PMID:28223972

Multilocus sequence typing of total-genome-sequenced bacteria.

PubMed

Larsen, Mette V; Cosentino, Salvatore; Rasmussen, Simon; Friis, Carsten; Hasman, Henrik; Marvig, Rasmus Lykke; Jelsbak, Lars; Sicheritz-Pontén, Thomas; Ussery, David W; Aarestrup, Frank M; Lund, Ole

2012-04-01

Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

Genomic library screening for viruses from the human dental plaque revealed pathogen-specific lytic phage sequences.

PubMed

Al-Jarbou, Ahmed Nasser

2012-01-01

Bacterial pathogenesis presents an astounding arsenal of virulence factors that allow them to conquer many different niches throughout the course of infection. Principally fascinating is the fact that some bacterial species are able to induce different diseases by expression of different combinations of virulence factors. Nevertheless, studies aiming at screening for the presence of bacteriophages in humans have been limited. Such screening procedures would eventually lead to identification of phage-encoded properties that impart increased bacterial fitness and/or virulence in a particular niche, and hence, would potentially be used to reverse the course of bacterial infections. As the human oral cavity represents a rich and dynamic ecosystem for several upper respiratory tract pathogens. However, little is known about virus diversity in human dental plaque which is an important reservoir. We applied the culture-independent approach to characterize virus diversity in human dental plaque making a library from a virus DNA fraction amplified using a multiple displacement method and sequenced 80 clones. The resulting sequence showed 44% significant identities to GenBank databases by TBLASTX analysis. TBLAST homology comparisons showed that 66% was viral; 18% eukarya; 10% bacterial; 6% mobile elements. These sequences were sorted into 6 contigs and 45 single sequences in which 4 contigs and a single sequence showed significant identity to a small region of a putative prophage in the Corynebacterium diphtheria genome. These findings interestingly highlight the uniqueness of over half of the sequences, whilst the dominance of a pathogen-specific prophage sequences imply their role in virulence.

Plants of the fynbos biome harbour host species-specific bacterial communities.

PubMed

Miyambo, Tsakani; Makhalanyane, Thulani P; Cowan, Don A; Valverde, Angel

2016-08-01

The fynbos biome in South Africa is globally recognised as a plant biodiversity hotspot. However, very little is known about the bacterial communities associated with fynbos plants, despite interactions between primary producers and bacteria having an impact on the physiology of both partners and shaping ecosystem diversity. This study reports on the structure, phylogenetic composition and potential roles of the endophytic bacterial communities located in the stems of three fynbos plants (Erepsia anceps, Phaenocoma prolifera and Leucadendron laureolum). Using Illumina MiSeq 16S rRNA sequencing we found that different subpopulations of Deinococcus-Thermus, Alphaproteobacteria, Acidobacteria and Firmicutes dominated the endophytic bacterial communities. Alphaproteobacteria and Actinobacteria were prevalent in P. prolifera, whereas Deinococcus-Thermus dominated in L. laureolum, revealing species-specific host-bacteria associations. Although a high degree of variability in the endophytic bacterial communities within hosts was observed, we also detected a core microbiome across the stems of the three plant species, which accounted for 72% of the sequences. Altogether, it seems that both deterministic and stochastic processes shaped microbial communities. Endophytic bacterial communities harboured putative plant growth-promoting bacteria, thus having the potential to influence host health and growth. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach

PubMed Central

Jacquiod, Samuel; Franqueville, Laure; Cécillon, Sébastien; M. Vogel, Timothy; Simonet, Pascal

2013-01-01

Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome. PMID:24278158

Comprehensive Molecular Characterization of Bacterial Communities in Feces of Pet Birds Using 16S Marker Sequencing.

PubMed

Garcia-Mazcorro, Jose F; Castillo-Carranza, Stephany A; Guard, Blake; Gomez-Vazquez, Jose P; Dowd, Scot E; Brigthsmith, Donald J

2017-01-01

Birds and other animals live and evolve in close contact with millions of microorganisms (microbiota). While the avian microbiota has been well characterized in domestic poultry, the microbiota of other bird species has been less investigated. The aim of this study was to describe the fecal bacterial communities of pet birds. Pooled fecal samples from 22 flocks representing over 150 individual birds of three different species (Melopsittacus undulatus or budgerigars, Nymphicus hollandicus or cockatiels, and Serinus canaria or domestic canaries) were used for analysis using the 16S rRNA gene sequencing in the MiSeq platform (Illumina). Firmicutes was the most abundant phylum (median 88.4 %; range 12.9-98.4 %) followed by other low-abundant phyla such as Proteobacteria (median 2.3 %; 0.1-85.3 %) and Actinobacteria (median 1.7 %; 0-18.3 %). Lactobacillaceae (mostly Lactobacillus spp.) was the most abundant family (median 78.1 %; 1.4-97.5 %), especially in budgerigars and canaries, and it deserves attention because of the ascribed beneficial properties of lactic acid bacteria. Importantly, feces from birds contain intestinal, urinary, and reproductive-associated microbiota thus posing a serious problem to study one anatomical region at a time. Other groups of interest include the family Clostridiaceae that showed very low abundance (overall median <0.1 %) with the exception of two samples from cockatiels (14 and 45.9 %) and one sample from budgerigars (19.9 %). Analysis of UniFrac metrics showed that overall, the microbial communities from the 22 flocks tended to cluster together for each bird species, meaning each species shed distinctive bacterial communities in feces. This descriptive analysis provides insight into the fecal microbiota of pet birds.

Harnessing CRISPR-Cas systems for bacterial genome editing.

PubMed

Selle, Kurt; Barrangou, Rodolphe

2015-04-01

Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity.

PubMed

Illeghems, Koen; De Vuyst, Luc; Papalexandratou, Zoi; Weckx, Stefan

2012-01-01

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.

Spatiotemporal changes in bacterial community and microbial activity in a full-scale drinking water treatment plant.

PubMed

Hou, Luanfeng; Zhou, Qin; Wu, Qingping; Gu, Qihui; Sun, Ming; Zhang, Jumei

2018-06-01

To gain insight into the bacterial dynamics present in drinking water treatment (DWT) systems, the microbial community and activity in a full-scale DWT plant (DWTP) in Guangzhou, South China, were investigated using Illumina Hiseq sequencing analyses combined with cultivation-based techniques during the wet and dry seasons. Illumina sequencing analysis of 16S rRNA genes revealed a large shift in the proportion of Actinobacteria, Proteobacteria and Firmicutes during the treatment process, with the proportion of Actinobacteria decreased sharply, whereas that of Proteobacteria and Firmicutes increased and predominated in treated water. Both microbial activity and bacterial diversity during the treatment process showed obvious spatial variation, with higher levels observed during the dry season and lower levels during the wet season. Clustering analysis and principal component analysis indicated dramatic shifts in the bacterial community after chlorination, suggesting that chlorination was highly effective at influencing the bacterial community. The bacterial community structure of finished water primarily comprised Pseudomonas, Citrobacter, and Acinetobacter, and interestingly showed high similarity to biofilms on granular activated carbon. Additionally, the abundance of bacterial communities was relatively stable in finished water and did not change with the season. A large number of unique operational taxonomic units were shared during treatment steps, indicating the presence of a diverse core microbiome throughout the treatment process. Opportunistic pathogens, including Pseudomonas, Acinetobacter, Citrobacter, Mycobacterium, Salmonella, Staphylococcus, Legionella, Streptococcus and Enterococcus, were detected in water including finished water, suggesting a potential threat to drinking-water safety. We also detected bacteria isolated from each treatment step using the pure-culture method. In particular, two isolates, identified as Mycobacterium sp. and Blastococcus

Spatial variations of bacterial community and its relationship with water chemistry in Sanya Bay, South China Sea as determined by DGGE fingerprinting and multivariate analysis.

PubMed

Ling, Juan; Zhang, Yan-Ying; Dong, Jun-De; Wang, You-Shao; Feng, Jing-Bing; Zhou, Wei-Hua

2015-10-01

Bacteria play important roles in the structure and function of marine food webs by utilizing nutrients and degrading the pollutants, and their distribution are determined by surrounding water chemistry to a certain extent. It is vital to investigate the bacterial community's structure and identifying the significant factors by controlling the bacterial distribution in the paper. Flow cytometry showed that the total bacterial abundance ranged from 5.27 × 10(5) to 3.77 × 10(6) cells/mL. Molecular fingerprinting technique, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing has been employed to investigate the bacterial community composition. The results were then interpreted through multivariate statistical analysis and tended to explain its relationship to the environmental factors. A total of 270 bands at 83 different positions were detected in DGGE profiles and 29 distinct DGGE bands were sequenced. The predominant bacteria were related to Phyla Protebacteria species (31 %, nine sequences), Cyanobacteria (37.9 %, eleven sequences) and Actinobacteria (17.2 %, five sequences). Other phylogenetic groups identified including Firmicutes (6.9 %, two sequences), Bacteroidetes (3.5 %, one sequences) and Verrucomicrobia (3.5 %, one sequences). Conical correspondence analysis was used to elucidate the relationships between the bacterial community compositions and environmental factors. The results showed that the spatial variations in the bacterial community composition was significantly related to phosphate (P = 0.002, P < 0.01), dissolved organic carbon (P = 0.004, P < 0.01), chemical oxygen demand (P = 0.010, P < 0.05) and nitrite (P = 0.016, P < 0.05). This study revealed the spatial variations of bacterial community and significant environmental factors driving the bacterial composition shift. These results may be valuable for further investigation on the functional microbial structure and expression quantitatively under the polluted

Dietary and developmental shifts in butterfly-associated bacterial communities

PubMed Central

2018-01-01

Bacterial communities associated with insects can substantially influence host ecology, evolution and behaviour. Host diet is a key factor that shapes bacterial communities, but the impact of dietary transitions across insect development is poorly understood. We analysed bacterial communities of 12 butterfly species across different developmental stages, using amplicon sequencing of the 16S rRNA gene. Butterfly larvae typically consume leaves of a single host plant, whereas adults are more generalist nectar feeders. Thus, we expected bacterial communities to vary substantially across butterfly development. Surprisingly, only few species showed significant dietary and developmental transitions in bacterial communities, suggesting weak impacts of dietary transitions across butterfly development. On the other hand, bacterial communities were strongly influenced by butterfly species and family identity, potentially due to dietary and physiological variation across the host phylogeny. Larvae of most butterfly species largely mirrored bacterial community composition of their diets, suggesting passive acquisition rather than active selection. Overall, our results suggest that although butterflies harbour distinct microbiomes across taxonomic groups and dietary guilds, the dramatic dietary shifts that occur during development do not impose strong selection to maintain distinct bacterial communities across all butterfly hosts. PMID:29892359

[The composition of the gastrointestinal bacterial flora of mouse embryos and the placenta tissue bacterial flora].

PubMed

Lei, D; Lin, Y; Jiang, X; Lan, L; Zhang, W; Wang, B X

2017-03-02

Objective: To explore the composition of the gastrointestinal bacterial flora of mouse embryos and the placenta tissue bacterial flora. Method: Twenty-four specimens were collected from pregnant Kunming mouse including 8 mice of early embryonic (12-13 days) gastrointestinal tissues, 8 cases of late embryonic (19-20 days)gastrointestinal tissues, 8 of late pregnancy placental tissues.The 24 samples were extracted by DNeasy Blood & Tissue kit for high-throughput DNA sequencing. Result: The level of Proteobacteria, Bacteroidetes, Actino-bacteria and Firmicutes were predominantin all specimens.The relative content of predominant bacterial phyla in each group: Proteobacteria (95.00%, 88.14%, 87.26%), Bacteroidetes(1.71%, 2.15%, 2.63%), Actino-Bacteria(1.16%, 4.10%, 3.38%), Firmicutes(0.75%, 2.62%, 2.01%). At the level of family, there were nine predominant bacterial families in which Enterobacteriaeae , Shewanel laceae and Moraxellaceae were dominant.The relative content of dominant bacterial family in eachgroup: Enterobacteriaeae (46.99%, 44.34%, 41.08%), Shewanellaceae (21.99%, 21.10%, 19.05%), Moraxellaceae (9.18%, 7.09%, 5.64%). From the species of flora, the flora from fetal gastrointestinal in early pregnancy and late pregnancy (65.44% and 62.73%) were the same as that from placenta tissue in the late pregnancy.From the abundance of bacteria, at the level of family, the same content of bacteria in three groups accounted for 78.16%, 72.53% and 65.78% respectively. Conclusion: It was proved that the gastrointestinal bacterial flora of mouse embryos and the placenta tissue bacterial flora were colonized. At the same time the bacteria are classified.

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PubMed

Nakano, Kazuma; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Minami, Maiko; Nakanishi, Tetsuhiro; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

2017-07-01

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II's sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.

Defining the Estimated Core Genome of Bacterial Populations Using a Bayesian Decision Model

PubMed Central

van Tonder, Andries J.; Mistry, Shilan; Bray, James E.; Hill, Dorothea M. C.; Cody, Alison J.; Farmer, Chris L.; Klugman, Keith P.; von Gottberg, Anne; Bentley, Stephen D.; Parkhill, Julian; Jolley, Keith A.; Maiden, Martin C. J.; Brueggemann, Angela B.

2014-01-01

The bacterial core genome is of intense interest and the volume of whole genome sequence data in the public domain available to investigate it has increased dramatically. The aim of our study was to develop a model to estimate the bacterial core genome from next-generation whole genome sequencing data and use this model to identify novel genes associated with important biological functions. Five bacterial datasets were analysed, comprising 2096 genomes in total. We developed a Bayesian decision model to estimate the number of core genes, calculated pairwise evolutionary distances (p-distances) based on nucleotide sequence diversity, and plotted the median p-distance for each core gene relative to its genome location. We designed visually-informative genome diagrams to depict areas of interest in genomes. Case studies demonstrated how the model could identify areas for further study, e.g. 25% of the core genes with higher sequence diversity in the Campylobacter jejuni and Neisseria meningitidis genomes encoded hypothetical proteins. The core gene with the highest p-distance value in C. jejuni was annotated in the reference genome as a putative hydrolase, but further work revealed that it shared sequence homology with beta-lactamase/metallo-beta-lactamases (enzymes that provide resistance to a range of broad-spectrum antibiotics) and thioredoxin reductase genes (which reduce oxidative stress and are essential for DNA replication) in other C. jejuni genomes. Our Bayesian model of estimating the core genome is principled, easy to use and can be applied to large genome datasets. This study also highlighted the lack of knowledge currently available for many core genes in bacterial genomes of significant global public health importance. PMID:25144616

Use of tuf Sequences for Genus-Specific PCR Detection and Phylogenetic Analysis of 28 Streptococcal Species

PubMed Central

Picard, François J.; Ke, Danbing; Boudreau, Dominique K.; Boissinot, Maurice; Huletsky, Ann; Richard, Dave; Ouellette, Marc; Roy, Paul H.; Bergeron, Michel G.

2004-01-01

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence. PMID:15297518

Characterizing bacterial communities in tilapia pond surface sediment and their responses to pond differences and temporal variations.

PubMed

Fan, Limin; Barry, Kamira; Hu, Gengdong; Meng, Shunlong; Song, Chao; Qiu, Liping; Zheng, Yao; Wu, Wei; Qu, Jianhong; Chen, Jiazhang; Xu, Pao

2017-01-01

Bacterial community compositions in the surface sediment of tilapia ponds and their responses to pond characteristics or seasonal variations were investigated. For that, three ponds with different stocking densities were selected to collect the samples. And the method of Illumina high-throughput sequencing was used to amplify the bacterial 16S rRNA genes. A total of 662, 876 valid reads and 5649 operational taxonomic units were obtained. Further analysis showed that the dominant phyla in all three ponds were Proteobacteria, Bacteroidetes, Chloroflexi, and Acidobacteria. The phyla Planctomycetes, Firmicutes, Chlorobi, and Spirochaetae were also relatively abundant. Among the eight phyla, the abundances of only Proteobacteria, Bacteroidetes, Firmicutes, and Spirochaetae were affected by seasonal variations, while seven of these (with the exception of Acidobacteria) were affected by pond differences. A comprehensive analysis of the richness and diversity of the bacterial 16S rRNA gene, and of the similarity in bacterial community composition in sediment also showed that the communities in tilapia pond sediment were shaped more by pond differences than by seasonal variations. Linear discriminant analysis further indicated that the influences of pond characteristics on sediment bacterial communities might be related to feed coefficients and stocking densities of genetically improved farmed tilapia (GIFT).

Global biogeographic sampling of bacterial secondary metabolism

PubMed Central

Charlop-Powers, Zachary; Owen, Jeremy G; Reddy, Boojala Vijay B; Ternei, Melinda A; Guimarães, Denise O; de Frias, Ulysses A; Pupo, Monica T; Seepe, Prudy; Feng, Zhiyang; Brady, Sean F

2015-01-01

Recent bacterial (meta)genome sequencing efforts suggest the existence of an enormous untapped reservoir of natural-product-encoding biosynthetic gene clusters in the environment. Here we use the pyro-sequencing of PCR amplicons derived from both nonribosomal peptide adenylation domains and polyketide ketosynthase domains to compare biosynthetic diversity in soil microbiomes from around the globe. We see large differences in domain populations from all except the most proximal and biome-similar samples, suggesting that most microbiomes will encode largely distinct collections of bacterial secondary metabolites. Our data indicate a correlation between two factors, geographic distance and biome-type, and the biosynthetic diversity found in soil environments. By assigning reads to known gene clusters we identify hotspots of biomedically relevant biosynthetic diversity. These observations not only provide new insights into the natural world, they also provide a road map for guiding future natural products discovery efforts. DOI: http://dx.doi.org/10.7554/eLife.05048.001 PMID:25599565

Bacterial community dynamic associated with autochthonous bioaugmentation for enhanced Cu phytoremediation of salt-marsh sediments.

PubMed

Almeida, C Marisa R; Oliveira, Tânia; Reis, Izabela; Gomes, Carlos R; Mucha, Ana P

2017-12-01

Autochthonous bioaugmentation for metal phytoremediation is still little explored, particularly its application to estuarine salt marshes, but results obtained so far are promising. Nevertheless, understanding the behaviour of the microbial communities in the process of bioaugmentation and their role in improving metal phytoremediation is very important to fully validate the application of this biological technology. This study aimed to characterize the bacterial community dynamic associated with the application of autochthonous bioaugmentation in an experimentation which showed that Phragmites australis rhizosphere microorganisms could increase this salt marsh plant potential to phytoremediate Cu contaminated sediments. Bacterial communities present in the autochthonous microbial consortium resistant to Cu added to the medium and in the sediment at the beginning and at the end of the experiment were characterized by ARISA. Complementarily, the consortium and the sediment used for its production were characterized by next generation sequencing using the pyrosequencing platform 454. The microbial consortium resistant to Cu obtained from non-vegetated sediment was dominated by the genus Lactococcus (46%), Raoultella (25%), Bacillus (12%) and Acinetobacter (11%), whereas the one obtained form rhizosediment was dominated by the genus Gluconacetobacter (77%), Bacillus (17%) and Dyella (3%). Results clearly showed that, after two months of experiment, Cu caused a shift in the bacterial community structure of sediments, an effect that was observed either with or without addition of the metal resistant microbial consortium. Therefore, bioaugmentation application improved the process of phytoremediation (metal translocation by the plant was increased) without inducing long term changes in the bacterial community structure of the sediments. So, phytoremediation combined with autochthonous bioaugmentation can be a suitable technology for the recovery of estuarine areas

Census of bacterial microbiota associated with the glacier ice worm Mesenchytraeus solifugus.

PubMed

Murakami, Takumi; Segawa, Takahiro; Bodington, Dylan; Dial, Roman; Takeuchi, Nozomu; Kohshima, Shiro; Hongoh, Yuichi

2015-03-01

The glacier ice worm, Mesenchytraeus solifugus, is a unique annelid, inhabiting only snow and ice in North American glaciers. Here, we analyzed the taxonomic composition of bacteria associated with M. solifugus based on the 16S rRNA gene. We analyzed four fixed-on-site and 10 starved ice worm individuals, along with glacier surface samples. In total, 1341 clones of 16S rRNA genes were analyzed for the ice worm samples, from which 65 bacterial phylotypes (99.0% cut-off) were identified. Of these, 35 phylotypes were closely related to sequences obtained from their habitat glacier and/or other components of cryosphere; whereas three dominant phylotypes were affiliated with animal-associated lineages of the class Mollicutes. Among the three, phylotype Ms-13 shared less than 89% similarity with database sequences and was closest to a gut symbiont of a terrestrial earthworm. Using fluorescence in situ hybridization, Ms-13 was located on the gut wall surface of the ice worms. We propose a novel genus and species, 'Candidatus Vermiplasma glacialis', for this bacterium. Our results raise the possibility that the ice worm has exploited indigenous glacier bacteria, while several symbiotic bacterial lineages have maintained their association with the ice worm during the course of adaptive evolution to the permanently cold environment. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Succession of the turkey gastrointestinal bacterial microbiome related to weight gain.

PubMed

Danzeisen, Jessica L; Calvert, Alamanda J; Noll, Sally L; McComb, Brian; Sherwood, Julie S; Logue, Catherine M; Johnson, Timothy J

2013-12-23

Because of concerns related to the use of antibiotics in animal agriculture, antibiotic-free alternatives are greatly needed to prevent disease and promote animal growth. One of the current challenges facing commercial turkey production in Minnesota is difficulty obtaining flock average weights typical of the industry standard, and this condition has been coined "Light Turkey Syndrome" or LTS. This condition has been identified in Minnesota turkey flocks for at least five years, and it has been observed that average flock body weights never approach their genetic potential. However, a single causative agent responsible for these weight reductions has not been identified despite numerous efforts to do so. The purpose of this study was to identify the bacterial community composition within the small intestines of heavy and light turkey flocks using 16S rRNA sequencing, and to identify possible correlations between microbiome and average flock weight. This study also sought to define the temporal succession of bacteria occurring in the turkey ileum. Based upon 2.7 million sequences across nine different turkey flocks, dominant operational taxonomic units (OTUs) were identified and compared between the flocks studied. OTUs that were associated with heavier weight flocks included those with similarity to Candidatus division Arthromitus and Clostridium bartlettii, while these flocks had decreased counts of several Lactobacillus species compared to lighter weight flocks. The core bacterial microbiome succession in commercial turkeys was also defined. Several defining markers of microbiome succession were identified, including the presence or abundance of Candidatus division Arthromitus, Lactobacillus aviarius, Lactobacillus ingluviei, Lactobacillus salivarius, and Clostridium bartlettii. Overall, the succession of the ileum bacterial microbiome in commercial turkeys proceeds in a predictable manner. Efforts to prevent disease and promote growth in the absence of

Succession of the turkey gastrointestinal bacterial microbiome related to weight gain

PubMed Central

Danzeisen, Jessica L.; Calvert, Alamanda J.; Noll, Sally L.; McComb, Brian; Sherwood, Julie S.; Logue, Catherine M.

2013-01-01

Because of concerns related to the use of antibiotics in animal agriculture, antibiotic-free alternatives are greatly needed to prevent disease and promote animal growth. One of the current challenges facing commercial turkey production in Minnesota is difficulty obtaining flock average weights typical of the industry standard, and this condition has been coined “Light Turkey Syndrome” or LTS. This condition has been identified in Minnesota turkey flocks for at least five years, and it has been observed that average flock body weights never approach their genetic potential. However, a single causative agent responsible for these weight reductions has not been identified despite numerous efforts to do so. The purpose of this study was to identify the bacterial community composition within the small intestines of heavy and light turkey flocks using 16S rRNA sequencing, and to identify possible correlations between microbiome and average flock weight. This study also sought to define the temporal succession of bacteria occurring in the turkey ileum. Based upon 2.7 million sequences across nine different turkey flocks, dominant operational taxonomic units (OTUs) were identified and compared between the flocks studied. OTUs that were associated with heavier weight flocks included those with similarity to Candidatus division Arthromitus and Clostridium bartlettii, while these flocks had decreased counts of several Lactobacillus species compared to lighter weight flocks. The core bacterial microbiome succession in commercial turkeys was also defined. Several defining markers of microbiome succession were identified, including the presence or abundance of Candidatus division Arthromitus, Lactobacillus aviarius, Lactobacillus ingluviei, Lactobacillus salivarius, and Clostridium bartlettii. Overall, the succession of the ileum bacterial microbiome in commercial turkeys proceeds in a predictable manner. Efforts to prevent disease and promote growth in the absence of

Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.

PubMed Central

Pena, S D; Barreto, G; Vago, A R; De Marco, L; Reinach, F C; Dias Neto, E; Simpson, A J

1994-01-01

Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA. Images PMID:8127912

Effect of disopyramide on bacterial diversity in drinking water

NASA Astrophysics Data System (ADS)

Wu, Qing; Zhao, Xiaofei; Tian, Qi; Wang, Lei; Zhao, Xinhua

2018-02-01

Disopyramide was detected in drinking water by LC-MS/MS and the microbial diversity was investigated by PCR and high-throughput sequencing. The results showed that bacteria community structure in drinking water changed a lot when added different concentrations of disopyramide. The results of Shannon index showed that the total number and abundance of bacterial community species in drinking water samples decreased significantly after the addition of disopyramide. However, the number and abundance of community structure did not change with the concentration of disopyramide. Disopyramide inhibits the activity of bacterial community in drinking water and also can reduce the bacterial community diversity in drinking water.

Bacterial Genome Instability

PubMed Central

Darmon, Elise

2014-01-01

SUMMARY Bacterial genomes are remarkably stable from one generation to the next but are plastic on an evolutionary time scale, substantially shaped by horizontal gene transfer, genome rearrangement, and the activities of mobile DNA elements. This implies the existence of a delicate balance between the maintenance of genome stability and the tolerance of genome instability. In this review, we describe the specialized genetic elements and the endogenous processes that contribute to genome instability. We then discuss the consequences of genome instability at the physiological level, where cells have harnessed instability to mediate phase and antigenic variation, and at the evolutionary level, where horizontal gene transfer has played an important role. Indeed, this ability to share DNA sequences has played a major part in the evolution of life on Earth. The evolutionary plasticity of bacterial genomes, coupled with the vast numbers of bacteria on the planet, substantially limits our ability to control disease. PMID:24600039

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys

PubMed Central

Werner, Jeffrey J; Koren, Omry; Hugenholtz, Philip; DeSantis, Todd Z; Walters, William A; Caporaso, J Gregory; Angenent, Largus T; Knight, Rob; Ley, Ruth E

2012-01-01

Taxonomic classification of the thousands–millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases. PMID:21716311

Composition and variation of sediment bacterial and nirS-harboring bacterial communities at representative sites of the Bohai Gulf coastal zone, China.

PubMed

Guan, Xiangyu; Zhu, Lingling; Li, Youxun; Xie, Yuxuan; Zhao, Mingzhang; Luo, Ximing

2014-04-01

With rapid urbanization, anthropogenic activities are increasingly influencing the natural environment of the Bohai Bay. In this study, the composition and variation of bacterial and nirS-harboring bacterial communities in the coastal zone sediments of the Bohai Gulf were analyzed using PCR-based clone libraries. A total of 95 genera were detected in the bacterial communities, with Proteobacteria (72.1 %), Acidobacteria (10.5 %), Firmicutes (1.7 %), Bacteroidetes (1.4 %), Chloroflexi (0.7 %) and Planctomycetes (0.7 %) being the dominated phyla. The NirS sequences were divided into nine Clusters (A-I). Canonical correlation analysis showed that the bacterial or denitrifying communities were correlated with different environmental factors, such as total organic carbon, total nitrogen, ammonium, sulfate, etc. Furthermore, bacterial communities' composition and diversity are influenced by oil exploration, sewage discharge and other anthropogenic activities in the coastal area of the Bohai Sea. Thus, this study provided useful information on further research on regional or global environmental control and restore.

Natural bacterial communities serve as quantitative geochemical biosensors.

PubMed

Smith, Mark B; Rocha, Andrea M; Smillie, Chris S; Olesen, Scott W; Paradis, Charles; Wu, Liyou; Campbell, James H; Fortney, Julian L; Mehlhorn, Tonia L; Lowe, Kenneth A; Earles, Jennifer E; Phillips, Jana; Techtmann, Steve M; Joyner, Dominique C; Elias, Dwayne A; Bailey, Kathryn L; Hurt, Richard A; Preheim, Sarah P; Sanders, Matthew C; Yang, Joy; Mueller, Marcella A; Brooks, Scott; Watson, David B; Zhang, Ping; He, Zhili; Dubinsky, Eric A; Adams, Paul D; Arkin, Adam P; Fields, Matthew W; Zhou, Jizhong; Alm, Eric J; Hazen, Terry C

2015-05-12

Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts. Copyright © 2015 Smith et al.

Natural bacterial communities serve as quantitative geochemical biosensors

DOE PAGES

Smith, Mark B.; Rocha, Andrea M.; Smillie, Chris S.; ...

2015-05-12

Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination,more » even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.« less