Genetic diversity assessment of anoxygenic photosynthetic bacteria by distance-based grouping analysis of pufM sequences.

PubMed

Zeng, Y H; Chen, X H; Jiao, N Z

2007-12-01

To assess how completely the diversity of anoxygenic phototrophic bacteria (APB) was sampled in natural environments. All nucleotide sequences of the APB marker gene pufM from cultures and environmental clones were retrieved from the GenBank database. A set of cutoff values (sequence distances 0.06, 0.15 and 0.48 for species, genus, and (sub)phylum levels, respectively) was established using a distance-based grouping program. Analysis of the environmental clones revealed that current efforts on APB isolation and sampling in natural environments are largely inadequate. Analysis of the average distance between each identified genus and an uncultured environmental pufM sequence indicated that the majority of cultured APB genera lack environmental representatives. The distance-based grouping method is fast and efficient for bulk functional gene sequences analysis. The results clearly show that we are at a relatively early stage in sampling the global richness of APB species. Periodical assessment will undoubtedly facilitate in-depth analysis of potential biogeographical distribution pattern of APB. This is the first attempt to assess the present understanding of APB diversity in natural environments. The method used is also useful for assessing the diversity of other functional genes.

Effects of Working Couple's Retirement Sequence on Satisfaction in Patriarchal Culture Country: Probing on Gender Difference.

PubMed

Lee, Ayoung; Cho, Joonmo

2017-01-01

We examined the effects of the differences in the retirement sequence (i.e., who retires first between spouses) on satisfaction in Korea of patriarchal culture. Our empirical study demonstrates that households where men retired first had a much lower satisfaction than households where women retired first. In addition, men were found to show lower satisfaction than wives in both households where women retire first and the households where men retire first. Retirement sequence affecting their satisfaction at the point when only one of the spouses is retired continues to affect their satisfaction after both of them are retired. This means that the difference in the couple's retirement sequence has an ongoing effect on their later happiness. The analysis of the effect of a couple's retirement sequence on the satisfaction in their old life may be useful for improving an individual and couples' quality of life in countries with similar cultures.

Virulence-Associated Genome Mutations of Murine Rotavirus Identified by Alternating Serial Passages in Mice and Cell Cultures

PubMed Central

Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

2014-01-01

ABSTRACT Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3′ consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. IMPORTANCE Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. PMID:24599996

Virulence-associated genome mutations of murine rotavirus identified by alternating serial passages in mice and cell cultures.

PubMed

Tsugawa, Takeshi; Tatsumi, Masatoshi; Tsutsumi, Hiroyuki

2014-05-01

Although significant clinical efficacy and safety of rotavirus vaccines were recently revealed in many countries, the mechanism of their attenuation is not well understood. We passaged serially a cell culture-adapted murine rotavirus EB strain in mouse pups or in cell cultures alternately and repeatedly and fully sequenced all 11 genes of 21 virus samples passaged in mice or in cell cultures. Sequence analysis revealed that mouse-passaged viruses that regained virulence almost consistently acquired four kinds of amino acid (aa) substitutions in VP4 and substitution in aa 37 (Val to Ala) in NSP4. In addition, they gained and invariably conserved the 3' consensus sequence in NSP1. The molecular changes occurred along with the acquisition of virulence during passages in mice and then disappeared following passages in cell cultures. Intraperitoneal injection of recombinant NSP4 proteins confirmed the aa 37 site as important for its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence. Serial passage of a virulent wild-type virus in vitro often results in loss of virulence of the virus in an original animal host, while serial passage of a cell culture-adapted avirulent virus in vivo often gains virulence in an animal host. Actually, live attenuated virus vaccines were originally produced by serial passage in cell cultures. Although clinical efficacy and safety of rotavirus vaccines were recently revealed, the mechanism of their attenuation is not well understood. We passaged serially a murine rotavirus by alternating switch of host (mice or cell cultures) repeatedly and sequenced the eleven genes of the passaged viruses to identify mutations associated with the emergence or disappearance of virulence. Sequence analysis revealed that changes in three genes (VP4, NSP1, and NSP4) were associated with virulence in mice. Intraperitoneal injection of recombinant NSP4 proteins confirmed its diarrheagenic activity in mice. These genome changes are likely to be correlated with rotavirus virulence.

Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

PubMed Central

Ehrenshaft, M; Daub, M E

1994-01-01

We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures. Images PMID:8085820

Dynamic assessment of microbial ecology (DAME): A shiny app for analysis and visualization of microbial sequencing data

USDA-ARS?s Scientific Manuscript database

A new renaissance in knowledge about the role of commensal microbiota in health and disease is well underway facilitated by culture-independent sequencing technologies; however, microbial sequencing data poses new challenges (e.g., taxonomic hierarchy, overdispersion) not generally seen in more trad...

PHYLOGENETIC ANALYSIS OF 16S RRNA GENE SEQUENCES REVEALS THE PREVALENCE OF MYCOBACTERIA SP., ALPHA-PROTEOBACTERIA, AND UNCULTURED BACTERIA IN DRINKING WATER MICROBIAL COMMUNITIES

EPA Science Inventory

Previous studies have shown that culture-based methods tend to underestimate the densities and diversity of bacterial populations inhabiting water distribution systems (WDS). In this study, the phylogenetic diversity of drinking water bacteria was assessed using sequence analysis...

Molecular Detection, Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge

PubMed Central

Watanabe, Kazuya; Teramoto, Maki; Futamata, Hiroyuki; Harayama, Shigeaki

1998-01-01

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge. PMID:9797297

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

PubMed

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2008-04-01

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.

Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting▿ †

PubMed Central

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2008-01-01

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs. PMID:18310426

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

PubMed Central

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

2011-01-01

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

Enrichment of denitrifying methane-oxidizing microorganisms using up-flow continuous reactors and batch cultures.

PubMed

Hatamoto, Masashi; Kimura, Masafumi; Sato, Takafumi; Koizumi, Masato; Takahashi, Masanobu; Kawakami, Shuji; Araki, Nobuo; Yamaguchi, Takashi

2014-01-01

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4±3.4 mg-N·L(-1)·day(-1) was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68% of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58% of total microbial cells were bacteria while archaeal cells accounted for 7% of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98% sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89-91% sequence similarity to DAMO bacteria in the inoculum. These results indicate that electron acceptor and cultivation method strongly affect the microbial community structures of DAMO consortia.

Comparative genome analysis of the candidate functional starter culture strains Lactobacillus fermentum 222 and Lactobacillus plantarum 80 for controlled cocoa bean fermentation processes.

PubMed

Illeghems, Koen; De Vuyst, Luc; Weckx, Stefan

2015-10-12

Lactobacillus fermentum 222 and Lactobacillus plantarum 80, isolates from a spontaneous Ghanaian cocoa bean fermentation process, proved to be interesting functional starter culture strains for cocoa bean fermentations. Lactobacillus fermentum 222 is a thermotolerant strain, able to dominate the fermentation process, thereby converting citrate and producing mannitol. Lactobacillus plantarum 80 is an acid-tolerant and facultative heterofermentative strain that is competitive during cocoa bean fermentation processes. In this study, whole-genome sequencing and comparative genome analysis was used to investigate the mechanisms of these strains to dominate the cocoa bean fermentation process. Through functional annotation and analysis of the high-coverage contigs obtained through 454 pyrosequencing, plantaricin production was predicted for L. plantarum 80. For L. fermentum 222, genes encoding a complete arginine deiminase pathway were attributed. Further, in-depth functional analysis revealed the capacities of these strains associated with carbohydrate and amino acid metabolism, such as the ability to use alternative external electron acceptors, the presence of an extended pyruvate metabolism, and the occurrence of several amino acid conversion pathways. A comparative genome sequence analysis using publicly available genome sequences of strains of the species L. plantarum and L. fermentum revealed unique features of both strains studied. Indeed, L. fermentum 222 possessed genes encoding additional citrate transporters and enzymes involved in amino acid conversions, whereas L. plantarum 80 is the only member of this species that harboured a gene cluster involved in uptake and consumption of fructose and/or sorbose. In-depth genome sequence analysis of the candidate functional starter culture strains L. fermentum 222 and L. plantarum 80 revealed their metabolic capacities, niche adaptations and functionalities that enable them to dominate the cocoa bean fermentation process. Further, these results offered insights into the cocoa bean fermentation ecosystem as a whole and will facilitate the selection of appropriate starter culture strains for controlled cocoa bean fermentation processes.

A core microbiome associated with the peritoneal tumors of pseudomyxoma peritonei

PubMed Central

2013-01-01

Background Pseudomyxoma peritonei (PMP) is a malignancy characterized by dissemination of mucus-secreting cells throughout the peritoneum. This disease is associated with significant morbidity and mortality and despite effective treatment options for early-stage disease, patients with PMP often relapse. Thus, there is a need for additional treatment options to reduce relapse rate and increase long-term survival. A previous study identified the presence of both typed and non-culturable bacteria associated with PMP tissue and determined that increased bacterial density was associated with more severe disease. These findings highlighted the possible role for bacteria in PMP disease. Methods To more clearly define the bacterial communities associated with PMP disease, we employed a sequenced-based analysis to profile the bacterial populations found in PMP tumor and mucin tissue in 11 patients. Sequencing data were confirmed by in situ hybridization at multiple taxonomic depths and by culturing. A pilot clinical study was initiated to determine whether the addition of antibiotic therapy affected PMP patient outcome. Main results We determined that the types of bacteria present are highly conserved in all PMP patients; the dominant phyla are the Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. A core set of taxon-specific sequences were found in all 11 patients; many of these sequences were classified into taxonomic groups that also contain known human pathogens. In situ hybridization directly confirmed the presence of bacteria in PMP at multiple taxonomic depths and supported our sequence-based analysis. Furthermore, culturing of PMP tissue samples allowed us to isolate 11 different bacterial strains from eight independent patients, and in vitro analysis of subset of these isolates suggests that at least some of these strains may interact with the PMP-associated mucin MUC2. Finally, we provide evidence suggesting that targeting these bacteria with antibiotic treatment may increase the survival of PMP patients. Conclusions Using 16S amplicon-based sequencing, direct in situ hybridization analysis and culturing methods, we have identified numerous bacterial taxa that are consistently present in all PMP patients tested. Combined with data from a pilot clinical study, these data support the hypothesis that adding antimicrobials to the standard PMP treatment could improve PMP patient survival. PMID:23844722

Culture adaptation of malaria parasites selects for convergent loss-of-function mutants.

PubMed

Claessens, Antoine; Affara, Muna; Assefa, Samuel A; Kwiatkowski, Dominic P; Conway, David J

2017-01-24

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

Identification of species of viridans group streptococci in clinical blood culture isolates by sequence analysis of the RNase P RNA gene, rnpB.

PubMed

Westling, Katarina; Julander, Inger; Ljungman, Per; Vondracek, Martin; Wretlind, Bengt; Jalal, Shah

2008-03-01

Viridans group streptococci (VGS) cause severe diseases such as infective endocarditis and septicaemia. Genetically, VGS species are very close to each other and it is difficult to identify them to species level with conventional methods. The aims of the present study were to use sequence analysis of the RNase P RNA gene (rnpB) to identify VGS species in clinical blood culture isolates, and to compare the results with the API 20 Strep system that is based on phenotypical characteristics. Strains from patients with septicaemia or endocarditis were analysed with PCR amplification and sequence analysis of the rnpB gene. Clinical data were registered as well. One hundred and thirty two VGS clinical blood culture isolates from patients with septicaemia (n=95) or infective endocarditis (n=36) were analysed; all but one were identified by rnpB. Streptococcus oralis, Streptococcus sanguinis and Streptococcus gordonii strains were most common in the patients with infective endocarditis. In the isolates from patients with haematological diseases, Streptococcus mitis and S. oralis dominated. In addition in 76 of the isolates it was possible to compare the results from rnpB analysis and the API 20 Strep system. In 39/76 (51%) of the isolates the results were concordant to species level; in 55 isolates there were no results from API 20 Strep. Sequence analysis of the RNase P RNA gene (rnpB) showed that almost all isolates could be identified. This could be of importance for evaluation of the portal of entry in patients with septicaemia or infective endocarditis.

A new arenavirus in a cluster of fatal transplant-associated diseases.

PubMed

Palacios, Gustavo; Druce, Julian; Du, Lei; Tran, Thomas; Birch, Chris; Briese, Thomas; Conlan, Sean; Quan, Phenix-Lan; Hui, Jeffrey; Marshall, John; Simons, Jan Fredrik; Egholm, Michael; Paddock, Christopher D; Shieh, Wun-Ju; Goldsmith, Cynthia S; Zaki, Sherif R; Catton, Mike; Lipkin, W Ian

2008-03-06

Three patients who received visceral-organ transplants from a single donor on the same day died of a febrile illness 4 to 6 weeks after transplantation. Culture, polymerase-chain-reaction (PCR) and serologic assays, and oligonucleotide microarray analysis for a wide range of infectious agents were not informative. We evaluated RNA obtained from the liver and kidney transplant recipients. Unbiased high-throughput sequencing was used to identify microbial sequences not found by means of other methods. The specificity of sequences for a new candidate pathogen was confirmed by means of culture and by means of PCR, immunohistochemical, and serologic analyses. High-throughput sequencing yielded 103,632 sequences, of which 14 represented an Old World arenavirus. Additional sequence analysis showed that this new arenavirus was related to lymphocytic choriomeningitis viruses. Specific PCR assays based on a unique sequence confirmed the presence of the virus in the kidneys, liver, blood, and cerebrospinal fluid of the recipients. Immunohistochemical analysis revealed arenavirus antigen in the liver and kidney transplants in the recipients. IgM and IgG antiviral antibodies were detected in the serum of the donor. Seroconversion was evident in serum specimens obtained from one recipient at two time points. Unbiased high-throughput sequencing is a powerful tool for the discovery of pathogens. The use of this method during an outbreak of disease facilitated the identification of a new arenavirus transmitted through solid-organ transplantation. Copyright 2008 Massachusetts Medical Society.

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 methods. The use of pyrosequencing allowed for the identification of low abundance bacteria in leaf salad vegetables not detected by culture-dependent methods. The presence of a range of bacterial populations as endophytes presents an interesting phenomenon as these microorganisms cannot be removed by washing and are thus ingested during salad consumption.

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 traditional culture-dependent methods. Conclusions The use of pyrosequencing allowed for the identification of low abundance bacteria in leaf salad vegetables not detected by culture-dependent methods. The presence of a range of bacterial populations as endophytes presents an interesting phenomenon as these microorganisms cannot be removed by washing and are thus ingested during salad consumption. PMID:24289725

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. PCR-DGGE applied to sequential enrichment cultures in DBT-containing sulfur-free basal salts medium prepared from the A and treated FSL soils revealed the selection of up to 10 distinct bands. Sequencing a subset of these bands provided evidence for the presence of organisms related to Pseudomonas putida, a Pseudomonas sp., Stenotrophomonas maltophilia, and Rhodococcus erythropolis. Several of 52 colonies obtained from the A and FSL soils on agar plates with DBT as the sole sulfur source produced bands that matched the migration of bands selected in the enrichment cultures. Evidence for the presence of dszB in 12 strains was obtained, whereas dszA and dszC genes were found in only 7 and 6 strains, respectively. Most of the strains carrying dszA or dszC were classified as R. erythropolis related, and all revealed the capacity to desulfurize DBT. A comparison of 37 dszA sequences, obtained via PCR from the A and FSL soils, from enrichments of these soils, and from isolates, revealed the great similarity of all sequences to the canonical (R. erythropolis strain IGTS8) dszA sequence and a large degree of internal conservation. The 37 sequences recovered were grouped in three clusters. One group, consisting of 30 sequences, was minimally 98% related to the IGTS8 sequence, a second group of 2 sequences was slightly different, and a third group of 5 sequences was 95% similar. The first two groups contained sequences obtained from both soil types and enrichment cultures (including isolates), but the last consisted of sequences obtained directly from the polluted A soil. PMID:11229891

Biodiversity of fungi in hot desert sands.

PubMed

Murgia, Manuela; Fiamma, Maura; Barac, Aleksandra; Deligios, Massimo; Mazzarello, Vittorio; Paglietti, Bianca; Cappuccinelli, Pietro; Al-Qahtani, Ahmed; Squartini, Andrea; Rubino, Salvatore; Al-Ahdal, Mohammed N

2018-03-05

The fungal community of six sand samples from Saudi Arabia and Jordan deserts was characterized by culture-independent analysis via next generation sequencing of the 18S rRNA genes and by culture-dependent methods followed by sequencing of internal transcribed spacer (ITS) region. By 18S sequencing were identified from 163 to 507 OTUs per sample, with a percentage of fungi ranging from 3.5% to 82.7%. The identified fungal Phyla were Ascomycota, Basal fungi, and Basidiomycota and the most abundant detected classes were Dothideomycetes, Pezizomycetes, and Sordariomycetes. A total of 11 colonies of filamentous fungi were isolated and cultured from six samples, and the ITS sequencing pointed toward five different species of the class Sordariomycetes, belonging to genera Fusarium (F. redolens, F. solani, F. equiseti), Chaetomium (C. madrasense), and Albifimbria (A. terrestris). The results of this study show an unexpectedly large fungal biodiversity in the Middle East desert sand and their possible role and implications on human health. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Detection and identification of cutaneous leishmaniasis isolates by culture, Polymerase chain reaction and sequence analyses in Syrian and Central Anatolia patients.

PubMed

Beyhan, Yunus E; Karakus, Mehmet; Karagoz, Alper; Mungan, Mesut; Ozkan, Aysegul T; Hokelek, Murat

2017-09-01

To characterize the cutaneous leishmaniasis (CL) isolates of Syrian and Central Anatolia patients at species levels. Methods: Skin scrapings of 3 patients (2 Syrian, 1 Turkish) were taken and examined by direct examination, culture in Novy-MacNeal-Nicole (NNN) medium, internal transcribed spacer polymerase chain reaction and sequence analysis (PCR). Results:According to microscopic examination, culture and PCR methods, 3 samples were detected positive. The sequencing results of all isolates in the study were identified as Leishmania tropica. The same genotypes were detected in the 3 isolates and nucleotide sequence submitted into GenBank with the accession number: KP689599. Conclusion: This finding could give information about the transmission of CL between Turkey and Syria. Because of the Syrian civil war, most of the Syrian citizens circulating in Turkey and different part of Europe, this can be increase the risk of spreading the disease. So, prevention measurements must be taken urgently.

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

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture.

PubMed

Nahku, Ranno; Peebo, Karl; Valgepea, Kaspar; Barrick, Jeffrey E; Adamberg, Kaarel; Vilu, Raivo

2011-09-01

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations.

Stock culture heterogeneity rather than new mutational variation complicates short-term cell physiology studies of Escherichia coli K-12 MG1655 in continuous culture

PubMed Central

Nahku, Ranno; Peebo, Karl; Valgepea, Kaspar; Barrick, Jeffrey E.; Adamberg, Kaarel

2011-01-01

Nutrient-limited continuous cultures in chemostats have been used to study microbial cell physiology for over 60 years. Genome instability and genetic heterogeneity are possible uncontrolled factors in continuous cultivation experiments. We investigated these issues by using high-throughput (HT) DNA sequencing to characterize samples from different phases of a glucose-limited accelerostat (A-stat) experiment with Escherichia coli K-12 MG1655 and a duration regularly used in cell physiology studies (20 generations of continuous cultivation). Seven consensus mutations from the reference sequence and five subpopulations characterized by different mutations were detected in the HT-sequenced samples. This genetic heterogeneity was confirmed to result from the stock culture by Sanger sequencing. All the subpopulations in which allele frequencies increased (betA, cspG/cspH, glyA) during the experiment were also present at the end of replicate A-stats, indicating that no new subpopulations emerged during our experiments. The fact that ~31 % of the cells in our initial cultures obtained directly from a culture stock centre were mutants raises concerns that even if cultivations are started from single colonies, there is a significant chance of picking a mutant clone with an altered phenotype. Our results show that current HT DNA sequencing technology allows accurate subpopulation analysis and demonstrates that a glucose-limited E. coli K-12 MG1655 A-stat experiment with a duration of tens of generations is suitable for studying cell physiology and collecting quantitative data for metabolic modelling without interference from new mutations. PMID:21700661

Identification of bacteria on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties by 16S rRNA gene sequencing and by microbiological culture

PubMed Central

Dempsey, Kate E; Riggio, Marcello P; Lennon, Alan; Hannah, Victoria E; Ramage, Gordon; Allan, David; Bagg, Jeremy

2007-01-01

It has been postulated that bacteria attached to the surface of prosthetic hip joints can cause localised inflammation, resulting in failure of the replacement joint. However, diagnosis of infection is difficult with traditional microbiological culture methods, and evidence exists that highly fastidious or non-cultivable organisms have a role in implant infections. The purpose of this study was to use culture and culture-independent methods to detect the bacteria present on the surface of prosthetic hip joints removed during revision arthroplasties. Ten consecutive revisions were performed by two surgeons, which were all clinically and radiologically loose. Five of the hip replacement revision surgeries were performed because of clinical infections and five because of aseptic loosening. Preoperative and perioperative specimens were obtained from each patient and subjected to routine microbiological culture. The prostheses removed from each patient were subjected to mild ultrasonication to dislodge adherent bacteria, followed by aerobic and anaerobic microbiological culture. Bacterial DNA was extracted from each sonicate and the 16S rRNA gene was amplified with the universal primer pair 27f/1387r. All 10 specimens were positive for the presence of bacteria by both culture and PCR. PCR products were then cloned, organised into groups by RFLP analysis and one clone from each group was sequenced. Bacteria were identified by comparison of the 16S rRNA gene sequences obtained with those deposited in public access sequence databases. A total of 512 clones were analysed by RFLP analysis, of which 118 were sequenced. Culture methods identified species from the genera Leifsonia (54.3%), Staphylococcus (21.7%), Proteus (8.7%), Brevundimonas (6.5%), Salibacillus (4.3%), Methylobacterium (2.2%) and Zimmermannella (2.2%). Molecular detection methods identified a more diverse microflora. The predominant genus detected was Lysobacter, representing 312 (60.9%) of 512 clones analysed. In all, 28 phylotypes were identified: Lysobacter enzymogenes was the most abundant phylotype (31.4%), followed by Lysobacter sp. C3 (28.3%), gamma proteobacterium N4-7 (6.6%), Methylobacterium SM4 (4.7%) and Staphylococcus epidermidis (4.7%); 36 clones (7.0%) represented uncultivable phylotypes. We conclude that a diverse range of bacterial species are found within biofilms on the surface of clinically infected and non-infected prosthetic hip joints removed during revision arthroplasties. PMID:17501992

Molecular analysis of microflora associated with dentoalveolar abscesses.

PubMed Central

Dymock, D; Weightman, A J; Scully, C; Wade, W G

1996-01-01

The microflora associated with three dentoalveolar abscesses was determined by cultural and molecular methods. 16S rRNA genes were randomly amplified by means of conserved eubacterial primers and cloned. Restriction fragment length polymorphism analysis of the clones and amplified genes encoding 16S rRNA from the cultured bacteria was used to detect putative unculturable bacteria. Clones representative of five predominant groups of uncultured organisms were sequenced. Two were identified as Porphyromonas gingivalis and Prevotella oris, and one was found to be closely related to Peptostreptococcus micros. The remaining two clones did not correspond to known, previously sequenced organisms. One was related to Zoogloea ramigera, a species of aerobic waterborne organisms, while the other was distantly related to the genus Prevotella. This study has demonstrated the possibility of the characterization of microflora associated with human infection by molecular methods without the inherent biases of culture. PMID:8904410

Phylogenetic analysis of rubella virus strains during an outbreak in São Paulo, 2007-2008.

PubMed

Figueiredo, C A; Oliveira, M I; Curti, S P; Afonso, A M S; Frugi Yu, A L; Gualberto, F; Durigon, E L

2012-10-01

Rubella virus (RV) is an important human pathogen that causes rubella, an acute contagious disease. It also causes severe birth defects collectively known as congenital rubella syndrome when infection occurs during the first trimester of pregnancy. Here, we present the phylogenetic analysis of RV that circulated in São Paulo during the 2007-2008 outbreak. Samples collected from patients diagnosed with rubella were isolated in cell culture and sequenced. RV RNA was obtained from samples or RV-infected cell cultures and amplified by reverse transcriptase-polymerase chain reaction. Sequences were assigned to genotypes by phylogenetic analysis using RV reference sequences. Seventeen sequences were analyzed, and three genotypes were identified: 1a, 1G, and 2B. Genotypes 1a and 1G, which were isolated in 2007, were responsible for sporadic rubella cases in São Paulo. Thereafter, in late 2007, the epidemiological conditions changed, resulting in a large RV outbreak with the clear dominance of genotype 2B. The results of this study provide new approaches for monitoring the progress of elimination of rubella from São Paulo, Brazil. Copyright © 2012 Wiley Periodicals, Inc.

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

PubMed

Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

2006-05-25

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir.

PubMed

Kaster, Krista M; Bonaunet, Kristin; Berland, Harald; Kjeilen-Eilertsen, Grethe; Brakstad, Odd Gunnar

2009-11-01

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.

Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

PubMed

Frantzen, Cyril A; Kleppen, Hans Petter; Holo, Helge

2018-02-01

Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp . lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR , a core gene, and epsD , present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk. IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product. Copyright © 2018 American Society for Microbiology.

[Methods, challenges and opportunities for big data analyses of microbiome].

PubMed

Sheng, Hua-Fang; Zhou, Hong-Wei

2015-07-01

Microbiome is a novel research field related with a variety of chronic inflamatory diseases. Technically, there are two major approaches to analysis of microbiome: metataxonome by sequencing the 16S rRNA variable tags, and metagenome by shot-gun sequencing of the total microbial (mainly bacterial) genome mixture. The 16S rRNA sequencing analyses pipeline includes sequence quality control, diversity analyses, taxonomy and statistics; metagenome analyses further includes gene annotation and functional analyses. With the development of the sequencing techniques, the cost of sequencing will decrease, and big data analyses will become the central task. Data standardization, accumulation, modeling and disease prediction are crucial for future exploit of these data. Meanwhile, the information property in these data, and the functional verification with culture-dependent and culture-independent experiments remain the focus in future research. Studies of human microbiome will bring a better understanding of the relations between the human body and the microbiome, especially in the context of disease diagnosis and therapy, which promise rich research opportunities.

Coupling Deep Transcriptome Analysis with Untargeted Metabolic Profiling in Ophiorrhiza pumila to Further the Understanding of the Biosynthesis of the Anti-Cancer Alkaloid Camptothecin and Anthraquinones

PubMed Central

Yamazaki, Mami; Mochida, Keiichi; Asano, Takashi; Nakabayashi, Ryo; Chiba, Motoaki; Udomson, Nirin; Yamazaki, Yasuyo; Goodenowe, Dayan B.; Sankawa, Ushio; Yoshida, Takuhiro; Toyoda, Atsushi; Totoki, Yasushi; Sakaki, Yoshiyuki; Góngora-Castillo, Elsa; Buell, C. Robin; Sakurai, Tetsuya; Saito, Kazuki

2013-01-01

The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila. PMID:23503598

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/- cells identified with next generation sequencing.

PubMed

Revollo, Javier; Wang, Yiying; McKinzie, Page; Dad, Azra; Pearce, Mason; Heflich, Robert H; Dobrovolsky, Vasily N

2017-12-01

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk +/- cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones. Published by Elsevier B.V.

Probing the Rare Biosphere of the North-West Mediterranean Sea: An Experiment with High Sequencing Effort.

PubMed

Crespo, Bibiana G; Wallhead, Philip J; Logares, Ramiro; Pedrós-Alió, Carlos

2016-01-01

High-throughput sequencing (HTS) techniques have suggested the existence of a wealth of species with very low relative abundance: the rare biosphere. We attempted to exhaustively map this rare biosphere in two water samples by performing an exceptionally deep pyrosequencing analysis (~500,000 final reads per sample). Species data were derived by a 97% identity criterion and various parametric distributions were fitted to the observed counts. Using the best-fitting Sichel distribution we estimate a total species richness of 1,568-1,669 (95% Credible Interval) and 5,027-5,196 for surface and deep water samples respectively, implying that 84-89% of the total richness in those two samples was sequenced, and we predict that a quadrupling of the present sequencing effort would suffice to observe 90% of the total richness in both samples. Comparing the HTS results with a culturing approach we found that most of the cultured taxa were not obtained by HTS, despite the high sequencing effort. Culturing therefore remains a useful tool for uncovering marine bacterial diversity, in addition to its other uses for studying the ecology of marine bacteria.

Analysis of the Gull Fecal Microbial Community Reveals the Dominance of Catellicoccus marimammalium in Relation to Culturable Enterococci

PubMed Central

Koskey, Amber M.; Fisher, Jenny C.; Traudt, Mary F.; Newton, Ryan J.

2014-01-01

Gulls are prevalent in beach environments and can be a major source of fecal contamination. Gulls have been shown to harbor a high abundance of fecal indicator bacteria (FIB), such as Escherichia coli and enterococci, which can be readily detected as part of routine beach monitoring. Despite the ubiquitous presence of gull fecal material in beach environments, the associated microbial community is relatively poorly characterized. We generated comprehensive microbial community profiles of gull fecal samples using Roche 454 and Illumina MiSeq platforms to investigate the composition and variability of the gull fecal microbial community and to measure the proportion of FIB. Enterococcaceae and Enterobacteriaceae were the two most abundant families in our gull samples. Sequence comparisons between short-read data and nearly full-length 16S rRNA gene clones generated from the same samples revealed Catellicoccus marimammalium as the most numerous taxon among all samples. The identification of bacteria from gull fecal pellets cultured on membrane-Enterococcus indoxyl-β-d-glucoside (mEI) plates showed that the dominant sequences recovered in our sequence libraries did not represent organisms culturable on mEI. Based on 16S rRNA gene sequencing of gull fecal isolates cultured on mEI plates, 98.8% were identified as Enterococcus spp., 1.2% were identified as Streptococcus spp., and none were identified as C. marimammalium. Illumina deep sequencing indicated that gull fecal samples harbor significantly higher proportions of C. marimammalium 16S rRNA gene sequences (>50-fold) relative to typical mEI culturable Enterococcus spp. C. marimammalium therefore can be confidently utilized as a genetic marker to identify gull fecal pollution in the beach environment. PMID:24242244

Complete genome sequence and phylogenetic analyses of an aquabirnavirus isolated from a diseased marbled eel culture in Taiwan.

PubMed

Wen, Chiu-Ming

2017-08-01

An aquabirnavirus was isolated from diseased marbled eels (Anguilla marmorata; MEIPNV1310) with gill haemorrhages and associated mortality. Its genome segment sequences were obtained through next-generation sequencing and compared with published aquabirnavirus sequences. The results indicated that the genome sequence of MEIPNV1310 contains segment A (3099 nucleotides) and segment B (2789 nucleotides). Phylogenetic analysis showed that MEIPNV1310 is closely related to the infectious pancreatic necrosis Ab strain within genogroup II. This genome sequence is beneficial for studying the geographic distribution and evolution of aquabirnaviruses.

Environmental, depositional and cultural changes in the upper Pleistocene and early Holocene; the Cinglera del Capello Sequence (Capellades, Spain)

USGS Publications Warehouse

Vaquero, Manuel; Allué, Ethel; Bischoff, James L.; Burjachs, Francesc; Vallverdú, Josep

2013-01-01

The correlation between environmental and cultural changes is one of the primary archeological and paleoanthropological research topics. Analysis of ice and marine cores has yielded a high-resolution record of millennial-scale changes during the Late Pleistocene and Holocene eras. However, cultural changes are documented in low-resolution continental deposits; thus, their correlation with the millennial-scale climatic sequence is often difficult. In this paper, we present a rare occurrence in which a thick archeological sequence is associated with a high-resolution environmental record. The Cinglera del Capello is a tufa-draped cliff located in the northeastern Iberian Peninsula, 50 km west of Barcelona. This cliff harbors several rock-shelters with Late Pleistocene and Early Holocene deposits. Together, the deposits of four rock-shelters span from 7000 to 70,000 years ago and provide a high-resolution record of the environmental and human dynamics during this timespan. This record allows the correlation of the cultural and environmental changes. The multiproxy approach to the Cinglera evidence indicates that the main cultural stages of the Late Pleistocene and Early Holocene (Middle Paleolithic, Upper Paleolithic and Mesolithic) are associated with significant changes in the environmental and depositional contexts.

Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem.

PubMed

Illeghems, Koen; De Vuyst, Luc; Weckx, Stefan

2013-08-01

Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process. The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition. Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.

Composition of the bacterial community in the gut of the pine engraver, Ips pini (Say) (Coloptera) colonizing red pine

Treesearch

Italo Jr. Delalibera; Archana Vasanthakumar; Benjamin J. Burwitz; Patrick D. Schloss; Kier D. Klepzig; Jo Handelsman; Kenneth F. Raffa

2007-01-01

The gut bacterial community of a bark beetle, the pine engraver Ips pini (Say), was characterized using culture-dependent and culture-independent methods. Bacteria from individual guts of larvae, pupae and adults were cultured and DNA was extracted from samples of pooled larval guts. Analysis of 16S rRNA gene sequences amplified directly from the gut...

Bacterial diversity of the Colombian fermented milk "Suero Costeño" assessed by culturing and high-throughput sequencing and DGGE analysis of 16S rRNA gene amplicons.

PubMed

Motato, Karina Edith; Milani, Christian; Ventura, Marco; Valencia, Francia Elena; Ruas-Madiedo, Patricia; Delgado, Susana

2017-12-01

"Suero Costeño" (SC) is a traditional soured cream elaborated from raw milk in the Northern-Caribbean coast of Colombia. The natural microbiota that characterizes this popular Colombian fermented milk is unknown, although several culturing studies have previously been attempted. In this work, the microbiota associated with SC from three manufacturers in two regions, "Planeta Rica" (Córdoba) and "Caucasia" (Antioquia), was analysed by means of culturing methods in combination with high-throughput sequencing and DGGE analysis of 16S rRNA gene amplicons. The bacterial ecosystem of SC samples was revealed to be composed of lactic acid bacteria belonging to the Streptococcaceae and Lactobacillaceae families; the proportions and genera varying among manufacturers and region of elaboration. Members of the Lactobacillus acidophilus group, Lactocococcus lactis, Streptococcus infantarius and Streptococcus salivarius characterized this artisanal product. In comparison with culturing, the use of molecular in deep culture-independent techniques provides a more realistic picture of the overall bacterial communities residing in SC. Besides the descriptive purpose, these approaches will facilitate a rational strategy to follow (culture media and growing conditions) for the isolation of indigenous strains that allow standardization in the manufacture of SC. Copyright © 2017 Elsevier Ltd. All rights reserved.

[Isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris in Yunnan Safari Park].

PubMed

Cao, Yanru; Jiang, Yi; Li, Youlong; Chen, Xiu; Jin, Rongxian; He, Wenxiang

2012-07-04

We studied the isolation methods and diversity of culturable fecal actinobacteria associated with Panthera tigris tigris by using culture-dependent approaches. Fresh fecal samples of healthy Panthera tigris tigris were collected from Yunnan Safari Park. Pretreatment of the samples, isolation media and inhibitors were tested for actinobacteria isolation. 16S rRNA genes of actinobacteria were sequenced and subjected to phylogenetic analysis. The abundance of culturable actinobacteria was 1.10 x 10(8) cfu/g colony forming units (CFU) per gram of feces (wet weight). We obtained 110 purified cultural actinobacterium strains. The analysis based on 16S rRNA gene sequences showed that these strains were distributed in 10 different families and 12 genera of actinobacteria at least, and most of them were non-filamentous, such as Arthrobacter, Dietzia, Kocuria, Corynebacterium and Microbacterium. Streptomyces was the mainly classical filamentous actinobacteria, and up to 64% of total. Drying and heating up the fecal samples can greatly increase the rate of the actinobacteria. Many kinds of inhibitors and chemical defined media are suitable for isolation of fecal actinobacteria. The culturable actinobacteria are abundant in Panthera tigris tigris feces. Our study found an effective method to isolate animals' fecal actinobacteria and it's useful for studying and exploiting animals' fecal actinobacteria.

Video Analysis in Cross-Cultural Environments and Methodological Issues

ERIC Educational Resources Information Center

Montandon, Christiane

2015-01-01

This paper addresses the use of videography combined with group interviews, as a way to better understand the informal learnings of 11-12 year old children in cross-cultural encounters during French-German school exchanges. The complete, consistent video data required the researchers to choose the most significant sequences to highlight the…

Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

NASA Technical Reports Server (NTRS)

Orihuela, C. J.; Mills, J.; Robb, C. W.; Wilson, C. J.; Watson, D. A.; Niesel, D. W.

2001-01-01

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

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

PubMed Central

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

2014-01-01

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

SxtA gene sequence analysis of dinoflagellate Alexandrium minutum

NASA Astrophysics Data System (ADS)

Norshaha, Safida Anira; Latib, Norhidayu Abdul; Usup, Gires; Yusof, Nurul Yuziana Mohd

2015-09-01

The dinoflagellate Alexandrium minutum is typically known for the production of potent neurotoxins such as saxitoxin, affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). These phenomena is related to the harmful algal blooms (HABs) that is believed to be influenced by environmental and nutritional factors. Previous study has revealed that SxtA gene is a starting gene that involved in the saxitoxin production pathway. The aim of this study was to analyse the sequence of the sxtA gene in A. minutum. The dinoflagellates culture was cultured at temperature 26°C with 16:8-hour light:dark photocycle. After the samples were harvested, RNA was extracted, complementary DNA (cDNA) was synthesised and amplified by polymerase chain reaction (PCR). The PCR products were then purified and cloned before sequenced. The SxtA sequence obtained was then analyzed in order to identify the presence of SxtA gene in Alexandrium minutum.

Multilocus sequence typing (MLST) analysis of Propionibacterium acnes isolates from radical prostatectomy specimens.

PubMed

Mak, Tim N; Yu, Shu-Han; De Marzo, Angelo M; Brüggemann, Holger; Sfanos, Karen S

2013-05-01

Inflammation is commonly observed in radical prostatectomy specimens, and evidence suggests that inflammation may contribute to prostate carcinogenesis. Multiple microorganisms have been implicated in serving as a stimulus for prostatic inflammation. The pro-inflammatory anaerobe, Propionibacterium acnes, is ubiquitously found on human skin and is associated with the skin disease acne vulgaris. Recent studies have shown that P. acnes can be detected in prostatectomy specimens by bacterial culture or by culture-independent molecular techniques. Radical prostatectomy tissue samples were obtained from 30 prostate cancer patients and subject to both aerobic and anaerobic culture. Cultured species were identified by 16S rDNA gene sequencing. Propionibacterium acnes isolates were typed using multilocus sequence typing (MLST). Our study confirmed that P. acnes can be readily cultured from prostatectomy tissues (7 of 30 cases, 23%). In some cases, multiple isolates of P. acnes were cultured as well as other Propionibacterium species, such as P. granulosum and P. avidum. Overall, 9 of 30 cases (30%) were positive for Propionibacterium spp. MLST analyses identified eight different sequence types (STs) among prostate-derived P. acnes isolates. These STs belong to two clonal complexes, namely CC36 (type I-2) and CC53/60 (type II), or are CC53/60-related singletons. MLST typing results indicated that prostate-derived P. acnes isolates do not fall within the typical skin/acne STs, but rather are characteristic of STs associated with opportunistic infections and/or urethral flora. The MLST typing results argue against the likelihood that prostatectomy-derived P. acnes isolates represent contamination from skin flora. Copyright © 2012 Wiley Periodicals, Inc.

A novel multiplex PCR assay for simultaneous detection of nine clinically significant bacterial pathogens associated with bovine mastitis.

PubMed

Ashraf, Aqeela; Imran, Muhammad; Yaqub, Tahir; Tayyab, Muhammad; Shehzad, Wasim; Thomson, Peter C

2017-06-01

For rapid and simultaneous detection of nine bovine mastitic pathogens, a sensitive and specific multiplex PCR assay was developed. The assay was standardized using reference strains and validated on mastitic milk cultures which were identified to species level based on 16S rRNA sequencing. Multiplex PCR assay also efficiently detected the target bacterial strains directly from milk. The detection limit of the assay was up to 50 pg for DNA isolated from pure cultures and 10 4  CFU/ml for spiked milk samples. As estimated by latent class analysis, the assay was sensitive up to 88% and specific up to 98% for targeted mastitic pathogens, compared with the bacterial culture method and the 16S rRNA sequence analysis. This novel molecular assay could be useful for monitoring and maintaining the bovine udder health, ensuring the bacteriological safety of milk, and conducting epidemiological studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Skin Microbiome Surveys Are Strongly Influenced by Experimental Design.

PubMed

Meisel, Jacquelyn S; Hannigan, Geoffrey D; Tyldsley, Amanda S; SanMiguel, Adam J; Hodkinson, Brendan P; Zheng, Qi; Grice, Elizabeth A

2016-05-01

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provides more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e., gastrointestinal) and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource and cost intensive, provides evidence of a community's functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This study highlights the importance of experimental design for downstream results in skin microbiome surveys. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Skin microbiome surveys are strongly influenced by experimental design

PubMed Central

Meisel, Jacquelyn S.; Hannigan, Geoffrey D.; Tyldsley, Amanda S.; SanMiguel, Adam J.; Hodkinson, Brendan P.; Zheng, Qi; Grice, Elizabeth A.

2016-01-01

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provide more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e. gastrointestinal), and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource- and cost-intensive, provides evidence of a community’s functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This work highlights the importance of experimental design for downstream results in skin microbiome surveys. PMID:26829039

Bioremediation potential of a highly mercury resistant bacterial strain Sphingobium SA2 isolated from contaminated soil.

PubMed

Mahbub, Khandaker Rayhan; Krishnan, Kannan; Megharaj, Mallavarapu; Naidu, Ravi

2016-02-01

A mercury resistant bacterial strain, SA2, was isolated from soil contaminated with mercury. The 16S rRNA gene sequence of this isolate showed 99% sequence similarity to the genera Sphingobium and Sphingomonas of α-proteobacteria group. However, the isolate formed a distinct phyletic line with the genus Sphingobium suggesting the strain belongs to Sphingobium sp. Toxicity studies indicated resistance to high levels of mercury with estimated EC50 values 4.5 mg L(-1) and 44.15 mg L(-1) and MIC values 5.1 mg L(-1) and 48.48 mg L(-1) in minimal and rich media, respectively. The strain SA2 was able to volatilize mercury by producing mercuric reductase enzyme which makes it potential candidate for remediating mercury. ICP-QQQ-MS analysis of Hg supplemented culture solutions confirmed that almost 79% mercury in the culture suspension was volatilized in 6 h. A very small amount of mercury was observed to accumulate in cell pellets which was also evident according to ESEM-EDX analysis. The mercuric reductase gene merA was amplified and sequenced. The deduced amino acid sequence demonstrated sequence homology with α-proteobacteria and Ascomycota group. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pond Crash Forensics: Presumptive identification of pond crash agents by next generation sequencing in replicate raceway mass cultures of Nannochloropsis salina

DOE PAGES

Carney, Laura T.; Wilkenfeld, Joshua S.; Lane, Pam D.; ...

2016-06-02

Productivity of algal mass culture can be severely reduced by contaminating organisms. It is, therefore, important to identify contaminants, determine their effect on productivity and, ultimately, develop countermeasures against such contamination. In this paper, we utilized microbiome analysis by second-generation sequencing of small subunit rRNA genes to characterize the predator and pathogen burden of open raceway cultures of Nannochloropsis salina. Samples were analyzed from replicate raceways before and after crashes. In one culture cycle, we identified two algivorous species, the rotifer Brachionus and gastrotrich Chaetonotus, the presence of which may have contributed to the loss of algal biomass. In themore » second culture cycle, the raceways were treated with hypochlorite in an unsuccessful attempt to interdict the crash. Finally, our analyses were shown to be an effective strategy for the identification of the biological contaminants and the characterization of intervention strategies.« less

A Multicultural Sequence of Humanities Electives.

ERIC Educational Resources Information Center

Anderson, Gwendolyn; Ewing, Dessa

In order to promote multi-cultural literacy among its students, Delaware County Community College (DCCC) developed a multi-cultural sequence of humanities electives. The sequence emerged as a response to the predominantly White student body's lack of knowledge or curiosity about other cultures. The first of the four courses in the sequence is…

The status of the species Enterobacter siamensisKhunthongpan et al. 2014. Request for an Opinion.

PubMed

Kämpfer, Peter; Doijad, Swapnil; Chakraborty, Trinad; Glaeser, Stefanie P

2016-01-01

In the course of a taxonomic study describing novel species of the genus Enterobacter it was found that the 16S rRNA gene sequence of the type strain of Enterobacter siamensis, obtained both directly from the authors of the publication on Enterobacter siamensis and from the Korean Collection for Type Cultures (C2361T and KCTC 23282T, respectively), was not congruent with the 16S rRNA gene sequence deposited in the GenBank database under the accession number HQ888848, which was applied for phylogenetic analysis in the species proposal. The remaining deposit in the Japanese type culture collection, NBRC 107138T, showed an identical 16S rRNA gene sequence to the other two cultures and overall, this sequence differed at 35 positions in comparison with the 1429 bp sequence published under the accession number HQ888848.Therefore, the type strain of this species cannot be included in any further scientific comparative study. It is proposed that the Judicial Commission of the International Committee on Systematics of Prokaryotes place the name Enterobacter siamensis on the list of rejected names, if a suitable replacement for the type strain is not found or a neotype strain is not proposed within two years following the publication of this Request for an Opinion.

Analysis of microbial community variation during the mixed culture fermentation of agricultural peel wastes to produce lactic acid.

PubMed

Liang, Shaobo; Gliniewicz, Karol; Gerritsen, Alida T; McDonald, Armando G

2016-05-01

Mixed cultures fermentation can be used to convert organic wastes into various chemicals and fuels. This study examined the fermentation performance of four batch reactors fed with different agricultural (orange, banana, and potato (mechanical and steam)) peel wastes using mixed cultures, and monitored the interval variation of reactor microbial communities with 16S rRNA genes using Illumina sequencing. All four reactors produced similar chemical profile with lactic acid (LA) as dominant compound. Acetic acid and ethanol were also observed with small fractions. The Illumina sequencing results revealed the diversity of microbial community decreased during fermentation and a community of largely lactic acid producing bacteria dominated by species of Lactobacillus developed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting.

PubMed

Andrés-Barrao, Cristina; Benagli, Cinzia; Chappuis, Malou; Ortega Pérez, Ruben; Tonolla, Mauro; Barja, François

2013-03-01

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species. Copyright © 2012 Elsevier GmbH. All rights reserved.

Development and application of a multilocus sequence analysis method for the identification of genotypes within genus Bradyrhizobium and for establishing nodule occupancy of soybean (Glycine max L. Merr)

USDA-ARS?s Scientific Manuscript database

A Multilocus Sequence Typing (MLST) method based on allelic variation of 7 chromosomal loci was developed for characterizing genotypes within the genus Bradyrhizobium. With the method 29 distinct multilocus genotypes (GTs) were identified among 191 culture collection soybean strains. The occupancy ...

Fusarium diversity in soil using a specific molecular approach and a cultural approach.

PubMed

Edel-Hermann, Véronique; Gautheron, Nadine; Mounier, Arnaud; Steinberg, Christian

2015-04-01

Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1α) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1α gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples. Published by Elsevier B.V.

The cel3 gene of Agaricus bisporus codes for a modular cellulase and is transcriptionally regulated by the carbon source.

PubMed Central

Chow, C M; Yagüe, E; Raguz, S; Wood, D A; Thurston, C F

1994-01-01

A 52-kDa protein, CEL3, has been separated from the culture filtrate of Agaricus bisporus during growth on cellulose. A PCR-derived probe was made, with a degenerate oligodeoxynucleotide derived from the amino acid sequence of a CEL3 CNBr cleavage product and was used to select cel3 cDNA clones from an A. bisporus cDNA library. Two allelic cDNAs were isolated. They showed 98.8% identity of their nucleotide sequences. The deduced amino acid sequence and domain architecture of CEL3 showed a high degree of similarity to those of cellobiohydrolase II of Trichoderma reesei. Functional expression of cel3 cDNA in Saccharomyces cerevisiae was achieved by placing it under the control of a constitutive promoter and fusing it to the yeast invertase signal sequence. Recombinant CEL3 secreted by yeast showed enzymatic activity towards crystalline cellulose. At long reaction times, CEL3 was also able to degrade carboxymethyl cellulose. Northern (RNA) analysis showed that cel3 gene expression was induced by cellulose and repressed by glucose, fructose, 2-deoxyglucose, and lactose. Glycerol, mannitol, sorbitol, and maltose were neutral carbon sources. Nuclear run-on analysis showed that the rate of synthesis of cel3 mRNA in cellulose-grown cultures was 13 times higher than that in glucose-grown cultures. A low basal rate of cel3 mRNA synthesis was observed in the nuclei isolated from glucose-grown mycelia. Images PMID:8085821

Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

PubMed

Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

2015-10-01

Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

Cellulose in Cyanobacteria. Origin of Vascular Plant Cellulose Synthase?

PubMed Central

Nobles, David R.; Romanovicz, Dwight K.; Brown, R. Malcolm

2001-01-01

Although cellulose biosynthesis among the cyanobacteria has been suggested previously, we present the first conclusive evidence, to our knowledge, of the presence of cellulose in these organisms. Based on the results of x-ray diffraction, electron microscopy of microfibrils, and cellobiohydrolase I-gold labeling, we report the occurrence of cellulose biosynthesis in nine species representing three of the five sections of cyanobacteria. Sequence analysis of the genomes of four cyanobacteria revealed the presence of multiple amino acid sequences bearing the DDD35QXXRW motif conserved in all cellulose synthases. Pairwise alignments demonstrated that CesAs from plants were more similar to putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and Nostoc punctiforme American Type Culture Collection 29133 than any other cellulose synthases in the database. Multiple alignments of putative cellulose synthases from Anabaena sp. Pasteur Culture Collection 7120 and N. punctiforme American Type Culture Collection 29133 with the cellulose synthases of other prokaryotes, Arabidopsis, Gossypium hirsutum, Populus alba × Populus tremula, corn (Zea mays), and Dictyostelium discoideum showed that cyanobacteria share an insertion between conserved regions U1 and U2 found previously only in eukaryotic sequences. Furthermore, phylogenetic analysis indicates that the cyanobacterial cellulose synthases share a common branch with CesAs of vascular plants in a manner similar to the relationship observed with cyanobacterial and chloroplast 16s rRNAs, implying endosymbiotic transfer of CesA from cyanobacteria to plants and an ancient origin for cellulose synthase in eukaryotes. PMID:11598227

Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing.

PubMed

Sahoo, Malaya K; Holubar, Marisa; Huang, ChunHong; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Waggoner, Jesse J; Troy, Stephanie B; Garcia-Garcia, Lourdes; Ferreyra-Reyes, Leticia; Maldonado, Yvonne; Pinsky, Benjamin A

2017-07-01

Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication. Copyright © 2017 Sahoo et al.

Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing

PubMed Central

Wang, Bin; Guo, Guangwu; Wang, Chao; Lin, Ying; Wang, Xiaoning; Zhao, Mouming; Guo, Yong; He, Minghui; Zhang, Yong; Pan, Li

2010-01-01

Aspergillus oryzae, an important filamentous fungus used in food fermentation and the enzyme industry, has been shown through genome sequencing and various other tools to have prominent features in its genomic composition. However, the functional complexity of the A. oryzae transcriptome has not yet been fully elucidated. Here, we applied direct high-throughput paired-end RNA-sequencing (RNA-Seq) to the transcriptome of A. oryzae under four different culture conditions. With the high resolution and sensitivity afforded by RNA-Seq, we were able to identify a substantial number of novel transcripts, new exons, untranslated regions, alternative upstream initiation codons and upstream open reading frames, which provide remarkable insight into the A. oryzae transcriptome. We were also able to assess the alternative mRNA isoforms in A. oryzae and found a large number of genes undergoing alternative splicing. Many genes and pathways that might be involved in higher levels of protein production in solid-state culture than in liquid culture were identified by comparing gene expression levels between different cultures. Our analysis indicated that the transcriptome of A. oryzae is much more complex than previously anticipated, and these results may provide a blueprint for further study of the A. oryzae transcriptome. PMID:20392818

Survey of the transcriptome of Aspergillus oryzae via massively parallel mRNA sequencing.

PubMed

Wang, Bin; Guo, Guangwu; Wang, Chao; Lin, Ying; Wang, Xiaoning; Zhao, Mouming; Guo, Yong; He, Minghui; Zhang, Yong; Pan, Li

2010-08-01

Aspergillus oryzae, an important filamentous fungus used in food fermentation and the enzyme industry, has been shown through genome sequencing and various other tools to have prominent features in its genomic composition. However, the functional complexity of the A. oryzae transcriptome has not yet been fully elucidated. Here, we applied direct high-throughput paired-end RNA-sequencing (RNA-Seq) to the transcriptome of A. oryzae under four different culture conditions. With the high resolution and sensitivity afforded by RNA-Seq, we were able to identify a substantial number of novel transcripts, new exons, untranslated regions, alternative upstream initiation codons and upstream open reading frames, which provide remarkable insight into the A. oryzae transcriptome. We were also able to assess the alternative mRNA isoforms in A. oryzae and found a large number of genes undergoing alternative splicing. Many genes and pathways that might be involved in higher levels of protein production in solid-state culture than in liquid culture were identified by comparing gene expression levels between different cultures. Our analysis indicated that the transcriptome of A. oryzae is much more complex than previously anticipated, and these results may provide a blueprint for further study of the A. oryzae transcriptome.

Contribution of PCR Denaturing Gradient Gel Electrophoresis Combined with Mixed Chromatogram Software Separation for Complex Urinary Sample Analysis.

PubMed

Kotásková, Iva; Mališová, Barbora; Obručová, Hana; Holá, Veronika; Peroutková, Tereza; Růžička, Filip; Freiberger, Tomáš

2017-01-01

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment. © 2017 S. Karger AG, Basel.

Cultivation of Hard-To-Culture Subsurface Mercury-Resistant Bacteria and Discovery of New merA Gene Sequences▿

PubMed Central

Rasmussen, L. D.; Zawadsky, C.; Binnerup, S. J.; Øregaard, G.; Sørensen, S. J.; Kroer, N.

2008-01-01

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments. PMID:18441111

Genomic Analysis of Vaccine-Derived Poliovirus Strains in Stool Specimens by Combination of Full-Length PCR and Oligonucleotide Microarray Hybridization

PubMed Central

Laassri, Majid; Dragunsky, Eugenia; Enterline, Joan; Eremeeva, Tatiana; Ivanova, Olga; Lottenbach, Kathleen; Belshe, Robert; Chumakov, Konstantin

2005-01-01

Sabin strains of poliovirus used in the manufacture of oral poliovirus vaccine (OPV) are prone to genetic variations that occur during growth in cell cultures and the organisms of vaccine recipients. Such derivative viruses often have increased neurovirulence and transmissibility, and in some cases they can reestablish chains of transmission in human populations. Monitoring for vaccine-derived polioviruses is an important part of the worldwide campaign to eradicate poliomyelitis. Analysis of vaccine-derived polioviruses requires, as a first step, their isolation in cell cultures, which takes significant time and may yield viral stocks that are not fully representative of the strains present in the original sample. Here we demonstrate that full-length viral cDNA can be PCR amplified directly from stool samples and immediately subjected to genomic analysis by oligonucleotide microarray hybridization and nucleotide sequencing. Most fecal samples from healthy children who received OPV were found to contain variants of Sabin vaccine viruses. Sequence changes in the 5′ untranslated region were common, as were changes in the VP1-coding region, including changes in a major antigenic site. Analysis of stool samples taken from cases of acute flaccid paralysis revealed the presence of mixtures of recombinant polioviruses, in addition to the emergence of new sequence variants. Avoiding the need for cell culture isolation dramatically shortened the time needed for identification and analysis of vaccine-derived polioviruses and could be useful for preliminary screening of clinical samples. The amplified full-length viral cDNA can be archived and used to recover live virus for further virological studies. PMID:15956413

Effects of Cr(III) and CR(VI) on nitrification inhibition as determined by SOUR, function-specific gene expression and 16S rRNA sequence analysis of wastewater nitrifying enrichments

EPA Science Inventory

The effect of Cr(III) and Cr(VI) on ammonia oxidation, the transcriptional responses of functional genes involved in nitrification and changes in 16S rRNA level sequences were examined in nitrifying enrichment cultures. The nitrifying bioreactor was operated as a continuous react...

Validation of Skeletal Muscle cis-Regulatory Module Predictions Reveals Nucleotide Composition Bias in Functional Enhancers

PubMed Central

Kwon, Andrew T.; Chou, Alice Yi; Arenillas, David J.; Wasserman, Wyeth W.

2011-01-01

We performed a genome-wide scan for muscle-specific cis-regulatory modules (CRMs) using three computational prediction programs. Based on the predictions, 339 candidate CRMs were tested in cell culture with NIH3T3 fibroblasts and C2C12 myoblasts for capacity to direct selective reporter gene expression to differentiated C2C12 myotubes. A subset of 19 CRMs validated as functional in the assay. The rate of predictive success reveals striking limitations of computational regulatory sequence analysis methods for CRM discovery. Motif-based methods performed no better than predictions based only on sequence conservation. Analysis of the properties of the functional sequences relative to inactive sequences identifies nucleotide sequence composition can be an important characteristic to incorporate in future methods for improved predictive specificity. Muscle-related TFBSs predicted within the functional sequences display greater sequence conservation than non-TFBS flanking regions. Comparison with recent MyoD and histone modification ChIP-Seq data supports the validity of the functional regions. PMID:22144875

Cultivable Anaerobic Microbiota of Severe Early Childhood Caries▿¶

PubMed Central

Tanner, A. C. R.; Mathney, J. M. J.; Kent, R. L.; Chalmers, N. I.; Hughes, C. V.; Loo, C. Y.; Pradhan, N.; Kanasi, E.; Hwang, J.; Dahlan, M. A.; Papadopolou, E.; Dewhirst, F. E.

2011-01-01

Severe early childhood caries (ECC), while strongly associated with Streptococcus mutans using selective detection (culture, PCR), has also been associated with a widely diverse microbiota using molecular cloning approaches. The aim of this study was to evaluate the microbiota of severe ECC using anaerobic culture. The microbial composition of dental plaque from 42 severe ECC children was compared with that of 40 caries-free children. Bacterial samples were cultured anaerobically on blood and acid (pH 5) agars. Isolates were purified, and partial sequences for the 16S rRNA gene were obtained from 5,608 isolates. Sequence-based analysis of the 16S rRNA isolate libraries from blood and acid agars of severe ECC and caries-free children had >90% population coverage, with greater diversity occurring in the blood isolate library. Isolate sequences were compared with taxon sequences in the Human Oral Microbiome Database (HOMD), and 198 HOMD taxa were identified, including 45 previously uncultivated taxa, 29 extended HOMD taxa, and 45 potential novel groups. The major species associated with severe ECC included Streptococcus mutans, Scardovia wiggsiae, Veillonella parvula, Streptococcus cristatus, and Actinomyces gerensceriae. S. wiggsiae was significantly associated with severe ECC children in the presence and absence of S. mutans detection. We conclude that anaerobic culture detected as wide a diversity of species in ECC as that observed using cloning approaches. Culture coupled with 16S rRNA identification identified over 74 isolates for human oral taxa without previously cultivated representatives. The major caries-associated species were S. mutans and S. wiggsiae, the latter of which is a candidate as a newly recognized caries pathogen. PMID:21289150

Sequencing, Analysis, and Annotation of Expressed Sequence Tags for Camelus dromedarius

PubMed Central

Al-Swailem, Abdulaziz M.; Shehata, Maher M.; Abu-Duhier, Faisel M.; Al-Yamani, Essam J.; Al-Busadah, Khalid A.; Al-Arawi, Mohammed S.; Al-Khider, Ali Y.; Al-Muhaimeed, Abdullah N.; Al-Qahtani, Fahad H.; Manee, Manee M.; Al-Shomrani, Badr M.; Al-Qhtani, Saad M.; Al-Harthi, Amer S.; Akdemir, Kadir C.; Otu, Hasan H.

2010-01-01

Despite its economical, cultural, and biological importance, there has not been a large scale sequencing project to date for Camelus dromedarius. With the goal of sequencing complete DNA of the organism, we first established and sequenced camel EST libraries, generating 70,272 reads. Following trimming, chimera check, repeat masking, cluster and assembly, we obtained 23,602 putative gene sequences, out of which over 4,500 potentially novel or fast evolving gene sequences do not carry any homology to other available genomes. Functional annotation of sequences with similarities in nucleotide and protein databases has been obtained using Gene Ontology classification. Comparison to available full length cDNA sequences and Open Reading Frame (ORF) analysis of camel sequences that exhibit homology to known genes show more than 80% of the contigs with an ORF>300 bp and ∼40% hits extending to the start codons of full length cDNAs suggesting successful characterization of camel genes. Similarity analyses are done separately for different organisms including human, mouse, bovine, and rat. Accompanying web portal, CAGBASE (http://camel.kacst.edu.sa/), hosts a relational database containing annotated EST sequences and analysis tools with possibility to add sequences from public domain. We anticipate our results to provide a home base for genomic studies of camel and other comparative studies enabling a starting point for whole genome sequencing of the organism. PMID:20502665

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China.

PubMed

Teng, Y; Liu, H; Lv, J Q; Fan, W H; Zhang, Q Y; Qin, Q W

2007-01-01

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

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.

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

Characterization of acid-tolerant H/CO-utilizing methanogenic enrichment cultures from an acidic peat bog in New York State.

PubMed

Bräuer, Suzanna L; Yashiro, Erika; Ueno, Norikiyo G; Yavitt, Joseph B; Zinder, Stephen H

2006-08-01

Two methanogenic cultures were enriched from acidic peat soil using a growth medium buffered to c. pH 5. One culture, 6A, was obtained from peat after incubation with H(2)/CO(2), whereas culture NTA was derived from a 10(-4) dilution of untreated peat into a modified medium. 16S rRNA gene clone libraries from each culture contained one methanogen and two bacterial sequences. The methanogen 16S rRNA gene sequences were 99% identical with each other and belonged to the novel "R-10/Fen cluster" family of the Methanomicrobiales, whereas their mcrA sequences were 96% identical. One bacterial 16S rRNA gene sequence from culture 6A belonged to the Bacteroidetes and showed 99% identity with sequences from methanogenic enrichments from German and Russian bogs. The other sequence belonged to the Firmicutes and was identical to a thick rod-shaped citrate-utilizing organism isolated from culture 6A, the numbers of which decreased when the Ti (III) chelator was switched from citrate to nitrilotriacetate. Bacterial clones from the NTA culture clustered in the Delta- and Betaproteobacteria. Both cultures contained thin rods, presumably the methanogens, as the predominant morphotype, and represent a significant advance in characterization of the novel acidiphilic R-10 family methanogens.

Depletion of Unwanted Nucleic Acid Templates by Selective Cleavage: LNAzymes, Catalytically Active Oligonucleotides Containing Locked Nucleic Acids, Open a New Window for Detecting Rare Microbial Community Members

PubMed Central

Dolinšek, Jan; Dorninger, Christiane; Lagkouvardos, Ilias; Wagner, Michael

2013-01-01

Many studies of molecular microbial ecology rely on the characterization of microbial communities by PCR amplification, cloning, sequencing, and phylogenetic analysis of genes encoding rRNAs or functional marker enzymes. However, if the established clone libraries are dominated by one or a few sequence types, the cloned diversity is difficult to analyze by random clone sequencing. Here we present a novel approach to deplete unwanted sequence types from complex nucleic acid mixtures prior to cloning and downstream analyses. It employs catalytically active oligonucleotides containing locked nucleic acids (LNAzymes) for the specific cleavage of selected RNA targets. When combined with in vitro transcription and reverse transcriptase PCR, this LNAzyme-based technique can be used with DNA or RNA extracts from microbial communities. The simultaneous application of more than one specific LNAzyme allows the concurrent depletion of different sequence types from the same nucleic acid preparation. This new method was evaluated with defined mixtures of cloned 16S rRNA genes and then used to identify accompanying bacteria in an enrichment culture dominated by the nitrite oxidizer “Candidatus Nitrospira defluvii.” In silico analysis revealed that the majority of publicly deposited rRNA-targeted oligonucleotide probes may be used as specific LNAzymes with no or only minor sequence modifications. This efficient and cost-effective approach will greatly facilitate tasks such as the identification of microbial symbionts in nucleic acid preparations dominated by plastid or mitochondrial rRNA genes from eukaryotic hosts, the detection of contaminants in microbial cultures, and the analysis of rare organisms in microbial communities of highly uneven composition. PMID:23263968

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.

[Blue-light induced expression of S-adenosy-L-homocysteine hydrolase-like gene in Mucor amphibiorum RCS1].

PubMed

Gao, Ya; Wang, Shu; Fu, Mingjia; Zhong, Guolin

2013-09-04

To determine blue-light induced expression of S-adenosyl-L-homocysteine hydrolase-like (sahhl) gene in fungus Mucor amphibiorum RCS1. In the random process of PCR, a sequence of 555 bp was obtained from M. amphibiorum RCS1. The 555 bp sequence was labeled with digoxin to prepare the probe for northern hybridization. By northern hybridization, the transcription of sahhl gene was analyzed in M. amphibiorum RCS1 mycelia culture process from darkness to blue light to darkness. Simultaneously real-time PCR method was used to the sahhl gene expression analysis. Compared with the sequence of sahh gene from Homo sapiens, Mus musculus and some fungi species, a high homology of the 555 bp sequence was confirmed. Therefore, the preliminary confirmation has supported that the 555 bp sequence should be sahhl gene from M. amphibiorum RCS1. Under the dark pre-culture in 24 h, a large amounts of transcript of sahhl gene in the mycelia can be detected by northern hybridization and real-time PCR in the condition of 24 h blue light. But a large amounts of transcript of sahhl gene were not found in other detection for the dark pre-culture of 48 h, even though M. amphibiorum RCS1 mycelia were induced by blue light. Blue light can induce the expression of sahhl gene in the vigorous growth of M. amphibiorum RCS1 mycelia.

Characterization of culturable anaerobic bacteria from the forestomach of an eastern grey kangaroo, Macropus giganteus.

PubMed

Ouwerkerk, D; Klieve, A V; Forster, R J; Templeton, J M; Maguire, A J

2005-01-01

To determine the culturable biodiversity of anaerobic bacteria isolated from the forestomach contents of an eastern grey kangaroo, Macropus giganteus, using phenotypic characterization and 16S rDNA sequence analysis. Bacteria from forestomach contents of an eastern grey kangaroo were isolated using anaerobic media containing milled curly Mitchell grass (Astrebla lappacea). DNA was extracted and the 16S rDNA sequenced for phylogenetic analysis. Forty bacterial isolates were obtained and placed in 17 groups based on phenotypic characteristics and restriction enzyme digestion of 16S rDNA PCR products. DNA sequencing revealed that the 17 groups comprised five known species (Clostridium butyricum, Streptococcus bovis, Clostridium sporogenes, Clostridium paraputrificum and Enterococcus avium) and 12 groups apparently representing new species, all within the phylum Firmicutes. Foregut contents from Australian macropod marsupials contain a microbial ecosystem with a novel bacterial biodiversity comprising a high percentage of previously unrecognized species. This study adds to knowledge of Australia's unique biodiversity, which may provide a future bioresource of genetic information and bacterial species of benefit to agriculture.

Microbial diversity of a Camembert-type cheese using freeze-dried Tibetan kefir coculture as starter culture by culture-dependent and culture-independent methods.

PubMed

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

PubMed Central

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese. PMID:25360757

Mycobacterium tuberculosis and whole genome sequencing: a practical guide and online tools available for the clinical microbiologist.

PubMed

Satta, G; Atzeni, A; McHugh, T D

2017-02-01

Whole genome sequencing (WGS) has the potential to revolutionize the diagnosis of Mycobacterium tuberculosis infection but the lack of bioinformatic expertise among clinical microbiologists is a barrier for adoption. Software products for analysis should be simple, free of charge, able to accept data directly from the sequencer (FASTQ files) and to provide the basic functionalities all-in-one. The main aim of this narrative review is to provide a practical guide for the clinical microbiologist, with little or no practical experience of WGS analysis, with a specific focus on software products tailor-made for M. tuberculosis analysis. With sequencing performed by an external provider, it is now feasible to implement WGS analysis in the routine clinical practice of any microbiology laboratory, with the potential to detect resistance weeks before traditional phenotypic culture methods, but the clinical microbiologist should be aware of the limitations of this approach. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Novel insect-specific flavivirus isolated from northern Europe

PubMed Central

Huhtamo, Eili; Moureau, Gregory; Cook, Shelley; Julkunen, Ora; Putkuri, Niina; Kurkela, Satu; Uzcátegui, Nathalie Y.; Harbach, Ralph E.; Gould, Ernest A.; Vapalahti, Olli; de Lamballerie, Xavier

2012-01-01

Mosquitoes collected in Finland were screened for flaviviral RNA leading to the discovery and isolation of a novel flavivirus designated Hanko virus (HANKV). Virus characterization, including phylogenetic analysis of the complete coding sequence, confirmed HANKV as a member of the “insect-specific” flavivirus (ISF) group. HANKV is the first member of this group isolated from northern Europe, and therefore the first northern European ISF for which the complete coding sequence has been determined. HANKV was not transcribed as DNA in mosquito cell culture, which appears atypical for an ISF. HANKV shared highest sequence homology with the partial NS5 sequence available for the recently discovered Spanish Ochlerotatus flavivirus (SOcFV). Retrospective analysis of mitochondrial sequences from the virus-positive mosquito pool suggested an Ochlerotatus mosquito species as the most likely host for HANKV. HANKV and SOcFV may therefore represent a novel group of Ochlerotatus-hosted insect-specific flaviviruses in Europe and further afield. PMID:22999256

Proteome reference maps of Medicago truncatula embryogenic cell cultures generated from single protoplasts.

PubMed

Imin, Nijat; De Jong, Femke; Mathesius, Ulrike; van Noorden, Giel; Saeed, Nasir A; Wang, Xin-Ding; Rose, Ray J; Rolfe, Barry G

2004-07-01

Using a combination of two-dimensional gel electrophoresis (2-DE) protein mapping and mass spectrometry (MS) analysis, we have established proteome reference maps of Medicago truncatula embryogenic tissue culture cells. The cultures were generated from single protoplasts, which provided a relatively homogeneous cell population. We used these to analyze protein expression at the globular stages of somatic embryogenesis, which is the earliest morphogenetic embryonic stage. Over 3000 proteins could reproducibly be resolved over a pI range of 4-11. Three hundred and twelve protein spots were extracted from colloidal Coomassie Blue-stained 2-DE gels and analyzed by matrix-assisted laser desorption/ionization-time of flight MS analysis and tandem MS sequencing. This enabled the identification of 169 protein spots representing 128 unique gene products using a publicly available expressed sequence tag database and the MASCOT search engine. These reference maps will be valuable for the investigation of the molecular events which occur during somatic embryogenesis in M. truncatula. The proteome reference maps and supplementary materials will be available and updated for public access at http://semele.anu.edu.au/.

Discovery of a trans-Dichloroethene-Respiring Dehalogenimonas Species in the 1,1,2,2-Tetrachloroethane-Dechlorinating WBC-2 Consortium

PubMed Central

Manchester, Marie J.; Hug, Laura A.; Zarek, Matt; Zila, Anna

2012-01-01

The WBC-2 consortium is an organohalide-respiring anaerobic microbial enrichment culture capable of dechlorinating 1,1,2,2-tetrachloroethane (TeCA) to ethene. In the WBC-2 culture, TeCA is first transformed to trans-dichloroethene (tDCE) by dichloroelimination; tDCE is subsequently transformed to vinyl chloride (VC) and then to ethene by hydrogenolysis. Analysis of 16S rRNA gene clone libraries from culture DNA revealed sequences from three putative dechlorinating organisms belonging to Dehalococcoides, Dehalobacter, and Dehalogenimonas genera. Quantitative PCR primers were designed for each of these sequences, and their abundance was quantified in enrichment cultures over time. These data revealed that complete dechlorination of TeCA to ethene involves all three organisms. Dehalobacter spp. grew during the dihaloelimination of TeCA to tDCE, while Dehalococcoides and Dehalogenimonas spp. grew during hydrogenolysis of tDCE to ethene. This is the first time a genus other than Dehalococcoides has been implicated in dechlorination of tDCE to VC. PMID:22635995

Identification of a mouse synaptic glycoprotein gene in cultured neurons.

PubMed

Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

2005-10-01

Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

SEPT9 Mutations and a Conserved 17q25 Sequence in Sporadic and Hereditary Brachial Plexus Neuropathy

PubMed Central

Klein, Christopher J.; Wu, Yanhong; Cunningham, Julie M.; Windebank, Anthony J.; Dyck, P. James B.; Friedenberg, Scott M.; Klein, Diane M.; Dyck, Peter J.

2009-01-01

Background The clinical characteristics of sporadic brachial plexus neuropathy (S-BPN) and hereditary brachial plexus neuropathy (H-BPN) are similar. At times of attack inflammation in brachial plexus nerves has been identified in both conditions. SEPT-9 mutations (Arg88Trp, Ser93Phe, 5UTR-131G to C) occur in some families with H-BPN. These mutations were not found in American H-BPN kindreds with a conserved 500 Kb sequence of DNA at 17q25 (the location of SEPT-9) where a founder mutation has been suggested. Objective To study 17q25 and SEPT-9 in S-BPN (56 patients) and H-BPN (13 kindreds). Methods Allele analysis at 17q25, SEPT-9 DNA sequencing and mRNA analysis from lymphoblast cultures. Results A conserved 17q25 sequence was found in 5 of 13 H-BPN kindreds and one S-BPN patient. This conserved sequence was not found in the family with a SEPT-9 mutation (Arg88Trp) or controls (182). SEPT-9 mRNA expression did not differ between forms of H-BPN and controls. No known mutations of SEPT-9 were found in S-BPN. Conclusions/Relevance Rare S-BPN patients have the same conserved 17q25 sequence found in many American H-BPN kindreds. BPN patients with this conserved sequence do not appear to have SEPT-9 mutations or alterations of its mRNA expression levels in lymphoblast cultures. BPN patients with this conserved sequence may have the most common genetic cause in the Americas by a founder effect mutation. PMID:19204161

Culture-Independent Analysis of Aerosol Microbiology in a Metropolitan Subway System

PubMed Central

Robertson, Charles E.; Baumgartner, Laura K.; Harris, J. Kirk; Peterson, Kristen L.; Stevens, Mark J.; Frank, Daniel N.

2013-01-01

The goal of this study was to determine the composition and diversity of microorganisms associated with bioaerosols in a heavily trafficked metropolitan subway environment. We collected bioaerosols by fluid impingement on several New York City subway platforms and associated sites in three sampling sessions over a 1.5-year period. The types and quantities of aerosolized microorganisms were determined by culture-independent phylogenetic analysis of small-subunit rRNA gene sequences by using both Sanger (universal) and pyrosequencing (bacterial) technologies. Overall, the subway bacterial composition was relatively simple; only 26 taxonomic families made up ∼75% of the sequences determined. The microbiology was more or less similar throughout the system and with time and was most similar to outdoor air, consistent with highly efficient air mixing in the system. Identifiable bacterial sequences indicated that the subway aerosol assemblage was composed of a mixture of genera and species characteristic of soil, environmental water, and human skin commensal bacteria. Eukaryotic diversity was mainly fungal, dominated by organisms of types associated with wood rot. Human skin bacterial species (at 99% rRNA sequence identity) included the Staphylococcus spp. Staphylococcus epidermidis (the most abundant and prevalent commensal of the human integument), S. hominis, S. cohnii, S. caprae, and S. haemolyticus, all well-documented human commensal bacteria. We encountered no organisms of public health concern. This study is the most extensive culture-independent survey of subway microbiota so far and puts in place pre-event information required for any bioterrorism surveillance activities or monitoring of the microbiological impact of recent subway flooding events. PMID:23542619

Culture-independent analysis of aerosol microbiology in a metropolitan subway system.

PubMed

Robertson, Charles E; Baumgartner, Laura K; Harris, J Kirk; Peterson, Kristen L; Stevens, Mark J; Frank, Daniel N; Pace, Norman R

2013-06-01

The goal of this study was to determine the composition and diversity of microorganisms associated with bioaerosols in a heavily trafficked metropolitan subway environment. We collected bioaerosols by fluid impingement on several New York City subway platforms and associated sites in three sampling sessions over a 1.5-year period. The types and quantities of aerosolized microorganisms were determined by culture-independent phylogenetic analysis of small-subunit rRNA gene sequences by using both Sanger (universal) and pyrosequencing (bacterial) technologies. Overall, the subway bacterial composition was relatively simple; only 26 taxonomic families made up ~75% of the sequences determined. The microbiology was more or less similar throughout the system and with time and was most similar to outdoor air, consistent with highly efficient air mixing in the system. Identifiable bacterial sequences indicated that the subway aerosol assemblage was composed of a mixture of genera and species characteristic of soil, environmental water, and human skin commensal bacteria. Eukaryotic diversity was mainly fungal, dominated by organisms of types associated with wood rot. Human skin bacterial species (at 99% rRNA sequence identity) included the Staphylococcus spp. Staphylococcus epidermidis (the most abundant and prevalent commensal of the human integument), S. hominis, S. cohnii, S. caprae, and S. haemolyticus, all well-documented human commensal bacteria. We encountered no organisms of public health concern. This study is the most extensive culture-independent survey of subway microbiota so far and puts in place pre-event information required for any bioterrorism surveillance activities or monitoring of the microbiological impact of recent subway flooding events.

Microbial composition analyses by 16S rRNA sequencing: A proof of concept approach to provenance determination of archaeological ochre.

PubMed

Lenehan, Claire E; Tobe, Shanan S; Smith, Renee J; Popelka-Filcoff, Rachel S

2017-01-01

Many archaeological science studies use the concept of "provenance", where the origins of cultural material can be determined through physical or chemical properties that relate back to the origins of the material. Recent studies using DNA profiling of bacteria have been used for the forensic determination of soils, towards determination of geographic origin. This manuscript presents a novel approach to the provenance of archaeological minerals and related materials through the use of 16S rRNA sequencing analysis of microbial DNA. Through the microbial DNA characterization from ochre and multivariate statistics, we have demonstrated the clear discrimination between four distinct Australian cultural ochre sites.

Song hybridization events during revolutionary song change provide insights into cultural transmission in humpback whales

PubMed Central

Garland, Ellen C.; Rendell, Luke; Lamoni, Luca; Poole, M. Michael; Noad, Michael J.

2017-01-01

Cultural processes occur in a wide variety of animal taxa, from insects to cetaceans. The songs of humpback whales are one of the most striking examples of the transmission of a cultural trait and social learning in any nonhuman animal. To understand how songs are learned, we investigate rare cases of song hybridization, where parts of an existing song are spliced with a new one, likely before an individual totally adopts the new song. Song unit sequences were extracted from over 9,300 phrases recorded during two song revolutions across the South Pacific Ocean, allowing fine-scale analysis of composition and sequencing. In hybrid songs the current and new songs were spliced together in two specific ways: (i) singers placed a single hybrid phrase, in which content from both songs were combined, between the two song types when transitioning from one to the other, and/or (ii) singers spliced complete themes from the revolutionary song into the current song. Sequence analysis indicated that both processes were governed by structural similarity rules. Hybrid phrases or theme substitutions occurred at points in the songs where both songs contained “similar sounds arranged in a similar pattern.” Songs appear to be learned as segments (themes/phrase types), akin to birdsong and human language acquisition, and these can be combined in predictable ways if the underlying structural pattern is similar. These snapshots of song change provide insights into the mechanisms underlying song learning in humpback whales, and comparative perspectives on the evolution of human language and culture. PMID:28739940

Song hybridization events during revolutionary song change provide insights into cultural transmission in humpback whales.

PubMed

Garland, Ellen C; Rendell, Luke; Lamoni, Luca; Poole, M Michael; Noad, Michael J

2017-07-24

Cultural processes occur in a wide variety of animal taxa, from insects to cetaceans. The songs of humpback whales are one of the most striking examples of the transmission of a cultural trait and social learning in any nonhuman animal. To understand how songs are learned, we investigate rare cases of song hybridization, where parts of an existing song are spliced with a new one, likely before an individual totally adopts the new song. Song unit sequences were extracted from over 9,300 phrases recorded during two song revolutions across the South Pacific Ocean, allowing fine-scale analysis of composition and sequencing. In hybrid songs the current and new songs were spliced together in two specific ways: ( i ) singers placed a single hybrid phrase, in which content from both songs were combined, between the two song types when transitioning from one to the other, and/or ( ii ) singers spliced complete themes from the revolutionary song into the current song. Sequence analysis indicated that both processes were governed by structural similarity rules. Hybrid phrases or theme substitutions occurred at points in the songs where both songs contained "similar sounds arranged in a similar pattern." Songs appear to be learned as segments (themes/phrase types), akin to birdsong and human language acquisition, and these can be combined in predictable ways if the underlying structural pattern is similar. These snapshots of song change provide insights into the mechanisms underlying song learning in humpback whales, and comparative perspectives on the evolution of human language and culture.

Application of cryopreservation to genetic analyses of a photosynthetic picoeukaryote community.

PubMed

Kawachi, Masanobu; Kataoka, Takafumi; Sato, Mayumi; Noël, Mary-Hélène; Kuwata, Akira; Demura, Mikihide; Yamaguchi, Haruyo

2016-02-01

Cryopreservation is useful for long-term maintenance of living strains in microbial culture collections. We applied this technique to environmental specimens from two monitoring sites at Sendai Bay, Japan and compared the microbial diversity of photosynthetic picoeukaryotes in samples before and after cryopreservation. Flow cytometry (FCM) showed no considerable differences between specimens. We used 2500 cells sorted with FCM for next-generation sequencing of 18S rRNA gene amplicons and after removing low-quality sequences obtained 10,088-37,454 reads. Cluster analysis and comparative correlation analysis of observed high-level operational taxonomic units indicated similarity between specimens before and after cryopreservation. The effects of cryopreservation on cells were assessed with representative culture strains, including fragile cryptophyte cells. We confirmed the usefulness of cryopreservation for genetic studies on environmental specimens, and found that small changes in FCM cytograms after cryopreservation may affect biodiversity estimation. Copyright © 2015 Elsevier B.V. All rights reserved.

[Molecular epidemiological analysis of rubella virus isolates from 2001 to 2011 in Shanghai, China].

PubMed

Li, Chong-Shan; Yang, Yu-Ying; Wang, Jian-Guo; Zhu, Zhen; Tang, Wei; Li, Zhi; Sun, Xiao-Dong; Xu, Wen-Bo

2012-03-01

Throat swabs collected from patients whose serum was measles IgM negative and rubella IgM positive during 2001-2011 were used to conduct cell culture for rubella virus. After identification of cell culture with RT-PCR, nucleotide of gene E1 of rubella virus was amplified and sequenced, followed by molecular epidemiological analysis. A total of 31 rubella viruses were isolated from 60 throat swabs. Compared 27 isolates with the WHO reference strains of all genotypes, phylogenetic tree was constructed based on the amplified 739 nucleotide fragment. These isolates belonged to two different genotypes respectively. Isolates 11009, 11052 and 11106 in 2011 belonged to genotype 2B, and others belonged to genotype 1E. Most of mutations were nonsense mutation, and sequence of amino acid was highly conserved. Amino acid sequence of most isolates of genotype 1E was identical, which suggested rubella viruses from same transmission chain might be transmitted continually since 2001. Rubella virus genotype 2B was found to be popular for the first time in Shanghai in 2011. The nucleotide sequences of these genotype 2B isolates showed 99% identity compared with that of isolates recently from Vietnam, Japan and Argentina. The resources of these strains were not confirmed due to the absence of rubella virus surveillance before.

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

Microbial diversity in an Armenian geothermal spring assessed by molecular and culture-based methods.

PubMed

Panosyan, Hovik; Birkeland, Nils-Kåre

2014-11-01

The phylogenetic diversity of the prokaryotic community thriving in the Arzakan hot spring in Armenia was studied using molecular and culture-based methods. A sequence analysis of 16S rRNA gene clone libraries demonstrated the presence of a diversity of microorganisms belonging to the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Epsilonproteobacteria, Firmicutes, Bacteroidetes phyla, and Cyanobacteria. Proteobacteria was the dominant group, representing 52% of the bacterial clones. Denaturing gradient gel electrophoresis profiles of the bacterial 16S rRNA gene fragments also indicated the abundance of Proteobacteria, Bacteroidetes, and Cyanobacteria populations. Most of the sequences were most closely related to uncultivated microorganisms and shared less than 96% similarity with their closest matches in GenBank, indicating that this spring harbors a unique community of novel microbial species or genera. The majority of the sequences of an archaeal 16S rRNA gene library, generated from a methanogenic enrichment, were close relatives of members of the genus Methanoculleus. Aerobic endospore-forming bacteria mainly belonging to Bacillus and Geobacillus were detected only by culture-dependent methods. Three isolates were successfully obtained having 99, 96, and 96% 16S rRNA gene sequence similarities to Arcobacter sp., Methylocaldum sp., and Methanoculleus sp., respectively. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of multilocus sequence typing and pulsed-field gel electrophoresis for Salmonella spp. identification in surface water

NASA Astrophysics Data System (ADS)

Kuo, Chun Wei; Hao Huang, Kuan; Hsu, Bing Mu; Tsai, Hsien Lung; Tseng, Shao Feng; Kao, Po Min; Shen, Shu Min; Chou Chiu, Yi; Chen, Jung Sheng

2013-04-01

Salmonella is one of the most important pathogens of waterborne diseases with outbreaks from contaminated water reported worldwide. In addition, Salmonella spp. can survive for long periods in aquatic environments. To realize genotypes and serovars of Salmonella in aquatic environments, we isolated the Salmonella strains by selective culture plates to identify the serovars of Salmonella by serological assay, and identify the genotypes by Multilocus sequence typing (MLST) based on the sequence data from University College Cork (UCC), respectively. The results show that 36 stream water samples (30.1%) and 18 drinking water samples (23.3%) were confirmed the existence of Salmonella using culture method combined PCR specific invA gene amplification. In this study, 24 cultured isolates of Salmonella from water samples were classified to fifteen Salmonella enterica serovars. In addition, we construct phylogenetic analysis using phylogenetic tree and Minimum spanning tree (MST) method to analyze the relationship of clinical, environmental, and geographical data. Phylogenetic tree showed that four main clusters and our strains can be distributed in all. The genotypes of isolates from stream water are more biodiversity while comparing the Salmonella strains genotypes from drinking water sources. According to MST data, we can found the positive correlation between serovars and genotypes of Salmonella. Previous studies revealed that the result of Pulsed field gel electrophoresis (PFGE) method can predict the serovars of Salmonella strain. Hence, we used the MLST data combined phylogenetic analysis to identify the serovars of Salmonella strain and achieved effectiveness. While using the geographical data combined phylogenetic analysis, the result showed that the dominant strains were existed in whole stream area in rainy season. Keywords: Salmonella spp., MLST, phylogenetic analysis, PFGE

Identification of a high frequency transposon induced by tissue culture, nDaiZ, a member of the hAT family in rice.

PubMed

Huang, Jian; Zhang, Kewei; Shen, Yi; Huang, Zejun; Li, Ming; Tang, Ding; Gu, Minghong; Cheng, Zhukuan

2009-03-01

Recent completion of rice genome sequencing has revealed that more than 40% of its genome consists of repetitive sequences, and most of them are related to inactive transposable elements. In the present study, a transposable element, nDaiZ0, which is induced by tissue culture with high frequency, was identified by sequence analysis of an allelic line of the golden hull and internode 2 (gh2) mutant, which was integrated into the forth exon of GH2. The 528-bp nDaiZ0 has 14-bp terminal inverted repeats (TIRs), and generates an 8-bp duplication of its target sites (TSD) during its mobilization. nDaiZs are non-autonomous transposons and have no coding capacity. Bioinformatics analysis and southern blot hybridization showed that at least 16 copies of nDaiZ elements exist in the japonica cultivar Nipponbare genome and 11 copies in the indica cultivar 93-11 genome. During tissue culture, only one copy, nDaiZ9, located on chromosome 5 in the genome of Nipponbare can be activated with its transposable frequency reaching 30%. However, nDaiZ9 was not present in the 93-11 genome. The larger elements, DaiZs, were further identified by database searching using nDaiZ0 as a query because they share similar TIRs and subterminal sequences. DaiZ can also generate an 8-bp TSD. DaiZ elements contain a conserved region with a high similarity to the hAT dimerization motif, suggesting that the nDaiZ-DaiZ transposon system probably belongs to the hAT superfamily of class II transposons. Phylogenetic analysis indicated that it is a new type of plant hAT-like transposon. Although nDaiZ is activated by tissue culture, the high transposable frequency indicates that it could become a useful gene tagging system for rice functional genomic studies. In addition, the mechanism of the high transposable ability of nDaiZ9 is discussed.

Moussa virus: a new member of the Rhabdoviridae family isolated from Culex decens mosquitoes in Côte d’Ivoire

PubMed Central

Quan, Phenix-Lan; Junglen, Sandra; Tashmukhamedova, Alla; Conlan, Sean; Hutchison, Stephen K.; Kurth, Andreas; Ellerbrok, Heinz; Egholm, Michael; Briese, Thomas; Leendertz, Fabian H.; Ian Lipkin, W

2009-01-01

Characterization of arboviruses at the interface of pristine habitats and anthropogenic landscapes is crucial to comprehensive emergent disease surveillance and forecasting efforts. In context of surveillance campaign in and around a West African rainforest, particles morphologically consistent with rhabdoviruses were identified in cell cultures infected with homogenates of trapped mosquitoes. RNA recovered from these cultures was used to derive the first complete genome sequence of a rhabdovirus isolated from Culex decens mosquitoes in Côte d’Ivoire, tentatively named Moussa virus (MOUV). MOUV shows the classical genome organization of rhabdoviruses, with five open reading frames (ORF) in a linear order. However, sequences show only limited conservation (12–33% identity at amino acid level), and ORF2 and ORF3 have no significant similarity to sequences deposited in GenBank. Phylogenetic analysis indicates a potential new species with distant relationship to Tupaia and Tibrogargan virus. PMID:19804801

The dynamics of genome replication using deep sequencing

PubMed Central

Müller, Carolin A.; Hawkins, Michelle; Retkute, Renata; Malla, Sunir; Wilson, Ray; Blythe, Martin J.; Nakato, Ryuichiro; Komata, Makiko; Shirahige, Katsuhiko; de Moura, Alessandro P.S.; Nieduszynski, Conrad A.

2014-01-01

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology. PMID:24089142

Moussa virus: a new member of the Rhabdoviridae family isolated from Culex decens mosquitoes in Côte d'Ivoire.

PubMed

Quan, Phenix-Lan; Junglen, Sandra; Tashmukhamedova, Alla; Conlan, Sean; Hutchison, Stephen K; Kurth, Andreas; Ellerbrok, Heinz; Egholm, Michael; Briese, Thomas; Leendertz, Fabian H; Lipkin, W Ian

2010-01-01

Characterization of arboviruses at the interface of pristine habitats and anthropogenic landscapes is crucial to comprehensive emergent disease surveillance and forecasting efforts. In context of a surveillance campaign in and around a West African rainforest, particles morphologically consistent with rhabdoviruses were identified in cell cultures infected with homogenates of trapped mosquitoes. RNA recovered from these cultures was used to derive the first complete genome sequence of a rhabdovirus isolated from Culex decens mosquitoes in Côte d'Ivoire, tentatively named Moussa virus (MOUV). MOUV shows the classical genome organization of rhabdoviruses, with five open reading frames (ORF) in a linear order. However, sequences show only limited conservation (12-33% identity at amino acid level), and ORF2 and ORF3 have no significant similarity to sequences deposited in GenBank. Phylogenetic analysis indicates a potential new species with distant relationship to Tupaia and Tibrogargan virus.

Metagenomic analysis of bloodstream infections in patients with acute leukemia and therapy-induced neutropenia

PubMed Central

Gyarmati, P.; Kjellander, C.; Aust, C.; Song, Y.; Öhrmalm, L.; Giske, C. G.

2016-01-01

Leukemic patients are often immunocompromised due to underlying conditions, comorbidities and the effects of chemotherapy, and thus at risk for developing systemic infections. Bloodstream infection (BSI) is a severe complication in neutropenic patients, and is associated with increased mortality. BSI is routinely diagnosed with blood culture, which only detects culturable pathogens. We analyzed 27 blood samples from 9 patients with acute leukemia and suspected BSI at different time points of their antimicrobial treatment using shotgun metagenomics sequencing in order to detect unculturable and non-bacterial pathogens. Our findings confirm the presence of bacterial, fungal and viral pathogens alongside antimicrobial resistance genes. Decreased white blood cell (WBC) counts were associated with the presence of microbial DNA, and was inversely proportional to the number of sequencing reads. This study could indicate the use of high-throughput sequencing for personalized antimicrobial treatments in BSIs. PMID:26996149

Microbial diversity of culturable heterotrophs in the rhizosphere of salt marsh grass, Porteresia coarctata (Tateoka) in a mangrove ecosystem.

PubMed

Bharathkumar, Srinivasan; Paul, Diby; Nair, Sudha

2008-02-01

A study was conducted to understand the complexity of bacterial diversity of rhizosphere of Porteresia coarctata based on culture dependent method. A large number of bacteria were isolated on nutrient agar medium supplemented with 1% NaCl and the dominant ones were further analyzed with PCR-RFLP method. The sequence analyses of the dominant strains revealed that most of the sequences belonged to members of gamma proteobacteria, firmicutes, bacteroidetes and uncultured bacteria. The phylogenetic analysis of 16S rRNA gene sequences revealed close relationships to a wide range of clones or bacterial species of various divisions. These results afford an understanding of the role of rhizobacteria in alleviating salt stress in Porteresia coarctata expected to contribute towards long-term goal of improving plant-microbe interactions for salinity affected fields. (c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Endophytic bacteria in plant tissue culture: differences between easy- and difficult-to-propagate Prunus avium genotypes.

PubMed

Quambusch, Mona; Pirttilä, Anna Maria; Tejesvi, Mysore V; Winkelmann, Traud; Bartsch, Melanie

2014-05-01

The endophytic bacterial communities of six Prunus avium L. genotypes differing in their growth patterns during in vitro propagation were identified by culture-dependent and culture-independent methods. Five morphologically distinct isolates from tissue culture material were identified by 16S rDNA sequence analysis. To detect and analyze the uncultivable fraction of endophytic bacteria, a clone library was established from the amplified 16S rDNA of total plant extract. Bacterial diversity within the clone libraries was analyzed by amplified ribosomal rDNA restriction analysis and by sequencing a clone for each identified operational taxonomic unit. The most abundant bacterial group was Mycobacterium sp., which was identified in the clone libraries of all analyzed Prunus genotypes. Other dominant bacterial genera identified in the easy-to-propagate genotypes were Rhodopseudomonas sp. and Microbacterium sp. Thus, the community structures in the easy- and difficult-to-propagate cherry genotypes differed significantly. The bacterial genera, which were previously reported to have plant growth-promoting effects, were detected only in genotypes with high propagation success, indicating a possible positive impact of these bacteria on in vitro propagation of P. avium, which was proven in an inoculation experiment. © The Author 2014. Published by Oxford University Press. All rights reserved.

Molecular cloning of the Coch gene of guinea pig inner ear and its expression analysis in cultured fibrocytes of the spiral ligament.

PubMed

Li, Lishu; Ikezono, Tetsuo; Sekine, Kuwon; Shindo, Susumu; Matsumura, Tomohiro; Pawankar, Ruby; Ichimiya, Issei; Yagi, Toshiaki

2010-08-01

We have cloned guinea pig Coch cDNA and the sequence information will be useful for future molecular study combined with physiological experiments. Proper Coch gene expression appears to be dependent on the unique extracellular micro-environment of the inner ear in vivo. These results provide insight into the Coch gene expression and its regulation. To characterize the guinea pig Coch gene, we performed molecular cloning and expression analysis in the inner ear and cultured fibrocytes of the spiral ligament. The Coch cDNA was isolated using RACE. Cochlin isofoms were studied by Western blot using three different types of mammalian inner ear. The cochlear fibrocytes were cultured and characterized by immunostaining. Coch gene expression in the fibrocytes was investigated and the influence of cytokine stimulation was evaluated. The full-length 1991 bp Coch cDNA that encodes a 553 amino acid protein was isolated. The sequence had significant homology with other mammals, and the sizes of the Cochlin isoforms were identical. In the cultured fibrocytes, Coch mRNA was expressed in a very small amount and the isoform production was different, compared with the results in vivo. Cytokine stimulation did not alter the level of mRNA expression or isoform formation.

Culturing of female bladder bacteria reveals an interconnected urogenital microbiota.

PubMed

Thomas-White, Krystal; Forster, Samuel C; Kumar, Nitin; Van Kuiken, Michelle; Putonti, Catherine; Stares, Mark D; Hilt, Evann E; Price, Travis K; Wolfe, Alan J; Lawley, Trevor D

2018-04-19

Metagenomic analyses have indicated that the female bladder harbors an indigenous microbiota. However, there are few cultured reference strains with sequenced genomes available for functional and experimental analyses. Here we isolate and genome-sequence 149 bacterial strains from catheterized urine of 77 women. This culture collection spans 78 species, representing approximately two thirds of the bacterial diversity within the sampled bladders, including Proteobacteria, Actinobacteria, and Firmicutes. Detailed genomic and functional comparison of the bladder microbiota to the gastrointestinal and vaginal microbiotas demonstrates similar vaginal and bladder microbiota, with functional capacities that are distinct from those observed in the gastrointestinal microbiota. Whole-genome phylogenetic analysis of bacterial strains isolated from the vagina and bladder in the same women identifies highly similar Escherichia coli, Streptococcus anginosus, Lactobacillus iners, and Lactobacillus crispatus, suggesting an interlinked female urogenital microbiota that is not only limited to pathogens but is also characteristic of health-associated commensals.

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

PubMed

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

2017-10-01

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

Application of the major capsid protein as a marker of the phylogenetic diversity of Emiliania huxleyi viruses.

PubMed

Rowe, Janet M; Fabre, Marie-Françoise; Gobena, Daniel; Wilson, William H; Wilhelm, Steven W

2011-05-01

Studies of the Phycodnaviridae have traditionally relied on the DNA polymerase (pol) gene as a biomarker. However, recent investigations have suggested that the major capsid protein (MCP) gene may be a reliable phylogenetic biomarker. We used MCP gene amplicons gathered across the North Atlantic to assess the diversity of Emiliania huxleyi-infecting Phycodnaviridae. Nucleotide sequences were examined across >6000 km of open ocean, with comparisons between concentrates of the virus-size fraction of seawater and of lysates generated by exposing host strains to these same virus concentrates. Analyses revealed that many sequences were only sampled once, while several were over-represented. Analyses also revealed nucleotide sequences distinct from previous coastal isolates. Examination of lysed cultures revealed a new richness in phylogeny, as MCP sequences previously unrepresented within the existing collection of E. huxleyi viruses (EhV) were associated with viruses lysing cultures. Sequences were compared with previously described EhV MCP sequences from the North Sea and a Norwegian Fjord, as well as from the Gulf of Maine. Principal component analysis indicates that location-specific distinctions exist despite the presence of sequences common across these environments. Overall, this investigation provides new sequence data and an assessment on the use of the MCP gene. © 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved.

Isolation and characterization of Scophthalmus maximus rhabdovirus.

PubMed

Zhang, Qi-Ya; Tao, Jian-Jun; Gui, Lang; Zhou, Guang-Zhou; Ruan, Hong-Mei; Li, Zhen-Qiu; Gui, Jian-Fang

2007-02-28

A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalmus maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.

16S rRNA analysis provides evidence of biofilms on all components of three infected periprosthetic knees including permanent braided suture.

PubMed

Swearingen, Matthew C; DiBartola, Alex C; Dusane, Devendra; Granger, Jeffrey; Stoodley, Paul

2016-10-01

Bacterial biofilms are the main etiological agent of periprosthetic joint infections (PJI); however, it is unclear if biofilms colonize one or multiple components. Because biofilms can colonize a variety of surfaces, we hypothesized that biofilms would be present on all components. 16S ribosomal RNA (rRNA) gene sequencing analysis was used to identify bacteria recovered from individual components and non-absorbable suture material recovered from three PJI total knee revision cases. Bray-Curtis non-metric multidimensional scaling analysis revealed no significant differences in similarity when factoring component, material type, or suture versus non-suture material, but did reveal significant differences in organism profile between patients (P < 0.001) and negative controls (P < 0.001). Confocal microscopy and a novel agar encasement culturing method also confirmed biofilm growth on a subset of components. While 16S sequencing suggested that the microbiology was more complex than revealed by culture contaminating, bacterial DNA generates a risk of false positives. This report highlights that biofilm bacteria may colonize all infected prosthetic components including braided suture material, and provides further evidence that clinical culture can fail to sufficiently identify the full pathogen profile in PJI cases. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Dissecting biological “dark matter” with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth

PubMed Central

Marcy, Yann; Ouverney, Cleber; Bik, Elisabeth M.; Lösekann, Tina; Ivanova, Natalia; Martin, Hector Garcia; Szeto, Ernest; Platt, Darren; Hugenholtz, Philip; Relman, David A.; Quake, Stephen R.

2007-01-01

We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community. PMID:17620602

Culture and the sequence of steps in theory of mind development.

PubMed

Shahaeian, Ameneh; Peterson, Candida C; Slaughter, Virginia; Wellman, Henry M

2011-09-01

To examine cultural contrasts in the ordered sequence of conceptual developments leading to theory of mind (ToM), we compared 135 3- to 6-year-olds (77 Australians; 58 Iranians) on an established 5-step ToM scale (Wellman & Liu, 2004). There was a cross-cultural difference in the sequencing of ToM steps but not in overall rates of ToM mastery. In line with our predictions, the children from Iran conformed to a distinctive sequence previously observed only in children in China. In contrast to the case with children from Australia (and the United States), knowledge access was understood earlier than opinion diversity in children from Iran, consistent with this collectivist culture's emphasis on filial respect, dispute avoidance, and acquiring knowledge. Having a sibling was linked with faster overall ToM progress in Australia only and was not related to scale sequences in either culture.

Fungal diversity in grape must and wine fermentation assessed by massive sequencing, quantitative PCR and DGGE

PubMed Central

Wang, Chunxiao; García-Fernández, David; Mas, Albert; Esteve-Zarzoso, Braulio

2015-01-01

The diversity of fungi in grape must and during wine fermentation was investigated in this study by culture-dependent and culture-independent techniques. Carignan and Grenache grapes were harvested from three vineyards in the Priorat region (Spain) in 2012, and nine samples were selected from the grape must after crushing and during wine fermentation. From culture-dependent techniques, 362 isolates were randomly selected and identified by 5.8S-ITS-RFLP and 26S-D1/D2 sequencing. Meanwhile, genomic DNA was extracted directly from the nine samples and analyzed by qPCR, DGGE and massive sequencing. The results indicated that grape must after crushing harbored a high species richness of fungi with Aspergillus tubingensis, Aureobasidium pullulans, or Starmerella bacillaris as the dominant species. As fermentation proceeded, the species richness decreased, and yeasts such as Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae successively occupied the must samples. The “terroir” characteristics of the fungus population are more related to the location of the vineyard than to grape variety. Sulfur dioxide treatment caused a low effect on yeast diversity by similarity analysis. Because of the existence of large population of fungi on grape berries, massive sequencing was more appropriate to understand the fungal community in grape must after crushing than the other techniques used in this study. Suitable target sequences and databases were necessary for accurate evaluation of the community and the identification of species by the 454 pyrosequencing of amplicons. PMID:26557110

High diversity of airborne fungi in the hospital environment as revealed by meta-sequencing-based microbiome analysis

PubMed Central

Tong, Xunliang; Xu, Hongtao; Zou, Lihui; Cai, Meng; Xu, Xuefeng; Zhao, Zuotao; Xiao, Fei; Li, Yanming

2017-01-01

Invasive fungal infections acquired in the hospital have progressively emerged as an important cause of life-threatening infection. In particular, airborne fungi in hospitals are considered critical pathogens of hospital-associated infections. To identify the causative airborne microorganisms, high-volume air samplers were utilized for collection, and species identification was performed using a culture-based method and DNA sequencing analysis with the Illumina MiSeq and HiSeq 2000 sequencing systems. Few bacteria were grown after cultivation in blood agar. However, using microbiome sequencing, the relative abundance of fungi, Archaea species, bacteria and viruses was determined. The distribution characteristics of fungi were investigated using heat map analysis of four departments, including the Respiratory Intensive Care Unit, Intensive Care Unit, Emergency Room and Outpatient Department. The prevalence of Aspergillus among fungi was the highest at the species level, approximately 17% to 61%, and the prevalence of Aspergillus fumigatus among Aspergillus species was from 34% to 50% in the four departments. Draft genomes of microorganisms isolated from the hospital environment were obtained by sequence analysis, indicating that investigation into the diversity of airborne fungi may provide reliable results for hospital infection control and surveillance. PMID:28045065

Analysis of a microbial community oxidizing inorganic sulfide and mercaptans.

PubMed

Duncan, K E; Sublette, K L; Rider, P A; Stepp, A; Beitle, R R; Conner, J A; Kolhatkar, R

2001-01-01

Successful treatment of refinery spent-sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio-Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacilli spp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR-amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands), Gram-positive cocci (Micrococcineae), alpha proteobacteria (3 bands), and an unidentified beta proteobacterium. Culturable microbes included several genera of fungi as well as various Gram-positive and Gram-negative heterotrophic bacteria not seen in techniques using direct DNA extraction.

Characterization of fungi in office dust: Comparing results of microbial secondary metabolites, fungal internal transcribed spacer region sequencing, viable culture and other microbial indices.

PubMed

Park, J-H; Sulyok, M; Lemons, A R; Green, B J; Cox-Ganser, J M

2018-05-04

Recent developments in molecular and chemical methods have enabled the analysis of fungal DNA and secondary metabolites, often produced during fungal growth, in environmental samples. We compared 3 fungal analytical methods by analysing floor dust samples collected from an office building for fungi using viable culture, internal transcribed spacer (ITS) sequencing and secondary metabolites using liquid chromatography-tandem mass spectrometry. Of the 32 metabolites identified, 29 had a potential link to fungi with levels ranging from 0.04 (minimum for alternariol monomethylether) to 5700 ng/g (maximum for neoechinulin A). The number of fungal metabolites quantified per sample ranged from 8 to 16 (average = 13/sample). We identified 216 fungal operational taxonomic units (OTUs) with the number per sample ranging from 6 to 29 (average = 18/sample). We identified 37 fungal species using culture, and the number per sample ranged from 2 to 13 (average = 8/sample). Agreement in identification between ITS sequencing and culturing was weak (kappa = -0.12 to 0.27). The number of cultured fungal species poorly correlated with OTUs, which did not correlate with the number of metabolites. These suggest that using multiple measurement methods may provide an improved understanding of fungal exposures in indoor environments and that secondary metabolites may be considered as an additional source of exposure. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular diversity, cultivation, and improved detection by fluorescent in situ hybridization of a dominant group of human gut bacteria related to Roseburia spp. or Eubacterium rectale.

PubMed

Aminov, Rustam I; Walker, Alan W; Duncan, Sylvia H; Harmsen, Hermie J M; Welling, Gjalt W; Flint, Harry J

2006-09-01

Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper oligonucleotide, each recognized an average of 7% of bacteria detected by the eubacterial probe Eub338 in feces from 10 healthy volunteers. Most of the diversity within this important group of butyrate-producing gut bacteria can apparently be retrieved through cultivation.

Molecular Diversity, Cultivation, and Improved Detection by Fluorescent In Situ Hybridization of a Dominant Group of Human Gut Bacteria Related to Roseburia spp. or Eubacterium rectale

PubMed Central

Aminov, Rustam I.; Walker, Alan W.; Duncan, Sylvia H.; Harmsen, Hermie J. M.; Welling, Gjalt W.; Flint, Harry J.

2006-01-01

Phylogenetic analysis was used to compare 16S rRNA sequences from 19 cultured human gut strains of Roseburia and Eubacterium rectale with 356 related sequences derived from clone libraries. The cultured strains were found to represent five of the six phylotypes identified. A new oligonucleotide probe, Rrec584, and the previous group probe Rint623, when used in conjunction with a new helper oligonucleotide, each recognized an average of 7% of bacteria detected by the eubacterial probe Eub338 in feces from 10 healthy volunteers. Most of the diversity within this important group of butyrate-producing gut bacteria can apparently be retrieved through cultivation. PMID:16957265

High-Throughput Sequencing and Metagenomics: Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology

PubMed Central

2013-01-01

Following recent trends in environmental microbiology, food microbiology has benefited from the advances in molecular biology and adopted novel strategies to detect, identify, and monitor microbes in food. An in-depth study of the microbial diversity in food can now be achieved by using high-throughput sequencing (HTS) approaches after direct nucleic acid extraction from the sample to be studied. In this review, the workflow of applying culture-independent HTS to food matrices is described. The current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described. PMID:23475615

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture-dependent and PCR-DGGE methods.

PubMed

Lin, Xiao-Li; Pan, Qin-Jian; Tian, Hong-Gang; Douglas, Angela E; Liu, Tong-Xian

2015-03-01

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture-dependent method and PCR-DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty-five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Cloning and Analysis of the Alternative Oxidase Gene of Neurospora Crassa

PubMed Central

Li, Q.; Ritzel, R. G.; McLean, LLT.; McIntosh, L.; Ko, T.; Bertrand, H.; Nargang, F. E.

1996-01-01

Mitochondria of Neurospora crassa contain a cyanide-resistant alternative respiratory pathway in addition to the cytochrome pathway. The alternative oxidase is present only when electron flow through the cytochrome chain is restricted. Both genomic and cDNA copies for the alternative oxidase gene have been isolated and analyzed. The sequence of the predicted protein is homologous to that of other species. The mRNA for the alternative oxidase is scarce in wild-type cultures grown under normal conditions, but it is abundant in cultures grown in the presence of chloramphenicol, an inhibitor of mitochondrial protein synthesis, or in mutants deficient in mitochondrial cytochromes. Thus, induction of alternative oxidase appears to be at the transcriptional level. Restriction fragment length polymorphism mapping of the isolated gene demonstrated that it is located in a position corresponding to the aod-1 locus. Sequence analysis of mutant aod-1 alleles reveals mutations affecting the coding sequence of the alternative oxidase. The level of aod-1 mRNA in an aod-2 mutant strain that had been grown in the presence of chloramphenicol was reduced several fold relative to wild-type, supporting the hypothesis that the product of aod-2 is required for optimal expression of aod-1. PMID:8770590

Isolation and characterization of a ranavirus from koi, Cyprinus carpio L., experiencing mass mortalities in India.

PubMed

George, M R; John, K R; Mansoor, M M; Saravanakumar, R; Sundar, P; Pradeep, V

2015-04-01

We investigated mass mortalities of koi, Cyprinus carpio Linnaeus, 1758, experienced in South Indian fish farms by virus isolation, electron microscopy, PCR detection, sequencing of capsid protein gene and transmission studies. Samples of moribund koi brought to the laboratory suffered continuous mortality exhibiting swimming abnormalities, intermittent surfacing and skin darkening. Irido-like virus was isolated from the infected fish in the indigenous snakehead kidney cell line (SNKD2a). Icosahedral virus particles of 100 to 120 nm were observed in the infected cell cultures, budding from the cell membrane. Virus transmission and pathogenicity studies revealed that horizontal transmission occurred associated with mortality. PCR analysis of infected fish and cell cultures confirmed the presence of Ranavirus capsid protein sequences. Sequence analysis of the major capsid protein gene showed an identity of 99.9% to that of largemouth bass virus isolated from North America. Detection and successful isolation of this viral agent becomes the first record of isolation of a virus resembling Santee-Cooper Ranavirus from a koi and from India. We propose the name koi ranavirus to this agent. © 2014 John Wiley & Sons Ltd.

Microbial diversity in ikaite tufa columns: an alkaline, cold ecological niche in Greenland.

PubMed

Stougaard, Peter; Jørgensen, Flemming; Johnsen, Mads G; Hansen, Ole C

2002-08-01

Ikaite tufa columns from the Ikka Fjord in south-western Greenland constitute a natural, stable environment at low temperature and with a pH ranging from neutral at the exterior to very alkaline (pH 10.4) at the interior of the column. Phylogenetic analysis of culturable organisms revealed ten different isolates representing three of the major bacterial divisions. Nine of the isolates showed 94-99% similarity to known sequences, whereas one isolate displayed a low degree of similarity (less than 90%) to a Cyclobacterium species. Seven of the isolates were shown to be cold active alkaliphiles, whereas three isolates showed optimal growth at neutral pH. Phylogenetic analysis of DNA isolated directly from the ikaite material demonstrated the presence of a microbial flora more diverse than the culturable isolates. Whereas approximately half of the phylotypes showed 90-99% similarity to known meso- or thermophilic alkaliphiles, the rest of the sequences displayed less than 90% similarity when compared to known 16S rRNA gene sequences in databases. Thus, in the present paper, we demonstrate that ikaite columns that host a specialized macroscopic flora and fauna also contain a unique, cold active, alkaliphilic microflora.

Phylogeny of culturable cyanobacteria from Brazilian mangroves.

PubMed

Silva, Caroline Souza Pamplona; Genuário, Diego Bonaldo; Vaz, Marcelo Gomes Marçal Vieira; Fiore, Marli Fátima

2014-03-01

The cyanobacterial community from Brazilian mangrove ecosystems was examined using a culture-dependent method. Fifty cyanobacterial strains were isolated from soil, water and periphytic samples collected from Cardoso Island and Bertioga mangroves using specific cyanobacterial culture media. Unicellular, homocytous and heterocytous morphotypes were recovered, representing five orders, seven families and eight genera (Synechococcus, Cyanobium, Cyanobacterium, Chlorogloea, Leptolyngbya, Phormidium, Nostoc and Microchaete). All of these novel mangrove strains had their 16S rRNA gene sequenced and BLAST analysis revealed sequence identities ranging from 92.5 to 99.7% when they were compared with other strains available in GenBank. The results showed a high variability of the 16S rRNA gene sequences among the genotypes that was not associated with the morphologies observed. Phylogenetic analyses showed several branches formed exclusively by some of these novel 16S rRNA gene sequences. BLAST and phylogeny analyses allowed for the identification of Nodosilinea and Oxynema strains, genera already known to exhibit poor morphological diacritic traits. In addition, several Nostoc and Leptolyngbya morphotypes of the mangrove strains may represent new generic entities, as they were distantly affiliated with true genera clades. The presence of non-ribosomal peptide synthetase, polyketide synthase, microcystin and saxitoxin genes were detected in 20.5%, 100%, 37.5% and 33.3%, respectively, of the 44 tested isolates. A total of 134 organic extracts obtained from 44 strains were tested against microorganisms, and 26% of the extracts showed some antimicrobial activity. This is the first polyphasic study of cultured cyanobacteria from Brazilian mangrove ecosystems using morphological, genetic and biological approaches. Copyright © 2014 Elsevier GmbH. All rights reserved.

Culturable bacteria present in the fluid of the hooded-pitcher plant Sarracenia minor based on 16S rDNA gene sequence data.

PubMed

Siragusa, Alex J; Swenson, Janice E; Casamatta, Dale A

2007-08-01

The culturable microbial community within the pitcher fluid of 93 Sarracenia minor carnivorous plants was examined over a 2-year study. Many aspects of the plant/bacterial/insect interaction within the pitcher fluid are minimally understood because the bacterial taxa present in these pitchers have not been identified. Thirteen isolates were characterized by 16S rDNA sequencing and subsequent phylogenetic analysis. The Proteobacteria were the most abundant taxa and included representatives from Serratia, Achromobacter, and Pantoea. The Actinobacteria Micrococcus was also abundant while Bacillus, Lactococcus, Chryseobacterium, and Rhodococcus were infrequently encountered. Several isolates conformed to species identifiers (>98% rDNA gene sequence similarity) including Serratia marcescens (isolates found in 27.5% of pitchers), Achromobacter xylosoxidans (37.6%), Micrococcus luteus (40.9%), Bacillus cereus (isolates found in 10.2%), Bacillus thuringiensis (5.4%), Lactococcus lactis (17.2%), and Rhodococcus equi (2.2%). Species-area curves suggest that sampling efforts were sufficient to recover a representative culturable bacterial community. The bacteria present represent a diverse community probably as a result of introduction by insect vectors, but the ecological significance remains under explored.

How mass spectrometric approaches applied to bacterial identification have revolutionized the study of human gut microbiota.

PubMed

Grégory, Dubourg; Chaudet, Hervé; Lagier, Jean-Christophe; Raoult, Didier

2018-03-01

Describing the human hut gut microbiota is one the most exciting challenges of the 21 st century. Currently, high-throughput sequencing methods are considered as the gold standard for this purpose, however, they suffer from several drawbacks, including their inability to detect minority populations. The advent of mass-spectrometric (MS) approaches to identify cultured bacteria in clinical microbiology enabled the creation of the culturomics approach, which aims to establish a comprehensive repertoire of cultured prokaryotes from human specimens using extensive culture conditions. Areas covered: This review first underlines how mass spectrometric approaches have revolutionized clinical microbiology. It then highlights the contribution of MS-based methods to culturomics studies, paying particular attention to the extension of the human gut microbiota repertoire through the discovery of new bacterial species. Expert commentary: MS-based approaches have enabled cultivation methods to be resuscitated to study the human gut microbiota and thus to fill in the blanks left by high-throughput sequencing methods in terms of culturing minority populations. Continued efforts to recover new taxa using culture methods, combined with their rapid implementation in genomic databases, would allow for an exhaustive analysis of the gut microbiota through the use of a comprehensive approach.

Longitudinal studies on maternal HIV-1 variants by biological phenotyping, sequence analysis and viral load.

PubMed

Renta, J Y; Cadilla, C L; Vega, M E; Hillyer, G V; Estrada, C; Jiménez, E; Abreu, E; Méndez, I; Gandía, J; Meléndez-Guerrero, L M

1997-11-01

In this study, the HIV-1 variant viruses from ten pregnant women and their infants were isolated and characterized longitudinally in order to determine the role that viral envelope (gp120-V3 loop) gene variation and viral tropism play in vertical transmission. Biological phenotyping of each HIV variant was accomplished by growth in MT-2, and macrophages from healthy and non-HIV-infected donors. Genetic characterization of the variants was accomplished by DNA sequence analysis. All the women enrolled in this study received ZDV therapy. Virus was cultured from eight out of ten env V3-PCR positive mothers. HIV-1 isolates were all non-syncitium inducing variants. None of the mothers were found to transmit HIV, as determined by DNA PCR and quantitative co-cultures on their infants which were seronegative for HIV-1 through one year after birth. Viral cultures from infant blood samples were negative and infants were all healthy. However, nested env V3-PCR detected proviral DNA in five out of ten infants. In contrast, conventional gag-PCR was negative in the same five infants. Sequences of the five maternal-infant pairs were different, suggesting unique infant HIV-1 variants. The three highest maternal viral load values corresponded to infants that were env V3-PCR positive. These results suggest that HIV-1 particles are transmitted from ZDV-treated mothers to infants. Infant follow up is recommended to determine if HIV-1 has been inhibited by the immune system of the infants.

Bacterial diversity of Taxus rhizosphere: culture-independent and culture-dependent approaches.

PubMed

Hao, Da Cheng; Ge, Guang Bo; Yang, Ling

2008-07-01

The regional variability of Taxus rhizosphere bacterial community composition and diversity was studied by comparative analysis of three large 16S rRNA gene clone libraries from the Taxus rhizosphere in different regions of China (subtropical and temperate regions). One hundred and forty-six clones were screened for three libraries. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the abundance of sequences affiliated with Gammaproteobacteria, Betaproteobacteria, and Actinobacteria was higher in the library from the T. xmedia rhizosphere of the temperate region compared with the subtropical Taxus mairei rhizosphere. On the other hand, Acidobacteria was more abundant in libraries from the subtropical Taxus mairei rhizosphere. Richness estimates and diversity indices of three libraries revealed major differences, indicating a higher richness in the Taxus rhizosphere bacterial communities of the subtropical region and considerable variability in the bacterial community composition within this region. By enrichment culture, a novel Actinobacteria strain DICP16 was isolated from the T. xmedia rhizosphere of the temperate region and was identified as Leifsonia shinshuensis sp. via 16S rRNA gene and gyrase B sequence analyses. DICP16 was able to remove the xylosyl group from 7-xylosyl-10-deacetylbaccatin III and 7-xylosyl-10-deacetylpaclitaxel, thereby making the xylosyltaxanes available as sources of 10-deacetylbaccatin III and the anticancer drug paclitaxel. Taken together, the present studies provide, for the first time, the knowledge of the biodiversity of microorganisms populating Taxus rhizospheres.

Microbial composition during Chinese soy sauce koji-making based on culture dependent and independent methods.

PubMed

Yan, Yin-zhuo; Qian, Yu-lin; Ji, Feng-di; Chen, Jing-yu; Han, Bei-zhong

2013-05-01

Koji-making is a key process for production of high quality soy sauce. The microbial composition during koji-making was investigated by culture-dependent and culture-independent methods to determine predominant bacterial and fungal populations. The culture-dependent methods used were direct culture and colony morphology observation, and PCR amplification of 16S/26S rDNA fragments followed by sequencing analysis. The culture-independent method was based on the analysis of 16S/26S rDNA clone libraries. There were differences between the results obtained by different methods. However, sufficient overlap existed between the different methods to identify potentially significant microbial groups. 16 and 20 different bacterial species were identified using culture-dependent and culture-independent methods, respectively. 7 species could be identified by both methods. The most predominant bacterial genera were Weissella and Staphylococcus. Both 6 different fungal species were identified using culture-dependent and culture-independent methods, respectively. Only 3 species could be identified by both sets of methods. The most predominant fungi were Aspergillus and Candida species. This work illustrated the importance of a comprehensive polyphasic approach in the analysis of microbial composition during soy sauce koji-making, the knowledge of which will enable further optimization of microbial composition and quality control of koji to upgrade Chinese traditional soy sauce product. Copyright © 2013 Elsevier Ltd. All rights reserved.

Culture and the Sequence of Steps in Theory of Mind Development

ERIC Educational Resources Information Center

Shahaeian, Ameneh; Peterson, Candida C.; Slaughter, Virginia; Wellman, Henry M.

2011-01-01

To examine cultural contrasts in the ordered sequence of conceptual developments leading to theory of mind (ToM), we compared 135 3- to 6-year-olds (77 Australians; 58 Iranians) on an established 5-step ToM scale (Wellman & Liu, 2004). There was a cross-cultural difference in the sequencing of ToM steps but not in overall rates of ToM mastery.…

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.

Rhodotorula minuta fungemia in a ewe lamb.

PubMed

Chitko-McKown, C G; Leymaster, K A; Heaton, M P; Griffin, D D; Veatch, J K; Jones, S A; Clawson, M L

2014-12-01

An 8-month-old crossbred ewe, normal upon physical examination, was humanely euthanized for tissue collection. After approximately 3 weeks in tissue culture, fungi began budding out of cells obtained from the choroid plexus. After an additional 3 weeks, budding was observed in kidney cell cultures and eventually in monocyte cultures as well. Serum from the lamb was submitted to the Veterinary Diagnostic Laboratory at Colorado State University for fungal diagnosis and was found negative for Aspergillus, Blastomyces, Coccidioidomycosis and Histoplasmosis. DNA was isolated from fungi collected from tissue culture supernatants and used in a set of pan-fungal PCR assays with DNA from Candida acting as a positive control. PCR products were sequenced and BLAST analysis performed. The unknown fungal sequence aligned with 100% identity to Rhodotorula minuta an emerging opportunistic pathogen. Samples were submitted to The Fungal Testing Laboratory at The University of Texas Health Science Center at San Antonio for additional validation. We believe this to be the first report of Rhodotorula fungemia in a sheep in the United States. © 2013 Blackwell Verlag GmbH.

Metaphor and Knowing: Analysis, Synthesis, Rationale.

ERIC Educational Resources Information Center

Rico, Gabriele Lusser

Evidence is presented to indicate that human knowing involves both a propositional mode stressing discourse, sequence, and logic and an appositional mode characterized by metaphoric constructs, holistic relationships, and the capacity to process many variables simultaneously. Separate sections discuss our culture's heavy emphasis on propositional…

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation.

PubMed

Kitimu, Shedrack R; Taylor, Julian; March, Timothy J; Tairo, Fred; Wilkinson, Mike J; Rodríguez López, Carlos M

2015-01-01

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation

PubMed Central

Kitimu, Shedrack R.; Taylor, Julian; March, Timothy J.; Tairo, Fred; Wilkinson, Mike J.; Rodríguez López, Carlos M.

2015-01-01

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops. PMID:26322052

Metagenomics analysis of microbial communities associated with a traditional rice wine starter culture (Xaj-pitha) of Assam, India.

PubMed

Bora, Sudipta Sankar; Keot, Jyotshna; Das, Saurav; Sarma, Kishore; Barooah, Madhumita

2016-12-01

This is the first report on the microbial diversity of xaj-pitha, a rice wine fermentation starter culture through a metagenomics approach involving Illumine-based whole genome shotgun (WGS) sequencing method. Metagenomic DNA was extracted from rice wine starter culture concocted by Ahom community of Assam and analyzed using a MiSeq ® System. A total of 2,78,231 contigs, with an average read length of 640.13 bp, were obtained. Data obtained from the use of several taxonomic profiling tools were compared with previously reported microbial diversity studies through the culture-dependent and culture-independent method. The microbial community revealed the existence of amylase producers, such as Rhizopus delemar, Mucor circinelloides, and Aspergillus sp. Ethanol producers viz., Meyerozyma guilliermondii, Wickerhamomyces ciferrii, Saccharomyces cerevisiae, Candida glabrata, Debaryomyces hansenii, Ogataea parapolymorpha, and Dekkera bruxellensis, were found associated with the starter culture along with a diverse range of opportunistic contaminants. The bacterial microflora was dominated by lactic acid bacteria (LAB). The most frequent occurring LAB was Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc lactis, Weissella cibaria, Lactococcus lactis, Weissella para mesenteroides, Leuconostoc pseudomesenteroides, etc. Our study provided a comprehensive picture of microbial diversity associated with rice wine fermentation starter and indicated the superiority of metagenomic sequencing over previously used techniques.

Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl-ortho-Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA

PubMed Central

Pulliam Holoman, Tracey R.; Elberson, Margaret A.; Cutter, Leah A.; May, Harold D.; Sowers, Kevin R.

1998-01-01

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the δ, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species. PMID:9726883

An Ecotype of Neorickettsia risticii Causing Potomac Horse Fever in Canada

PubMed Central

Xiong, Qingming; Bekebrede, Hannah; Sharma, Pratibha; Arroyo, Luis G.; Baird, John D.

2016-01-01

ABSTRACT Neorickettsia (formerly Ehrlichia) risticii is an obligatory intracellular bacterium of digenetic trematodes. When a horse accidentally ingests aquatic insects containing encysted trematodes infected with N. risticii, the bacterium is transmitted from trematodes to horse cells and causes an acute and often fatal disease called Potomac horse fever (PHF). Since the discovery of N. risticii in the United States in 1984, using immunofluorescence and PCR assays, PHF has been increasingly recognized throughout North America and South America. However, so far, there exist only a few stable N. risticii culture isolates, all of which are from horses within the United States, and the strain diversity and environmental spreading and distribution of pathogenic N. risticii strains remain poorly understood. This paper reports the isolation of N. risticii from the blood of a horse with acute PHF in Ontario, Canada. Intracellular N. risticii colonies were detected in P388D1 cells after 47 days of culturing and 8 days after the addition of rapamycin. Molecular phylogenetic analysis based on amino acid sequences of major surface proteins P51 and Ssa1 showed that this isolate is distinct from any previously sequenced strains but closely related to midwestern U.S. strains. This is the first Canadian strain cultured, and a new method was developed to reactivate dormant N. risticii to improve culture isolation. IMPORTANCE Neorickettsia risticii is an environmental bacterium that lives inside flukes that are parasitic to aquatic snails, insects, and bats. When a horse accidentally ingests insects harboring flukes infected with N. risticii, the bacterium is transmitted to the horse and causes an acute and often fatal disease called Potomac horse fever. Although the disease has been increasingly recognized throughout North and South America, N. risticii has not been cultured outside the United States. This paper reports the first Canadian strain cultured and a new method to effectively culture isolate N. risticii from the horse blood sample. Molecular analysis showed that the genotype of this Canadian strain is distinct from previously sequenced strains but closely related to midwestern U.S. strains. Culture isolation of N. risticii strains would confirm the geographic presence of pathogenic N. risticii, help elucidate N. risticii strain diversity and environmental spreading and distribution, and improve diagnosis and development of vaccines for this dreadful disease. PMID:27474720

An Ecotype of Neorickettsia risticii Causing Potomac Horse Fever in Canada.

PubMed

Xiong, Qingming; Bekebrede, Hannah; Sharma, Pratibha; Arroyo, Luis G; Baird, John D; Rikihisa, Yasuko

2016-10-01

Neorickettsia (formerly Ehrlichia) risticii is an obligatory intracellular bacterium of digenetic trematodes. When a horse accidentally ingests aquatic insects containing encysted trematodes infected with N. risticii, the bacterium is transmitted from trematodes to horse cells and causes an acute and often fatal disease called Potomac horse fever (PHF). Since the discovery of N. risticii in the United States in 1984, using immunofluorescence and PCR assays, PHF has been increasingly recognized throughout North America and South America. However, so far, there exist only a few stable N. risticii culture isolates, all of which are from horses within the United States, and the strain diversity and environmental spreading and distribution of pathogenic N. risticii strains remain poorly understood. This paper reports the isolation of N. risticii from the blood of a horse with acute PHF in Ontario, Canada. Intracellular N. risticii colonies were detected in P388D1 cells after 47 days of culturing and 8 days after the addition of rapamycin. Molecular phylogenetic analysis based on amino acid sequences of major surface proteins P51 and Ssa1 showed that this isolate is distinct from any previously sequenced strains but closely related to midwestern U.S. strains. This is the first Canadian strain cultured, and a new method was developed to reactivate dormant N. risticii to improve culture isolation. Neorickettsia risticii is an environmental bacterium that lives inside flukes that are parasitic to aquatic snails, insects, and bats. When a horse accidentally ingests insects harboring flukes infected with N. risticii, the bacterium is transmitted to the horse and causes an acute and often fatal disease called Potomac horse fever. Although the disease has been increasingly recognized throughout North and South America, N. risticii has not been cultured outside the United States. This paper reports the first Canadian strain cultured and a new method to effectively culture isolate N. risticii from the horse blood sample. Molecular analysis showed that the genotype of this Canadian strain is distinct from previously sequenced strains but closely related to midwestern U.S. strains. Culture isolation of N. risticii strains would confirm the geographic presence of pathogenic N. risticii, help elucidate N. risticii strain diversity and environmental spreading and distribution, and improve diagnosis and development of vaccines for this dreadful disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis

DOE PAGES

Woebken, Dagmar; Burow, Luke C.; Prufert-Bebout, Leslie; ...

2012-01-12

N 2 fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N 2 and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N 2 at night indicated that cyanobacterialmore » nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of 15N2-incubated samples revealed significant incorporation of 15N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining 15N 2 tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N 2 fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. Here, this work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.« less

Metagenomics workflow analysis of endophytic bacteria from oil palm fruits

NASA Astrophysics Data System (ADS)

Tanjung, Z. A.; Aditama, R.; Sudania, W. M.; Utomo, C.; Liwang, T.

2017-05-01

Next-Generation Sequencing (NGS) has become a powerful sequencing tool for microbial study especially to lead the establishment of the field area of metagenomics. This study described a workflow to analyze metagenomics data of a Sequence Read Archive (SRA) file under accession ERP004286 deposited by University of Sao Paulo. It was a direct sequencing data generated by 454 pyrosequencing platform originated from oil palm fruits endophytic bacteria which were cultured using oil-palm enriched medium. This workflow used SortMeRNA to split ribosomal reads sequence, Newbler (GS Assembler and GS Mapper) to assemble and map reads into genome reference, BLAST package to identify and annotate contigs sequence, and QualiMap for statistical analysis. Eight bacterial species were identified in this study. Enterobacter cloacae was the most abundant species followed by Citrobacter koseri, Seratia marcescens, Latococcus lactis subsp. lactis, Klebsiella pneumoniae, Citrobacter amalonaticus, Achromobacter xylosoxidans, and Pseudomonas sp. respectively. All of these species have been reported as endophyte bacteria in various plant species and each has potential as plant growth promoting bacteria or another application in agricultural industries.

Relevant principal factors affecting the reproducibility of insect primary culture.

PubMed

Ogata, Norichika; Iwabuchi, Kikuo

2017-06-01

The primary culture of insect cells often suffers from problems with poor reproducibility in the quality of the final cell preparations. The cellular composition of the explants (cell number and cell types), surgical methods (surgical duration and surgical isolation), and physiological and genetic differences between donors may be critical factors affecting the reproducibility of culture. However, little is known about where biological variation (interindividual differences between donors) ends and technical variation (variance in replication of culture conditions) begins. In this study, we cultured larval fat bodies from the Japanese rhinoceros beetle, Allomyrina dichotoma, and evaluated, using linear mixed models, the effect of interindividual variation between donors on the reproducibility of the culture. We also performed transcriptome analysis of the hemocyte-like cells mainly seen in the cultures using RNA sequencing and ultrastructural analyses of hemocytes using a transmission electron microscope, revealing that the cultured cells have many characteristics of insect hemocytes.

[Molecular characterization of pathogenic bacteria of the respiratory tract in peruvian patients with cystic fibrosis].

PubMed

Aquino, Ruth; Gonzáles, Emely; Samaniego, Sol; Rivera, Juan; Cedeño, Virna; Urbina, Yrene; Diringer, Benoit

2017-01-01

To molecularly characterize the pathogenic bacteria of the respiratory tract isolated from patients with cystic fibrosis (CF) in Peru. Bacterial communities cultured from sputum samples of pediatric and adult patients with CF admitted to the Edgardo Rebagliati Martins National Hospital and the National Institute of Child Health were characterized. Standard microbiological techniques were used for bacterial culture, and gene sequencing of 16S rRNA and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and tandem MALDI-TOF mass spectrometry (MALDI TOF/TOF) were used for molecular characterization. Seventeen bacterial strains were characterized by 16S rRNA sequencing, and the identified pathogenic bacteria were Pseudomonas aeruginosa (31.5%), Staphylococcus aureus (12.6%), Pseudomonas spp. (11.8%), and Klebsiella oxytoca (3.1%). MALDI-TOF analysis generated a series of spectra representative of each isolated bacterial species, whereas MALDI TOF/TOF analysis identified the peptides and proteins of the most common strains and provided data on pathogenicity and sensitivity to antibiotics. The primary pathogenic microorganisms found in the respiratory tract of patients with CF in Peru were the same as those found in other countries. This study is the first to perform 16S rRNA sequencing as well as MALDI-TOF and MALDI-TOF/TOF analysis of the bacterial pathogens circulating in Peru. The inclusion of proteomic analysis further allowed for the identification of native microorganisms involved in CF.

Transcriptional enhancer from milk protein genes

DOEpatents

Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

1999-01-01

The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

Impact of antimicrobial use during beef production on fecal occurrence of antimicrobial resistance

USDA-ARS?s Scientific Manuscript database

Objective: To determine the impact of typical antimicrobial use during cattle production on fecal occurrence of antimicrobial resistance by culture, quantitative PCR, and metagenomic sequencing. Experimental Design & Analysis: Feces were recovered from colons of 36 lots of "conventional" (CONV) ca...

Probability from a Socio-Cultural Perspective

ERIC Educational Resources Information Center

Sharma, Sashi

2016-01-01

There exists considerable and rich literature on students' misconceptions about probability; less attention has been paid to the development of students' probabilistic thinking in the classroom. Grounded in an analysis of the literature, this article offers a lesson sequence for developing students' probabilistic understanding. In particular, a…

Performance of culture media for the isolation and identification of Staphylococcus aureus from bovine mastitis.

PubMed

Bautista-Trujillo, G U; Solorio-Rivera, J L; Rentería-Solórzano, I; Carranza-Germán, S I; Bustos-Martínez, J A; Arteaga-Garibay, R I; Baizabal-Aguirre, V M; Cajero-Juárez, M; Bravo-Patiño, A; Valdez-Alarcón, J J

2013-03-01

Rapid isolation and identification of pathogens is a major goal of diagnostic microbiology. In order to isolate and identify Staphylococcus aureus, a number of authors have used a variety of selective and/or differential culture media. However, to date, there are no reports comparing the efficacy of selective and differential culture media for S. aureus isolation from bovine mastitis cases using the 16S rRNA (rrs) gene sequence as a gold standard test. In the present study, we evaluated the efficacy of four selective and/or differential culture media for the isolation of S. aureus from milk samples collected from cows suffering from bovine mastitis. Four hundred and forty isolates were obtained using salt-mannitol agar (SMA, Bioxon), Staphylococcus-110 agar (S110, Bioxon), CHROMAgar Staph aureus (CSA, BD-BBL) and sheep's blood agar (SBA, BD-BBL). All bacterial isolates were identified by their typical colony morphology in the respective media, by secondary tests (for coagulase and β-haemolysis) and by partial 16S rRNA (rrs) gene sequencing as a gold standard test. Sensitivity, positive predictive and negative predictive values were higher for SMA (86.96, 52.63 and 95.95%, respectively) compared with S110 (70.00, 23.73 and 90.91%, respectively), CSA (69.23, 28.13 and 95.74%, respectively) and SBA (68.75, 37.93 and 89.58%, respectively) while specificity values were similar for all media. Data indicated that the use of culture media for S. aureus isolation combined with determination of coagulase activity and haemolysis as secondary tests improved accuracy of the identification and was in accordance with rrs gene sequence-analysis compared with the use of the culture media alone.

Towards the ecology of hyperthermophiles: biotopes, new isolation strategies and novel metabolic properties.

PubMed

Huber, R; Huber, H; Stetter, K O

2000-12-01

Ecological studies have shown that water-containing terrestrial, subterranean and submarine high-temperature environments harbor a great diversity of hyperthermophilic prokaryotes, growing fastest at temperatures of 80 degrees C or above. The investigations included cultivation, isolation and detailed analysis of these hyperthermophiles as well as in situ 16S rRNA gene sequence analysis and in situ hybridization studies. For a safe and fast isolation of novel hyperthermophiles from mixed cultures, a new, plating-independent isolation technique was developed, based on the use of a laser microscope ('optical tweezers'). This method, combined with 16S rRNA gene sequence analysis and whole-cell hybridization using fluorescently labelled oligonucleotide probes, even allows the recovery of pure cultures of phylogenetically predicted organisms harboring novel 16S rRNA gene sequences. In their natural habitats, hyperthermophiles form complex food webs, consisting of primary producers and consumers of organic material. Their metabolic potential includes various types of aerobic and anaerobic respiration and different modes of fermentation. In hydrothermal and geothermal environments, hyperthermophiles have important ecological functions in biogeochemical processes. Members of the Sulfolobales are able to mobilize heavy metals from sulfidic ores like pyrite or chalcopyrite. Biomineralization processes of hyperthermophiles include the formation of magnetite from iron or the precipitation of arsenate as realgar, a reaction performed by a novel hyperthermophile that was isolated from Pisciarelli Solfatara, Naples, Italy.

Characterisation of the microbiota of rice sourdoughs and description of Lactobacillus spicheri sp. nov.

PubMed

Meroth, Christiane B; Hammes, Walter P; Hertel, Christian

2004-03-01

The microbiota of two industrially processed rice sourdoughs was characterised by bacteriological culture in combination with PCR-denaturing gradient gel electrophoresis (DGGE) and 16S/28S rDNA sequence analysis. Rice sourdough I was continuously propagated for several years by back-slopping every week, whereas sourdough II was processed by using a commercial starter culture and back-slopping daily for three days. In rice sourdough II Candida krusei and Saccharomyces cerevisiae as well as Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus kimchii, Lactobacillus plantarum, and Lactobacillus pontis dominated at the first day of fermentation. RAPD analysis of lactobacilli revealed identical profiles for each of the species except for L. fermentum and L. pontis indicating the presence of different strains. Fluctuations within the LAB community during fermentation were monitored by PCR-DGGE. L. pontis decreased in numbers over time and L. curvatus became dominant after 3 days of fermentation. Rice sourdough I contained S. cerevisiae, Lactobacillus paracasei (present with three different RAPD types), Lactobacillus paralimentarius, and a Lactobacillus strain which could not be allotted to any valid species. Phylogenetic analysis based on 16S rDNA sequences revealed Lactobacillus brevis as the closest relative (97.3% sequence similarity). Differences in some phenotypic characteristics and DNA-DNA relatedness indicated that the strain represents a new Lactobacillus species, for which the name Lactobacillus spicheri is proposed.

Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam. nov. on the basis of 16S rRNA sequence analyses

NASA Technical Reports Server (NTRS)

Zhao, H.; Yang, D.; Woese, C. R.; Bryant, M. P.

1993-01-01

After enrichment from Chinese rural anaerobic digestor sludge, anaerobic, sporing and nonsporing, saturated fatty acid-beta-oxidizing syntrophic bacteria were isolated as cocultures with H2- and formate-utilizing Methanospirillum hungatei or Desulfovibrio sp. strain G-11. The syntrophs degraded C4 to C8 saturated fatty acids, including isobutyrate and 2-methylbutyrate. They were adapted to grow on crotonate and were isolated as pure cultures. The crotonate-grown pure cultures alone did not grow on butyrate in either the presence or the absence of some common electron acceptors. However, when they were reconstituted with M. hungatei, growth on butyrate again occurred. In contrast, crotonate-grown Clostridium kluyveri and Clostridium sticklandii, as well as Clostridium sporogenes, failed to grow on butyrate when these organisms were cocultured with M. hungatei. The crotonate-grown pure subcultures of the syntrophs described above were subjected to 16S rRNA sequence analysis. Several previously documented fatty acid-beta-oxidizing syntrophs grown in pure cultures with crotonate were also subjected to comparative sequence analyses. The sequence analyses revealed that the new sporing and nonsporing isolates and other syntrophs that we sequenced, which had either gram-negative or gram-positive cell wall ultrastructure, all belonged to the phylogenetically gram-positive phylum. They were not closely related to any of the previously known subdivisions in the gram-positive phylum with which they were compared, but were closely related to each other, forming a new subdivision in the phylum. We recommend that this group be designated Syntrophomonadaceae fam. nov.; a description is given.

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.

Bacillus pumilus SAFR-032 isolate

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri J. (Inventor)

2007-01-01

The present invention relates to discovery and isolation of a biologically pure culture of a Bacillus pumilus SAFR-032 isolate with UV sterilization resistant properties. This novel strain has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus. The GenBank accession number for the 16S rDNA sequence of the Bacillus pumilus SAFR-032 isolate is AY167879.

Comparative analysis of microbial community of novel lactic acid fermentation inoculated with different undefined mixed cultures.

PubMed

Liang, Shaobo; Gliniewicz, Karol; Mendes-Soares, Helena; Settles, Matthew L; Forney, Larry J; Coats, Erik R; McDonald, Armando G

2015-03-01

Three undefined mixed cultures (activated sludge) from different municipal wastewater treatment plants were used as seeds in a novel lactic acid fermentation process fed with potato peel waste (PPW). Anaerobic sequencing batch fermenters were run under identical conditions to produce predominantly lactic acid. Illumina sequencing was used to examine the 16S rRNA genes of bacteria in the three seeds and fermenters. Results showed that the structure of microbial communities of three seeds were different. All three fermentation products had unique community structures that were dominated (>96%) by species of the genus Lactobacillus, while members of this genus constituted <0.1% in seeds. The species of Lactobacillus sp. differed among the three fermentations. Results of this study suggest the structure of microbial communities in lactic acid fermentation of PPW with undefined mixed cultures were robust and resilient, which provided engineering prospects for the microbial utilization of carbohydrate wastes to produce lactic acid. Copyright © 2014 Elsevier Ltd. All rights reserved.

Isolation, pure culture and characterization of Serratia symbiotica sp. nov., the R-type of secondary endosymbiont of the black bean aphid Aphis fabae.

PubMed

Sabri, Ahmed; Leroy, Pascal; Haubruge, Eric; Hance, Thierry; Frère, Isabelle; Destain, Jacqueline; Thonart, Philippe

2011-09-01

An intracellular symbiotic bacterium was isolated from the flora of a natural clone of the black bean aphid Aphis fabae. The strain was able to grow freely in aerobic conditions on a rich medium containing 1 % of each of the following substrates: glucose, yeast extract and casein peptone. Pure culture was achieved through the use of solid-phase culture on the same medium and the strain was designated CWBI-2.3(T). 16S rRNA gene sequence analysis revealed that strain CWBI-2.3(T) was a member of the class Gammaproteobacteria, having high sequence similarity (>99 %) with 'Candidatus Serratia symbiotica', the R-type of secondary endosymbiont that is found in several aphid species. As strain CWBI-2.3(T) ( = LMG 25624(T) = DSM 23270(T)) was the first R-type symbiont to be isolated and characterized, it was designated as the type strain of Serratia symbiotica sp. nov.

Molecular diagnostics and ITS-based phylogenic analysis of Streptococcus suis serotype 2 in central Vietnam.

PubMed

Nguyen, Bach Hoang; Phan, Dieu Hong Nu; Nguyen, Hien Xuan; Le, An Van; Alberti, Alberto

2015-07-04

Streptococcus suis (S. suis) serotype 2 has recently become the most prevalent cause of meningitis in adults in many areas of Vietnam. This study provides data on S. suis molecular diagnosis in central Vietnam using a real-time polymerase chain reaction (PCR) assay targeting the S. suis serotype 2 cps2J gene. Additionally, 16S-23S rDNA intragenic spacer (ITS)-based phylogenic analysis of strains isolated from cerebrospinal fluid (CSF) in Thua Thien Hue Province, Vietnam, is presented and discussed. Pathogenic bacteria were isolated from 40 CSF samples, and 18 were identified as S. suis by culture-dependent methods. Capsular serotyping was assessed by real-time PCR. ITS sequences were obtained after traditional PCR and were used in phylogenic analyses. Pathogenic bacteria were isolated from 36 out of 40 CSF samples. A total of 18 S. suis strains were isolated and assigned to serotype 2 by real-time PCR. One CSF sample, negative when tested by culture-dependent methods, was positive to S. suis serotype 2 by real-time PCR. Pairwise alignments of the 18 ITS sequences did not reveal any variable nucleotide position, and resulted in a single sequence type. Sequences were similar to S. suis serotype 2 reference ITS sequences (> 98.1%), and there was no lack of an ITS spacer region in the isolates. S. suis serotype 2 is the most prevalent serotype in central Vietnam. Real-time PCR assay proved to be a reliable diagnostic method for early detection of S. suis 2 in CSF samples.

Cultivating microbial dark matter in benzene-degrading methanogenic consortia.

PubMed

Luo, Fei; Devine, Cheryl E; Edwards, Elizabeth A

2016-09-01

The microbes responsible for anaerobic benzene biodegradation remain poorly characterized. In this study, we identified and quantified microbial populations in a series of 16 distinct methanogenic, benzene-degrading enrichment cultures using a combination of traditional 16S rRNA clone libraries (four cultures), pyrotag 16S rRNA amplicon sequencing (11 cultures), metagenome sequencing (1 culture) and quantitative polymerase chain reaction (qPCR; 12 cultures). An operational taxonomic unit (OTU) from the Deltaproteobacteria designated ORM2 that is only 84% to 86% similar to Syntrophus or Desulfobacterium spp. was consistently identified in all enrichment cultures, and typically comprised more than half of the bacterial sequences. In addition to ORM2, a sequence belonging to Parcubacteria (candidate division OD1) identified from the metagenome data was the only other OTU common to all the cultures surveyed. Culture transfers (1% and 0.1%) were made in the presence and absence of benzene, and the abundance of ORM2, OD1 and other OTUs was tracked over 415 days using qPCR. ORM2 sequence abundance increased only when benzene was present, while the abundance of OD1 and other OTUs increased even in the absence of benzene. Deltaproteobacterium ORM2 is unequivocally the benzene-metabolizing population. This study also hints at laboratory cultivation conditions for a member of the widely distributed yet uncultivated Parcubacteria (OD1). © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

Assessment of a Pan-Dermatophyte Nested-PCR Compared with Conventional Methods for Direct Detection and Identification of Dermatophytosis Agents in Animals.

PubMed

Piri, Fahimeh; Zarei Mahmoudabadi, Ali; Ronagh, Ali; Ahmadi, Bahram; Makimura, Koichi; Rezaei-Matehkolaei, Ali

2018-06-26

Conventional direct microscopy with potassium hydroxide (KOH) and culture were found to lack the ability to establish a fast and specific diagnosis of dermatophytosis. A pan-dermatophyte nested-PCR assay was developed using a novel primer pair targeting the translation elongation factor 1-α (Tef-1α) sequences for direct detection and identification of most veterinary relevant dermatophytes in animal samples suspected to dermatophytosis. A total of 140 animal skin and hair samples were subjected to direct microscopy, culture, and ITS-RFLP/ITS-sequencing of culture isolates for the detection and identification of dermatophytosis agents. Nested-PCR sequencing was performed on all the extracted DNAs using a commercial kit after dissolving the specimens by mechanical beating. Nested-PCR was positive in 90% of samples, followed by direct microscopy (85.7%) and culture (75%). The degree of agreement between nested-PCR and direct microscopy (94.4%) was higher than with culture (83.3%). In 105 culture positive cases, the measures of agreement for the identification of dermatophytosis agents were as follows: 100% between nested-PCR sequencing and ITS-RFLP/ITS-sequencing and 63.8% between nested-PCR sequencing and culture. The developed nested-PCR was faster as well as more sensitive and specific than conventional methods for detection and identification of dermatophytes in clinical samples, which was particularly suitable for epidemiological studies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1[W

PubMed Central

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-01-01

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159

Genetic Regulation in the Aiptasia pallida Symbiosis - Performance Report, Year 1.

DTIC Science & Technology

1997-02-01

and symbiotic zooxanthellae is one developed for serial analysis of gene expression (SAGE). We initially tested the SAGE protocol with cDNA generated...technically difficult. We are now focusing on constructing representative cDNA libraries from cultured and symbiotic zooxanthellae and will sequence

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of pakistan and their industrial importance

PubMed Central

Akhtar, Nasrin; Ghauri, Muhammad A.; Iqbal, Aamira; Anwar, Munir A.; Akhtar, Kalsoom

2008-01-01

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99% 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97%, 98% and 99% 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications. PMID:24031194

Molecular Methods To Improve Diagnosis and Identification of Mucormycosis▿

PubMed Central

Hammond, Sarah P.; Bialek, Ralf; Milner, Danny A.; Petschnigg, Eva M.; Baden, Lindsey R.; Marty, Francisco M.

2011-01-01

Mucormycosis is difficult to diagnose. Samples from suspected cases often fail to grow Mucorales in microbiologic cultures. We identified all hematologic malignancy and stem cell transplant patients diagnosed with proven mucormycosis between 2001 and 2009 at Brigham and Women's Hospital/Dana-Farber Cancer Institute. Seminested PCR targeting Mucorales 18S ribosomal DNA and sequencing were performed on formalin-fixed paraffin-embedded tissue samples. Of 29 cases of mucormycosis, 27 had tissue samples available for PCR and sequencing. Mucorales PCR was positive in 22. Among 12 culture-positive cases, 10 were PCR positive and sequencing was concordant with culture results to the genus level in 9. Among 15 culture-negative cases, PCR was positive and sequencing allowed genus identification in 12. Mucorales PCR is useful for confirmation of the diagnosis of mucormycosis and for further characterization of the infection in cases where cultures are negative. PMID:21508149

Molecular methods to improve diagnosis and identification of mucormycosis.

PubMed

Hammond, Sarah P; Bialek, Ralf; Milner, Danny A; Petschnigg, Eva M; Baden, Lindsey R; Marty, Francisco M

2011-06-01

Mucormycosis is difficult to diagnose. Samples from suspected cases often fail to grow Mucorales in microbiologic cultures. We identified all hematologic malignancy and stem cell transplant patients diagnosed with proven mucormycosis between 2001 and 2009 at Brigham and Women's Hospital/Dana-Farber Cancer Institute. Seminested PCR targeting Mucorales 18S ribosomal DNA and sequencing were performed on formalin-fixed paraffin-embedded tissue samples. Of 29 cases of mucormycosis, 27 had tissue samples available for PCR and sequencing. Mucorales PCR was positive in 22. Among 12 culture-positive cases, 10 were PCR positive and sequencing was concordant with culture results to the genus level in 9. Among 15 culture-negative cases, PCR was positive and sequencing allowed genus identification in 12. Mucorales PCR is useful for confirmation of the diagnosis of mucormycosis and for further characterization of the infection in cases where cultures are negative.

Variability of ribosomal RNA genes in Rauwolfia species: parallelism between tissue culture-induced rearrangements and interspecies polymorphism.

PubMed

Andreev, I O; Spiridonova, K V; Solovyan, V T; Kunakh, V A

2005-01-01

An analysis of 18S-25S and 5S rRNA genes in intact plants and cultured tissues of some Rauwolfia species was performed to compare these sequences variability occurred as a result of the species evolution in nature and that induced by tissue culture. The restriction fragment length polymorphism of 18S-25S and 5S rDNA was found both in intact plants of various Rauwolfia species and in long-term Rauwolfia serpentina tissue cultures. In addition, changes in the amount of 18S-25S rRNA genes were observed in long-term R. serpentina tissue cultures. The results demonstrate that rDNA variability observed in intact plants as well as in long-term cultures is attributed to differences in the same regions of ribosomal RNA genes.

Diagnostics of Tree Diseases Caused by Phytophthora austrocedri Species.

PubMed

Mulholland, Vincent; Elliot, Matthew; Green, Sarah

2015-01-01

We present methods for the detection and quantification of four Phytophthora species which are pathogenic on trees; Phytophthora ramorum, Phytophthora kernoviae, Phytophthora lateralis, and Phytophthora austrocedri. Nucleic acid extraction methods are presented for phloem tissue from trees, soil, and pure cultures on agar plates. Real-time PCR methods are presented and include primer and probe sets for each species, general advice on real-time PCR setup and data analysis. A method for sequence-based identification, useful for pure cultures, is also included.

Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis

PubMed Central

Wise, Mark G.; McArthur, J Vaun; Shimkets, Lawrence J.

1999-01-01

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described. PMID:10543800

Severe chronic osteomyelitis caused by Morganella morganii with high population diversity.

PubMed

Zhu, Jialiang; Li, Haifeng; Feng, Li; Yang, Min; Yang, Ronggong; Yang, Lin; Li, Li; Li, Ruoyan; Liu, Minshan; Hou, Shuxun; Ke, Yuehua; Li, Wenfeng; Bai, Fan

2016-09-01

A case of chronic osteomyelitis probably caused by Morganella morganii, occurring over a period of 30 years, is reported. The organism was identified through a combination of sample culture, direct sequencing, and 16S RNA gene amplicon sequencing. Further whole-genome sequencing and population structure analysis of the isolates from the patient showed the bacterial population to be highly diverse. This case provides a valuable example of a long-term infection caused by an opportunistic pathogen, M. morganii, with high diversity, which might evolve during replication within the host. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies.

PubMed

Réfega, Susana; Girard-Misguich, Fabienne; Bourdieu, Christiane; Péry, Pierre; Labbé, Marie

2003-04-02

Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.

Distinctive archaebacterial species associated with anaerobic rumen protozoan Entodinium caudatum.

PubMed

Tóthová, T; Piknová, M; Kisidayová, S; Javorský, P; Pristas, P

2008-01-01

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India.

PubMed

Pudupakam, Raghavendra Sumanth; Raghunath, Shobana; Pudupakam, Meghanath; Daggupati, Sreenivasulu

2017-03-01

Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India

PubMed Central

Pudupakam, Raghavendra Sumanth; Raghunath, Shobana; Pudupakam, Meghanath; Daggupati, Sreenivasulu

2017-01-01

Aim: Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates. Materials and Methods: The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established. Results: The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains. Conclusion: Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability. PMID:28435199

Identification and Antimicrobial Resistance of Bacteria Isolated from Probiotic Products Used in Shrimp Culture.

PubMed

Noor Uddin, Gazi Md; Larsen, Marianne Halberg; Christensen, Henrik; Aarestrup, Frank M; Phu, Tran Minh; Dalsgaard, Anders

2015-01-01

Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds. Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing. We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing. Only 6/60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.

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.

Isolation, identification and fibrolytic characteristics of rumen fungi grown with indigenous methanogen from yaks (Bos grunniens) grazing on the Qinghai-Tibetan Plateau.

PubMed

Wei, Y-Q; Yang, H-J; Luan, Y; Long, R-J; Wu, Y-J; Wang, Z-Y

2016-03-01

To obtain co-cultures of anaerobic fungi and their indigenously associated methanogens from the rumen of yaks grazing on the Qinghai-Tibetan Plateau and investigate their morphology features and ability to degrade lignocellulose. Twenty fungus-methanogen co-cultures were obtained by Hungate roll-tube technique. The fungi were identified as Orpinomyces, Neocallimastix and Piromyces genera based on the morphological characteristics and internal transcribed spacer 1 sequences analysis. All methanogens were identified as Methanobrevibacter sp. by 16S rRNA gene sequencing. There were four types of co-cultures: Neocallimastix with Methanobrevibacter ruminantium, Orpinomyces with M. ruminantium, Orpinomyces with Methanobrevibacter millerae and Piromyces with M. ruminantium among 20 co-cultures. In vitro studies with wheat straw as substrate showed that the Neocallimastix with M. ruminantium co-cultures and Piromyces with M. ruminantium co-cultures exhibited higher xylanase, filter paper cellulase (FPase), ferulic acid esterase, acetyl esterase activities, in vitro dry matter digestibility, gas, CH4 , acetate production, ferulic acid and p-coumaric acid releases. The Neocallimastix frontalis Yak16 with M. ruminantium co-culture presented the strongest lignocellulose degradation ability among 20 co-cultures. Twenty fungus-methanogen co-cultures were obtained from the rumen of grazing yaks. The N. frontalis with M. ruminantium co-cultures were highly effective combination for developing a fermentative system that bioconverts lignocellulose to high activity fibre-degrading enzyme, CH4 and acetate. The N. frontalis with M. ruminantium co-cultures from yaks grazing on the Qinghai-Tibetan Plateau present great potential in lignocellulose biodegradation industry. © 2015 The Society for Applied Microbiology.

Transcription analysis of peloric mutants of Phalaenopsis orchids derived from tissue culture.

PubMed

Chen, Ya Huei; Tsai, Yi Jung; Huang, Jian Zhi; Chen, Fure Chyi

2005-08-01

Tissue culture has been widely used for mass propagation of Phalaenopsis. However, somaclonal variation occurred during micropropagation process posed a severe problem by affecting product quality. In this study, wild type and peloric flower buds of Phalaenopsis hybrids derived from flower stalk nodal culture were used for cDNA-RAPD and cDNA suppression subtractive hybridization analyses in order to study their genetic difference in terms of expressed sequence tags. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced. These ESTs sequences can be grouped into several functional categories involved in different cellular processes including metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein localization, and into a subcategory of proteins with unknown function. Cymbidium mosaic virus transcript was surprisingly found expressed frequently in the peloric mutant of P. Little Mary. Real-time RT-PCR analysis on selected ESTs showed that in mutant flower buds, a bZIP transcription factor (TGA1a-like protein) was down-regulated, while up-regulated genes include auxin-regulated protein kinase, cyclophilin, and TCP-like genes. A retroelement clone was also preferentially expressed in the peloric mutant flowers. On the other hand, ESTs involved in DNA methylation, chromatin remodeling and post-transcriptional regulation, such as DNA methyltransferase, histone acetyltransferase, ERECTA, and DEAD/DEAH RNA helicase, were enriched in normal flower buds than the mutants. The enriched transcripts in the wild type indicate the down regulation of these transcripts in the mutants, and vice versa. The potential roles of the analyzed transcripts in the development of Phalaenopsis flowers are discussed.

Phylogenetic Diversity of Bacteria Associated with the Marine Sponge Rhopaloeides odorabile†

PubMed Central

Webster, Nicole S.; Wilson, Kate J.; Blackall, Linda L.; Hill, Russell T.

2001-01-01

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the β- and γ-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge. PMID:11133476

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.

Influence of Geographical Origin and Flour Type on Diversity of Lactic Acid Bacteria in Traditional Belgian Sourdoughs▿ †

PubMed Central

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2007-01-01

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough. PMID:17675431

Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs.

PubMed

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2007-10-01

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.

Has the use of molecular methods for the characterization of the human oral microbiome changed our understanding of the role of bacteria in the pathogenesis of periodontal disease?

PubMed

Wade, William Geoffrey

2011-03-01

Only around half of oral bacteria can be grown in the laboratory using conventional culture methods. Molecular methods based on 16S rRNA gene sequence are now available and are being used to characterize the periodontal microbiota in its entirety. This review describes the cultural characterization of the oral and periodontal microbiotas and explores the influence of the additional data now available from culture-independent molecular analyses on current thinking on the role of bacteria in periodontitis. Culture-independent molecular analysis of the periodontal microbiota has shown it to be far more diverse than previously thought. A number of species including some that have yet to be cultured are as strongly associated with disease as those organisms traditionally regarded as periodontal pathogens. Sequencing of bacterial genomes has revealed a high degree of intra-specific genetic diversity. The use of molecular methods for the characterization of the periodontal microbiome has greatly expanded the range of bacterial species known to colonize this habitat. Understanding the interactions between the human host and its commensal bacterial community at the functional level is a priority. © 2011 John Wiley & Sons A/S.

Cloning and expression of UDP-glucose: flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis.

PubMed

Hirotani, M; Kuroda, R; Suzuki, H; Yoshikawa, T

2000-05-01

A cDNA encoding UDP-glucose: baicalein 7-O-glucosyltransferase (UBGT) was isolated from a cDNA library from hairy root cultures of Scutellaria baicalensis Georgi probed with a partial-length cDNA clone of a UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) from grape (Vitis vinifera L.). The heterologous probe contained a glucosyltransferase consensus amino acid sequence which was also present in the Scutellaria cDNA clones. The complete nucleotide sequence of the 1688-bp cDNA insert was determined and the deduced amino acid sequences are presented. The nucleotide sequence analysis of UBGT revealed an open reading frame encoding a polypeptide of 476 amino acids with a calculated molecular mass of 53,094 Da. The reaction product for baicalein and UDP-glucose catalyzed by recombinant UBGT in Escherichia coli was identified as authentic baicalein 7-O-glucoside using high-performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. The enzyme activities of recombinant UBGT expressed in E. coli were also detected towards flavonoids such as baicalein, wogonin, apigenin, scutellarein, 7,4'-dihydroxyflavone and kaempferol, and phenolic compounds. The accumulation of UBGT mRNA in hairy roots was in response to wounding or salicylic acid treatments.

Nonuniform multiview color texture mapping of image sequence and three-dimensional model for faded cultural relics with sift feature points

NASA Astrophysics Data System (ADS)

Li, Na; Gong, Xingyu; Li, Hongan; Jia, Pengtao

2018-01-01

For faded relics, such as Terracotta Army, the 2D-3D registration between an optical camera and point cloud model is an important part for color texture reconstruction and further applications. This paper proposes a nonuniform multiview color texture mapping for the image sequence and the three-dimensional (3D) model of point cloud collected by Handyscan3D. We first introduce nonuniform multiview calibration, including the explanation of its algorithm principle and the analysis of its advantages. We then establish transformation equations based on sift feature points for the multiview image sequence. At the same time, the selection of nonuniform multiview sift feature points is introduced in detail. Finally, the solving process of the collinear equations based on multiview perspective projection is given with three steps and the flowchart. In the experiment, this method is applied to the color reconstruction of the kneeling figurine, Tangsancai lady, and general figurine. These results demonstrate that the proposed method provides an effective support for the color reconstruction of the faded cultural relics and be able to improve the accuracy of 2D-3D registration between the image sequence and the point cloud model.

Anaerobic degradation of propane and butane by sulfate-reducing bacteria enriched from marine hydrocarbon cold seeps.

PubMed

Jaekel, Ulrike; Musat, Niculina; Adam, Birgit; Kuypers, Marcel; Grundmann, Olav; Musat, Florin

2013-05-01

The short-chain, non-methane hydrocarbons propane and butane can contribute significantly to the carbon and sulfur cycles in marine environments affected by oil or natural gas seepage. In the present study, we enriched and identified novel propane and butane-degrading sulfate reducers from marine oil and gas cold seeps in the Gulf of Mexico and Hydrate Ridge. The enrichment cultures obtained were able to degrade simultaneously propane and butane, but not other gaseous alkanes. They were cold-adapted, showing highest sulfate-reduction rates between 16 and 20 °C. Analysis of 16S rRNA gene libraries, followed by whole-cell hybridizations with sequence-specific oligonucleotide probes showed that each enrichment culture was dominated by a unique phylotype affiliated with the Desulfosarcina-Desulfococcus cluster within the Deltaproteobacteria. These phylotypes formed a distinct phylogenetic cluster of propane and butane degraders, including sequences from environments associated with hydrocarbon seeps. Incubations with (13)C-labeled substrates, hybridizations with sequence-specific probes and nanoSIMS analyses showed that cells of the dominant phylotypes were the first to become enriched in (13)C, demonstrating that they were directly involved in hydrocarbon degradation. Furthermore, using the nanoSIMS data, carbon assimilation rates were calculated for the dominant cells in each enrichment culture.

Prevalence and distribution of White Spot Syndrome Virus in cultured shrimp.

PubMed

Hossain, A; Nandi, S P; Siddique, M A; Sanyal, S K; Sultana, M; Hossain, M A

2015-02-01

White Spot Syndrome Virus (WSSV) is a dsDNA virus causing White Spot Syndrome Disease (WSSD) in shrimp with almost 100% morality rate within 3-10 days. In Bangladesh, WSSD is one of the major impediments of shrimp farming. This study first investigated the prevalence and distribution of WSSV in cultured shrimps of the coastal regions in Bangladesh. A total of 60 shrimp samples, collected from the 25 shrimp farms of different coastal regions (Satkhira, Khulna, Bagerhat and Cox's Bazar), were analysed during 2013-2014 by conventional PCR using VP28 and VP664 gene-specific primers; 39 of 60 samples were found WSSV positive. SYBR green real-time PCR using 71-bp amplicon for VP664 gene correlated well with conventional PCR data. The prevalence rates of WSSV among the collected 60 samples were Satkhira 79%, Khulna 50%, Bagerhat 38% and Cox's Bazar 25%. Sequencing of WSSV-positive PCR amplicons of VP28 showed 99% similarity with WSSV NCBI Ref/Seq Sequences. Molecular analysis of the VP28 gene sequences of WSSV revealed that Bangladeshi strains phylogenetically affiliated to the strains belong to India. This work concluded that WSSV infections are widely distributed in the coastal regions cultured shrimp in Bangladesh. © 2014 The Society for Applied Microbiology.

Sexual and asexual states of some endophytic Phialocephala species of Picea.

PubMed

Tanney, Joey B; Douglas, Brian; Seifert, Keith A

2016-01-01

Unidentified DNA sequences in isolation-based or culture-free studies of conifer endophytes are a persistent problem that requires a field approach to resolve. An investigation of foliar endophytes of Picea glauca, P. mariana, P. rubens and Pinus strobus in eastern Canada, using a combined field, morphological, cultural and DNA sequencing approach, resulted in the frequent isolation of Phialocephala spp. and the first verified discovery of their mollisia-like sexual states in the field. Phialocephala scopiformis and Ph. piceae were the most frequent species isolated as endophytes from healthy conifer needles. Corresponding Mollisia or mollisioid sexual states for Ph. scopiformis, Ph. piceae and several undescribed species in a clade containing Ph. dimorphospora were collected in the sampling area and characterized by analysis of the nuc internal transcribed spacer rDNA (ITS) and gene for the largest subunit of RNA polymerase II (RPB1) loci. Four novel species and one new combination in a clade containing Ph. dimorphospora, the type of Phialocephala, are presented, accompanied by descriptions of apothecia and previously undocumented synanamorphs. An epitype culture and corresponding reference sequences for Phialocephala dimorphospora are proposed. The resulting ITS barcodes linked with robust taxonomic species concepts are an important resource for future research on forest ecosystems and endophytes. © 2016 by The Mycological Society of America.

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment.

PubMed

García-Mena, Jaime; Cano-Ramirez, Claudia; Garibay-Orijel, Claudio; Ramirez-Canseco, Sergio; Poggi-Varaldo, Héctor M

2005-06-01

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 microg-1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

Comparative analysis of the Flavobacterium columnare genomovar I and II genomes

USDA-ARS?s Scientific Manuscript database

Columnaris disease caused by Gram-negative rod Flavobacterium columnare is one of the most common diseases of catfish. F. columnare is also a common problem in other cultured fish species worldwide. F. columnare has three major genomovars; we have sequenced a representative strain from genomovar I (...

Osteomyelitis associated with Nocardiopsis composta in a dog.

PubMed

Salas, Elisa N; Royal, Debra; Kurz, Lance; Loy, J Dustin

2015-05-01

We report the first detection of Nocardiopsis composta in association with osteomyelitis in a young male miniature Australian shepherd dog. Findings included suppurative osteomyelitis containing intralesional Fite's acid fast bacilli, aerobic culture of branching Gram-positive rods, and positive identification via phenotypic analysis and 16S rDNA sequencing.

Comparative transcriptome analysis of Aspergillus flavus isolates under different oxidative stresses and culture media

USDA-ARS?s Scientific Manuscript database

Aspergillus flavus and aflatoxin contamination in the field are known to be influenced by numerous stress factors, particularly drought and heat stress. However, the purpose of aflatoxin production is unknown. Here, we report transcriptome analyses comprised of 282.6 Gb of sequencing data describing...

Bacterial diversity of autotrophic enriched cultures from remote, glacial Antarctic, Alpine and Andean aerosol, snow and soil samples

NASA Astrophysics Data System (ADS)

González-Toril, E.; Amils, R.; Delmas, R. J.; Petit, J.-R.; Komárek, J.; Elster, J.

2009-01-01

Four different communities and one culture of autotrophic microbial assemblages were obtained by incubation of samples collected from high elevation snow in the Alps (Mt. Blanc area) and the Andes (Nevado Illimani summit, Bolivia), from Antarctic aerosol (French station Dumont d'Urville) and a maritime Antarctic soil (King George Island, South Shetlands, Uruguay Station Artigas), in a minimal mineral (oligotrophic) media. Molecular analysis of more than 200 16S rRNA gene sequences showed that all cultured cells belong to the Bacteria domain. Phylogenetic comparison with the currently available rDNA database allowed sequences belonging to Proteobacteria Alpha-, Beta- and Gamma-proteobacteria), Actinobacteria and Bacteroidetes phyla to be identified. The Andes snow culture was the richest in bacterial diversity (eight microorganisms identified) and the marine Antarctic soil the poorest (only one). Snow samples from Col du Midi (Alps) and the Andes shared the highest number of identified microorganisms (Agrobacterium, Limnobacter, Aquiflexus and two uncultured Alphaproteobacteria clones). These two sampling sites also shared four sequences with the Antarctic aerosol sample (Limnobacter, Pseudonocardia and an uncultured Alphaproteobacteriaclone). The only microorganism identified in the Antarctica soil (Brevundimonas sp.) was also detected in the Antarctic aerosol. Most of the identified microorganisms had been detected previously in cold environments, marine sediments soils and rocks. Air current dispersal is the best model to explain the presence of very specific microorganisms, like those identified in this work, in environments very distant and very different from each other.

The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo

PubMed Central

Schageman, Jeoffrey; Li, Mu; Barta, Tim; Lea, Kristi; Gu, Jian; Magdaleno, Susan; Setterquist, Robert; Vlassov, Alexander V.

2013-01-01

Exosomes are small (30–150 nm) vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform. PMID:24205503

Hydrogenotrophic culture enrichment reveals rumen Lachnospiraceae and Ruminococcaceae acetogens and hydrogen-responsive Bacteroidetes from pasture-fed cattle.

PubMed

Gagen, Emma J; Padmanabha, Jagadish; Denman, Stuart E; McSweeney, Christopher S

2015-07-01

Molecular information suggests that there is a broad diversity of acetogens in the rumen, distinct from any currently isolated acetogens. We combined molecular analysis with enrichment culture techniques to investigate this diversity further. Methane-inhibited, hydrogenotrophic enrichment cultures produced acetate as the dominant end product. Acetyl-CoA synthase gene analysis revealed putative acetogens in the cultures affiliated with the Lachnospiraceae and Ruminococcaceae as has been found in other rumen studies. No formyltetrahydrofolate synthetase genes affiliating with acetogens or with 'homoacetogen similarity' scores >90% were identified. To further investigate the hydrogenotrophic populations in these cultures and link functional gene information with 16S rRNA gene identity, cultures were subcultured quickly, twice, through medium without exogenous hydrogen, followed by incubation without exogenous hydrogen. Comparison of cultures lacking hydrogen and their parent cultures revealed novel Lachnospiraceae and Ruminococcaceae that diminished in the absence of hydrogen, supporting the hypothesis that they were likely the predominant acetogens in the enrichments. Interestingly, a range of Bacteroidetes rrs sequences that demonstrated <86% identity to any named isolate also diminished in cultures lacking hydrogen. Acetogens or sulphate reducers from the Bacteroidetes have not been reported previously; therefore this observation requires further investigation. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Genome Sequence of African Swine Fever Virus BA71, the Virulent Parental Strain of the Nonpathogenic and Tissue-Culture Adapted BA71V.

PubMed

Rodríguez, Javier M; Moreno, Leticia Tais; Alejo, Alí; Lacasta, Anna; Rodríguez, Fernando; Salas, María L

2015-01-01

The strain BA71V has played a key role in African swine fever virus (ASFV) research. It was the first genome sequenced, and remains the only genome completely determined. A large part of the studies on the function of ASFV genes, viral transcription, replication, DNA repair and morphogenesis, has been performed using this model. This avirulent strain was obtained by adaptation to grow in Vero cells of the highly virulent BA71 strain. We report here the analysis of the genome sequence of BA71 in comparison with that of BA71V. They possess the smallest genomes for a virulent or an attenuated ASFV, and are essentially identical except for a relatively small number of changes. We discuss the possible contribution of these changes to virulence. Analysis of the BA71 sequence allowed us to identify new similarities among ASFV proteins, and with database proteins including two ASFV proteins that could function as a two-component signaling network.

Molecular analysis of leptospires from serogroup Sejroe obtained from asymptomatic cattle in Rio de Janeiro - Brazil reveals genetic proximity to serovar Guaricura.

PubMed

Loureiro, A P; Hamond, C; Pinto, P; Bremont, S; Bourhy, P; Lilenbaum, W

2016-04-01

Bovine leptospirosis causes substantial reproductive failure in cattle, mainly due to infections with serovar (sv) Hardjo infection. Notwithstanding, other serovars from the serogroup (sg) Sejroe could also have important roles in bovine leptospirosis. The objective was to investigate genetic diversity of serogroup Sejroe isolates obtained from asymptomatic cattle in the state of Rio de Janeiro, Brazil. Urine and vaginal fluid (VF) were collected from clinically healthy cattle immediately after slaughter. Five isolates were recovered and characterized (serogrouping) as belonging to sg Sejroe. Sequencing of rrs and secY genes further identified them as Leptospira santarosai. Analysis of secY sequences indicated a high level of sequence homology to sv Guaricura strains. Based on culture and sequence data, we inferred that other members of sg Sejroe may be important in bovine leptospiral infection, particularly genotypes of L. santarosai serovar Guaricura. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Diversity and enzyme-producing activity of culturable halophilic bacteria in Daishan Saltern of East China].

PubMed

Yang, Dan-Dan; Li, Qian; Huang, Jing-Jing; Chen, Min

2012-11-01

Soil and saline water samples were collected from the Daishan Saltern of East China, and the halophilic bacteria were isolated and cultured by using selective media, aimed to investigate the diversity and enzyme-producing activity of culturable halophilic bacteria in saltern environment. A total of 181 strains were isolated by culture-dependent method. Specific primers were used to amplify the 16S rRNA gene of bacteria and archaea. The operation taxonomy units (OTUs) were determined by ARDRA method, and the representative strain of each OTU was sequenced. The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The results showed that the isolated 181 strains displayed 21 operational taxonomic units (OTUs), of which, 12 OTUs belonged to halophilic bacteria, and the others belonged to halophilic archaea. Phylogenetic analysis indicated that there were 7 genera presented among the halophilic bacteria group, and 4 genera presented among the halophilic archaea group. The dominant halophilic strains were of Halomonas and Haloarcula, with 46.8% in halophilic bacteria and 49.1% in halophilic archaea group, respectively. Enzyme-producing analysis indicated that most strains displayed enzyme-producing activity, including the activities of producing amylase, proteinase and lipase, and the dominant strains capable of enzyme-producing were of Haloarcula. Our results showed that in the environment of Daishan Saltern, there existed a higher diversity of halophilic bacteria, being a source sink for screening enzyme-producing bacterial strains.

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling

PubMed Central

França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S.

2015-01-01

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes. PMID:26512991

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System, and Antibiotic Sensitivity Profiling.

PubMed

França, Luís; Simões, Catarina; Taborda, Marco; Diogo, Catarina; da Costa, Milton S

2015-01-01

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.

Detection and Whole-Genome Sequencing of Carbapenemase-Producing Aeromonas hydrophila Isolates from Routine Perirectal Surveillance Culture

PubMed Central

Hughes, Heather Y.; Lau, Anna F.; Dekker, John P.; Michelin, Angela V.; Youn, Jung-Ho; Henderson, David K.; Frank, Karen M.; Segre, Julia A.

2016-01-01

Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the blaKPC-2 gene in one isolate prompted inclusion of non-Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes. PMID:26888898

Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans

PubMed Central

Liu, Junyan; Deng, Yang; Peters, Brian M.; Li, Lin; Li, Bing; Chen, Lequn; Xu, Zhenbo; Shirtliff, Mark E.

2016-01-01

Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria regardless of beer type, and thus pose significant problems for the brewery industry. The aim of this study was to investigate the genetic mechanisms involved in the ability of the hard-to-culture beer-spoilage bacterium Lactobacillus acetotolerans to enter into the viable putative non-culturable (VPNC) state. A genome-wide transcriptional analysis of beer-spoilage L. acetotolerans strains BM-LA14526, BM-LA14527, and BM-LA14528 under normal, mid-term and VPNC states were performed using RNA-sequencing (RNA-seq) and further bioinformatics analyses. GO function, COG category, and KEGG pathway enrichment analysis were conducted to investigate functional and related metabolic pathways of the differentially expressed genes. Functional and pathway enrichment analysis indicated that heightened stress response and reduction in genes associated with transport, metabolic process, and enzyme activity might play important roles in the formation of the VPNC state. This is the first transcriptomic analysis on the formation of the VPNC state of beer spoilage L. acetotolerans. PMID:27819317

Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans.

PubMed

Liu, Junyan; Deng, Yang; Peters, Brian M; Li, Lin; Li, Bing; Chen, Lequn; Xu, Zhenbo; Shirtliff, Mark E

2016-11-07

Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria regardless of beer type, and thus pose significant problems for the brewery industry. The aim of this study was to investigate the genetic mechanisms involved in the ability of the hard-to-culture beer-spoilage bacterium Lactobacillus acetotolerans to enter into the viable putative non-culturable (VPNC) state. A genome-wide transcriptional analysis of beer-spoilage L. acetotolerans strains BM-LA14526, BM-LA14527, and BM-LA14528 under normal, mid-term and VPNC states were performed using RNA-sequencing (RNA-seq) and further bioinformatics analyses. GO function, COG category, and KEGG pathway enrichment analysis were conducted to investigate functional and related metabolic pathways of the differentially expressed genes. Functional and pathway enrichment analysis indicated that heightened stress response and reduction in genes associated with transport, metabolic process, and enzyme activity might play important roles in the formation of the VPNC state. This is the first transcriptomic analysis on the formation of the VPNC state of beer spoilage L. acetotolerans.

A novel spotted fever group Rickettsia infecting Amblyomma parvitarsum (Acari: Ixodidae) in highlands of Argentina and Chile.

PubMed

Ogrzewalska, Maria; Nieri-Bastos, Fernanda A; Marcili, Arlei; Nava, Santiago; González-Acuña, Daniel; Muñoz-Leal, Sebastián; Ruiz-Arrondo, Ignacio; Venzal, José M; Mangold, Atilio; Labruna, Marcelo B

2016-04-01

The tick Amblyomma parvitarsum (Acari: Ixodidae) has established populations in Andean and Patagonic environments of South America. For the present study, adults of A. parvitarsum were collected in highland areas (elevation >3500 m) of Argentina and Chile during 2009-2013, and tested by PCR for rickettsial infection in the laboratory, and isolation of rickettsiae in Vero cell culture by the shell vial technique. Overall, 51 (62.2%) out of 82 A. parvitarsum adult ticks were infected by spotted fever group (SFG) rickettsiae, which generated DNA sequences 100% identical to each other, and when submitted to BLAST analysis, they were 99.3% identical to corresponding sequence of the ompA gene of Rickettsia sp. strain Atlantic rainforest. Rickettsiae were successfully isolated in Vero cell culture from two ticks, one from Argentina and one from Chile. DNA extracted from the third passage of the isolates of Argentina and Chile were processed by PCR, resulting in partial sequences for three rickettsial genes (gltA, ompB, ompA). These sequences were concatenated and aligned with rickettsial corresponding sequences available in GenBank. Phylogenetic analysis revealed that the A. pavitarsum rickettsial agent grouped under high bootstrap support in a clade composed by the SFG pathogens R. sibirica, R. africae, R. parkeri, Rickettsia sp. strain Atlantic rainforest, and two unnamed SFG agents of unknown pathogenicty, Rickettsia sp. strain NOD, and Rickettsia sp. strain ApPR. The pathogenic role of this A. parvitarsum rickettsia cannot be discarded, since several species of tick-borne rickettsiae that were considered nonpathogenic for decades are now associated with human infections. Copyright © 2016. Published by Elsevier GmbH.

Occurrence and Phylogenetic Diversity of Sphingomonas Strains in Soils Contaminated with Polycyclic Aromatic Hydrocarbons

PubMed Central

Leys, Natalie M. E. J.; Ryngaert, Annemie; Bastiaens, Leen; Verstraete, Willy; Top, Eva M.; Springael, Dirk

2004-01-01

Bacterial strains of the genus Sphingomonas are often isolated from contaminated soils for their ability to use polycyclic aromatic hydrocarbons (PAH) as the sole source of carbon and energy. The direct detection of Sphingomonas strains in contaminated soils, either indigenous or inoculated, is, as such, of interest for bioremediation purposes. In this study, a culture-independent PCR-based detection method using specific primers targeting the Sphingomonas 16S rRNA gene combined with denaturing gradient gel electrophoresis (DGGE) was developed to assess Sphingomonas diversity in PAH-contaminated soils. PCR using the new primer pair on a set of template DNAs of different bacterial genera showed that the method was selective for bacteria belonging to the family Sphingomonadaceae. Single-band DGGE profiles were obtained for most Sphingomonas strains tested. Strains belonging to the same species had identical DGGE fingerprints, and in most cases, these fingerprints were typical for one species. Inoculated strains could be detected at a cell concentration of 104 CFU g of soil−1. The analysis of Sphingomonas population structures of several PAH-contaminated soils by the new PCR-DGGE method revealed that soils containing the highest phenanthrene concentrations showed the lowest Sphingomonas diversity. Sequence analysis of cloned PCR products amplified from soil DNA revealed new 16S rRNA gene Sphingomonas sequences significantly different from sequences from known cultivated isolates (i.e., sequences from environmental clones grouped phylogenetically with other environmental clone sequences available on the web and that possibly originated from several potential new species). In conclusion, the newly designed Sphingomonas-specific PCR-DGGE detection technique successfully analyzed the Sphingomonas communities from polluted soils at the species level and revealed different Sphingomonas members not previously detected by culture-dependent detection techniques. PMID:15066784

Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosis of pertussis using the polymerase chain reaction.

PubMed Central

Glare, E M; Paton, J C; Premier, R R; Lawrence, A J; Nisbet, I T

1990-01-01

A tandemly repeated 1,046-base-pair (bp) ClaI DNA fragment from Bordetella pertussis was cloned into Escherichia coli by using the vector pUC19. This fragment, when isolated, hybridized strongly to DNA from all 100 clinical isolates of B. pertussis tested. It was shown to have homology to single-copy sequences in Bordetella bronchiseptica but not Bordetella parapertussis and did not hybridize to lysate blots of a wide range of other bacteria, including members of the closely related genera Pasteurella, Alcaligenes, and Haemophilus. The 1,046-bp fragment was sequenced, and complementary synthetic oligonucleotides flanking a 153-bp region within the repeated element were used as primers for specific amplification of this region using the polymerase chain reaction (PCR). This procedure was then applied to the rapid (5-h) detection of B. pertussis in nasopharyngeal secretions collected from 332 children with suspected pertussis. The test yielded positive results in a total of 98 samples, compared with 66 for culture and 33 for direct immunofluorescence (IF). All of the IF-positive samples were PCR positive, as were 63 of the samples from which B. pertussis was eventually cultured. Two hundred thirty-one specimens which were negative by IF and culture were also negative in the PCR assay. However, 33 culture- and IF-negative specimens were positive by PCR assay. Several of these specimens were collected from close contacts of culture-proven pertussis patients, were follow-up specimens from such patients, or were from patients with serological evidence of pertussis and therefore may be true-rather than false-positives. Images PMID:2229381

Enterohemorrhagic Escherichia coli O157 in milk and dairy products from Libya: Isolation and molecular identification by partial sequencing of 16S rDNA

PubMed Central

Garbaj, Aboubaker M.; Awad, Enas M.; Azwai, Salah M.; Abolghait, Said K.; Naas, Hesham T.; Moawad, Ashraf A.; Gammoudi, Fatim T.; Barbieri, Ilaria; Eldaghayes, Ibrahim M.

2016-01-01

Aim: The aim of this work was to isolate and molecularly identify enterohemorrhagic Escherichia coli (EHEC) O157 in milk and dairy products in Libya, in addition; to clear the accuracy of cultural and biochemical identification as compared with molecular identification by partial sequencing of 16S rDNA for the existing isolates. Materials and Methods: A total of 108 samples of raw milk (cow, she-camel, and goat) and locally made dairy products (fermented cow’s milk, Maasora, Ricotta and ice cream) were collected from some regions (Janzour, Tripoli, Kremiya, Tajoura and Tobruk) in Libya. Samples were subjected to microbiological analysis for isolation of E. coli that was detected by conventional cultural and molecular method using polymerase chain reaction and partial sequencing of 16S rDNA. Results: Out of 108 samples, only 27 isolates were found to be EHEC O157 based on their cultural characteristics (Tellurite-Cefixime-Sorbitol MacConkey) that include 3 isolates from cow’s milk (11%), 3 isolates from she-camel’s milk (11%), two isolates from goat’s milk (7.4%) and 7 isolates from fermented raw milk samples (26%), isolates from fresh locally made soft cheeses (Maasora and Ricotta) were 9 (33%) and 3 (11%), respectively, while none of the ice cream samples revealed any growth. However, out of these 27 isolates, only 11 were confirmed to be E. coli by partial sequencing of 16S rDNA and E. coli O157 Latex agglutination test. Phylogenetic analysis revealed that majority of local E. coli isolates were related to E. coli O157:H7 FRIK944 strain. Conclusion: These results can be used for further studies on EHEC O157 as an emerging foodborne pathogen and its role in human infection in Libya. PMID:27956766

Pathogenesis, Molecular Genetics, and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne’s Disease

PubMed Central

Rathnaiah, Govardhan; Zinniel, Denise K.; Bannantine, John P.; Stabel, Judith R.; Gröhn, Yrjö T.; Collins, Michael T.; Barletta, Raúl G.

2017-01-01

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants causing chronic diarrhea, malnutrition, and muscular wasting. Neonates and young animals are infected primarily by the fecal–oral route. MAP attaches to, translocates via the intestinal mucosa, and is phagocytosed by macrophages. The ensuing host cellular immune response leads to granulomatous enteritis characterized by a thick and corrugated intestinal wall. We review various tissue culture systems, ileal loops, and mice, goats, and cattle used to study MAP pathogenesis. MAP can be detected in clinical samples by microscopy, culturing, PCR, and an enzyme-linked immunosorbent assay. There are commercial vaccines that reduce clinical disease and shedding, unfortunately, their efficacies are limited and may not engender long-term protective immunity. Moreover, the potential linkage with Crohn’s disease and other human diseases makes MAP a concern as a zoonotic pathogen. Potential therapies with anti-mycobacterial agents are also discussed. The completion of the MAP K-10 genome sequence has greatly improved our understanding of MAP pathogenesis. The analysis of this sequence has identified a wide range of gene functions involved in virulence, lipid metabolism, transcriptional regulation, and main metabolic pathways. We also review the transposons utilized to generate random transposon mutant libraries and the recent advances in the post-genomic era. This includes the generation and characterization of allelic exchange mutants, transcriptomic analysis, transposon mutant banks analysis, new efforts to generate comprehensive mutant libraries, and the application of transposon site hybridization mutagenesis and transposon sequencing for global analysis of the MAP genome. Further analysis of candidate vaccine strains development is also provided with critical discussions on their benefits and shortcomings, and strategies to develop a highly efficacious live-attenuated vaccine capable of differentiating infected from vaccinated animals. PMID:29164142

Genomics and metagenomics in medical microbiology.

PubMed

Padmanabhan, Roshan; Mishra, Ajay Kumar; Raoult, Didier; Fournier, Pierre-Edouard

2013-12-01

Over the last two decades, sequencing tools have evolved from laborious time-consuming methodologies to real-time detection and deciphering of genomic DNA. Genome sequencing, especially using next generation sequencing (NGS) has revolutionized the landscape of microbiology and infectious disease. This deluge of sequencing data has not only enabled advances in fundamental biology but also helped improve diagnosis, typing of pathogen, virulence and antibiotic resistance detection, and development of new vaccines and culture media. In addition, NGS also enabled efficient analysis of complex human micro-floras, both commensal, and pathological, through metagenomic methods, thus helping the comprehension and management of human diseases such as obesity. This review summarizes technological advances in genomics and metagenomics relevant to the field of medical microbiology. Copyright © 2013 Elsevier B.V. All rights reserved.

Draft Genome Sequence of Muricauda sp. Strain K001 Isolated from a Marine Cyanobacterial Culture.

PubMed

Vizzotto, Carla S; Lopes, Fabyano A C; Green, Stefan J; Steindorff, Andrei S; Walter, Juline M; Thompson, Fabiano L; Krüger, Ricardo H

2018-05-31

We report the whole-genome sequence of Muricauda sp. strain K001 isolated from a marine cyanobacterial culture. This genome sequence will improve our understanding of the influence of heterotrophic bacteria on the physiology of cyanobacteria and may contribute to the development of new natural products. Copyright © 2018 Vizzotto et al.

Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses

PubMed Central

Jazaeri Farsani, Seyed Mohammad; Deijs, Martin; Dijkman, Ronald; Molenkamp, Richard; Jeeninga, Rienk E; Ieven, Margareta; Goossens, Herman; van der Hoek, Lia

2015-01-01

Background Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state-of-the-art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses. Method Three respiratory clinical samples were tested on well-differentiated pseudostratified tracheobronchial human airway epithelial (HAE) cultures grown at an air–liquid interface, which resemble the airway epithelium. Cells were stained with convalescent serum of the patients to identify infected cells and apical washes were analyzed by VIDISCA-454, a next-generation sequencing virus discovery technique. Results Infected cells were observed for all three samples. Sequencing subsequently indicated that the cells were infected by either human coronavirus OC43, influenzavirus B, or influenzavirus A. The sequence reads covered a large part of the genome (52%, 82%, and 57%, respectively). Conclusion We present here a new method for virus discovery that requires a virus culture on primary cells and an antibody detection. The virus in the harvest can be used to characterize the viral genome sequence and cell tropism, but also provides progeny virus to initiate experiments to fulfill the Koch's postulates. PMID:25482367

Computational analysis and functional expression of ancestral copepod luciferase.

PubMed

Takenaka, Yasuhiro; Noda-Ogura, Akiko; Imanishi, Tadashi; Yamaguchi, Atsushi; Gojobori, Takashi; Shigeri, Yasushi

2013-10-10

We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species. © 2013.

Osteomyelitis associated with Nocardiopsis composta in a dog

PubMed Central

Salas, Elisa N.; Royal, Debra; Kurz, Lance; Loy, J. Dustin

2015-01-01

We report the first detection of Nocardiopsis composta in association with osteomyelitis in a young male miniature Australian shepherd dog. Findings included suppurative osteomyelitis containing intralesional Fite’s acid fast bacilli, aerobic culture of branching Gram-positive rods, and positive identification via phenotypic analysis and 16S rDNA sequencing. PMID:25969577

Assessing Global Awareness over Short-Term Study Abroad Sequence: A Factor Analysis

ERIC Educational Resources Information Center

Kurt, Mark R.; Olitsky, Neal H.; Geis, Paul

2013-01-01

Academic study abroad programs are uniquely equipped to give students the opportunities to achieve outcomes for global citizenship (Langran, Langran, and Ozment 2009). These programs take students outside the confines of their home institutions and expose students to new cultures and languages while integrating academic content to enhance the…

Phylogeny and active ingredients of artificial Ophiocordyceps lanpingensis ascomata

NASA Astrophysics Data System (ADS)

Chen, Zihong; Xu, Ling; Yu, Hong; Zeng, Wenbo; Dai, Yongdong; Wang, Yuanbing

2018-04-01

To evaluate the morphological character, phylogenesis and functional components of artificial Ophiocordyceps lanpingensis, a related species of O. sinensis. The ascomata of O. lanpingensis was induced with its asexual strain, HLANY0707 and its microscopic feature was described. Phylogenesis was analyzed with ITS-5.8S sequences of HLANY0707, its cultured stroma, and 39 relative sequences of Hirsutella and Ophiocordyceps based on the maximum likelihood tree. Six nucleosides of artificial O. lanpingensis, natural O. lanpingensis and natural O. sinensis were compared with HPLC analysis. Artificial ascomata of O. lanpingensis could be massively produced with HLANY0707 and had similar microscopic features as the nature specimens. Phylogenetic analysis showed that both the artificial and natural O. lanpingensis had closer relationship with O. sinensis, O. xuefengensis, H. uncinata and O. robertsii, the species whose massively cultured ascomata being not reported. Nucleosides of artificial O. lanpingensis were very similar to natural O. sinensis, implying a promising application prospect of artificial O. lanpingensis as an alternative to O. sinensis. It showed a promising way to develop artificial O. lanpingensis and conserve the rare and endangered species, O. sinensis.

The Cognitive Advantages of Counting Specifically: A Representational Analysis of Verbal Numeration Systems in Oceanic Languages.

PubMed

Bender, Andrea; Schlimm, Dirk; Beller, Sieghard

2015-10-01

The domain of numbers provides a paradigmatic case for investigating interactions of culture, language, and cognition: Numerical competencies are considered a core domain of knowledge, and yet the development of specifically human abilities presupposes cultural and linguistic input by way of counting sequences. These sequences constitute systems with distinct structural properties, the cross-linguistic variability of which has implications for number representation and processing. Such representational effects are scrutinized for two types of verbal numeration systems-general and object-specific ones-that were in parallel use in several Oceanic languages (English with its general system is included for comparison). The analysis indicates that the object-specific systems outperform the general systems with respect to counting and mental arithmetic, largely due to their regular and more compact representation. What these findings reveal on cognitive diversity, how the conjectures involved speak to more general issues in cognitive science, and how the approach taken here might help to bridge the gap between anthropology and other cognitive sciences is discussed in the conclusion. Copyright © 2015 Cognitive Science Society, Inc.

Indentification of huperzine A-producing endophytic fungi isolated from Huperzia serrata.

PubMed

Dong, Li-Hui; Fan, San-Wei; Ling, Qing-Zhi; Huang, Bei-Bei; Wei, Zhao-Jun

2014-03-01

This present study was designed to investigate the production of huperzine A (HupA), an acetylcholine inhibitor, which was produced by an endophytic fungi isolated from Huperzia serrata. Screening of 94 endophytic fungal isolates obtained from plant H. serrata was carried out for the production of HupA. Their morphological characteristics were studied and rDNA sequence analysis was carried out. The cultures were grown in liquid culture medium and the extracted metabolites were analyzed by thin layer chromatography and high performance liquid chromatograph for the presence of HupA. The DPPH scavenging ratio and inhibition ratio of acetylcholinesterase (AchE) of the same were determined. 3 out of 94 strains i.e. S29, L44 and S94 showed significant AchE-inhibitory activity and antioxidant activity. Strain L44 which exhibited maximum yield of HupA (37.63 μg/g on dry weight basis) was identified as Trichoderma species by ITS sequence analysis. In conclusion, endophytic fungi from H. serrata can be used as a new resource of HupA.

In vivo evolution of antimicrobial resistance in a series of Staphylococcus aureus patient isolates: the entire picture or a cautionary tale?

PubMed Central

van Hal, Sebastiaan J.; Steen, Jason A.; Espedido, Björn A.; Grimmond, Sean M.; Cooper, Matthew A.; Holden, Matthew T. G.; Bentley, Stephen D.; Gosbell, Iain B.; Jensen, Slade O.

2014-01-01

Objectives To obtain an expanded understanding of antibiotic resistance evolution in vivo, particularly in the context of vancomycin exposure. Methods The whole genomes of six consecutive methicillin-resistant Staphylococcus aureus blood culture isolates (ST239-MRSA-III) from a single patient exposed to various antimicrobials (over a 77 day period) were sequenced and analysed. Results Variant analysis revealed the existence of non-susceptible sub-populations derived from a common susceptible ancestor, with the predominant circulating clone(s) selected for by type and duration of antimicrobial exposure. Conclusions This study highlights the dynamic nature of bacterial evolution and that non-susceptible sub-populations can emerge from clouds of variation upon antimicrobial exposure. Diagnostically, this has direct implications for sample selection when using whole-genome sequencing as a tool to guide clinical therapy. In the context of bacteraemia, deep sequencing of bacterial DNA directly from patient blood samples would avoid culture ‘bias’ and identify mutations associated with circulating non-susceptible sub-populations, some of which may confer cross-resistance to alternate therapies. PMID:24047554

In vivo evolution of antimicrobial resistance in a series of Staphylococcus aureus patient isolates: the entire picture or a cautionary tale?

PubMed

van Hal, Sebastiaan J; Steen, Jason A; Espedido, Björn A; Grimmond, Sean M; Cooper, Matthew A; Holden, Matthew T G; Bentley, Stephen D; Gosbell, Iain B; Jensen, Slade O

2014-02-01

To obtain an expanded understanding of antibiotic resistance evolution in vivo, particularly in the context of vancomycin exposure. The whole genomes of six consecutive methicillin-resistant Staphylococcus aureus blood culture isolates (ST239-MRSA-III) from a single patient exposed to various antimicrobials (over a 77 day period) were sequenced and analysed. Variant analysis revealed the existence of non-susceptible sub-populations derived from a common susceptible ancestor, with the predominant circulating clone(s) selected for by type and duration of antimicrobial exposure. This study highlights the dynamic nature of bacterial evolution and that non-susceptible sub-populations can emerge from clouds of variation upon antimicrobial exposure. Diagnostically, this has direct implications for sample selection when using whole-genome sequencing as a tool to guide clinical therapy. In the context of bacteraemia, deep sequencing of bacterial DNA directly from patient blood samples would avoid culture 'bias' and identify mutations associated with circulating non-susceptible sub-populations, some of which may confer cross-resistance to alternate therapies.

Evaluation of microbial community in hydrothermal field by direct DNA sequencing

NASA Astrophysics Data System (ADS)

Kawarabayasi, Y.; Maruyama, A.

2002-12-01

Many extremophiles have been discovered from terrestrial and marine hydrothermal fields. Some thermophiles can grow beyond 90°C in culture, while direct microscopic analysis occasionally indicates that microbes may survive in much hotter hydrothermal fluids. However, it is very difficult to isolate and cultivate such microbes from the environments, i.e., over 99% of total microbes remains undiscovered. Based on experiences of entire microbial genome analysis (Y.K.) and microbial community analysis (A.M.), we started to find out unique microbes/genes in hydrothermal fields through direct sequencing of environmental DNA fragments. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected by an in situ filtration system from low-temperature fluids at RM24 in the Southern East Pacific Rise (S-EPR). A gene amplification (PCR) technique was not used for preventing mutation in the process. The nucleotide sequences of 285 clones indicated that no sequence had identical data in public databases. Among 27 clones determined entire sequences, no ORF was identified on 14 clones like intron in Eukaryote. On four clones, tetra-nucleotide-long multiple tandem repetitive sequences were identified. This type of sequence was identified in some familiar disease in human. The result indicates that living/dead materials with eukaryotic features may exist in this low temperature field. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. In randomly-selected 143 clones used for sequencing, no known sequence was identified. Unlike the clones in S-EPR library, clear ORFs were identified on all nine clones determined the entire sequence. It was found that one clone, H4052, contained the complete Aspartyl-tRNA synthetase. Phylogenetic analysis using amino acid sequences of this gene indicated that this gene was separated from other Euryarchaea before the differentiation of species. Thus, some novel archaeal species are expected to be in this field. The present direct cloning and sequencing technique is now opening a window to the new world in hydrothermal microbial community analysis.

PCR-DGGE assessment of the bacterial diversity of breast milk in women with lactational infectious mastitis

PubMed Central

Delgado, Susana; Arroyo, Rebeca; Martín, Rocío; Rodríguez, Juan M

2008-01-01

Background Infectious mastitis is a common condition during lactation and in fact, represents one of the main causes leading to a precocious weaning. The number of studies dealing with lactational mastitis is low and, up to now, the etiological diagnosis is frequently made on the basis of unspecific clinical signs. The aim of this study was to investigate the microbial diversity of breast milk in 20 women with lactational mastitis employing culture-dependent and culture-independent (PCR-DGGE) approaches. Methods Breast milk samples were cultured in different media to investigate the presence of bacteria and/or yeasts, and a total of 149 representative isolates were identified to the species level by 16S rRNA gene PCR sequencing. The microorganisms recovered were compared with those found by PCR-DGGE analysis. To identify the DGGE profiles two reference markers of different microbial species were constructed. Sequence analysis of unknown bands was also performed. Results Staphylococci were the dominant bacterial group and Staphylococcus epidermidis was the dominant species. In a lower number of samples, other bacteria (mainly streptococci and a few gram-negative species) were also identified. Globally, PCR-DGGE results showed a good correlation with those obtained by culture-based methods. However, although DNA bands corresponding to different lactic acid bacteria were detected, such bacteria could not be isolated from the milk samples. Conclusion Staphylococci seem to be the main etiological agents of human lactational mastitis. The combined use of culture and molecular techniques allowed a better characterization of the bacterial diversity in milk from women suffering from infectious mastitis. Our results suggest that this condition could be the result of a disbiotic process where some of the bacterial species usually present in human milk outgrow (staphylococci) while others disappear (lactobacilli or lactococci). PMID:18423017

PCR-DGGE assessment of the bacterial diversity of breast milk in women with lactational infectious mastitis.

PubMed

Delgado, Susana; Arroyo, Rebeca; Martín, Rocío; Rodríguez, Juan M

2008-04-18

Infectious mastitis is a common condition during lactation and in fact, represents one of the main causes leading to a precocious weaning. The number of studies dealing with lactational mastitis is low and, up to now, the etiological diagnosis is frequently made on the basis of unspecific clinical signs. The aim of this study was to investigate the microbial diversity of breast milk in 20 women with lactational mastitis employing culture-dependent and culture-independent (PCR-DGGE) approaches. Breast milk samples were cultured in different media to investigate the presence of bacteria and/or yeasts, and a total of 149 representative isolates were identified to the species level by 16S rRNA gene PCR sequencing. The microorganisms recovered were compared with those found by PCR-DGGE analysis. To identify the DGGE profiles two reference markers of different microbial species were constructed. Sequence analysis of unknown bands was also performed. Staphylococci were the dominant bacterial group and Staphylococcus epidermidis was the dominant species. In a lower number of samples, other bacteria (mainly streptococci and a few gram-negative species) were also identified. Globally, PCR-DGGE results showed a good correlation with those obtained by culture-based methods. However, although DNA bands corresponding to different lactic acid bacteria were detected, such bacteria could not be isolated from the milk samples. Staphylococci seem to be the main etiological agents of human lactational mastitis. The combined use of culture and molecular techniques allowed a better characterization of the bacterial diversity in milk from women suffering from infectious mastitis. Our results suggest that this condition could be the result of a disbiotic process where some of the bacterial species usually present in human milk outgrow (staphylococci) while others disappear (lactobacilli or lactococci).

From cultured to uncultured genome sequences: metagenomics and modeling microbial ecosystems.

PubMed

Garza, Daniel R; Dutilh, Bas E

2015-11-01

Microorganisms and the viruses that infect them are the most numerous biological entities on Earth and enclose its greatest biodiversity and genetic reservoir. With strength in their numbers, these microscopic organisms are major players in the cycles of energy and matter that sustain all life. Scientists have only scratched the surface of this vast microbial world through culture-dependent methods. Recent developments in generating metagenomes, large random samples of nucleic acid sequences isolated directly from the environment, are providing comprehensive portraits of the composition, structure, and functioning of microbial communities. Moreover, advances in metagenomic analysis have created the possibility of obtaining complete or nearly complete genome sequences from uncultured microorganisms, providing important means to study their biology, ecology, and evolution. Here we review some of the recent developments in the field of metagenomics, focusing on the discovery of genetic novelty and on methods for obtaining uncultured genome sequences, including through the recycling of previously published datasets. Moreover we discuss how metagenomics has become a core scientific tool to characterize eco-evolutionary patterns of microbial ecosystems, thus allowing us to simultaneously discover new microbes and study their natural communities. We conclude by discussing general guidelines and challenges for modeling the interactions between uncultured microorganisms and viruses based on the information contained in their genome sequences. These models will significantly advance our understanding of the functioning of microbial ecosystems and the roles of microbes in the environment.

Generation and analysis of expressed sequence tags from the bone marrow of Chinese Sika deer.

PubMed

Yao, Baojin; Zhao, Yu; Zhang, Mei; Li, Juan

2012-03-01

Sika deer is one of the best-known and highly valued animals of China. Despite its economic, cultural, and biological importance, there has not been a large-scale sequencing project for Sika deer to date. With the ultimate goal of sequencing the complete genome of this organism, we first established a bone marrow cDNA library for Sika deer and generated a total of 2,025 reads. After processing the sequences, 2,017 high-quality expressed sequence tags (ESTs) were obtained. These ESTs were assembled into 1,157 unigenes, including 238 contigs and 919 singletons. Comparative analyses indicated that 888 (76.75%) of the unigenes had significant matches to sequences in the non-redundant protein database, In addition to highly expressed genes, such as stearoyl-CoA desaturase, cytochrome c oxidase, adipocyte-type fatty acid-binding protein, adiponectin and thymosin beta-4, we also obtained vascular endothelial growth factor-A and heparin-binding growth-associated molecule, both of which are of great importance for angiogenesis research. There were 244 (21.09%) unigenes with no significant match to any sequence in current protein or nucleotide databases, and these sequences may represent genes with unknown function in Sika deer. Open reading frame analysis of the sequences was performed using the getorf program. In addition, the sequences were functionally classified using the gene ontology hierarchy, clusters of orthologous groups of proteins and Kyoto encyclopedia of genes and genomes databases. Analysis of ESTs described in this paper provides an important resource for the transcriptome exploration of Sika deer, and will also facilitate further studies on functional genomics, gene discovery and genome annotation of Sika deer.

Quantitative mutant analysis of viral quasispecies by chip-based matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

PubMed Central

Amexis, Georgios; Oeth, Paul; Abel, Kenneth; Ivshina, Anna; Pelloquin, Francois; Cantor, Charles R.; Braun, Andreas; Chumakov, Konstantin

2001-01-01

RNA viruses exist as quasispecies, heterogeneous and dynamic mixtures of mutants having one or more consensus sequences. An adequate description of the genomic structure of such viral populations must include the consensus sequence(s) plus a quantitative assessment of sequence heterogeneities. For example, in quality control of live attenuated viral vaccines, the presence of even small quantities of mutants or revertants may indicate incomplete or unstable attenuation that may influence vaccine safety. Previously, we demonstrated the monitoring of oral poliovirus vaccine with the use of mutant analysis by PCR and restriction enzyme cleavage (MAPREC). In this report, we investigate genetic variation in live attenuated mumps virus vaccine by using both MAPREC and a platform (DNA MassArray) based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Mumps vaccines prepared from the Jeryl Lynn strain typically contain at least two distinct viral substrains, JL1 and JL2, which have been characterized by full length sequencing. We report the development of assays for characterizing sequence variants in these substrains and demonstrate their use in quantitative analysis of substrains and sequence variations in mixed virus cultures and mumps vaccines. The results obtained from both the MAPREC and MALDI-TOF methods showed excellent correlation. This suggests the potential utility of MALDI-TOF for routine quality control of live viral vaccines and for assessment of genetic stability and quantitative monitoring of genetic changes in other RNA viruses of clinical interest. PMID:11593021

Real-Time Culture Change Improves Lean Success: Sequenced Culture Change Gets Failing Grades.

PubMed

Kusy, Mitchell; Diamond, Marty; Vrchota, Scott

2015-01-01

Success with the Lean management system is rooted in a culture of stakeholder engagement and commitment. Unfortunately, many leaders view Lean as an "add-on" tool instead of one that requires a new way of thinking and approaching culture. This article addresses the "why, how, and what" to promote a Lean culture that works. We present a five-phased approach grounded in evidence-based practices of real-time culture change. We further help healthcare leaders understand the differences between traditional "sequenced" approaches to culture change and "real-time" methods--and why these real-time practices are more sustainable and ultimately more successful than traditional culture change methods.

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, David B.; Lao, Guifang

1998-01-01

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

Baseline Survey of Root-Associated Microbes of Taxus chinensis (Pilger) Rehd

PubMed Central

Sun, Guiling; Wilson, Iain W.; Wu, Jianqiang; Hoffman, Angela; Cheng, Junwen; Qiu, Deyou

2015-01-01

Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis. PMID:25821956

Baseline survey of root-associated microbes of Taxus chinensis (Pilger) Rehd.

PubMed

Zhang, Qian; Liu, Hongwei; Sun, Guiling; Wilson, Iain W; Wu, Jianqiang; Hoffman, Angela; Cheng, Junwen; Qiu, Deyou

2015-01-01

Taxol (paclitaxel) a diterpenoid is one of the most effective anticancer drugs identified. Biosynthesis of taxol was considered restricted to the Taxus genera until Stierle et al. discovered that an endophytic fungus isolated from Taxus brevifolia could independently synthesize taxol. Little is known about the mechanism of taxol biosynthesis in microbes, but it has been speculated that its biosynthesis may differ from plants. The microbiome from the roots of Taxus chinensis have been extensively investigated with culture-dependent methods to identify taxol synthesizing microbes, but not using culture independent methods.,Using bar-coded high-throughput sequencing in combination with a metagenomics approach, we surveyed the microbial diversity and gene composition of the root-associated microbiomefrom Taxus chinensis (Pilger) Rehd. High-throughput amplicon sequencing revealed 187 fungal OTUs which is higher than any previously reported fungal number identified with the culture-dependent method, suggesting that T. chinensis roots harbor novel and diverse fungi. Some operational taxonomic units (OTU) identified were identical to reported microbe strains possessing the ability to synthesis taxol and several genes previously associated with taxol biosynthesis were identified through metagenomics analysis.

Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

PubMed

Martínez-Herrero, M C; Garijo-Toledo, M M; Liebhart, D; Ganas, P; Martínez-Díaz, R A; Ponce-Gordo, F; Carrero-Ruiz, A; Hess, M; Gómez-Muñoz, M T

2017-11-01

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur. Copyright © 2017 Elsevier B.V. All rights reserved.

Making the Leap from Research Laboratory to Clinic: Challenges and Opportunities for Next-Generation Sequencing in Infectious Disease Diagnostics

PubMed Central

Goldberg, Brittany; Sichtig, Heike; Geyer, Chelsie; Ledeboer, Nathan

2015-01-01

ABSTRACT Next-generation DNA sequencing (NGS) has progressed enormously over the past decade, transforming genomic analysis and opening up many new opportunities for applications in clinical microbiology laboratories. The impact of NGS on microbiology has been revolutionary, with new microbial genomic sequences being generated daily, leading to the development of large databases of genomes and gene sequences. The ability to analyze microbial communities without culturing organisms has created the ever-growing field of metagenomics and microbiome analysis and has generated significant new insights into the relation between host and microbe. The medical literature contains many examples of how this new technology can be used for infectious disease diagnostics and pathogen analysis. The implementation of NGS in medical practice has been a slow process due to various challenges such as clinical trials, lack of applicable regulatory guidelines, and the adaptation of the technology to the clinical environment. In April 2015, the American Academy of Microbiology (AAM) convened a colloquium to begin to define these issues, and in this document, we present some of the concepts that were generated from these discussions. PMID:26646014

Molecular analysis of microbial community in a groundwater sample polluted by landfill leachate and seawater*

PubMed Central

Tian, Yang-jie; Yang, Hong; Wu, Xiu-juan; Li, Dao-tang

2005-01-01

Seashore landfill aquifers are environments of special physicochemical conditions (high organic load and high salinity), and microbes in leachate-polluted aquifers play a significant role for intrinsic bioremediation. In order to characterize microbial diversity and look for clues on the relationship between microbial community structure and hydrochemistry, a culture-independent examination of a typical groundwater sample obtained from a seashore landfill was conducted by sequence analysis of 16S rDNA clone library. Two sets of universal 16S rDNA primers were used to amplify DNA extracted from the groundwater so that problems arising from primer efficiency and specificity could be reduced. Of 74 clones randomly selected from the libraries, 30 contained unique sequences whose analysis showed that the majority of them belonged to bacteria (95.9%), with Proteobacteria (63.5%) being the dominant division. One archaeal sequence and one eukaryotic sequence were found as well. Bacterial sequences belonging to the following phylogenic groups were identified: Bacteroidetes (20.3%), β, γ, δ and ε-subdivisions of Proteobacteria (47.3%, 9.5%, 5.4% and 1.3%, respectively), Firmicutes (1.4%), Actinobacteria (2.7%), Cyanobacteria (2.7%). The percentages of Proteobacteria and Bacteroides in seawater were greater than those in the groundwater from a non-seashore landfill, indicating a possible influence of seawater. Quite a few sequences had close relatives in marine or hypersaline environments. Many sequences showed affiliations with microbes involved in anaerobic fermentation. The remarkable abundance of sequences related to (per)chlorate-reducing bacteria (ClRB) in the groundwater was significant and worthy of further study. PMID:15682499

Metatranscriptome of an Anaerobic Benzene-Degrading, Nitrate-Reducing Enrichment Culture Reveals Involvement of Carboxylation in Benzene Ring Activation

PubMed Central

Luo, Fei; Gitiafroz, Roya; Devine, Cheryl E.; Gong, Yunchen; Hug, Laura A.; Raskin, Lutgarde

2014-01-01

The enzymes involved in the initial steps of anaerobic benzene catabolism are not known. To try to elucidate this critical step, a metatranscriptomic analysis was conducted to compare the genes transcribed during the metabolism of benzene and benzoate by an anaerobic benzene-degrading, nitrate-reducing enrichment culture. RNA was extracted from the mixed culture and sequenced without prior mRNA enrichment, allowing simultaneous examination of the active community composition and the differential gene expression between the two treatments. Ribosomal and mRNA sequences attributed to a member of the family Peptococcaceae from the order Clostridiales were essentially only detected in the benzene-amended culture samples, implicating this group in the initial catabolism of benzene. Genes similar to each of two subunits of a proposed benzene-carboxylating enzyme were transcribed when the culture was amended with benzene. Anaerobic benzoate degradation genes from strict anaerobes were transcribed only when the culture was amended with benzene. Genes for other benzoate catabolic enzymes and for nitrate respiration were transcribed in both samples, with those attributed to an Azoarcus species being most abundant. These findings indicate that the mineralization of benzene starts with its activation by a strict anaerobe belonging to the Peptococcaceae, involving a carboxylation step to form benzoate. These data confirm the previously hypothesized syntrophic association between a benzene-degrading Peptococcaceae strain and a benzoate-degrading denitrifying Azoarcus strain for the complete catabolism of benzene with nitrate as the terminal electron acceptor. PMID:24795366

Microbial Ecology of the Hive and Pollination Landscape: Bacterial Associates from Floral Nectar, the Alimentary Tract and Stored Food of Honey Bees (Apis mellifera)

PubMed Central

Mott, Brendon M.; Maes, Patrick; Snyder, Lucy; Schwan, Melissa R.; Walton, Alexander; Jones, Beryl M.; Corby-Harris, Vanessa

2013-01-01

Nearly all eukaryotes are host to beneficial or benign bacteria in their gut lumen, either vertically inherited, or acquired from the environment. While bacteria core to the honey bee gut are becoming evident, the influence of the hive and pollination environment on honey bee microbial health is largely unexplored. Here we compare bacteria from floral nectar in the immediate pollination environment, different segments of the honey bee (Apis mellifera) alimentary tract, and food stored in the hive (honey and packed pollen or “beebread”). We used cultivation and sequencing to explore bacterial communities in all sample types, coupled with culture-independent analysis of beebread. We compare our results from the alimentary tract with both culture-dependent and culture-independent analyses from previous studies. Culturing the foregut (crop), midgut and hindgut with standard media produced many identical or highly similar 16S rDNA sequences found with 16S rDNA clone libraries and next generation sequencing of 16S rDNA amplicons. Despite extensive culturing with identical media, our results do not support the core crop bacterial community hypothesized by recent studies. We cultured a wide variety of bacterial strains from 6 of 7 phylogenetic groups considered core to the honey bee hindgut. Our results reveal that many bacteria prevalent in beebread and the crop are also found in floral nectar, suggesting frequent horizontal transmission. From beebread we uncovered a variety of bacterial phylotypes, including many possible pathogens and food spoilage organisms, and potentially beneficial bacteria including Lactobacillus kunkeei, Acetobacteraceae and many different groups of Actinobacteria. Contributions of these bacteria to colony health may include general hygiene, fungal and pathogen inhibition and beebread preservation. Our results are important for understanding the contribution to pollinator health of both environmentally vectored and core microbiota, and the identification of factors that may affect bacterial detection and transmission, colony food storage and disease susceptibility. PMID:24358254

Microbial ecology of the hive and pollination landscape: bacterial associates from floral nectar, the alimentary tract and stored food of honey bees (Apis mellifera).

PubMed

Anderson, Kirk E; Sheehan, Timothy H; Mott, Brendon M; Maes, Patrick; Snyder, Lucy; Schwan, Melissa R; Walton, Alexander; Jones, Beryl M; Corby-Harris, Vanessa

2013-01-01

Nearly all eukaryotes are host to beneficial or benign bacteria in their gut lumen, either vertically inherited, or acquired from the environment. While bacteria core to the honey bee gut are becoming evident, the influence of the hive and pollination environment on honey bee microbial health is largely unexplored. Here we compare bacteria from floral nectar in the immediate pollination environment, different segments of the honey bee (Apis mellifera) alimentary tract, and food stored in the hive (honey and packed pollen or "beebread"). We used cultivation and sequencing to explore bacterial communities in all sample types, coupled with culture-independent analysis of beebread. We compare our results from the alimentary tract with both culture-dependent and culture-independent analyses from previous studies. Culturing the foregut (crop), midgut and hindgut with standard media produced many identical or highly similar 16S rDNA sequences found with 16S rDNA clone libraries and next generation sequencing of 16S rDNA amplicons. Despite extensive culturing with identical media, our results do not support the core crop bacterial community hypothesized by recent studies. We cultured a wide variety of bacterial strains from 6 of 7 phylogenetic groups considered core to the honey bee hindgut. Our results reveal that many bacteria prevalent in beebread and the crop are also found in floral nectar, suggesting frequent horizontal transmission. From beebread we uncovered a variety of bacterial phylotypes, including many possible pathogens and food spoilage organisms, and potentially beneficial bacteria including Lactobacillus kunkeei, Acetobacteraceae and many different groups of Actinobacteria. Contributions of these bacteria to colony health may include general hygiene, fungal and pathogen inhibition and beebread preservation. Our results are important for understanding the contribution to pollinator health of both environmentally vectored and core microbiota, and the identification of factors that may affect bacterial detection and transmission, colony food storage and disease susceptibility.

Cyanobacterial diversity in extreme environments in Baja California, Mexico: a polyphasic study.

PubMed

López-Cortés, A; García-Pichel, F; Nübel, U; Vázquez-Juárez, R

2001-12-01

Cyanobacterial diversity from two geographical areas of Baja California Sur, Mexico, were studied: Bahia Concepcion, and Ensenada de Aripez. The sites included hypersaline ecosystems, sea bottom, hydrothermal springs, and a shrimp farm. In this report we describe four new morphotypes, two are marine epilithic from Bahia Concepcion, Dermocarpa sp. and Hyella sp. The third, Geitlerinema sp., occurs in thermal springs and in shrimp ponds, and the fourth, Tychonema sp., is from a shrimp pond. The partial sequences of the 16S rRNA genes and the phylogenetic relationship of four cyanobacterial strains (Synechococcus cf. elongatus, Leptolyngbya cf. thermalis, Leptolyngbya sp., and Geitlerinema sp.) are also presented. Polyphasic studies that include the combination of light microscopy, cultures and the comparative analysis of 16S rRNA gene sequences provide the most powerful approach currently available to establish the diversity of these oxygenic photosynthetic microorganisms in culture and in nature.

Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31.

PubMed

Yin, Yajuan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2014-10-01

The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

Diversity and Distribution of Phenol Oxidase Producing Fungi from Soda Lake and Description of Curvularia lonarensis sp. nov.

PubMed

Sharma, Rahul; Prakash, Om; Sonawane, Mahesh S; Nimonkar, Yogesh; Golellu, Priyanka B; Sharma, Rohit

2016-01-01

Soda lake is hyper alkaline and saline habitat located in closed craters with high evaporation rate. In current study fungal diversity from water and sediment samples of a soda lake (Lonar lake) located in Buldhana district of Maharashtra, India was investigated using extensive culturomics approach and mimicking the natural conditions of Lonar lake in culture media. A total of 104 diverse isolates of extremophilic fungi were recovered from this study and phylogenetically characterized by internal transcribed spacer (ITS) region sequencing. In addition, due to important role of phenol oxidase, and peroxidase in degradation of toxic phenol, lignin, etc., all isolated pure cultures were also screened for extracellular phenol oxidase and peroxidase production potential. Diversity analysis indicated that different groups of extremophilic fungi are present in the water and sediment samples of Lonar lake. A total of 38 species of fungi belonging to 18-different genera were recovered. Out of 104 isolates 32 showed ≤97% sequences similarity, which were morphologically different and could be potential novel isolates of extremophilic fungi. However, out of 104 isolates only 14 showed the extracellular phenol oxidase production potentials at alkaline pH. Curvularia sp. strain MEF018 showed highest phenol oxidase production at alkaline condition and had low sequence similarity with previously characterized species (96% with Curvularia pseudorobusta ). Taxonomic characterization (morphological and physiological) and multi locus sequence analysis (MLSA) using combined alignment of ITS-LSU- gpd of strain MEF018 showed that it is a novel species of the genus Curvularia and hence proposed as Curvularia lonarensis sp. nov.

Diversity and Distribution of Phenol Oxidase Producing Fungi from Soda Lake and Description of Curvularia lonarensis sp. nov.

PubMed Central

Sharma, Rahul; Prakash, Om; Sonawane, Mahesh S.; Nimonkar, Yogesh; Golellu, Priyanka B.; Sharma, Rohit

2016-01-01

Soda lake is hyper alkaline and saline habitat located in closed craters with high evaporation rate. In current study fungal diversity from water and sediment samples of a soda lake (Lonar lake) located in Buldhana district of Maharashtra, India was investigated using extensive culturomics approach and mimicking the natural conditions of Lonar lake in culture media. A total of 104 diverse isolates of extremophilic fungi were recovered from this study and phylogenetically characterized by internal transcribed spacer (ITS) region sequencing. In addition, due to important role of phenol oxidase, and peroxidase in degradation of toxic phenol, lignin, etc., all isolated pure cultures were also screened for extracellular phenol oxidase and peroxidase production potential. Diversity analysis indicated that different groups of extremophilic fungi are present in the water and sediment samples of Lonar lake. A total of 38 species of fungi belonging to 18-different genera were recovered. Out of 104 isolates 32 showed ≤97% sequences similarity, which were morphologically different and could be potential novel isolates of extremophilic fungi. However, out of 104 isolates only 14 showed the extracellular phenol oxidase production potentials at alkaline pH. Curvularia sp. strain MEF018 showed highest phenol oxidase production at alkaline condition and had low sequence similarity with previously characterized species (96% with Curvularia pseudorobusta). Taxonomic characterization (morphological and physiological) and multi locus sequence analysis (MLSA) using combined alignment of ITS-LSU-gpd of strain MEF018 showed that it is a novel species of the genus Curvularia and hence proposed as Curvularia lonarensis sp. nov. PMID:27920761

Rhodotorula svalbardensis sp. nov., a novel yeast species isolated from cryoconite holes of Ny-Ålesund, Arctic.

PubMed

Singh, Purnima; Singh, Shiv M; Tsuji, Masaharu; Prasad, Gandham S; Hoshino, Tamotsu

2014-02-01

A psychrophilic yeast species was isolated from glacier cryoconite holes of Svalbard. Nucleotide sequences of the strains were studied using D1/D2 domain, ITS region and partial sequences of mitochondrial cytochrome b gene. The strains belonged to a clade of psychrophilic yeasts, but showed marked differences from related species in the D1/D2 domain and biochemical characters. Effects of temperature, salt and media on growth of the cultures were also studied. Screening of the cultures for amylase, cellulase, protease, lipase, urease and catalase activities was carried out. The strains expressed high amylase and lipase activities. Freeze tolerance ability of the isolates indicated the formation of unique hexagonal ice crystal structures due to presence of 'antifreeze proteins' (AFPs). FAME analysis of cultures showed a unique trend of increase in unsaturated fatty acids with decrease in temperature. The major fatty acids recorded were oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, myristic acid and pentadecanoic acid. Based on sequence data and, physiological and morphological properties of the strains, we propose a novel species, Rhodotorula svalbardensis and designate strains MLB-I (CCP-II) and CRY-YB-1 (CBS 12863, JCM 19699, JCM 19700, MTCC 10952) as its type strains (Etymology: sval.bar.den'sis. N.L. fem. adj. svalbardensis pertaining to Svalbard). Copyright © 2014 Elsevier Inc. All rights reserved.

Phylogenentic and enzymatic characterization of psychrophilic and psychrotolerant marine bacteria belong to γ-Proteobacteria group isolated from the sub-Antarctic Beagle Channel, Argentina.

PubMed

Cristóbal, Héctor A; Benito, Juliana; Lovrich, Gustavo A; Abate, Carlos M

2015-05-01

The phylogenetic and physiological characteristics of cultivable-dependent approaches were determined to establish the diversity of marine bacteria associated with the intestines of benthonic organisms and seawater samples from the Argentina's Beagle Channel. A total of 737 isolates were classified as psychrophlic and psychrotolerant culturable marine bacteria. These cold-adapted microorganisms are capable of producing cold-active glycosyl hydrolases, such as β-glucosidases, celulases, β-galactosidases, xylanases, chitinases, and proteases. These enzymes could have potential biotechnological applications for use in low-temperature manufacturing processes. According to polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S ribosomal DNA (ARDRA) and DNA gyrase subunit B (gyrB-RFLP), 11 operational taxonomic units (OTU) were identified and clustered in known genera using InfoStat software. The 50 isolates selected were sequenced based on near full sequence analysis of 16S rDNA and gyrB sequences and identified by their nearest neighbors ranging between 96 and 99 % of identities. Phylogenetic analyses using both genes allowed relationships between members of the cultured marine bacteria belonging to the γ-Proteobacteria group (Aeromonas, Halteromonas, Pseudomonas, Pseudoalteromonas, Shewanella, Serratia, Colwellia, Glacielocola, and Psychrobacter) to be evaluated. Our research reveals a high diversity of hydrolytic bacteria, and their products actuality has an industrial use in several bioprocesses at low-temperature manufacturing.

Natural and cultured populations of the mangrove oyster Saccostrea palmula from Sinaloa, Mexico, infected by Perkinsus marinus.

PubMed

Cáceres-Martínez, Jorge; Ortega, Mauricio García; Vásquez-Yeomans, Rebeca; García, Teresa de Jesús Pineda; Stokes, Nancy A; Carnegie, Ryan B

2012-07-01

The mangrove oyster Saccostrea palmula coexists with the pleasure oyster Crassostrea corteziensis in coastal lagoons of northwest Mexico. Recent discovery of Perkinsus marinus infecting the pleasure oyster in the region prompted evaluation of S. palmula as an alternative P. marinus host. An analysis to determine the possible presence of P. marinus in natural and cultured populations of S. palmula at four coastal lagoons in Sinaloa, Mexico was carried out during October-November 2010. Tissues from apparently healthy S. palmula were evaluated using Ray's fluid thioglycollate method (RFTM), which revealed a Perkinsus sp. to be present in all four locations at 6.7-20.0% prevalence. Histopathological analysis of these specimens showed tissue alterations and parasite forms consistent with moderate P. marinus infection, which was confirmed by ribosomal non-transcribed spacer (NTS)-based PCR assays on DNA samples from oysters positive by RFTM and histology. DNA sequencing of amplified NTS fragments (307 bp) produced a sequence 98-100% similar to GenBank-deposited sequences of the NTS from P. marinus. Fluorescent in situ hybridization for Perkinsus spp. and P. marinus corroborated the PCR results, showing clear hybridization of P. marinus in host tissues. This is the first record of P. marinus infecting a species from genus Saccostrea and the first record of the parasite from coastal lagoons in Sinaloa, Mexico. Copyright © 2012 Elsevier Inc. 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.

The assessment of epiphytic yeast diversity in sugarcane phyllosphere in Thailand by culture-independent method.

PubMed

Nasanit, Rujikan; Tangwong-O-Thai, Apirat; Tantirungkij, Manee; Limtong, Savitree

2015-12-01

The diversity of epiphytic yeasts from sugarcane (Saccharum officinarum Linn.) phyllospheres in Thailand was investigated by culture-independent method based on the analysis of the D1/D2 domains of the large subunit rRNA gene sequences. Forty-five samples of sugarcane leaf were collected randomly from ten provinces in Thailand. A total of 1342 clones were obtained from 45 clone libraries. 426 clones (31.7 %) were closely related to yeast strains in the GenBank database, and they were clustered into 31 operational taxonomic units (OTUs) with a similarity threshold of 99 %. All OTU sequences were classified in phylum Basidiomycota which were closely related to 11 yeast species in seven genera including Cryptococcus flavus, Hannaella coprosmaensis, Rhodotorula taiwanensis, Jaminaea angkoreiensis, Malassezia restricta, Pseudozyma antarctica, Pseudozyma aphidis, Pseudozyma hubeiensis, Pseudozyma prolifica, Pseudozyma shanxiensis, and Sporobolomyces vermiculatus. The most predominant yeasts detected belonged to Ustilaginales with 89.4 % relative frequency and the prevalent yeast genus was Pseudozyma. However, the majority were unable to be identified as known yeast species and these sequences may represent the sequences of new yeast taxa. In addition, The OTU that closely related to P. prolifica was commonly detected in sugarcane phyllosphere. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Determination of lactic microflora of kefir grains and kefir beverage by using culture-dependent and culture-independent methods.

PubMed

Kesmen, Zülal; Kacmaz, Nazife

2011-01-01

In this study, we investigated the bacterial compositions of kefir grains and kefir beverages collected from different regions of Turkey by using culture-independent and culture-dependent methods. In the culture-independent detection, 10 different species of bacteria were detected in total by using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the 16S rRNA gene V3 region. Among these species, Lactobacillus kefiranofaciens was the most dominant one in the kefir grains, while Lactococcus lactis was found to be significantly prevalent in the kefir beverages. In the culture-dependent detection, the primary differentiation and grouping of the isolates from kefir beverages and kefir grains were performed using repetitive sequence-based PCR (rep-PCR) fingerprinting, and the results were validated by 16S rDNA full-length sequencing. According to the results of culture-dependent methods, the most frequently isolated species were L. lactis, Leuconostoc mesenteroides, and Lactobacillus kefiri, respectively. Only 3 species, which are L. lactis, Lactobacillus acidophilus, and Streptococcus thermophilus, were detected with both culture-dependent and culture-independent methods. This study showed that the combination of both methods is necessary for a detailed and reliable investigation of microbial communities in kefir grains and kefir beverages. Due to their artisan- and region-dependent microflora, kefir products can be a source of interesting lactic acid bacteria, either new taxa or strains with specific functional properties, which might be used for the development of new starter cultures and innovative food products. Therefore, an increasing demand exists for new strains that show desirable effects on the product characteristics Artisan dairy products are a candidate source of such microorganisms. For this reason, in this study, the bacterial compositions of kefir grains and kefir beverages obtained from different regions of Turkey were studied using culture-dependent and culture-independent molecular methods. © 2011 Institute of Food Technologists®

Nucleotide sequences encoding a thermostable alkaline protease

DOEpatents

Wilson, D.B.; Lao, G.

1998-01-06

Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

Study of the Microbial Diversity of a Newly Discovered East Antarctic Freshwater Lake, L27C, and of a Perennially Ice-Covered Lake Untersee

NASA Technical Reports Server (NTRS)

Huang, Jonathan P.; Hoover, Richard B.; Andersen, Dale; Bej, Asim K.

2010-01-01

The microbial communities that reside within freshwater lakes of Schirmacher and Untersee Oases in East Antarctica must cope with extreme conditions that may include cold temperature, annual freeze-thaw cycles, exposure to UV radiation, especially during the austral summer months, low light beneath thick ice-cover, followed by seasonal darkness. The objective of this study was to assess the microbial biodiversity and distribution from samples taken from two freshwater lakes (L27C and Lake Untersee) that were collected during the Tawani 2008 International Antarctic Expedition that conducted research in this region of Antarctica. L27C is a small, previously unreported lake residing 2 km WNW of Maitri Station at Schirmacher Oasis. Biodiversity and distribution of microorganisms within the lake were studied using both culture-independent and culture-dependent methodologies based upon the analysis of eubacterial 16S rRNA gene sequences. Lake Untersee, a perennially ice-covered, ultra-oligotrophic, lake in the Otto-von-Gruber-Gebirge (Gruber Mountains) of central Dronning Maud Land was also sampled and the microbial diversity was analyzed by eubacterial 16S rRNA gene sequences derived from pure cultures. Direct culturing of water samples from each lake on separate R2A growth medium exhibited a variety of microorganisms including: Janthinobacterium, Hymenobacter, Sphingamonas, Subtercola, Deinococcus, Arthrobacter, Flavobacterium, Polaromonas, Rhodoferax and Duganella. The evaluation of samples from L27C through culture-independent methodology identified a rich microbial diversity consisting of six different phyla of bacteria. The culture-independent analysis also displayed the majority of bacteria (56%) belonged to the Class gamma-proteobacteria within the phylum Proteobacteria. Within the Class gamma-proteobacteria, Acinetobacter dominated (48%) the total microbial load. Overall, L27C exhibited 7 different phyla of bacteria and 20 different genera. Statistical analysis (Shannon-Weaver Diversity Index and Simpson Diversity Index) of the biodiversity of L27C displayed a moderately rich and diverse community. Investigations of the biodiversity and distribution of microorganisms in these lakes will help further our understanding of how the physical environment impact the structure and function within these microbially dominated ecosystems.

First identification of Francisella noatunensis subsp. orientalis causing mortality in Mexican tilapia Oreochromis spp.

PubMed

Ortega, Cesar; Mancera, Gerardo; Enríquez, Ricardo; Vargas, Augusto; Martínez, Simón; Fajardo, Raúl; Avendaño-Herrera, Ruben; Navarrete, María José; Romero, Alex

2016-08-09

Francisellosis, an emerging disease in tilapia Oreochromis spp., is caused by the facultative, intracellular bacterium Francisella noatunensis subsp. orientalis, which is present in various countries where tilapia farming is commercially important. We confirmed the presence of francisellosis in Mexican tilapia cultures in association with an outbreak during the second semester of 2012. Broodstock fish presented a mortality rate of approximately 40%, and disease was characterized by histologically classified granulomas, or whitish nodules, in different organs, mainly the spleen and kidney. Through DNA obtained from infected tissue and pure cultures in a cysteine heart medium supplemented with hemoglobin, F. noatunensis subsp. orientalis was initially confirmed through the amplification and analysis of the 16S rRNA gene and the internal transcribed spacer region. Phylogenetic analysis of these genes demonstrated close similarity with previously reported F. noatunensis subsp. orientalis sequences obtained from infected tilapia from various countries. The identification of this subspecies as the causative agent of the outbreak was confirmed using the iglC gene as a target sequence, which showed 99.5% identity to 2 F. noatunensis subsp. orientalis strains (Ethime-1 and Toba04). These findings represent the first documented occurrence of francisellosis in Mexican tilapia cultures, which highlights the importance of establishing preventative measures to minimize the spread of this disease within the Mexican aquaculture industry.

Gene Scanning of an Internalin B Gene Fragment Using High-Resolution Melting Curve Analysis as a Tool for Rapid Typing of Listeria monocytogenes

PubMed Central

Pietzka, Ariane T.; Stöger, Anna; Huhulescu, Steliana; Allerberger, Franz; Ruppitsch, Werner

2011-01-01

The ability to accurately track Listeria monocytogenes strains involved in outbreaks is essential for control and prevention of listeriosis. Because current typing techniques are time-consuming, cost-intensive, technically demanding, and difficult to standardize, we developed a rapid and cost-effective method for typing of L. monocytogenes. In all, 172 clinical L. monocytogenes isolates and 20 isolates from culture collections were typed by high-resolution melting (HRM) curve analysis of a specific locus of the internalin B gene (inlB). All obtained HRM curve profiles were verified by sequence analysis. The 192 tested L. monocytogenes isolates yielded 15 specific HRM curve profiles. Sequence analysis revealed that these 15 HRM curve profiles correspond to 18 distinct inlB sequence types. The HRM curve profiles obtained correlated with the five phylogenetic groups I.1, I.2, II.1, II.2, and III. Thus, HRM curve analysis constitutes an inexpensive assay and represents an improvement in typing relative to classical serotyping or multiplex PCR typing protocols. This method provides a rapid and powerful screening tool for simultaneous preliminary typing of up to 384 samples in approximately 2 hours. PMID:21227395

Pathogenicity, sequence and phylogenetic analysis of Malaysian Chicken anaemia virus obtained after low and high passages in MSB-1 cells.

PubMed

Chowdhury, S M Z H; Omar, A R; Aini, I; Hair-Bejo, M; Jamaluddin, A A; Md-Zain, B M; Kono, Y

2003-12-01

Specific-pathogen-free (SPF) chickens inoculated with low passage Chicken anaemia virus (CAV), SMSC-1 and 3-1 isolates produced lesions suggestive of CAV infection. Repeated passages of the isolates in cell culture until passage 60 (P60) and passage 123 produced viruses that showed a significantly reduced level of pathogenicity in SPF chickens compared to the low passage isolates. Sequence comparison indicated that nucleotide changes in only the coding region of the P60 passage isolates were thought to contribute to virus attenuation. Phylogenetic analysis indicated that SMSC-1 and 3-1 were highly divergent, but their P60 passage derivatives shared significant homology to a Japanese isolate A2.

Clinical identification of bacteria in human chronic wound infections: culturing vs. 16S ribosomal DNA sequencing

PubMed Central

2012-01-01

Background Chronic wounds affect millions of people and cost billions of dollars in the United States each year. These wounds harbor polymicrobial biofilm communities, which can be difficult to elucidate using culturing methods. Clinical molecular microbiological methods are increasingly being employed to investigate the microbiota of chronic infections, including wounds, as part of standard patient care. However, molecular testing is more sensitive than culturing, which results in markedly different results being reported to clinicians. This study compares the results of aerobic culturing and molecular testing (culture-free 16S ribosomal DNA sequencing), and it examines the relative abundance score that is generated by the molecular test and the usefulness of the relative abundance score in predicting the likelihood that the same organism would be detected by culture. Methods Parallel samples from 51 chronic wounds were studied using aerobic culturing and 16S DNA sequencing for the identification of bacteria. Results One hundred forty-five (145) unique genera were identified using molecular methods, and 68 of these genera were aerotolerant. Fourteen (14) unique genera were identified using aerobic culture methods. One-third (31/92) of the cultures were determined to be < 1% of the relative abundance of the wound microbiota using molecular testing. At the genus level, molecular testing identified 85% (78/92) of the bacteria that were identified by culture. Conversely, culturing detected 15.7% (78/497) of the aerotolerant bacteria and detected 54.9% of the collective aerotolerant relative abundance of the samples. Aerotolerant bacterial genera (and individual species including Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) with higher relative abundance scores were more likely to be detected by culture as demonstrated with regression modeling. Conclusion Discordance between molecular and culture testing is often observed. However, culture-free 16S ribosomal DNA sequencing and its relative abundance score can provide clinicians with insight into which bacteria are most abundant in a sample and which are most likely to be detected by culture. PMID:23176603

Screening, Characterization and In Vitro Evaluation of Probiotic Properties Among Lactic Acid Bacteria Through Comparative Analysis.

PubMed

Devi, Sundru Manjulata; Archer, Ann Catherine; Halami, Prakash M

2015-09-01

The present work aimed to identify probiotic bacteria from healthy human infant faecal and dairy samples. Subsequently, an assay was developed to evaluate the probiotic properties using comparative genetic approach for marker genes involved in adhesion to the intestinal epithelial layer. Several in vitro properties including tolerance to biological barriers (such as acid and bile), antimicrobial spectrum, resistance to simulated digestive fluids and cellular hydrophobicity were assessed. The potential probiotic cultures were rapidly characterized by morphological, physiological and molecular-based methods [such as RFLP, ITS, RAPD and (GTG)5]. Further analysis by 16S rDNA sequencing revealed that the selected isolates belong to Lactobacillus, Pediococcus and Enterococcus species. Two cultures of non-lactic, non-pathogenic Staphylococcus spp. were also isolated. The native isolates were able to survive under acidic, bile and simulated intestinal conditions. In addition, these cultures inhibited the growth of tested bacterial pathogens. Further, no correlation was observed between hydrophobicity and adhesion ability. Sequencing of probiotic marker genes such as bile salt hydrolase (bsh), fibronectin-binding protein (fbp) and mucin-binding protein (mub) for selected isolates revealed nucleotide variation. The probiotic binding domains were detected by several bioinformatic tools. The approach used in the study enabled the identification of potential probiotic domains responsible for adhesion of bacteria to intestinal epithelial layer, which may further assist in screening of novel probiotic bacteria. The rapid detection of binding domains will help in revealing the beneficial properties of the probiotic cultures. Further, studies will be performed to develop a novel probiotic product which will contribute in food and feed industry.

Comprehensive Analysis of Secondary Dental Root Canal Infections: A Combination of Culture and Culture-Independent Approaches Reveals New Insights

PubMed Central

Anderson, Annette Carola; Hellwig, Elmar; Vespermann, Robin; Wittmer, Annette; Schmid, Michael; Karygianni, Lamprini; Al-Ahmad, Ali

2012-01-01

Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and –independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis. PMID:23152922

Draft genome sequence of multidrug-resistant Staphylococcus haemolyticus IPK_TSA25 harbouring a Staphylococcus aureus plasmid, pS0385-1.

PubMed

Kim, Hyung Jun; Jang, Soojin

2017-12-01

Staphylococcus haemolyticus is the second most frequently isolated coagulase-negative staphylococci from blood cultures. Moreover, multidrug resistance associated with the genome flexibility of S. haemolyticus has been increasingly reported worldwide. Here we report the draft genome sequence of multidrug-resistant S. haemolyticus IPK_TSA25 isolated from a building surface in South Korea. Genomic DNA of S. haemolyticus IPK_TSA25 was sequenced using the PacBio RS II sequencing platform. Generated reads were assembled using PacBio SMRT Analysis 2.3.0. The draft genome was annotated and antibiotic resistance genes were identified. The genome of 2517398bp contains various antibiotic resistance genes associated with resistance to β-lactams, aminoglycosides and macrolides. Genome analysis also revealed chromosomal integration of the full-length Staphylococcus aureus plasmid pS0385-1 containing a tetracycline resistance gene. The genome sequence reported in this study will provide valuable information to understand the flexibility of the S. haemolyticus genome, which facilitates acquisition of antibiotic resistance genes and contributes to the dissemination of antibiotic resistance by this emerging pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity.

PubMed

Nam, Bo-Hye; Seo, Jung-Kil; Lee, Min Jeong; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Park, Nam Gyu

2015-07-01

An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

PlasFlow: predicting plasmid sequences in metagenomic data using genome signatures

PubMed Central

Lipinski, Leszek; Dziembowski, Andrzej

2018-01-01

Abstract Plasmids are mobile genetics elements that play an important role in the environmental adaptation of microorganisms. Although plasmids are usually analyzed in cultured microorganisms, there is a need for methods that allow for the analysis of pools of plasmids (plasmidomes) in environmental samples. To that end, several molecular biology and bioinformatics methods have been developed; however, they are limited to environments with low diversity and cannot recover large plasmids. Here, we present PlasFlow, a novel tool based on genomic signatures that employs a neural network approach for identification of bacterial plasmid sequences in environmental samples. PlasFlow can recover plasmid sequences from assembled metagenomes without any prior knowledge of the taxonomical or functional composition of samples with an accuracy up to 96%. It can also recover sequences of both circular and linear plasmids and can perform initial taxonomical classification of sequences. Compared to other currently available tools, PlasFlow demonstrated significantly better performance on test datasets. Analysis of two samples from heavy metal-contaminated microbial mats revealed that plasmids may constitute an important fraction of their metagenomes and carry genes involved in heavy-metal homeostasis, proving the pivotal role of plasmids in microorganism adaptation to environmental conditions. PMID:29346586

Detection of Bacterial Pathogens from Broncho-Alveolar Lavage by Next-Generation Sequencing.

PubMed

Leo, Stefano; Gaïa, Nadia; Ruppé, Etienne; Emonet, Stephane; Girard, Myriam; Lazarevic, Vladimir; Schrenzel, Jacques

2017-09-20

The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus , Corynebacterium jeikeium and Rothia dentocariosa , the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.

Microbial Characterization of Qatari Barchan Sand Dunes

PubMed Central

Chatziefthimiou, Aspassia D.; Nguyen, Hanh; Richer, Renee; Louge, Michel; Sultan, Ali A.; Schloss, Patrick; Hay, Anthony G.

2016-01-01

This study represents the first characterization of sand microbiota in migrating barchan sand dunes. Bacterial communities were studied through direct counts and cultivation, as well as 16S rRNA gene and metagenomic sequence analysis to gain an understanding of microbial abundance, diversity, and potential metabolic capabilities. Direct on-grain cell counts gave an average of 5.3 ± 0.4 x 105 cells g-1 of sand. Cultured isolates (N = 64) selected for 16S rRNA gene sequencing belonged to the phyla Actinobacteria (58%), Firmicutes (27%) and Proteobacteria (15%). Deep-sequencing of 16S rRNA gene amplicons from 18 dunes demonstrated a high relative abundance of Proteobacteria, particularly enteric bacteria, and a dune-specific-pattern of bacterial community composition that correlated with dune size. Shotgun metagenome sequences of two representative dunes were analyzed and found to have similar relative bacterial abundance, though the relative abundances of eukaryotic, viral and enterobacterial sequences were greater in sand from the dune closer to a camel-pen. Functional analysis revealed patterns similar to those observed in desert soils; however, the increased relative abundance of genes encoding sporulation and dormancy are consistent with the dune microbiome being well-adapted to the exceptionally hyper-arid Qatari desert. PMID:27655399

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences

EPA Science Inventory

A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

Analysis of the gut microbiome in beef cattle and its association with feed intake, growth, and efficiency

USDA-ARS?s Scientific Manuscript database

Next-generation sequencing has taken a central role in studies of microbial ecology, especially with regard to culture-independent methods based on molecular phylogenies of the small-subunit ribosomal RNA gene (16S rRNA gene). The ability to relate trends at the species or genus level to host/envir...

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.

Draft genome sequence and annotation of Lactobacillus acetotolerans BM-LA14527, a beer-spoilage bacteria.

PubMed

Liu, Junyan; Li, Lin; Peters, Brian M; Li, Bing; Deng, Yang; Xu, Zhenbo; Shirtliff, Mark E

2016-09-01

Lactobacillus acetotolerans is a hard-to-culture beer-spoilage bacterium capable of entering into the viable putative nonculturable (VPNC) state. As part of an initial strategy to investigate the phenotypic behavior of L. acetotolerans, draft genome sequencing was performed. Results demonstrated a total of 1824 predicted annotated genes, with several potential VPNC- and beer-spoilage-associated genes identified. Importantly, this is the first genome sequence of L. acetotolerans as beer-spoilage bacteria and it may aid in further analysis of L. acetotolerans and other beer-spoilage bacteria, with direct implications for food safety control in the beer brewing industry. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Molecular markers for identifying a new selected variety of Pacific white shrimp Litopenaeus vannamei

NASA Astrophysics Data System (ADS)

Yu, Yang; Zhang, Xiaojun; Liu, Jingwen; Li, Fuhua; Huang, Hao; Li, Yijun; Liu, Xiaolin; Xiang, Jianhai

2015-01-01

Selective breeding of the Pacific white shrimp Litopenaeus vannamei during the last decade has produced new varieties exhibiting high growth rates and disease resistance. However, the identification of new varieties of shrimps from their phenotypic characters is difficult. This study introduces a new approach for identifying varieties of shrimps using molecular markers of microsatellites and mitochondrial control region sequences. The method was employed to identify a new selected variety, Kehai No. 1 (KH-1), from three representative stocks (control group): Zhengda; Tongwei; and a stock collected from Fujian Province, which is now cultured in mainland China. By pooled genotyping of KH-1 and the control group, five microsatellites showing differences between KH-1 and the control group were screened out. Individual genotyping data confirmed the results from pooled genotyping. The genotyping data for the five microsatellites were applied to the assignment analysis of the KH-1 group and the control group using the partial Bayesian assignment method in GENECLASS2. By sequencing the mitochondrial control regions of individuals from the KH-1 and control group, four haplotypes were observed in the KH-1 group, whereas 14 haplotypes were obtained in the control group. By combining the microsatellite assignment analysis with mitochondrial control region analysis, the average accuracy of identification of individuals in the KH-1 group and control group reached 89%. The five selected microsatellite loci and mitochondrial control region sequences were highly polymorphic and could be used to distinguish new selected varieties of L. vannamei from other populations cultured in China.

Genome Sequence of African Swine Fever Virus BA71, the Virulent Parental Strain of the Nonpathogenic and Tissue-Culture Adapted BA71V

PubMed Central

Rodríguez, Javier M.; Moreno, Leticia Tais; Alejo, Alí; Lacasta, Anna; Rodríguez, Fernando; Salas, María L.

2015-01-01

The strain BA71V has played a key role in African swine fever virus (ASFV) research. It was the first genome sequenced, and remains the only genome completely determined. A large part of the studies on the function of ASFV genes, viral transcription, replication, DNA repair and morphogenesis, has been performed using this model. This avirulent strain was obtained by adaptation to grow in Vero cells of the highly virulent BA71 strain. We report here the analysis of the genome sequence of BA71 in comparison with that of BA71V. They possess the smallest genomes for a virulent or an attenuated ASFV, and are essentially identical except for a relatively small number of changes. We discuss the possible contribution of these changes to virulence. Analysis of the BA71 sequence allowed us to identify new similarities among ASFV proteins, and with database proteins including two ASFV proteins that could function as a two-component signaling network. PMID:26618713

Draft Genome Sequences of Two Fusarium oxysporum Isolates Cultured from Infected Zinnia hybrida Plants Grown on the International Space Station

PubMed Central

Urbaniak, Camilla; Massa, Gioia; Hummerick, Mary; Khodadad, Christina; Schuerger, Andrew

2018-01-01

ABSTRACT Here, we present the whole-genome sequences of two Fusarium oxysporum isolates cultured from infected Zinnia hybrida plants that were grown onboard the International Space Station (ISS). PMID:29773617

Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain

PubMed Central

Zuljan, Federico; Espariz, Martín; Blancato, Victor S.; Esteban, Luis; Alarcón, Sergio

2016-01-01

We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains. PMID:26847906

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea.

PubMed

Sun, Wei; Dai, Shikun; Jiang, Shumei; Wang, Guanghua; Liu, Guohui; Wu, Houbo; Li, Xiang

2010-06-01

In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks.

PubMed

Valletta, Elisa; Kučera, Lukáš; Prokeš, Lubomír; Amato, Filippo; Pivetta, Tiziana; Hampl, Aleš; Havel, Josef; Vaňhara, Petr

2016-01-01

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general.

Multivariate Calibration Approach for Quantitative Determination of Cell-Line Cross Contamination by Intact Cell Mass Spectrometry and Artificial Neural Networks

PubMed Central

Prokeš, Lubomír; Amato, Filippo; Pivetta, Tiziana; Hampl, Aleš; Havel, Josef; Vaňhara, Petr

2016-01-01

Cross-contamination of eukaryotic cell lines used in biomedical research represents a highly relevant problem. Analysis of repetitive DNA sequences, such as Short Tandem Repeats (STR), or Simple Sequence Repeats (SSR), is a widely accepted, simple, and commercially available technique to authenticate cell lines. However, it provides only qualitative information that depends on the extent of reference databases for interpretation. In this work, we developed and validated a rapid and routinely applicable method for evaluation of cell culture cross-contamination levels based on mass spectrometric fingerprints of intact mammalian cells coupled with artificial neural networks (ANNs). We used human embryonic stem cells (hESCs) contaminated by either mouse embryonic stem cells (mESCs) or mouse embryonic fibroblasts (MEFs) as a model. We determined the contamination level using a mass spectra database of known calibration mixtures that served as training input for an ANN. The ANN was then capable of correct quantification of the level of contamination of hESCs by mESCs or MEFs. We demonstrate that MS analysis, when linked to proper mathematical instruments, is a tangible tool for unraveling and quantifying heterogeneity in cell cultures. The analysis is applicable in routine scenarios for cell authentication and/or cell phenotyping in general. PMID:26821236

Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae

PubMed Central

Huang, Youhua; Huang, Xiaohong; Liu, Hong; Gong, Jie; Ouyang, Zhengliang; Cui, Huachun; Cao, Jianhao; Zhao, Yingtao; Wang, Xiujie; Jiang, Yulin; Qin, Qiwei

2009-01-01

Background Soft-shelled turtle iridovirus (STIV) is the causative agent of severe systemic diseases in cultured soft-shelled turtles (Trionyx sinensis). To our knowledge, the only molecular information available on STIV mainly concerns the highly conserved STIV major capsid protein. The complete sequence of the STIV genome is not yet available. Therefore, determining the genome sequence of STIV and providing a detailed bioinformatic analysis of its genome content and evolution status will facilitate further understanding of the taxonomic elements of STIV and the molecular mechanisms of reptile iridovirus pathogenesis. Results We determined the complete nucleotide sequence of the STIV genome using 454 Life Science sequencing technology. The STIV genome is 105 890 bp in length with a base composition of 55.1% G+C. Computer assisted analysis revealed that the STIV genome contains 105 potential open reading frames (ORFs), which encode polypeptides ranging from 40 to 1,294 amino acids and 20 microRNA candidates. Among the putative proteins, 20 share homology with the ancestral proteins of the nuclear and cytoplasmic large DNA viruses (NCLDVs). Comparative genomic analysis showed that STIV has the highest degree of sequence conservation and a colinear arrangement of genes with frog virus 3 (FV3), followed by Tiger frog virus (TFV), Ambystoma tigrinum virus (ATV), Singapore grouper iridovirus (SGIV), Grouper iridovirus (GIV) and other iridovirus isolates. Phylogenetic analysis based on conserved core genes and complete genome sequence of STIV with other virus genomes was performed. Moreover, analysis of the gene gain-and-loss events in the family Iridoviridae suggested that the genes encoded by iridoviruses have evolved for favoring adaptation to different natural host species. Conclusion This study has provided the complete genome sequence of STIV. Phylogenetic analysis suggested that STIV and FV3 are strains of the same viral species belonging to the Ranavirus genus in the Iridoviridae family. Given virus-host co-evolution and the phylogenetic relationship among vertebrates from fish to reptiles, we propose that iridovirus might transmit between reptiles and amphibians and that STIV and FV3 are strains of the same viral species in the Ranavirus genus. PMID:19439104

Metavir 2: new tools for viral metagenome comparison and assembled virome analysis

PubMed Central

2014-01-01

Background Metagenomics, based on culture-independent sequencing, is a well-fitted approach to provide insights into the composition, structure and dynamics of environmental viral communities. Following recent advances in sequencing technologies, new challenges arise for existing bioinformatic tools dedicated to viral metagenome (i.e. virome) analysis as (i) the number of viromes is rapidly growing and (ii) large genomic fragments can now be obtained by assembling the huge amount of sequence data generated for each metagenome. Results To face these challenges, a new version of Metavir was developed. First, all Metavir tools have been adapted to support comparative analysis of viromes in order to improve the analysis of multiple datasets. In addition to the sequence comparison previously provided, viromes can now be compared through their k-mer frequencies, their taxonomic compositions, recruitment plots and phylogenetic trees containing sequences from different datasets. Second, a new section has been specifically designed to handle assembled viromes made of thousands of large genomic fragments (i.e. contigs). This section includes an annotation pipeline for uploaded viral contigs (gene prediction, similarity search against reference viral genomes and protein domains) and an extensive comparison between contigs and reference genomes. Contigs and their annotations can be explored on the website through specifically developed dynamic genomic maps and interactive networks. Conclusions The new features of Metavir 2 allow users to explore and analyze viromes composed of raw reads or assembled fragments through a set of adapted tools and a user-friendly interface. PMID:24646187

Complete Genome Sequence of Petrimonas sp. Strain IBARAKI, Assembled from the Metagenome Data of a Culture Containing Dehalococcoides spp.

PubMed

Ikegami, Kentaro; Aita, Yuto; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Shinzato, Misuzu; Ohki, Shun; Nakano, Kazuma; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi; Yohda, Masafumi

2018-05-03

The complete genome sequence of Petrimonas sp. strain IBARAKI in a Dehalococcoides -containing culture was determined using the PacBio RS II platform. The genome is a single circular chromosome of 3,693,233 nucleotides (nt), with a GC content of 44%. This is the first genome sequence of a Petrimonas species. Copyright © 2018 Ikegami et al.

Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi.

PubMed

Song, Zhangyong; Yin, Youping; Jiang, Shasha; Liu, Juanjuan; Chen, Huan; Wang, Zhongkang

2013-06-19

Nomuraea rileyi is used as an environmental-friendly biopesticide. However, mass production and commercialization of this organism are limited due to its fastidious growth and sporulation requirements. When cultured in amended medium, we found that N. rileyi could produce microsclerotia bodies, replacing conidiophores as the infectious agent. However, little is known about the genes involved in microsclerotia development. In the present study, the transcriptomes were analyzed using next-generation sequencing technology to find the genes involved in microsclerotia development. A total of 4.69 Gb of clean nucleotides comprising 32,061 sequences was obtained, and 20,919 sequences were annotated (about 65%). Among the annotated sequences, only 5928 were annotated with 34 gene ontology (GO) functional categories, and 12,778 sequences were mapped to 165 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we assessed the transcriptomic differences between cultures grown in minimal and amended medium. In total, 4808 sequences were found to be differentially expressed; 719 differentially expressed unigenes were assigned to 25 GO classes and 1888 differentially expressed unigenes were assigned to 161 KEGG pathways, including 25 enrichment pathways. Subsequently, we examined the up-regulation or uniquely expressed genes following amended medium treatment, which were also expressed on the enrichment pathway, and found that most of them participated in mediating oxidative stress homeostasis. To elucidate the role of oxidative stress in microsclerotia development, we analyzed the diversification of unigenes using quantitative reverse transcription-PCR (RT-qPCR). Our findings suggest that oxidative stress occurs during microsclerotia development, along with a broad metabolic activity change. Our data provide the most comprehensive sequence resource available for the study of N. rileyi. We believe that the transcriptome datasets will serve as an important public information platform to accelerate studies on N. rileyi microsclerotia.

Noncytopathogenic Pestivirus Strains Generated by Nonhomologous RNA Recombination: Alterations in the NS4A/NS4B Coding Region

PubMed Central

Gallei, Andreas; Orlich, Michaela; Thiel, Heinz-Juergen; Becher, Paul

2005-01-01

Several studies have demonstrated that cytopathogenic (cp) pestivirus strains evolve from noncytopathogenic (noncp) viruses by nonhomologous RNA recombination. In addition, two recent reports showed the rapid emergence of noncp Bovine viral diarrhea virus (BVDV) after a few cell culture passages of cp BVDV strains by homologous recombination between identical duplicated viral sequences. To allow the identification of recombination sites from noncp BVDV strains that evolve from cp viruses, we constructed the cp BVDV strains CP442 and CP552. Both harbor duplicated viral sequences of different origin flanking the cellular insertion Nedd8*; the latter is a prerequisite for their cytopathogenicity. In contrast to the previous studies, isolation of noncp strains was possible only after extensive cell culture passages of CP442 and CP552. Sequence analysis of 15 isolated noncp BVDVs confirmed that all recombinant strains lack at least most of Nedd8*. Interestingly, only one strain resulted from homologous recombination while the other 14 strains were generated by nonhomologous recombination. Accordingly, our data suggest that the extent of sequence identity between participating sequences influences both frequency and mode (homologous versus nonhomologous) of RNA recombination in pestiviruses. Further analyses of the noncp recombinant strains revealed that a duplication of 14 codons in the BVDV nonstructural protein 4B (NS4B) gene does not interfere with efficient viral replication. Moreover, an insertion of viral sequences between the NS4A and NS4B genes was well tolerated. These findings thus led to the identification of two genomic loci which appear to be suited for the insertion of heterologous sequences into the genomes of pestiviruses and related viruses. PMID:16254361

Identification and characterization of unrecognized viruses in stool samples of non-polio acute flaccid paralysis children by simplified VIDISCA.

PubMed

Shaukat, Shahzad; Angez, Mehar; Alam, Muhammad Masroor; Jebbink, Maarten F; Deijs, Martin; Canuti, Marta; Sharif, Salmaan; de Vries, Michel; Khurshid, Adnan; Mahmood, Tariq; van der Hoek, Lia; Zaidi, Syed Sohail Zahoor

2014-08-12

The use of sequence independent methods combined with next generation sequencing for identification purposes in clinical samples appears promising and exciting results have been achieved to understand unexplained infections. One sequence independent method, Virus Discovery based on cDNA Amplified Fragment Length Polymorphism (VIDISCA) is capable of identifying viruses that would have remained unidentified in standard diagnostics or cell cultures. VIDISCA is normally combined with next generation sequencing, however, we set up a simplified VIDISCA which can be used in case next generation sequencing is not possible. Stool samples of 10 patients with unexplained acute flaccid paralysis showing cytopathic effect in rhabdomyosarcoma cells and/or mouse cells were used to test the efficiency of this method. To further characterize the viruses, VIDISCA-positive samples were amplified and sequenced with gene specific primers. Simplified VIDISCA detected seven viruses (70%) and the proportion of eukaryotic viral sequences from each sample ranged from 8.3 to 45.8%. Human enterovirus EV-B97, EV-B100, echovirus-9 and echovirus-21, human parechovirus type-3, human astrovirus probably a type-3/5 recombinant, and tetnovirus-1 were identified. Phylogenetic analysis based on the VP1 region demonstrated that the human enteroviruses are more divergent isolates circulating in the community. Our data support that a simplified VIDISCA protocol can efficiently identify unrecognized viruses grown in cell culture with low cost, limited time without need of advanced technical expertise. Also complex data interpretation is avoided thus the method can be used as a powerful diagnostic tool in limited resources. Redesigning the routine diagnostics might lead to additional detection of previously undiagnosed viruses in clinical samples of patients.

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 Elsevier B.V.

[Molecular and prenatal diagnosis of a family with Fanconi anemia by next generation sequencing].

PubMed

Gong, Zhuwen; Yu, Yongguo; Zhang, Qigang; Gu, Xuefan

2015-04-01

To provide prenatal diagnosis for a pregnant woman who had given birth to a child with Fanconi anemia with combined next-generation sequencing (NGS) and Sanger sequencing. For the affected child, potential mutations of the FANCA gene were analyzed with NGS. Suspected mutation was verified with Sanger sequencing. For prenatal diagnosis, genomic DNA was extracted from cultured fetal amniotic fluid cells and subjected to analysis of the same mutations. A low-frequency frameshifting mutation c.989_995del7 (p.H330LfsX2, inherited from his father) and a truncating mutation c.3971C>T (p.P1324L, inherited from his mother) have been identified in the affected child and considered to be pathogenic. The two mutations were subsequently verified by Sanger sequencing. Upon prenatal diagnosis, the fetus was found to carry two mutations. The combined next-generation sequencing and Sanger sequencing can reduce the time for diagnosis and identify subtypes of Fanconi anemia and the mutational sites, which has enabled reliable prenatal diagnosis of this disease.

Draft Genome Sequences of Two Fusarium oxysporum Isolates Cultured from Infected Zinnia hybrida Plants Grown on the International Space Station.

PubMed

Urbaniak, Camilla; Massa, Gioia; Hummerick, Mary; Khodadad, Christina; Schuerger, Andrew; Venkateswaran, Kasthuri

2018-05-17

Here, we present the whole-genome sequences of two Fusarium oxysporum isolates cultured from infected Zinnia hybrida plants that were grown onboard the International Space Station (ISS). Copyright © 2018 Urbaniak et al.

Microbial community analysis of a coastal hot spring in Kagoshima, Japan, using molecular- and culture-based approaches.

PubMed

Nishiyama, Minako; Yamamoto, Shuichi; Kurosawa, Norio

2013-08-01

Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared <92% sequence similarity with their closest sequences in a public database, suggesting that the Ibusuki hot spring may harbor a unique and novel bacterial community. By contrast, 77 clones from the Site 2 archaeal library contained only three OTUs, most of which were affiliated with Thaumarchaeota.

Strain variation in Mycobacterium marinum fish isolates.

PubMed

Ucko, M; Colorni, A; Kvitt, H; Diamant, A; Zlotkin, A; Knibb, W R

2002-11-01

A molecular characterization of two Mycobacterium marinum genes, 16S rRNA and hsp65, was carried out with a total of 21 isolates from various species of fish from both marine and freshwater environments of Israel, Europe, and the Far East. The nucleotide sequences of both genes revealed that all M. marinum isolates from fish in Israel belonged to two different strains, one infecting marine (cultured and wild) fish and the other infecting freshwater (cultured) fish. A restriction enzyme map based on the nucleotide sequences of both genes confirmed the divergence of the Israeli marine isolates from the freshwater isolates and differentiated the Israeli isolates from the foreign isolates, with the exception of one of three Greek isolates from marine fish which was identical to the Israeli marine isolates. The second isolate from Greece exhibited a single base alteration in the 16S rRNA sequence, whereas the third isolate was most likely a new Mycobacterium species. Isolates from Denmark and Thailand shared high sequence homology to complete identity with reference strain ATCC 927. Combined analysis of the two gene sequences increased the detection of intraspecific variations and was thus of importance in studying the taxonomy and epidemiology of this aquatic pathogen. Whether the Israeli M. marinum strain infecting marine fish is endemic to the Red Sea and found extremely susceptible hosts in the exotic species imported for aquaculture or rather was accidentally introduced with occasional imports of fingerlings from the Mediterranean Sea could not be determined.

Outbreak Investigation Using High-Throughput Genome Sequencing within a Diagnostic Microbiology Laboratory

PubMed Central

Sherry, Norelle L.; Porter, Jessica L.; Seemann, Torsten; Watkins, Andrew; Stinear, Timothy P.

2013-01-01

Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the blaCTX-M-15 extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications. PMID:23408689

Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection.

PubMed

Ruan, Yi Jun; Wei, Chia Lin; Ee, Ai Ling; Vega, Vinsensius B; Thoreau, Herve; Su, Se Thoe Yun; Chia, Jer-Ming; Ng, Patrick; Chiu, Kuo Ping; Lim, Landri; Zhang, Tao; Peng, Chan Kwai; Lin, Ean Oon Lynette; Lee, Ng Mah; Yee, Sin Leo; Ng, Lisa F P; Chee, Ren Ee; Stanton, Lawrence W; Long, Philip M; Liu, Edison T

2003-05-24

The cause of severe acute respiratory syndrome (SARS) has been identified as a new coronavirus. Whole genome sequence analysis of various isolates might provide an indication of potential strain differences of this new virus. Moreover, mutation analysis will help to develop effective vaccines. We sequenced the entire SARS viral genome of cultured isolates from the index case (SIN2500) presenting in Singapore, from three primary contacts (SIN2774, SIN2748, and SIN2677), and one secondary contact (SIN2679). These sequences were compared with the isolates from Canada (TOR2), Hong Kong (CUHK-W1 and HKU39849), Hanoi (URBANI), Guangzhou (GZ01), and Beijing (BJ01, BJ02, BJ03, BJ04). We identified 129 sequence variations among the 14 isolates, with 16 recurrent variant sequences. Common variant sequences at four loci define two distinct genotypes of the SARS virus. One genotype was linked with infections originating in Hotel M in Hong Kong, the second contained isolates from Hong Kong, Guangzhou, and Beijing with no association with Hotel M (p<0.0001). Moreover, other common sequence variants further distinguished the geographical origins of the isolates, especially between Singapore and Beijing. Despite the recent onset of the SARS epidemic, genetic signatures are emerging that partition the worldwide SARS viral isolates into groups on the basis of contact source history and geography. These signatures can be used to trace sources of infection. In addition, a common variant associated with a non-conservative aminoacid change in the S1 region of the spike protein, suggests that immunological pressures might be starting to influence the evolution of the SARS virus in human populations.

Heterologous Array Analysis in Pinaceae: Hybridization of Pinus Taeda cDNA Arrays With cDNA From Needles and Embryogenic Cultures of P. Taeda, P. Sylvestris or Picea Abies

PubMed Central

van Zyl, Leonel; von Arnold, Sara; Bozhkov, Peter; Chen, Yongzhong; Egertsdotter, Ulrika; MacKay, John; Sederoff, Ronald R.; Shen, Jing; Zelena, Lyubov

2002-01-01

Hybridization of labelled cDNA from various cell types with high-density arrays of expressed sequence tags is a powerful technique for investigating gene expression. Few conifer cDNA libraries have been sequenced. Because of the high level of sequence conservation between Pinus and Picea we have investigated the use of arrays from one genus for studies of gene expression in the other. The partial cDNAs from 384 identifiable genes expressed in differentiating xylem of Pinus taeda were printed on nylon membranes in randomized replicates. These were hybridized with labelled cDNA from needles or embryogenic cultures of Pinus taeda, P. sylvestris and Picea abies, and with labelled cDNA from leaves of Nicotiana tabacum. The Spearman correlation of gene expression for pairs of conifer species was high for needles (r2 = 0.78 − 0.86), and somewhat lower for embryogenic cultures (r2 = 0.68 − 0.83). The correlation of gene expression for tobacco leaves and needles of each of the three conifer species was lower but sufficiently high (r2 = 0.52 − 0.63) to suggest that many partial gene sequences are conserved in angiosperms and gymnosperms. Heterologous probing was further used to identify tissue-specific gene expression over species boundaries. To evaluate the significance of differences in gene expression, conventional parametric tests were compared with permutation tests after four methods of normalization. Permutation tests after Z-normalization provide the highest degree of discrimination but may enhance the probability of type I errors. It is concluded that arrays of cDNA from loblolly pine are useful for studies of gene expression in other pines or spruces. PMID:18629264

Genome Sequence of Lactobacillus sakei subsp. sakei LS25, a Commercial Starter Culture Strain for Fermented Sausage.

PubMed

McLeod, Anette; Brede, Dag Anders; Rud, Ida; Axelsson, Lars

2013-07-11

Lactobacillus sakei is a lactic acid bacterium associated primarily with fermented meat and fish. Here, we present the draft genome sequence of L. sakei subsp. sakei strain LS25, a commercial starter culture strain for fermented sausage.

Longitudinal analysis of microbiota in microalga Nannochloropsis salina cultures

DOE PAGES

Geng, Haifeng; Sale, Kenneth L.; Tran-Gyamfi, Mary Bao; ...

2016-03-08

Here, large-scale open microalgae cultivation has tremendous potential to make a significant contribution to replacing petroleum-based fuels with biofuels. Open algal cultures are unavoidably inhabited with a diversity of microbes that live on, influence, and shape the fate of these ecosystems. However, there is little understanding of the resilience and stability of the microbial communities in engineered semicontinuous algal systems. To evaluate the dynamics and resilience of the microbial communities in microalgae biofuel cultures, we conducted a longitudinal study on open systems to compare the temporal profiles of the microbiota from two multigenerational algal cohorts, which include one seeded withmore » the microbiota from an in-house culture and the other exogenously seeded with a natural-occurring consortia of bacterial species harvested from the Pacific Ocean. From these month-long, semicontinuous open microalga Nannochloropsis salina cultures, we sequenced a time-series of 46 samples, yielding 8804 operational taxonomic units derived from 9,160,076 high-quality partial 16S rRNA sequences. We provide quantitative evidence that clearly illustrates the development of microbial community is associated with microbiota ancestry. In addition, N. salina growth phases were linked with distinct changes in microbial phylotypes. Alteromonadeles dominated the community in the N. salina exponential phase whereas Alphaproteobacteria and Flavobacteriia were more prevalent in the stationary phase. We also demonstrate that the N. salina-associated microbial community in open cultures is diverse, resilient, and dynamic in response to environmental perturbations. This knowledge has general implications for developing and testing design principles of cultivated algal systems.« less

Internet-accessible DNA sequence database for identifying fusaria from human and animal infections.

PubMed

O'Donnell, Kerry; Sutton, Deanna A; Rinaldi, Michael G; Sarver, Brice A J; Balajee, S Arunmozhi; Schroers, Hans-Josef; Summerbell, Richard C; Robert, Vincent A R G; Crous, Pedro W; Zhang, Ning; Aoki, Takayuki; Jung, Kyongyong; Park, Jongsun; Lee, Yong-Hwan; Kang, Seogchan; Park, Bongsoo; Geiser, David M

2010-10-01

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate's clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.

Presence of glucose, xylose, and glycerol fermenting bacteria in the deep biosphere of the former Homestake gold mine, South Dakota

USDA-ARS?s Scientific Manuscript database

Eight fermentative bacterial strains were isolated from mixed enrichment cultures of a composite soil sample collected at 1.34 km depth from the former Homestake gold mine in Lead, SD, USA. Phylogenetic analysis of their 16S rRNA gene sequences revealed that these isolates were affiliated with the p...

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees.

PubMed

Adams, Gerard C; Surve-Iyer, Rupa S; Iezzoni, Amy F

2002-01-01

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.

Characterization and in-vivo evaluation of potential probiotics of the bacterial flora within the water column of a healthy shrimp larviculture system

NASA Astrophysics Data System (ADS)

Xue, Ming; Liang, Huafang; He, Yaoyao; Wen, Chongqing

2016-05-01

A thorough understanding of the normal bacterial flora associated with shrimp larviculture systems contributes to probiotic screening and disease control. The bacterial community of the water column over a commercial Litopenaeus vannamei larval rearing run was characterized with both culture-dependent and culture-independent methods. A total of 27 phylotypes at the species level were isolated and identified based on 16S rDNA sequence analysis. Denaturing gradient gel electrophoresis (DGGE) analysis of the V3-V5 region of 16S rRNA genes showed a dynamic bacterial community with major changes occurred from stages zoea to mysis during the rearing run. The sequences retrieved were affiliated to four phyla, Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, with the family Rhodobacteraceae being the most frequently recovered one. Subsequently, 13 representative strains conferred higher larval survival than the control when evaluated in the in-vivo experiments; in particular, three candidates, assigned to Phaeobacter sp., Arthrobacter sp., and Microbacterium sp., significantly improved larval survival ( P < 0.05). Therefore, the healthy shrimp larviculture system harbored a diverse and favorable bacterial flora, which contribute to larval development and are of great importance in exploiting novel probiotics.

Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

PubMed

Chao, Mei; Lin, Chia-Chi; Lin, Feng-Ming; Li, Hsin-Pai; Iang, Shan-Bei

2015-12-01

Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

New MCM8 mutation associated with premature ovarian insufficiency and chromosomal instability in a highly consanguineous Tunisian family.

PubMed

Bouali, Nouha; Francou, Bruno; Bouligand, Jérôme; Imanci, Dilek; Dimassi, Sarra; Tosca, Lucie; Zaouali, Monia; Mougou, Soumaya; Young, Jacques; Saad, Ali; Guiochon-Mantel, Anne

2017-10-01

To identify the gene(s) involved in the etiology of premature ovarian insufficiency in a highly consanguineous Tunisian family. Genetic analysis of a large consanguineous family with several affected siblings. University hospital-based cytogenetics and molecular genetics laboratories. A highly consanguineous Tunisian family with several affected siblings born to healthy second-degree cousins. None. Targeted exome sequencing was performed by next-generation sequencing for affected family members. Mutations were validated by Sanger sequencing. Functional experiments were performed to explore the deleterious effects of the identified mutation. DNA damage was induced by increasing mitomycin C (MMC) concentrations on cultured peripheral lymphocytes. Analysis of the next-generation sequencing data revealed a new homozygous missense mutation in the minichromosome maintenance 8 gene (MCM8).This homozygous mutation (c. 482A>C; p.His161Pro) was predicted to be deleterious and segregated with the disease in the family. MCM8 participates in homologous recombination during meiosis and DNA double-stranded break repair by dimerizing with MCM9. Mcm8 knock out results in an early block in follicle development and small gonads. Given this, we tested the chromosomal breakage repair capacity of homozygous and heterozygous MCM8 p.His161Pro mutation on cultured peripheral lymphocytes exposed to increasing MMC concentrations. We found that chromosomal breakage after MMC exposure was significantly higher in cells from homozygously affected individuals than in those from a healthy control. Our findings provide additional support to the view that MCM8 mutations are involved in the primary ovarian insufficiency phenotype. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Skin lesion-associated pathogens from Octopus vulgaris: first detection of Photobacterium swingsii, Lactococcus garvieae and betanodavirus.

PubMed

Fichi, G; Cardeti, G; Perrucci, S; Vanni, A; Cersini, A; Lenzi, C; De Wolf, T; Fronte, B; Guarducci, M; Susini, F

2015-07-23

The common octopus Octopus vulgaris Cuvier, 1798 is extremely important in fisheries and is a useful protein source in most Mediterranean countries. Here we investigated pathogens associated with skin lesions in 9 naturally deceased specimens that included both cultured and wild common octopus. Within 30 min after death, each octopus was stored at 4°C and microbiologically examined within 24 h. Bacterial colonies, cultured from swabs taken from the lesions, were examined using taxonomical and biochemical analyses. Vibrio alginolyticus and V. parahaemolyticus were only isolated from cultured animals. A conventional PCR targeting the 16S ribosomal RNA (rRNA) gene and sequencing were performed on 2 bacterial isolates that remained unidentified after taxonomical and biochemical analysis. The sequence results indicated that the bacteria had a 99% identity with Lactococcus garvieae and Photobacterium swingsii. L. garvieae was confirmed using a specific PCR based on the 16S-23S rRNA internal transcribed spacer region, while P. swingsii was confirmed by phylogenetic analyses. Although all animals examined were found to be infected by the protozoan species Aggregata octopiana localised in the intestines, it was also present in skin lesions of 2 of the animals. Betanodavirus was detected in both cultured and wild individuals by cell culture, PCR and electron microscopy. These findings are the first report of L. garvieae and betanodavirus from skin lesions of common octopus and the first identification of P. swingsii both in octopus skin lesions and in marine invertebrates in Italy.

Cellular and molecular characterization of gametogenic progression in ex vivo cultured prepuberal mouse testes.

PubMed

Isoler-Alcaraz, J; Fernández-Pérez, D; Larriba, E; Del Mazo, J

2017-10-18

Recently, an effective testis culture method using a gas-liquid interphase, capable of differentiate male germ cells from neonatal spermatogonia to spermatozoa has been developed. Nevertheless, this methodology needs deep analyses that allow future experimental approaches in basic, pathologic and/or reprotoxicologic studies. Because of this, we characterized at cellular and molecular levels the entire in vitro spermatogenic progression, in order to understand and evaluate the characteristics that define the spermatogenic process in ex vivo cultured testes compared to the in vivo development. Testicular explants of CD1 mice aged 6 and 10 days post-partum were respectively cultured during 55 and 89 days. Cytological and molecular approaches were performed, analyzing germ cell proportion at different time culture points, meiotic markers immunodetecting synaptonemal complex protein SYCP3 by immunocytochemistry and the relative expression of different marker genes along the differentiation process by Reverse Transcription - quantitative Polymerase Chain Reaction. In addition, microRNA and piwi-interactingRNA profiles were also evaluated by Next Generation Sequencing and bioinformatic approaches. The method promoted and maintained the spermatogenic process during 89 days. At a cytological level we detected spermatogenic development delays of cultured explants compared to the natural in vivo process. The expression of different spermatogenic stages gene markers correlated with the proportion of different cell types detected in the cytological preparations. In vitro progression analysis of the different spermatogenic cell types, from both 6.5 dpp and 10.5 dpp testes explants, has revealed a relative delay in relation to in vivo process. The expression of the genes studied as biomarkers correlates with the cytologically and functional detected progression and differential expression identified in vivo. After a first analysis of deep sequencing data it has been observed that as long as cultures progress, the proportion of microRNAs declined respect to piwi-interactingRNAs levels that increased, showing a similar propensity than which happens in in vivo spermatogenesis. Our study allows to improve and potentially to control the ex vivo spermatogenesis development, opening new perspectives in the reproductive biology fields including male fertility.

[Cloning and characterization of genes differentially expressed in human dental pulp cells and gingival fibroblasts].

PubMed

Wang, Zhong-dong; Wu, Ji-nan; Zhou, Lin; Ling, Jun-qi; Guo, Xi-min; Xiao, Ming-zhen; Zhu, Feng; Pu, Qin; Chai, Yu-bo; Zhao, Zhong-liang

2007-02-01

To study the biological properties of human dental pulp cells (HDPC) by cloning and analysis of genes differentially expressed in HDPC in comparison with human gingival fibroblasts (HGF). HDPC and HGF were cultured and identified by immunocytochemistry. HPDC and HGF subtractive cDNA library was established by PCR-based modified subtractive hybridization, genes differentially expressed by HPDC were cloned, sequenced and compared to find homogeneous sequence in GenBank by BLAST. Cloning and sequencing analysis indicate 12 genes differentially expressed were obtained, in which two were unknown genes. Among the 10 known genes, 4 were related to signal transduction, 2 were related to trans-membrane transportation (both cell membrane and nuclear membrane), and 2 were related to RNA splicing mechanisms. The biological properties of HPDC are determined by the differential expression of some genes and the growth and differentiation of HPDC are associated to the dynamic protein synthesis and secretion activities of the cell.

Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2 ▿

PubMed Central

Borman, Andrew M.; Linton, Christopher J.; Oliver, Debra; Palmer, Michael D.; Szekely, Adrien; Johnson, Elizabeth M.

2010-01-01

Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures. PMID:20702674

Diversity of Streptomyces spp. in Eastern Himalayan region – computational RNomics approach to phylogeny

PubMed Central

Bhattacharjee, Kaushik; Banerjee, Subhro; Joshi, Santa Ram

2012-01-01

Isolation and characterization of actinomycetes from soil samples from altitudinal gradient of North-East India were investigated for computational RNomics based phylogeny. A total of 52 diverse isolates of Streptomyces from the soil samples were isolated on four different media and from these 6 isolates were selected on the basis of cultural characteristics, microscopic and biochemical studies. Sequencing of 16S rDNA of the selected isolates identified them to belong to six different species of Streptomyces. The molecular morphometric and physico-kinetic analysis of 16S rRNA sequences were performed to predict the diversity of the genus. The computational RNomics study revealed the significance of the structural RNA based phylogenetic analysis in a relatively diverse group of Streptomyces. PMID:22829729

Production of Functional Proteins: Balance of Shear Stress and Gravity

NASA Technical Reports Server (NTRS)

Goodwin, Thomas John (Inventor); Hammond, Timothy Grant (Inventor); Haysen, James Howard (Inventor)

2005-01-01

The present invention provides for a method of culturing cells and inducing the expression of at least one gene in the cell culture. The method provides for contacting the cell with a transcription factor decoy oligonucleotide sequence directed against a nucleotide sequence encoding a shear stress response element.

Constitutive expression of human pancreatic lipase-related protein 1 in Pichia pastoris.

PubMed

Aloulou, Ahmed; Grandval, Philippe; De Caro, Josiane; De Caro, Alain; Carrière, Frédéric

2006-06-01

High-level constitutive expression of the human pancreatic lipase-related protein 1 (HPLRP1) was achieved using the methylotrophic yeast Pichia pastoris. The HPLRP1 cDNA, including its original leader sequence, was subcloned into the pGAPZB vector and further integrated into the genome of P. pastoris X-33 under the control of the glyceraldehyde 3-phosphate dehydrogenase (GAP) constitutive promoter. A major protein with a molecular mass of 50 kDa was found to be secreted into the culture medium and was identified using anti-HPLRP1 polyclonal antibodies as HPLRP1 recombinant protein. The level of expression reached 100-120 mg of HPLRP1 per liter of culture medium after 40 h, as attested by specific and quantitative enzyme-linked immunosorbent assay. A single cation-exchange chromatography sufficed to obtain a highly purified recombinant HPLRP1 after direct batch adsorption onto S-Sepharose of the HPLRP1 present in the culture medium, at pH 5.5. N-terminal sequencing and mass spectrometry analysis were carried out to monitor the production of the mature protein and to confirm that its signal peptide was properly processed.

Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

PubMed Central

Manske, Magnus; Miotto, Olivo; Campino, Susana; Auburn, Sarah; Almagro-Garcia, Jacob; Maslen, Gareth; O’Brien, Jack; Djimde, Abdoulaye; Doumbo, Ogobara; Zongo, Issaka; Ouedraogo, Jean-Bosco; Michon, Pascal; Mueller, Ivo; Siba, Peter; Nzila, Alexis; Borrmann, Steffen; Kiara, Steven M.; Marsh, Kevin; Jiang, Hongying; Su, Xin-Zhuan; Amaratunga, Chanaki; Fairhurst, Rick; Socheat, Duong; Nosten, Francois; Imwong, Mallika; White, Nicholas J.; Sanders, Mandy; Anastasi, Elisa; Alcock, Dan; Drury, Eleanor; Oyola, Samuel; Quail, Michael A.; Turner, Daniel J.; Rubio, Valentin Ruano; Jyothi, Dushyanth; Amenga-Etego, Lucas; Hubbart, Christina; Jeffreys, Anna; Rowlands, Kate; Sutherland, Colin; Roper, Cally; Mangano, Valentina; Modiano, David; Tan, John C.; Ferdig, Michael T.; Amambua-Ngwa, Alfred; Conway, David J.; Takala-Harrison, Shannon; Plowe, Christopher V.; Rayner, Julian C.; Rockett, Kirk A.; Clark, Taane G.; Newbold, Chris I.; Berriman, Matthew; MacInnis, Bronwyn; Kwiatkowski, Dominic P.

2013-01-01

Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. 1,2 Here we describe methods for large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short term culture. Analysis of 86,158 exonic SNPs that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome. PMID:22722859

Molecular Analysis of Methanogen Richness in Landfill and Marshland Targeting 16S rDNA Sequences

PubMed Central

Yadav, Shailendra; Kundu, Sharbadeb; Ghosh, Sankar K.; Maitra, S. S.

2015-01-01

Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about “methanogenic archaea composition” and “abundance” in the contrasting ecosystems like “landfill” and “marshland” may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process. PMID:26568700

Pattern of elemental release during the granite dissolution can be changed by aerobic heterotrophic bacterial strains isolated from Damma Glacier (central Alps) deglaciated granite sand.

PubMed

Lapanje, Aleš; Wimmersberger, Celine; Furrer, Gerhard; Brunner, Ivano; Frey, Beat

2012-05-01

Colonisation and weathering of freshly deglaciated granite are key processes in initial soil formation and development. We have obtained 438 isolates from granite sand covering glacial toe, 284 isolates at 22°C and 154 at 4°C incubation temperatures, respectively, to obtain cultures for the investigation of their weathering capabilities under laboratory conditions. The isolation of bacteria from granite sand was performed on rich-, intermediate- and low-nutrient-content solid media. Isolates were identified by 16S rRNA gene sequencing. According to the genera-associated weathering capabilities described in the literature and according to their abundance in our culture collection, we selected eight strains to analyse their effects on the weathering dynamics of granite sand during the batch culture experiment. Analysis of culturable bacteria showed higher species richness among isolates from 22°C than from 4°C incubations. In the R2A and 1/100 Ravan media, we observed the highest species richness of isolates obtained at 22°C and 4°C incubation temperatures, respectively. The obtained 16S rRNA sequences revealed the presence of alpha-, beta- and gamma-proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The most numerous group of isolates was distantly related to Collimonas representatives, and according to the sequences of the 16S rRNA genes, they can form a new genus. Isolates from this group had the capability of causing increased dissolution rates for Fe, W, Ni and Rb. In general, at each sampling during the 30-day experiment, every strain showed a unique weathering profile resulting from differential rates of the dissolution and the precipitation of different minerals in the batch culture. Consequently, the presence of different strains, their growth stage and changes in proportions of strains in the bacterial community can affect further soil development and the successive colonisation by plants.

Propionibacterium acnes endocarditis: a case series.

PubMed

Banzon, J M; Rehm, S J; Gordon, S M; Hussain, S T; Pettersson, G B; Shrestha, N K

2017-06-01

Propionibacterium acnes remains a rare cause of infective endocarditis (IE). It is challenging to diagnose due to the organism's fastidious nature and the indolent presentation of the disease. The purpose of this study was to describe the clinical presentation and management of P. acnes IE with an emphasis on the methods of diagnosis. We identified patients from the Cleveland Clinic Infective Endocarditis Registry who were admitted from 2007 to 2015 with definite IE by Duke Criteria. Propionibacterium acnes was defined as the causative pathogen if it was identified in at least two culture specimens, or identified with at least two different modalities: blood culture, valve culture, valve sequencing or histopathological demonstration of microorganisms. We identified 24 cases of P. acnes IE, 23 (96%) of which were either prosthetic valve endocarditis or IE on an annuloplasty ring. Invasive disease (71%) and embolic complications (29%) were common. All but one patient underwent surgery. Propionibacterium acnes was identified in 12.5% of routine blood cultures, 75% of blood cultures with extended incubation, 55% of valve cultures, and 95% of valve sequencing specimens. In 11 of 24 patients (46%), no causative pathogen would have been identified without valve sequencing. Propionibacterium acnes almost exclusively causes prosthetic valve endocarditis and patients often present with advanced disease. The organism may not be readily cultured, and extended cultures appear to be necessary. In patients who have undergone surgery, valve sequencing is most reliable in establishing the diagnosis. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

A Natural View of Microbial Biodiversity within Hot Spring Cyanobacterial Mat Communities

PubMed Central

Ward, David M.; Ferris, Michael J.; Nold, Stephen C.; Bateson, Mary M.

1998-01-01

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats. PMID:9841675

A natural view of microbial biodiversity within hot spring cyanobacterial mat communities

NASA Technical Reports Server (NTRS)

Ward, D. M.; Ferris, M. J.; Nold, S. C.; Bateson, M. M.

1998-01-01

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.

Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

PubMed

Raphael, Brian H; Bradshaw, Marite; Kalb, Suzanne R; Joseph, Lavin A; Lúquez, Carolina; Barr, John R; Johnson, Eric A; Maslanka, Susan E

2014-05-01

Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

Exploring Nitrilase Sequence Space for Enantioselective Catalysis†

PubMed Central

Robertson, Dan E.; Chaplin, Jennifer A.; DeSantis, Grace; Podar, Mircea; Madden, Mark; Chi, Ellen; Richardson, Toby; Milan, Aileen; Miller, Mark; Weiner, David P.; Wong, Kelvin; McQuaid, Jeff; Farwell, Bob; Preston, Lori A.; Tan, Xuqiu; Snead, Marjory A.; Keller, Martin; Mathur, Eric; Kretz, Patricia L.; Burk, Mark J.; Short, Jay M.

2004-01-01

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 106 to 1010 members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses. PMID:15066841

Identification of interleukin-8 converting enzyme as cathepsin L.

PubMed

Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

2003-06-26

IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla)

PubMed Central

2010-01-01

Adenoviruses (AdV) broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL), preterminal protein (pTP) and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2 - 10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B). Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses. PMID:21054831

A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla).

PubMed

Wevers, Diana; Leendertz, Fabian H; Scuda, Nelly; Boesch, Christophe; Robbins, Martha M; Head, Josephine; Ludwig, Carsten; Kühn, Joachim; Ehlers, Bernhard

2010-11-05

Adenoviruses (AdV) broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL), preterminal protein (pTP) and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2-10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B). Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

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.

A generic assay for whole-genome amplification and deep sequencing of enterovirus A71

PubMed Central

Tan, Le Van; Tuyen, Nguyen Thi Kim; Thanh, Tran Tan; Ngan, Tran Thuy; Van, Hoang Minh Tu; Sabanathan, Saraswathy; Van, Tran Thi My; Thanh, Le Thi My; Nguyet, Lam Anh; Geoghegan, Jemma L.; Ong, Kien Chai; Perera, David; Hang, Vu Thi Ty; Ny, Nguyen Thi Han; Anh, Nguyen To; Ha, Do Quang; Qui, Phan Tu; Viet, Do Chau; Tuan, Ha Manh; Wong, Kum Thong; Holmes, Edward C.; Chau, Nguyen Van Vinh; Thwaites, Guy; van Doorn, H. Rogier

2015-01-01

Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples. PMID:25704598

Comparative analysis of Gram's stain, PNA-FISH and Sepsityper with MALDI-TOF MS for the identification of yeast direct from positive blood cultures.

PubMed

Gorton, Rebecca L; Ramnarain, P; Barker, K; Stone, N; Rattenbury, S; McHugh, T D; Kibbler, C C

2014-10-01

Fungaemia diagnosis could be improved by reducing the time to identification of yeast from blood cultures. This study aimed to evaluate three rapid methods for the identification of yeast direct from blood cultures; Gram's stain analysis, the AdvanDX Peptide Nucleic Acid in Situ Hybridisation Yeast Traffic Light system (PNA-FISH YTL) and Bruker Sepsityper alongside matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Fifty blood cultures spiked with a known single yeast strain were analysed by blinded operators experienced in each method. Identifications were compared with MALDI-TOF MS CHROMagar Candida culture and ITS rRNA sequence-based identifications. On first attempt, success rates of 96% (48/50) and 76% (36/50) were achieved using PNA-FISH YTL and Gram's stain respectively. MALDI-TOF MS demonstrated a success rate of 56% (28/50) when applying manufacturer's species log score thresholds and 76% (38/50) using in-house parameters, including lowering the species log score threshold to >1.5. In conclusion, PNA-FISH YTL demonstrated a high success rate successfully identifying yeast commonly encountered in fungaemia. Sepsityper(™) with MALDI-TOF MS was accurate but increased sensitivity is required. Due to the misidentification of commonly encountered yeast Gram's stain analysis demonstrated limited utility in this setting. © 2014 Blackwell Verlag GmbH.

Amplicon Sequencing of the slpH Locus Permits Culture-Independent Strain Typing of Lactobacillus helveticus in Dairy Products

PubMed Central

Moser, Aline; Wüthrich, Daniel; Bruggmann, Rémy; Eugster-Meier, Elisabeth; Meile, Leo; Irmler, Stefan

2017-01-01

The advent of massive parallel sequencing technologies has opened up possibilities for the study of the bacterial diversity of ecosystems without the need for enrichment or single strain isolation. By exploiting 78 genome data-sets from Lactobacillus helveticus strains, we found that the slpH locus that encodes a putative surface layer protein displays sufficient genetic heterogeneity to be a suitable target for strain typing. Based on high-throughput slpH gene sequencing and the detection of single-base DNA sequence variations, we established a culture-independent method to assess the biodiversity of the L. helveticus strains present in fermented dairy food. When we applied the method to study the L. helveticus strain composition in 15 natural whey cultures (NWCs) that were collected at different Gruyère, a protected designation of origin (PDO) production facilities, we detected a total of 10 sequence types (STs). In addition, we monitored the development of a three-strain mix in raclette cheese for 17 weeks. PMID:28775722

An implementation of next generation sequencing for prevention and diagnosis of urinary tract infection in urology.

PubMed

Mouraviev, Vladimir; McDonald, Michael

2018-06-01

The changing face of current infection phenotypes from planktonic to biofilm type has been developed implicating bacterial biofilms in recurrent infection. To date, no specific medical treatment exists to specifically target biofilms in the human host. Similarly, the identification of a biofilm has relied upon the analysis of tissue samples with electron microscopy or DNA identification with polymerase chain reaction (PCR) and sequencing. Standard culture and sensitivity test is not able to detect a presence of biofilms. Two types of molecular microbial diagnostic testing 'levels' are performed as noted below. In both types of analysis, the microbial DNA is extracted from the patient's sample. The patient report contains information about the pathogenic bacterial and fungal microorganisms detected, bacterial load and resistance genes to different antibiotics. Once the bacteria have been identified antibiotic recommendations are made based on research confirming the effectiveness of treatment. The technique was tested in 112 patients in different areas of urology for prevention and treatment purpose. The clinical application of next generation sequence in different clinical phase I-II trials (acute cystitis in 56 patients, rectal swabs before transrectal prostate biopsy in 32 men, neurogenic bladder in 13 patients, chronic bacterial prostatitis in 17 men) demonstrated that this novel approach extends our knowledge about the microbiome of the urogenital tract in both men and women. DNA sequence has a high sensitivity to detect a bacterial and fungal association with resistant genes to antibiotics revealed allowing to implement a targeted and individual prevention and treatment of urinary tract infection (UTI) with improved efficacy compared to standard culture and sensitivity technique. The next generation DNA sequence technology enables the discovery of new concepts regarding the role of microorganisms in diseases of the urinary tract with an individualized approach for a more accurate diagnosis, prevention, prophylaxis and treatment of UTI.

Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya.

PubMed

Azwai, S M; Alfallani, E A; Abolghait, S K; Garbaj, A M; Naas, H T; Moawad, A A; Gammoudi, F T; Rayes, H M; Barbieri, I; Eldaghayes, I M

2016-01-01

The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×10(4) CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×10(4) CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.

Ancient mitochondrial DNA and ancestry of Paquimé inhabitants, Casas Grandes (A.D. 1200-1450).

PubMed

Morales-Arce, Ana Y; Snow, Meradeth H; Kelley, Jane H; Anne Katzenberg, M

2017-07-01

The Casas Grandes (Paquimé) culture, located in the Northwest of Chihuahua, Mexico reached its apogee during the Medio Period (A.D. 1200-1450). Paquimé was abandoned by the end of the Medio Period (A.D. 1450), and the ancestry of its inhabitants remains unsolved. Some authors suggest that waves of Mesoamerican immigrants, possibly merchants, stimulated Paquimé's development during the Medio Period. Archaeological evidence suggests possible ties to groups that inhabited the Southwestern US cultures. This study uses ancient DNA analysis from fourteen samples to estimate genetic affinities of ancient Paquimé inhabitants. DNA was extracted from 14 dental ancient samples from Paquimé. PCR and Sanger sequencing were used to obtain mitochondrial control region sequences. Networks, PCoA, and Nei genetic distances were estimated to compare Paquimé haplotypes against available past haplotypes data from Southwestern and Mesoamerican groups. Haplogroups were characterized for 11 of the samples, and the results revealed the presence of four distinct Amerindian mitochondrial lineages: B (n = 5; 45%), A (n = 3; 27%), C (n = 2; 18%) and D (n = 1; 10%). Statistical analysis of the haplotypes, haplogroup frequencies, and Nei genetic distances showed close affinity of Paquimé with Mimbres. Although our results provide strong evidence of genetic affinities between Paquimé and Mimbres, with the majority of haplotypes shared or derived from ancient Southwest populations, the causes of cultural development at Paquimé still remain a question. These preliminary results provide evidence in support of other bioarchaeological studies, which have shown close biological affinities between Paquimé and Mimbres, a Puebloan culture, in the Southwestern US. © 2017 Wiley Periodicals, Inc.

Yeast community in traditional Portuguese Serpa cheese by culture-dependent and -independent DNA approaches.

PubMed

Gonçalves Dos Santos, Maria Teresa P; Benito, María José; Córdoba, María de Guía; Alvarenga, Nuno; Ruiz-Moyano Seco de Herrera, Santiago

2017-12-04

This study investigated the yeast community present in the traditional Portuguese cheese, Serpa, by culture-dependent and -independent methods. Sixteen batches of Serpa cheeses from various regional industries registered with the Protected Designation of Origin (PDO) versus non-PDO registered, during spring and winter, were used. Irrespective of the producer, the yeast counts were around 5log CFU/g in winter and, overall, were lower in spring. The yeast species identified at the end of ripening (30days), using PCR-RFLP analysis and sequencing of the 26S rRNA, mainly corresponded to Debaryomyces hansenii and Kluyveromyces marxianus, with Candida spp. and Pichia spp. present to a lesser extent. The culture-independent results, obtained using high-throughput sequencing analysis, confirmed the prevalence of Debaryomyces spp. and Kluyveromyces spp. but, also, that Galactomyces spp. was relevant for three of the five producers, which indicates its importance during the early stages of the cheese ripening process, considering it was not found among the dominant viable yeast species. In addition, differences between the identified yeast isolated from cheeses obtained from PDO and non-PDO registered industries, showed that the lack of regulation of the cheese-making practice, may unfavourably influence the final yeast microbiota. The new knowledge provided by this study of the yeast diversity in Serpa cheese, could be used to modify the cheese ripening conditions, to favour desirable yeast species. Additionally, the prevalent yeast isolates identified, Debaryomyces hansenii and Kluyveromyces spp., may have an important role during cheese ripening and in the final sensorial characteristics. Thus, the study of their technological and functional properties could be relevant, in the development of an autochthonous starter culture, to ensure final quality and safety of the cheese. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

PubMed Central

Azwai, S.M.; Alfallani, E.A.; Abolghait, S.K.; Garbaj, A.M.; Naas, H.T.; Moawad, A.A.; Gammoudi, F.T.; Rayes, H.M.; Barbieri, I.; Eldaghayes, I.M.

2016-01-01

The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6 % of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 ×104 CFU\\g. Chicken burger samples showed the highest bacterial count with 6.5 ×104 CFU\\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products. PMID:27004169

Isolation and molecular cloning of a fast-growing strain of human hepatitis A virus from its double-stranded replicative form.

PubMed Central

Venuti, A; Di Russo, C; del Grosso, N; Patti, A M; Ruggeri, F; De Stasio, P R; Martiniello, M G; Pagnotti, P; Degener, A M; Midulla, M

1985-01-01

A fast-growing strain of human hepatitis A virus was selected and characterized. The virus has the unusual property of developing a strong cytopathic effect in tissue culture in 7 to 10 days. Sequences of the viral genome were cloned into recombinant plasmids with the double-stranded replicative form as a template for the reverse transcription of cDNA. Restriction analysis and direct sequencing indicate that this strain is different from that described by Ticehurst et al. (Proc. Natl. Acad. Sci. USA 80:5885-5889, 1983) in the region that presumptively codes for the major capsid protein VP1, but both isolates have conserved large areas of homology in the untranslated 5'-terminal sequences of the genome. Images PMID:2997478

Identification of cDNAs encoding HSP70 and HSP90 in the abalone Haliotis tuberculata: Transcriptional induction in response to thermal stress in hemocyte primary culture.

PubMed

Farcy, Emilie; Serpentini, Antoine; Fiévet, Bruno; Lebel, Jean-Marc

2007-04-01

Heat-shock proteins are a multigene family of proteins whose expression is induced by a variety of stress factors. This work reports the cloning and sequencing of HSP70 and HSP90 cDNAs in the gastropod Haliotis tuberculata. The deduced amino acid sequences of both HSP70 and HSP90 from H. tuberculata shared a high degree of homology with their homologues in other species, including typical eukaryotic HSP70 and HSP90 signature sequences. We examined their transcription expression pattern in abalone hemocytes exposed to thermal stress. Real-time PCR analysis indicated that both HSP70 and HSP90 mRNA were expressed in control animals but rapidly increased after heat-shock.

Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

NASA Astrophysics Data System (ADS)

Chen, K.

2017-01-01

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens

PubMed Central

Pardo, Belén G; Fernández, Carlos; Millán, Adrián; Bouza, Carmen; Vázquez-López, Araceli; Vera, Manuel; Alvarez-Dios, José A; Calaza, Manuel; Gómez-Tato, Antonio; Vázquez, María; Cabaleiro, Santiago; Magariños, Beatriz; Lemos, Manuel L; Leiro, José M; Martínez, Paulino

2008-01-01

Background The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. Results A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value ≤ 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. Conclusion A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot. PMID:18817567

Diversity of Cultured Thermophilic Anaerobes in Hot Springs of Yunnan Province, China

NASA Astrophysics Data System (ADS)

Lin, L.; Lu, Y.; Dong, X.; Liu, X.; Wei, Y.; Ji, X.; Zhang, C.

2010-12-01

Thermophilic anaerobes including Archaea and Bacteria refer to those growing optimally at temperatures above 50°C and do not use oxygen as the terminal electron acceptor for growth. Study on thermophilic anaerobes will help to understand how life thrives under extreme conditions. Meanwhile thermophilic anaerobes are of importance in potential application and development of thermophilic biotechnology. We have surveyed culturable thermophilic anaerobes in hot springs (pH6.5-7.5; 70 - 94°C) in Rehai of Tengchong, Bangnazhang of Longlin, Eryuan of Dali,Yunnan, China. 50 strains in total were cultured from the hot springs water using Hungate anaerobic technique, and 30 strains were selected based on phenotypic diversity for analysis of 16S rDNA sequences. Phylogenetic analysis showed that 28 strains belonged to the members of five genera: Caldanaerobacter, Calaramator, Thermoanaerobacter, Dictyoglomus and Fervidobacterium, which formed five branches on the phylogenetic tree. Besides, 2 strains of methanogenic archaea were obtained. The majority of the isolates were the known species, however, seven strains were identified as novel species affiliated to the five genera based on the lower 16S rDNA sequence similarities (less than 93 - 97%) with the described species. This work would provide the future study on their diversity, distribution among different regions and the potential application of thermophilic enzyme. Supported by State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences(SKLMR-080605)and the Foundation of State Natural Science (30660009, 30960022, 31081220175).

A multi-laboratory profile of Mycoplasma contamination in Lawsonia intracellularis cultures

PubMed Central

2012-01-01

Background During the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem. When Mycoplasma contamination occurs in laboratories that study L. intracellularis, the cultures must be discarded for 4 reasons: 1) Mycoplasma is inevitably concentrated along with L. intracellularis during the passage of L. intracellularis; 2) Mycoplasma inhibits the growth of L. intracellularis; and 3) it is impossible to selectively eliminate Mycoplasma in L. intracellularis cultures. In this study, we observed the contamination of Mycoplasma species during L. intracellularis cultivation among multiple laboratories. Results The presence of a Mycoplasma infection in the L. intracellularis cultures was verified using polymerase chain reaction (PCR), and a sequence analysis of the partial 16S rRNA and 23S rRNA genes was performed. A PCR-based assay using genus-specific universal primers revealed that 29 (85.3%) of the 34 cultures were contaminated with Mycoplasma, including 26 with M. hyorhinis (89.2%), 2 with M. orale (6.9%), and 1 with M. fermentans (3.4%). The Mycoplasma contamination was not the result of infection with material of pig origin. McCoy cells, which are required for the cultivation of L. intracellularis, were also ruled out as the source of the Mycoplasma contamination. Conclusions In this study, M. hyorhinis was identified as the most common mollicute that contaminated L. intracellularis cultures. Whether L. intracellularis enhances the biological properties of Mycoplasma to promote infection in McCoy cells is not known. Because the McCoy cell line stocks that were used simultaneously were all negative for Mycoplasma, and the same worker handled both the McCoy cells to maintain the bacteria and the L. intracellularis cultures, it is possible that the L. intracellularis cultures are more vulnerable to Mycoplasma contamination. Taken together, these results suggest that continuous cultures of L. intracellularis must be tested for Mycoplasma contamination at regular intervals. The GenBank accession numbers for the sequences reported in this paper are JN689375 to JN689377. PMID:22284165

Phylogenetic analysis of Fusobacterium prausnitzii based upon the 16S rRNA gene sequence and PCR confirmation.

PubMed

Wang, R F; Cao, W W; Cerniglia, C E

1996-01-01

In order to develop a PCR method to detect Fusobacterium prausnitzii in human feces and to clarify the phylogenetic position of this species, its 16S rRNA gene sequence was determined. The sequence described in this paper is different from the 16S rRNA gene sequence is specific for F. prausnitzii, and the results of this assay confirmed that F. prausnitzii is the most common species in human feces. However, a PCR assay based on the original GenBank sequence was negative when it was performed with two strains of F. prausnitzii obtained from the American Type Culture Collection. A phylogenetic tree based on the new 16S rRNA gene sequence was constructed. On this tree F. prausnitzii was not a member of the Fusobacterium group but was closer to some Eubacterium spp. and located between Clostridium "clusters III and IV" (M.D. Collins, P.A. Lawson, A. Willems, J.J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J.A.E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994).

Isolation of Corynebacterium tuscaniae sp. nov. from Blood Cultures of a Patient with Endocarditis

PubMed Central

Riegel, Philippe; Creti, Roberta; Mattei, Romano; Nieri, Alfredo; von Hunolstein, Christina

2006-01-01

A strain of an unknown coryneform bacterium was repeatedly isolated in pure culture from the blood of a patient affected by endocarditis. Comparative 16S rRNA gene sequence analysis revealed that this isolate represented a new subline within the genus Corynebacterium. This new taxon can be identified by the presence of corynomycolic acids and its enzymatic activities and fermentation of sugars. Acid production from glucose and maltose, pyrazinamidase and alkaline phoshatase activities, and hippurate hydrolysis were the most characteristic phenotypic features of the bacterium. On the basis of both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species, Corynebacterium tuscaniae sp. nov. The type strain, ISS-5309, has been deposited in the American Type Culture Collection (ATCC BAA-1141) and in the Culture Collection of the University of Göteborg (CCUG 51321). PMID:16455875

Assessing the diversity of AM fungi in arid gypsophilous plant communities.

PubMed

Alguacil, M M; Roldán, A; Torres, M P

2009-10-01

In the present study, we used PCR-Single-Stranded Conformation Polymorphism (SSCP) techniques to analyse arbuscular mycorrhizal fungi (AMF) communities in four sites within a 10 km(2) gypsum area in Southern Spain. Four common plant species from these ecosystems were selected. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, SSCP analysis, sequencing and phylogenetic analyses. A total of 1443 SSU rRNA sequences were analysed, for 21 AM fungal types: 19 belonged to the genus Glomus, 1 to the genus Diversispora and 1 to the Scutellospora. Four sequence groups were identified, which showed high similarity to sequences of known glomalean species or isolates: Glo G18 to Glomus constrictum, Glo G1 to Glomus intraradices, Glo G16 to Glomus clarum, Scut to Scutellospora dipurpurescens and Div to one new genus in the family Diversisporaceae identified recently as Otospora bareai. There were three sequence groups that received strong support in the phylogenetic analysis, and did not seem to be related to any sequences of AM fungi in culture or previously found in the database; thus, they could be novel taxa within the genus Glomus: Glo G4, Glo G2 and Glo G14. We have detected the presence of both generalist and potential specialist AMF in gypsum ecosystems. The AMF communities were different in the plant studied suggesting some degree of preference in the interactions between these symbionts.

Enterocin TW21, a novel bacteriocin from dochi-isolated Enterococcus faecium D081821.

PubMed

Chang, S-Y; Chen, Y-S; Pan, S-F; Lee, Y-S; Chang, C-H; Chang, C-H; Yu, B; Wu, H-C

2013-09-01

Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821. Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300·6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2 -ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin. Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin. A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative. © 2013 The Society for Applied Microbiology.

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

Lagrimini, L.M.

Since this manuscript was submitted we have conducted a more thorough physiological analysis of water relations in wild-type and peroxidase overproducing plants. These experiments include pressure bomb, plasmolysis, and membrane integrity analysis. We are also in the process of analyzing other phenotypes in peroxidase overproducer plants such as excessive browning of tissue, the rapid death of tissue in culture, and poor germination of seed. Transformed plants of Nicotiana tabacum and Nicotiana sylvestris were obtained which have peroxidase activity 3--7 fold lower than wild-type plants. This was done by introducing a chimeric gene composed of the CaMV 35S promoter and themore » 5' half of the tobacco anionic peroxidase cDNA in the antisense RNA configuration. A manuscript which describes this work is being written, and will be submitted for publication in January 1990. The anionic peroxidase gene has been cloned by hybridization to the cloned cDNA. The entire gene is contained on an 8.7kb fragment within a lambda phage clone. Several smaller DNA fragments have been subcloned, and some have been sequenced. One exon within the coding sequence has been sequenced, along with the partial sequence of two introns. Further sequencing is being carried-out to identify the promoter, which will be later joined to a reporter gene. 6 figs.« less

Development of PCR for screening of enteroaggregative Escherichia coli.

PubMed Central

Schmidt, H; Knop, C; Franke, S; Aleksic, S; Heesemann, J; Karch, H

1995-01-01

In this study, we determined the sequence of the EcoRI-PstI fragment of the plasmid pCVD432, also termed the enteroaggregative Escherichia coli (EAggEC) probe. A primer pair complementary to this probe was designed for PCR amplification of a 630-bp region. Comparison of the analysis of the EAggEC probe sequence with those in database libraries revealed no significant similarity to any known bacterial gene. Pure cultures of E. coli cells, as well as mixed cultures from stool specimens, were investigated with the PCR assay, the EAggEC probe test, and the adherence test. Of 50 E. coli strains which demonstrated aggregative adherence to HEp-2 cells, 43 (86%) were positive with the EAggEC PCR. All 43 of these strains reacted with the EAggEC probe. Six EAggEC strains gave negative results by both molecular techniques. In contrast, only 4 of 418 (0.96%) strains representing other categories of diarrheagenic E. coli demonstrated a positive PCR result. The PCR was also successful in screening for the presence of EAggEC in enriched cultures grown from stool specimens. Compared with cell culture assays and colony hybridization, our findings revealed that the PCR assay was more rapid, simple, and highly sensitive and can therefore be recommended as a screening method for EAggEC in the clinical laboratory. PMID:7751380

The Potential Impact of Directionality, Colour Perceptions and Cultural Associations on Disaster Messages During Heatwaves in the UK

PubMed Central

Tang, Chris; Rundblad, Gabriella

2015-01-01

The health risks posed by heatwaves have been well documented. In the UK, before and during a heatwave, alerts are issued to the general public based on a tiered warning system integrating the use of colour and number sequences. There has of yet been no formal assessment of the public response to these messages. Cultural and language barriers make some members of ethnic minority communities particularly hard to reach. These may be less challenging amongst younger community members, who may be well placed to instigate the circulation of warning information to those less able or willing to use conventional channels. This qualitative study assesses the role of age and ethnic and cultural background in the conceptualisation of the number and colour systems used as part of the Heat-Health Watch System (HHWS) and the National Severe Weather Warning Service (NSWWS). Young and older participants were recruited from the Bangladeshi and white British populations of Tower Hamlets. All participants were given a cognitive task that required them to identify and draw associations between 12 cards depicting the four colours and numbers used in the warning system and four pictures providing contextualisation in terms of heatwave risk. A qualitative analysis of the heuristics used in the group discussions provided insights into the conceptualisations basic to interpreting colour and number sequences as representations of risk graduations, and how interpretation might be influenced by age and ethnic and cultural background. There were considerable differences in the interpretation of young Bangladeshi and older white British participants, on the one hand, and older Bangladeshi participants, on the other. Young Bangladeshis and older white British participants conceptualised the colours and numbers as a vertical scale, with the numbers/colours at “the top” corresponding to representations of higher temperature. This conceptualisation was mainly based on strong associations between colour and temperature, with risk only associated with the upper limit of the scale. Older Bangladeshi participants, on the other hand, conceptualised the numbers and pictures as a narrative sequence and disassociated the colours from the other cards. The differences between groups suggest potential cultural barriers to the “intended” interpretation of the colour and number sequences for older Bangladeshis but not for young Bangladeshis. The fact that the predominant association for the colour sequence for both young Bangladeshis and older white British participants was with graduations of temperature rather than risk raises questions about the applicability of using colours in a tiered warning system. PMID:25932346

Molecular Microbial Analyses of the Mars Exploration Rovers Assembly Facility

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; LaDuc, Myron T.; Newcombe, David; Kempf, Michael J.; Koke, John. A.; Smoot, James C.; Smoot, Laura M.; Stahl, David A.

2004-01-01

During space exploration, the control of terrestrial microbes associated with robotic space vehicles intended to land on extraterrestrial solar system bodies is necessary to prevent forward contamination and maintain scientific integrity during the search for life. Microorganisms associated with the spacecraft assembly environment can be a source of contamination for the spacecraft. In this study, we have monitored the microbial burden of air samples of the Mars Exploration Rovers' assembly facility at the Kennedy Space Center utilizing complementary diagnostic tools. To estimate the microbial burden and identify potential contaminants in the assembly facility, several microbiological techniques were used including culturing, cloning and sequencing of 16S rRNA genes, DNA microarray analysis, and ATP assays to assess viable microorganisms. Culturing severely underestimated types and amounts of contamination since many of the microbes implicated by molecular analyses were not cultivable. In addition to the cultivation of Agrobacterium, Burkholderia and Bacillus species, the cloning approach retrieved 16s rDNA sequences of oligotrophs, symbionts, and y-proteobacteria members. DNA microarray analysis based on rational probe design and dissociation curves complemented existing molecular techniques and produced a highly parallel, high resolution analysis of contaminating microbial populations. For instance, strong hybridization signals to probes targeting the Bacillus species indicated that members of this species were present in the assembly area samples; however, differences in dissociation curves between perfect-match and air sample sequences showed that these samples harbored nucleotide polymorphisms. Vegetative cells of several isolates were resistant when subjected to treatments of UVC (254 nm) and vapor H202 (4 mg/L). This study further validates the significance of non-cultivable microbes in association with spacecraft assembly facilities, as our analyses have identified several non-cultivable microbes likely to contaminate the surfaces of spacecraft hardware.

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups

PubMed Central

Khan, Mohammad Adil; Knox, Natalie; Prashar, Akriti; Alexander, David; Abdel-Nour, Mena; Duncan, Carla; Tang, Patrick; Amatullah, Hajera; Dos Santos, Claudia C.; Tijet, Nathalie; Low, Donald E.; Pourcel, Christine; Van Domselaar, Gary; Terebiznik, Mauricio; Ensminger, Alexander W.; Guyard, Cyril

2013-01-01

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence. PMID:23826259

Transcriptome analysis of the honey bee fungal pathogen, Ascosphaera apis: implications for host pathogenesis

PubMed Central

2012-01-01

Background We present a comprehensive transcriptome analysis of the fungus Ascosphaera apis, an economically important pathogen of the Western honey bee (Apis mellifera) that causes chalkbrood disease. Our goals were to further annotate the A. apis reference genome and to identify genes that are candidates for being differentially expressed during host infection versus axenic culture. Results We compared A. apis transcriptome sequence from mycelia grown on liquid or solid media with that dissected from host-infected tissue. 454 pyrosequencing provided 252 Mb of filtered sequence reads from both culture types that were assembled into 10,087 contigs. Transcript contigs, protein sequences from multiple fungal species, and ab initio gene predictions were included as evidence sources in the Maker gene prediction pipeline, resulting in 6,992 consensus gene models. A phylogeny based on 12 of these protein-coding loci further supported the taxonomic placement of Ascosphaera as sister to the core Onygenales. Several common protein domains were less abundant in A. apis compared with related ascomycete genomes, particularly cytochrome p450 and protein kinase domains. A novel gene family was identified that has expanded in some ascomycete lineages, but not others. We manually annotated genes with homologs in other fungal genomes that have known relevance to fungal virulence and life history. Functional categories of interest included genes involved in mating-type specification, intracellular signal transduction, and stress response. Computational and manual annotations have been made publicly available on the Bee Pests and Pathogens website. Conclusions This comprehensive transcriptome analysis substantially enhances our understanding of the A. apis genome and its expression during infection of honey bee larvae. It also provides resources for future molecular studies of chalkbrood disease and ultimately improved disease management. PMID:22747707

Piaget Versus Materialism: A Concordance of the Discordance.

ERIC Educational Resources Information Center

Burger, Henry G.

The purpose of this paper is to disconfirm Piaget's theory of an invariant sequence of intellectual maturation for all youths in all cultures. The author's view is that the kinds and sequences of human development vary greatly, depending on the cultural milieu. The author derives 36 propositions from Piaget's theory, dismisses 5 of them as…

Draft Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a Citrate-Fermenting Strain.

PubMed

Zuljan, Federico; Espariz, Martín; Blancato, Victor S; Esteban, Luis; Alarcón, Sergio; Magni, Christian

2016-02-04

We report the draft genome sequence of Lactococcus lactis subsp. lactis bv. diacetylactis CRL264, a natural strain isolated from artisanal cheese from northwest Argentina. L. lactis subsp. lactis bv. diacetylactis is one of the most important microorganisms used as starter culture around the world. The CRL264 strain constitutes a model microorganism in the studies on the generation of aroma compounds (diacetyl, acetoin, and 2,3-butanediol) by lactic acid bacteria. Our genome analysis shows similar genetic organization to other available genomes of L. lactis bv. diacetylactis strains. Copyright © 2016 Zuljan et al.

OPTIMIZATION OF ALKALINE Α-AMYLASE PRODUCTION BY THERMOPHILIC BACILL US SUBTILIS.

PubMed

Al-Johani, Nuha Bakeet; Al-Seeni, Madeha N; Ahmed, Youssri Mohamed

2017-01-01

Starch-degrading amylase enzyme is important in biotechnological applications as food, fermentation, textile, paper and pharmaceutical purposes. The aim of current study to isolate alkaline thermostable α-amylase bacteria and then study the composition of medium and culture conditions to optimize cells growth and a-amylase production. Thermophilic amylase producing bacterium was isolated from local hot water-springs in Gazan city Saudi Arabia. Phylogenetic analysis of 16 S rRNA sequence for the strain revealed that the strain have the same sequence of Bacillus subtilis . Maximum amylase production was observed, when B. subtilis cultured in medium containing starch at concentration 0.5%, and 10 g/L peptones as nitrogen source at pH 8.5 in when it was incubated for 48 h at 45°C. An amylase-producing bacterium were isolated from hot-spring water and was identified as B. subtilis . Amylase produced from B.subtilis had optimum temperature 45°C and pH 8.5 in shaking media.

Reorganization of low-molecular-weight fraction of plasma proteins in the annual cycle of cyprinidae.

PubMed

Andreeva, A M; Lamas, N E; Serebryakova, M V; Ryabtseva, I P; Bolshakov, V V

2015-02-01

Reorganization of the low-molecular-weight fraction of cyprinid plasma was analyzed using various electrophoretic techniques (disc electrophoresis, electrophoresis in polyacrylamide concentration gradient, in polyacrylamide with urea, and in SDS-polyacrylamide). The study revealed coordinated changes in the low-molecular-weight protein fractions with seasonal dynamics and related reproductive rhythms of fishes. We used cultured species of the Cyprinidae family with sequenced genomes for the detection of these interrelations in fresh-water and anadromous cyprinid species. The common features of organization of fish low-molecular-weight plasma protein fractions made it possible to make reliable identification of their proteins. MALDI mass-spectrometry analysis revealed the presence of the same proteins (hemopexin, apolipoproteins, and serpins) in the low-molecular-weight plasma fraction in wild species and cultured species with sequenced genomes (carp, zebrafish). It is found that the proteins of the first two classes are organized as complexes made of protein oligomers. Stoichiometry of these complexes changes in concordance with the seasonal and reproductive rhythms.

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA.

PubMed

Lemons, Angela R; Lindsley, William G; Green, Brett J

2018-05-02

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis.

PubMed

Huber, R; Burggraf, S; Mayer, T; Barns, S M; Rossnagel, P; Stetter, K O

1995-07-06

A variety of hyperthermophilic bacteria and archaea have been isolated from high-temperature environments by plating and serial dilutions. However, these techniques allow only the small percentage of organisms able to form colonies, or those that are predominant within environmental samples, to be obtained in pure culture. Recently, in situ 16S ribosomal RNA analyses of samples from the Obsidian hot pool at Yellowstone National Park, Wyoming, revealed a variety of archaeal sequences, which were all different from those of previously isolated species. This suggests substantial diversity of archaea with so far unknown morphological, physiological and biochemical features, which may play an important part within high-temperature ecosystems. Here we describe a procedure to obtain pure cultures of unknown organisms harbouring specific 16S rRNA sequences identified previously within the environment. It combines visual recognition of single cells by phylogenetic staining and cloning by 'optical tweezers'. Our result validates polymerase chain reaction data on the existence of large archael communities.

Identification and bioinformatics analysis of microRNAs from the sporophyte and gametophyte of Pyropia haitanensis

NASA Astrophysics Data System (ADS)

Huang, Aiyou; Wang, Guangce

2016-05-01

Pyropia haitanensis (T. J. Chang et B. F. Zheng) N. Kikuchi et M. Miyata ( Porphyra haitanensis) is an economically important genus that is cultured widely in China. P. haitanensis is cultured on a larger scale than Pyropia yezoensis, making up an important part of the total production of cultivated Pyropia in China. However, the majority of molecular mechanisms underlying the physiological processes of P. haitanensis remain unknown. P. haitanensis could utilize inorganic carbon and the sporophytes of P. haitanensis might possess a PCK-type C4-like carbon-fixation pathway. To identify microRNAs and their probable roles in sporophyte and gametophyte development, we constructed and sequenced small RNA libraries from sporophytes and gametophytes of P. haitanensis. Five microRNAs were identified that shared no sequence homology with known microRNAs. Our results indicated that P. haitanensis might posses a complex sRNA processing system in which the novel microRNAs act as important regulators of the development of different generations of P. haitanensis.

Effects of imatinib and nilotinib on the whole transcriptome of cultured murine osteoblasts.

PubMed

Kirschner, Gyöngyi; Balla, Bernadett; Horváth, Péter; Kövesdi, Andrea; Lakatos, Gergely; Takács, István; Nagy, Zsolt; Tóbiás, Bálint; Árvai, Kristóf; Kósa, János Pál; Lakatos, Péter

2016-09-01

Numerous clinical observations have confirmed that breakpoint cluster region-abelson fusion oncoprotein tyrosine kinase inhibitors used in leukemia treatment alter bone physiology in a complex manner. The aim of the present study was to analyze the whole transcriptome of cultured murine osteoblasts and determine the changes following treatment with imatinib and nilotinib using Sequencing by Oligonucleotide Ligation and Detection next generation RNA sequencing. This study also aimed to identify candidate signaling pathways and network regulators by multivariate Ingenuity Pathway Analysis. Based on the right-tailed Fisher's exact test, significantly altered pathways including upstream regulators were defined for each drug. The correlation between these pathways and bone metabolism was also examined. The preliminary results suggest the two drugs have different mechanisms of action on osteoblasts, and imatinib was shown to have a greater effect on gene expression. Data also indicated the potential role of a number of genes and signaling cascades that may contribute to identifying novel targets for the treatment of metabolic bone diseases.

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.

A novel approach to enhance biological nutrient removal using a culture supernatant from Micrococcus luteus containing resuscitation-promoting factor (Rpf) in SBR process.

PubMed

Liu, Yindong; Su, Xiaomei; Lu, Lian; Ding, Linxian; Shen, Chaofeng

2016-03-01

A culture supernatant from Micrococcus luteus containing resuscitation-promoting factor (SRpf) was used to enhance the biological nutrient removal of potentially functional bacteria. The obtained results suggest that SRpf accelerated the start-up process and significantly enhanced the biological nutrient removal in sequencing batch reactor (SBR). PO4 (3-)-P removal efficiency increased by over 12 % and total nitrogen removal efficiency increased by over 8 % in treatment reactor acclimated by SRpf compared with those without SRpf addition. The Illumina high-throughput sequencing analysis showed that SRpf played an essential role in shifts in the composition and diversity of bacterial community. The phyla of Proteobacteria and Actinobacteria, which were closely related to biological nutrient removal, were greatly abundant after SRpf addition. This study demonstrates that SRpf acclimation or addition might hold great potential as an efficient and cost-effective alternative for wastewater treatment plants (WWTPs) to meet more stringent operation conditions and legislations.

Rapid diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis using a molecular-based diagnostic algorithm.

PubMed

Simons, S O; van der Laan, T; Mulder, A; van Ingen, J; Rigouts, L; Dekhuijzen, P N R; Boeree, M J; van Soolingen, D

2014-10-01

There is an urgent need for rapid and accurate diagnosis of pyrazinamide-resistant multidrug-resistant tuberculosis (MDR-TB). No diagnostic algorithm has been validated in this population. We hypothesized that pncA sequencing added to rpoB mutation analysis can accurately identify patients with pyrazinamide-resistant MDR-TB. We identified from the Dutch national database (2007-11) patients with a positive Mycobacterium tuberculosis culture containing a mutation in the rpoB gene. In these cases, we prospectively sequenced the pncA gene. Results from the rpoB and pncA mutation analysis (pncA added to rpoB) were compared with phenotypic susceptibility testing results to rifampicin, isoniazid and pyrazinamide (reference standard) using the Mycobacterial Growth Indicator Tube 960 system. We included 83 clinical M. tuberculosis isolates containing rpoB mutations in the primary analysis. Rifampicin resistance was seen in 72 isolates (87%), isoniazid resistance in 73 isolates (88%) and MDR-TB in 65 isolates (78%). Phenotypic reference testing identified pyrazinamide-resistant MDR-TB in 31 isolates (48%). Sensitivity of pncA sequencing added to rpoB mutation analysis for detecting pyrazinamide-resistant MDR-TB was 96.8%, the specificity was 94.2%, the positive predictive value was 90.9%, the negative predictive value was 98.0%, the positive likelihood was 16.8 and the negative likelihood was 0.03. In conclusion, pyrazinamide-resistant MDR-TB can be accurately detected using pncA sequencing added to rpoB mutation analysis. We propose to include pncA sequencing in every isolate with an rpoB mutation, allowing for stratification of MDR-TB treatment according to pyrazinamide susceptibility. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

New Approaches to Attenuated Hepatitis a Vaccine Development: Cloning and Sequencing of Cell-Culture Adapted Viral cDNA.

DTIC Science & Technology

1987-10-13

after multiple passages in vivo and in vitro. J. Gen. Virol. 67, 1741- 1744. Sabin , A.B. (1985). Oral poliovirus vaccine : history of its development...IN (N NEW APPROACHES TO ATTENUATED HEPATITIS A VACCINE DEVELOPMENT: Q) CLONING AND SEQUENCING OF CELL-CULTURE ADAPTED VIRAL cDNA I ANNUAL REPORT...6ll02Bsl0 A 055 11. TITLE (Include Security Classification) New Approaches to Attenuated Hepatitis A Vaccine Development: Cloning and Sequencing of Cell

Ribosomal RNA gene detection and targeted culture of novel nitrogen-responsive fungal taxa from temperate pine forest soil.

PubMed

Hesse, Cedar N; Torres-Cruz, Terry J; Tobias, Terri Billingsley; Al-Matruk, Maryam; Porras-Alfaro, Andrea; Kuske, Cheryl R

Soil fungal communities are responsible for carbon and nitrogen (N) cycling. The high complexity of the soil fungal community and the high proportion of taxonomically unidentifiable sequences confound ecological interpretations in field studies because physiological information is lacking for many organisms known only by their rRNA sequences. This situation forces experimental comparisons to be made at broader taxonomic racks where functions become difficult to infer. The objective of this study was to determine OTU (operational taxonomic units) level responses of the soil fungal community to N enrichment in a temperate pine forest experiment and to use the sequencing data to guide culture efforts of novel N-responsive fungal taxa. Replicate samples from four soil horizons (up to 10 cm depth) were obtained from ambient, enriched CO 2 and N-fertilization plots. Through a fungal large subunit rRNA gene (LSU) sequencing survey, we identified two novel fungal clades that were abundant in our soil sampling (representing up to 27% of the sequences in some samples) and responsive to changes in soil N. The two N-responsive taxa with no predicted taxonomic association were targeted for isolation and culturing from specific soil samples where their sequences were abundant. Representatives of both OTUs were successfully cultured using a filtration approach. One taxon (OTU6) was most closely related to Saccharomycotina; the second taxon (OTU69) was most closely related to Mucoromycotina. Both taxa likely represent novel species. This study shows how observation of specific OTUs level responses to altered N status in a large rRNA gene field survey provided the impetus to design targeted culture approaches for isolation of novel N-responsive fungal taxa.

Identification, characterization and description of Arcobacter faecis sp. nov., isolated from a human waste septic tank.

PubMed

Whiteduck-Léveillée, Kerri; Whiteduck-Léveillée, Jenni; Cloutier, Michel; Tambong, James T; Xu, Renlin; Topp, Edward; Arts, Michael T; Chao, Jerry; Adam, Zaky; Lévesque, C André; Lapen, David R; Villemur, Richard; Khan, Izhar U H

2016-03-01

A study on the taxonomic classification of Arcobacter species was performed on the cultures isolated from various fecal sources where an Arcobacter strain AF1078(T) from human waste septic tank near Ottawa, Ontario, Canada was characterized using a polyphasic approach. Genetic investigations including 16S rRNA, atpA, cpn60, gyrA, gyrB and rpoB gene sequences of strain AF1078(T) are unique in comparison with other arcobacters. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is most closely related to Arcobacter lanthieri and Arcobacter cibarius. Analyses of atpA, cpn60, gyrA, gyrB and rpoB gene sequences suggested that strain AF1078(T) formed a phylogenetic lineage independent of other species in the genus. Whole-genome sequence, DNA-DNA hybridization, fatty acid profile and phenotypic analysis further supported the conclusion that strain AF1078(T) represents a novel Arcobacter species, for which the name Arcobacter faecis sp. nov. is proposed, with type strain AF1078(T) (=LMG 28519(T); CCUG 66484(T)). Crown Copyright © 2015. Published by Elsevier GmbH. All rights reserved.

A large scale analysis of cDNA in Arabidopsis thaliana: generation of 12,028 non-redundant expressed sequence tags from normalized and size-selected cDNA libraries.

PubMed

Asamizu, E; Nakamura, Y; Sato, S; Tabata, S

2000-06-30

For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.

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.

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

Comparison of two methods for purification of enterocin B, a bacteriocin produced by Enterococcus faecium W3.

PubMed

Dündar, Halil; Atakay, Mehmet; Çelikbıçak, Ömür; Salih, Bekir; Bozoğlu, Faruk

2015-01-01

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

Managing a Mycotic Thoracoabdominal Aneurysm: The Importance of Molecular Diagnostics.

PubMed

Bayliss, Christopher D; Booth, Karen L; Williams, Robin; Dark, John H; Gould, Kate F

2017-11-01

Pneumococcal mycotic aneurysms are rare and associated with significant mortality and morbidity. Early intravenous antibiotic therapy and surgical intervention is the mainstay of treatment. Pneumococci frequently autolyze in blood cultures, making microbiological diagnosis challenging. We present the case of a man in his mid 70s with multiple thoracoabdominal mycotic aneurysms. Surgery was performed to a threatening saccular aortic arch aneurysm. Samples were sent for microbiological analysis and all were culture negative. The samples were then referred for bacterial 16S ribosomal RNA sequencing, which revealed evidence of infection with Streptococcus pneumoniae. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation.

PubMed

Gimenez, Magalí Diana; Yañez-Santos, Anahí Mara; Paz, Rosalía Cristina; Quiroga, Mariana Paola; Marfil, Carlos Federico; Conci, Vilma Cecilia; García-Lampasona, Sandra Claudia

2016-01-01

This is the first report assessing epigenetic variation in garlic. High genetic and epigenetic polymorphism during in vitro culture was detected.Sequencing of MSAP fragments revealed homology with ESTs. Garlic (Allium sativum) is a worldwide crop of economic importance susceptible to viral infections that can cause significant yield losses. Meristem tissue culture is the most employed method to sanitize elite cultivars.Often the virus-free garlic plants obtained are multiplied in vitro (micro propagation). However, it was reported that micro-propagation frequently produces somaclonal variation at the phenotypic level, which is an undesirable trait when breeders are seeking to maintain varietal stability. We employed amplification fragment length polymorphism and methylation sensitive amplified polymorphism (MSAP) methodologies to assess genetic and epigenetic modifications in two culture systems: virus-free plants obtained by meristem culture followed by in vitro multiplication and field culture. Our results suggest that garlic exhibits genetic and epigenetic polymorphism under field growing conditions. However, during in vitro culture system both kinds of polymorphisms intensify indicating that this system induces somaclonal variation. Furthermore, while genetic changes accumulated along the time of in vitro culture, epigenetic polymorphism reached the major variation at 6 months and then stabilize, being demethylation and CG methylation the principal conversions.Cloning and sequencing differentially methylated MSAP fragments allowed us to identify coding and unknown sequences of A. sativum, including sequences belonging to LTR Gypsy retrotransposons. Together, our results highlight that main changes occur in the initial 6 months of micro propagation. For the best of our knowledge, this is the first report on epigenetic assessment in garlic.

Microevolution in prehistoric Andean populations: chronologic mtDNA variation in the desert valleys of northern Chile.

PubMed

Moraga, Mauricio; Santoro, Calogero M; Standen, Vivien G; Carvallo, Pilar; Rothhammer, Francisco

2005-06-01

Archeological evidence suggests that the iconographic and technological developments that took place in the highlands around Lake Titicaca in the Central Andean region had an influence on the cultural elaborations of the human groups in the valleys and the Pacific coast of northern Chile. In a previous communication, we were able to show, by means of a distance analysis, that a craniofacial differentiation accompanied the process of cultural evolution in the valleys (Rothhammer and Santoro [2001] Lat. Am. Antiq. 12:59-66). Recently, numerous South Amerindian mtDNA studies were published, and more accurate molecular techniques to study ancient mtDNA are available. In view of these recent developments, we decided 1) to study chronological changes of ancient mtDNA haplogroup frequencies in the nearby Lluta, Azapa, and Camarones Valleys, 2) to identify microevolutionary forces responsible for such changes, and 3) to compare ancient mtDNA haplogroup frequencies with previous data in order to validate craniometrical results and to reconstruct the biological history of the prehistoric valley groups in the context of their interaction with culturally more developed highland populations. From a total of 97 samples from 83 individuals, 68 samples (61 individuals) yielded amplifications for the fragments that harbor classical mtDNA markers. The haplogroup distribution among the total sample was as follows: 26.2%, haplogroup A; 34.4%, haplogroup B; 14.8%, haplogroup C; 3.3%, haplogroup D; and 21.3%, other haplogroups. Haplogroup B tended to increase, and haplogroup A to decrease during a 3,900-year time interval. The sequence data are congruent with the haplogroup analysis. In fact, the sequencing of hypervariable region I of 30 prehistoric individuals revealed 43 polymorphic sites. Sequence alignment and subsequent phylogenetic tree construction showed two major clusters associated with the most common restriction haplogroups. Individuals belonging to haplogroups C and D tended to cluster together with nonclassical lineages. 2004 Wiley-Liss, Inc.

Characterisation of prototype Nurmi cultures using culture-based microbiological techniques and PCR-DGGE.

PubMed

Waters, Sinéad M; Murphy, Richard A; Power, Ronan F G

2006-08-01

Undefined Nurmi-type cultures (NTCs) have been used successfully to prevent salmonella colonisation in poultry for decades. Such cultures are derived from the caecal contents of specific-pathogen-free birds and are administered via drinking water or spray application onto eggs in the hatchery. These cultures consist of many non-culturable and obligately anaerobic bacteria. Due to their undefined nature it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials for poultry. In this study, 10 batches of prototype NTCs were produced using an identical protocol over a period of 2 years. Traditional microbiological techniques and a molecular culture-independent methodology, polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), were applied to characterise these cultures and also to examine if the constituents of the NTCs were identical. Culture-dependent analysis of these cultures included plating on a variety of selective and semi-selective agars, examination of colony morphology, Gram-staining and a series of biochemical tests (API, BioMerieux, France). Two sets of PCR-DGGE studies were performed. These involved the amplification of universal and subsequently lactic acid bacteria (LAB)-specific hypervariable regions of a 16S rRNA gene by PCR. Resultant amplicons were subjected to DGGE. Sequence analysis was performed on subsequent bands present in resultant DGGE profiles using the Basic Local Alignment Search Tool (BLAST). Microbiological culturing techniques tended to isolate common probiotic bacterial species from the genera Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Clostridium, Escherichia, Pediococcus and Enterobacterium as well as members of the genera, Actinomyces, Bacteroides, Propionibacterium, Capnocytophaga, Proteus, and Klebsiella. Bacteroides, Enterococcus, Escherichia, Brevibacterium, Klebsiella, Lactobacillus, Clostridium, Bacillus, Eubacterium, Serratia, Citrobacter, Enterobacter, Pectobacterium and Pantoea spp. in addition to unculturable bacteria were identified as constituents of the NTCs using universal PCR-DGGE analysis. A number of the sequences detected by LAB-specific PCR-DGGE were homologous to those of a number of Lactobacillus spp., including L. fermentum, L. pontis, L. crispatus, L. salivarius, L. casei, L. suntoryeus, L. vaginalis, L. gasseri, L. aviaries, L. johnsonii, L. acidophilus, and L. mucosae in addition to a range of unculturable lactobacilli. While NTCs are successful due to their complexity, the presence of members of Lactobacillus spp. amongst other probiotic genera, in these samples possibly lends to the success of the NTC cultures as probiotics or competitive exclusion products in poultry over the decades. PCR-DGGE proved to be an effective tool in detecting non-culturable organisms present in these complex undefined cultures. In conclusion, while the culture-dependent identification methods or PCR-DGGE alone cannot comprehensively elucidate the bacterial species present in such complex cultures, their complementarity provides useful information on the identity of the constituents of NTCs and will aid in future development of defined probiotics. Moreover, for the purpose of analysing prototype NTCs during their development, PCR-DGGE overcomes the limitations associated with conventional culturing methods including their low sensitivities, inability to detect unculturable bacteria and unknown species, very slow turnabout time and poor reproducibility. This study demonstrated that PCR-DGGE is indeed more valuable in detecting predominant microbial populations between various NTCs than as an identification methodology, being more applicable as a quality control method used to analyse for batch-to-batch variation during NTC production.

A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture.

PubMed

Zhang, Jue; Schwartz, Michael P; Hou, Zhonggang; Bai, Yongsheng; Ardalani, Hamisha; Swanson, Scott; Steill, John; Ruotti, Victor; Elwell, Angela; Nguyen, Bao Kim; Bolin, Jennifer; Stewart, Ron; Thomson, James A; Murphy, William L

2017-04-11

A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for "3D-like" vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

PubMed

Adewumi, Gbenga A; Oguntoyinbo, Folarin A; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2012-01-01

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

Evidence for two koi herpesvirus (KHV) genotypes in South Korea.

PubMed

Kim, Hyoung Jun; Kwon, Se Ryun

2013-06-13

The geographic distribution of koi herpesvirus (KHV) has recently been analyzed by polymerase chain reaction (PCR, based on the alleles of 3 domains) and sequence analysis using 3 regions of KHV genomic DNA (SphI-5, 9/5, and the thymidine kinase gene). In this study, samples from 6 carp showing symptoms of KHV infection in 2008 were examined for the presence of KHV by using PCR and cell culture isolation methods. KHV was detected in 2 (Pyeongtaek and Buan) of the samples. Sequence analysis revealed that the genotype of the KHV PT-08 isolate was Asia genotype variant 1 (A1), and the genotype of the KHV BA-08 isolate was European genotype variant 4 (E4). In addition, PCR patterns and sequence analysis based on the alleles of 3 domains of an alternate KHV classification system confirmed that the genotype of the KHV PT-08 isolate was CyHV3-J, and the genotype of the KHV BA-08 isolate was CyHV3-third genotype. To our knowledge, this is the first study to demonstrate the presence of 2 genotypes of KHV (genotype A1/CyHV3-J; genotype E4/CyHV3-third genotype) in South Korea.

Metabarcoding of the kombucha microbial community grown in different microenvironments.

PubMed

Reva, Oleg N; Zaets, Iryna E; Ovcharenko, Leonid P; Kukharenko, Olga E; Shpylova, Switlana P; Podolich, Olga V; de Vera, Jean-Pierre; Kozyrovska, Natalia O

2015-12-01

Introducing of the DNA metabarcoding analysis of probiotic microbial communities allowed getting insight into their functioning and establishing a better control on safety and efficacy of the probiotic communities. In this work the kombucha poly-microbial probiotic community was analysed to study its flexibility under different growth conditions. Environmental DNA sequencing revealed a complex and flexible composition of the kombucha microbial culture (KMC) constituting more bacterial and fungal organisms in addition to those found by cultural method. The community comprised bacterial and yeast components including cultured and uncultivable microorganisms. Culturing the KMC under different conditions revealed the core part of the community which included acetobacteria of two genera Komagataeibacter (former Gluconacetobacter) and Gluconobacter, and representatives of several yeast genera among which Brettanomyces/Dekkera and Pichia (including former Issatchenkia) were dominant. Herbaspirillum spp. and Halomonas spp., which previously had not been described in KMC, were found to be minor but permanent members of the community. The community composition was dependent on the growth conditions. The bacterial component of KMC was relatively stable, but may include additional member-lactobacilli. The yeast species composition was significantly variable. High-throughput sequencing showed complexity and variability of KMC that may affect the quality of the probiotic drink. It was hypothesized that the kombucha core community might recruit some environmental bacteria, particularly lactobacilli, which potentially may contribute to the fermentative capacity of the probiotic drink. As many KMC-associated microorganisms cannot be cultured out of the community, a robust control for community composition should be provided by using DNA metabarcoding.

Genome-Scale Transcriptome Analysis in Response to Nitric Oxide in Birch Cells: Implications of the Triterpene Biosynthetic Pathway

PubMed Central

Zeng, Fansuo; Sun, Fengkun; Li, Leilei; Liu, Kun; Zhan, Yaguang

2014-01-01

Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10−5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis. PMID:25551661

IMG/M: integrated genome and metagenome comparative data analysis system

DOE PAGES

Chen, I-Min A.; Markowitz, Victor M.; Chu, Ken; ...

2016-10-13

The Integrated Microbial Genomes with Microbiome Samples (IMG/M: https://img.jgi.doe.gov/m/) system contains annotated DNA and RNA sequence data of (i) archaeal, bacterial, eukaryotic and viral genomes from cultured organisms, (ii) single cell genomes (SCG) and genomes from metagenomes (GFM) from uncultured archaea, bacteria and viruses and (iii) metagenomes from environmental, host associated and engineered microbiome samples. Sequence data are generated by DOE's Joint Genome Institute (JGI), submitted by individual scientists, or collected from public sequence data archives. Structural and functional annotation is carried out by JGI's genome and metagenome annotation pipelines. A variety of analytical and visualization tools provide support formore » examining and comparing IMG/M's datasets. IMG/M allows open access interactive analysis of publicly available datasets, while manual curation, submission and access to private datasets and computationally intensive workspace-based analysis require login/password access to its expert review(ER) companion system (IMG/M ER: https://img.jgi.doe.gov/ mer/). Since the last report published in the 2014 NAR Database Issue, IMG/M's dataset content has tripled in terms of number of datasets and overall protein coding genes, while its analysis tools have been extended to cope with the rapid growth in the number and size of datasets handled by the system.« less

IMG/M: integrated genome and metagenome comparative data analysis system

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

Chen, I-Min A.; Markowitz, Victor M.; Chu, Ken

The Integrated Microbial Genomes with Microbiome Samples (IMG/M: https://img.jgi.doe.gov/m/) system contains annotated DNA and RNA sequence data of (i) archaeal, bacterial, eukaryotic and viral genomes from cultured organisms, (ii) single cell genomes (SCG) and genomes from metagenomes (GFM) from uncultured archaea, bacteria and viruses and (iii) metagenomes from environmental, host associated and engineered microbiome samples. Sequence data are generated by DOE's Joint Genome Institute (JGI), submitted by individual scientists, or collected from public sequence data archives. Structural and functional annotation is carried out by JGI's genome and metagenome annotation pipelines. A variety of analytical and visualization tools provide support formore » examining and comparing IMG/M's datasets. IMG/M allows open access interactive analysis of publicly available datasets, while manual curation, submission and access to private datasets and computationally intensive workspace-based analysis require login/password access to its expert review(ER) companion system (IMG/M ER: https://img.jgi.doe.gov/ mer/). Since the last report published in the 2014 NAR Database Issue, IMG/M's dataset content has tripled in terms of number of datasets and overall protein coding genes, while its analysis tools have been extended to cope with the rapid growth in the number and size of datasets handled by the system.« less

IMG/M: integrated genome and metagenome comparative data analysis system

PubMed Central

Chen, I-Min A.; Markowitz, Victor M.; Chu, Ken; Palaniappan, Krishna; Szeto, Ernest; Pillay, Manoj; Ratner, Anna; Huang, Jinghua; Andersen, Evan; Huntemann, Marcel; Varghese, Neha; Hadjithomas, Michalis; Tennessen, Kristin; Nielsen, Torben; Ivanova, Natalia N.; Kyrpides, Nikos C.

2017-01-01

The Integrated Microbial Genomes with Microbiome Samples (IMG/M: https://img.jgi.doe.gov/m/) system contains annotated DNA and RNA sequence data of (i) archaeal, bacterial, eukaryotic and viral genomes from cultured organisms, (ii) single cell genomes (SCG) and genomes from metagenomes (GFM) from uncultured archaea, bacteria and viruses and (iii) metagenomes from environmental, host associated and engineered microbiome samples. Sequence data are generated by DOE's Joint Genome Institute (JGI), submitted by individual scientists, or collected from public sequence data archives. Structural and functional annotation is carried out by JGI's genome and metagenome annotation pipelines. A variety of analytical and visualization tools provide support for examining and comparing IMG/M's datasets. IMG/M allows open access interactive analysis of publicly available datasets, while manual curation, submission and access to private datasets and computationally intensive workspace-based analysis require login/password access to its expert review (ER) companion system (IMG/M ER: https://img.jgi.doe.gov/mer/). Since the last report published in the 2014 NAR Database Issue, IMG/M's dataset content has tripled in terms of number of datasets and overall protein coding genes, while its analysis tools have been extended to cope with the rapid growth in the number and size of datasets handled by the system. PMID:27738135

Social Anxiety among Chinese People

PubMed Central

Fan, Qianqian; Chang, Weining C.

2015-01-01

The experience of social anxiety has largely been investigated among Western populations; much less is known about social anxiety in other cultures. Unlike the Western culture, the Chinese emphasize interdependence and harmony with social others. In addition, it is unclear if Western constructed instruments adequately capture culturally conditioned conceptualizations and manifestations of social anxiety that might be specific to the Chinese. The present study employed a sequence of qualitative and quantitative approaches to examine the assessment of social anxiety among the Chinese people. Interviews and focus group discussions with Chinese participants revealed that some items containing the experience of social anxiety among the Chinese are not present in existing Western measures. Factor analysis was employed to examine the factor structure of the more comprehensive scale. This approach revealed an “other concerned anxiety” factor that appears to be specific to the Chinese. Subsequent analysis found that the new factor—other concerned anxiety—functioned the same as other social anxiety factors in their association with risk factors of social anxiety, such as attachment, parenting, behavioral inhibition/activation, and attitude toward group. The implications of these findings for a more culturally sensitive assessment tool of social anxiety among the Chinese were discussed. PMID:26380367

Social Anxiety among Chinese People.

PubMed

Fan, Qianqian; Chang, Weining C

2015-01-01

The experience of social anxiety has largely been investigated among Western populations; much less is known about social anxiety in other cultures. Unlike the Western culture, the Chinese emphasize interdependence and harmony with social others. In addition, it is unclear if Western constructed instruments adequately capture culturally conditioned conceptualizations and manifestations of social anxiety that might be specific to the Chinese. The present study employed a sequence of qualitative and quantitative approaches to examine the assessment of social anxiety among the Chinese people. Interviews and focus group discussions with Chinese participants revealed that some items containing the experience of social anxiety among the Chinese are not present in existing Western measures. Factor analysis was employed to examine the factor structure of the more comprehensive scale. This approach revealed an "other concerned anxiety" factor that appears to be specific to the Chinese. Subsequent analysis found that the new factor-other concerned anxiety-functioned the same as other social anxiety factors in their association with risk factors of social anxiety, such as attachment, parenting, behavioral inhibition/activation, and attitude toward group. The implications of these findings for a more culturally sensitive assessment tool of social anxiety among the Chinese were discussed.

Phylogenetic analysis of bacterial isolates from man-made high-pH, high-salt environments and identification of gene-cassette-associated open reading frames.

PubMed

Ghauri, Muhammad A; Khalid, Ahmad M; Grant, Susan; Grant, William D; Heaphy, Shaun

2006-06-01

Environmental samples were collected from high-pH sites in Pakistan, including a uranium heap set up for carbonate leaching, the lime unit of a tannery, and the Khewra salt mine. Another sample was collected from a hot spring on the shore of the soda lake, Magadi, in Kenya. Microbial cultures were enriched from Pakistani samples. Phylogenetic analysis of isolates was carried out by sequencing 16S rRNA genes. Genomic DNA was amplified by polymerase chain reaction using integron gene-cassette-specific primers. Different gene-cassette-linked genes were recovered from the cultured strains related to Halomonas magadiensis, Virgibacillus halodenitrificans, and Yania flava and from the uncultured environmental DNA sample. The usefulness of this technique as a tool for gene mining is indicated.

Phylogenetic diversity and biological activity of culturable Actinobacteria isolated from freshwater fish gut microbiota.

PubMed

Jami, Mansooreh; Ghanbari, Mahdi; Kneifel, Wolfgang; Domig, Konrad J

2015-06-01

The diversity of Actinobacteria isolated from the gut microbiota of two freshwater fish species namely Schizothorax zarudnyi and Schizocypris altidorsalis was investigated employing classical cultivation techniques, repetitive sequence-based PCR (rep-PCR), partial and full 16S rDNA sequencing followed by phylogenetic analysis. A total of 277 isolates were cultured by applying three different agar media. Based on rep-PCR profile analysis a subset of 33 strains was selected for further phylogenetic investigations, antimicrobial activity testing and diversity analysis of secondary-metabolite biosynthetic genes. The identification based on 16S rRNA gene sequencing revealed that the isolates belong to eight genera distributed among six families. At the family level, 72% of the 277 isolates belong to the family Streptomycetaceae. Among the non-streptomycetes group, the most dominant group could be allocated to the family of Pseudonocardiaceae followed by the members of Micromonosporaceae. Phylogenetic analysis clearly showed that many of the isolates in the genera Streptomyces, Saccharomonospora, Micromonospora, Nocardiopsis, Arthrobacter, Kocuria, Microbacterium and Agromyces formed a single and distinct cluster with the type strains. Notably, there is no report so far about the occurrence of these Actinobacteria in the microbiota of freshwater fish. Of the 33 isolates, all the strains exhibited antibacterial activity against a set of tested human and fish pathogenic bacteria. Then, to study their associated potential capacity to synthesize diverse bioactive natural products, diversity of genes associated with secondary-metabolite biosynthesis including PKS I, PKS II, NRPS, the enzyme PhzE of the phenazine pathways, the enzyme dTGD of 6-deoxyhexoses glycosylation pathway, the enzyme Halo of halogenation pathway and the enzyme CYP in polyene polyketide biosynthesis were investigated among the isolates. All the strains possess at least two types of the investigated biosynthetic genes, one-fourth of them harbours more than four. This study demonstrates the significant diversity of Actinobacteria in the fish gut microbiota and it's potential to produce biologically active compounds. Copyright © 2015 Elsevier GmbH. All rights reserved.

Production and Characterization of Ehrlichia risticii, the Agent of Potomac Horse Fever, from Snails (Pleuroceridae: Juga spp.) in Aquarium Culture and Genetic Comparison to Equine Strains

PubMed Central

Reubel, Gerhard H.; Barlough, Jeffrey E.; Madigan, John E.

1998-01-01

We report on the production and characterization of Ehrlichia risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae: Juga spp.) maintained in aquarium culture and compare it genetically to equine strains. Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF is enzootic and were maintained for several weeks in freshwater aquaria in the laboratory. Upon exposure to temperatures above 22°C the snails released trematode cercariae tentatively identified as virgulate cercariae. Fragments of three different genes (genes for 16S rRNA, the groESL heat shock operon, and the 51-kDa major antigen) were amplified from cercaria lysates by PCR and sequenced. Genetic information was also obtained from E. risticii strains from horses with PHF. The PCR positivity of snail secretions was associated with the presence of trematode cercariae. Sequence analysis of the three genes indicated that the source organism closely resembled E. risticii, and the sequences of all three genes were virtually identical to those of the genes of an equine E. risticii strain from a property near the snail collection site. Phylogenetic analyses of the three genes indicated the presence of geographical E. risticii strain clusters. PMID:9620368

Production and characterization of Ehrlichia risticii, the agent of Potomac horse fever, from snails (Pleuroceridae: Juga spp.) in aquarium culture and genetic comparison to equine strains.

PubMed

Reubel, G H; Barlough, J E; Madigan, J E

1998-06-01

We report on the production and characterization of Ehrlichia risticii, the agent of Potomac horse fever (PHF), from snails (Pleuroceridae: Juga spp.) maintained in aquarium culture and compare it genetically to equine strains. Snails were collected from stream waters on a pasture in Siskiyou County, Calif., where PHF is enzootic and were maintained for several weeks in freshwater aquaria in the laboratory. Upon exposure to temperatures above 22 degrees C the snails released trematode cercariae tentatively identified as virgulate cercariae. Fragments of three different genes (genes for 16S rRNA, the groESL heat shock operon, and the 51-kDa major antigen) were amplified from cercaria lysates by PCR and sequenced. Genetic information was also obtained from E. risticii strains from horses with PHF. The PCR positivity of snail secretions was associated with the presence of trematode cercariae. Sequence analysis of the three genes indicated that the source organism closely resembled E. risticii, and the sequences of all three genes were virtually identical to those of the genes of an equine E. risticii strain from a property near the snail collection site. Phylogenetic analyses of the three genes indicated the presence of geographical E. risticii strain clusters.

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

PubMed

Wan, Dongjin; Liu, Yongde; Niu, Zhenhua; Xiao, Shuhu; Li, Daorong

2016-02-01

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.

Apigenin Impacts the Growth of the Gut Microbiota and Alters the Gene Expression of Enterococcus.

PubMed

Wang, Minqian; Firrman, Jenni; Zhang, Liqing; Arango-Argoty, Gustavo; Tomasula, Peggy; Liu, LinShu; Xiao, Weidong; Yam, Kit

2017-08-03

Apigenin is a major dietary flavonoid with many bioactivities, widely distributed in plants. Apigenin reaches the colon region intact and interacts there with the human gut microbiota, however there is little research on how apigenin affects the gut bacteria. This study investigated the effect of pure apigenin on human gut bacteria, at both the single strain and community levels. The effect of apigenin on the single gut bacteria strains Bacteroides galacturonicus , Bifidobacterium catenulatum , Lactobacillus rhamnosus GG, and Enterococcus caccae , was examined by measuring their anaerobic growth profiles. The effect of apigenin on a gut microbiota community was studied by culturing a fecal inoculum under in vitro conditions simulating the human ascending colon. 16S rRNA gene sequencing and GC-MS analysis quantified changes in the community structure. Single molecule RNA sequencing was used to reveal the response of Enterococcus caccae to apigenin. Enterococcus caccae was effectively inhibited by apigenin when cultured alone, however, the genus Enterococcus was enhanced when tested in a community setting. Single molecule RNA sequencing found that Enterococcus caccae responded to apigenin by up-regulating genes involved in DNA repair, stress response, cell wall synthesis, and protein folding. Taken together, these results demonstrate that apigenin affects both the growth and gene expression of Enterococcus caccae .

Bacterial diversity in Adélie penguin, Pygoscelis adeliae, guano: molecular and morpho-physiological approaches.

PubMed

Zdanowski, Marek K; Weglenski, Piotr; Golik, Pawel; Sasin, Joanna M; Borsuk, Piotr; Zmuda, Magdalena J; Stankovic, Anna

2004-11-01

The total number of bacteria and culturable bacteria in Adélie penguin (Pygoscelis adeliae) guano was determined during 42 days of decomposition in a location adjacent to the rookery in Admiralty Bay, King George Island, Antarctica. Of the culturable bacteria, 72 randomly selected colonies were described using 49 morpho-physiological tests, 27 of which were subsequently considered significant in characterizing and differentiating the isolates. On the basis of the nucleotide sequence of a fragment of the 16S rRNA gene in each of 72 pure isolates, three major phylogenetic groups were identified, namely the Moraxellaceae/Pseudomonadaceae (29 isolates), the Flavobacteriaceae (14), and the Micrococcaceae (29). Grouping of the isolates on the basis of morpho-physiological tests (whether 49 or 27 parameters) showed similar results to those based on 16S rRNA gene sequences. Clusters were characterized by considerable intra-cluster variation in both 16S rRNA gene sequences and morpho-physiological responses. High diversity in abundance and morphometry of total bacterial communities during penguin guano decomposition was supported by image analysis of epifluorescence micrographs. The results indicate that the bacterial community in penguin guano is not only one of the richest in Antarctica, but is extremely diverse, both phylogenetically and morpho-physiologically.

PCR amplification and sequence analysis of the major capsid protein gene of megalocytiviruses isolated in Taiwan.

PubMed

Wang, C S; Chao, S Y; Ku, C C; Wen, C M; Shih, H H

2009-06-01

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Outbreaks of iridovirus disease have been reported among various fish species in Taiwan. However, the genotypes of these iridoviruses have not yet been determined. In this study, seven megalocytivirus isolates from four fish species: king grouper, Epinephelus lanceolatus (Bloch), barramundi perch, Lates calcarifer (Bloch), silver sea bream, Rhabdosargus sarba (Forsskal), and common ponyfish, Leiognathus equulus (Forsskal), cultured in three different regions of Taiwan were collected. The full open reading frame encoding the viral major capsid protein gene was amplified using PCR. The PCR products of approximately 1581 bp were cloned and the nucleotide sequences were phylogenetically analysed. Results showed that all seven PCR products contained a unique open reading frame with 1362 nucleotides and encoded a structural protein with 453 amino acids. Even though the nucleotide sequences were not identical, these seven megalocytiviruses were classified into one cluster and showed very high homology with red sea bream iridovirus (RSIV) with more than 97% identity. Thus, the seven iridovirus strains isolated from cultured marine fish in Taiwan were closer to the RSIV genotype than the infectious spleen and kidney necrosis virus genotype.

A comparison of microbial communities in deep-sea polymetallic nodules and the surrounding sediments in the Pacific Ocean

NASA Astrophysics Data System (ADS)

Wu, Yue-Hong; Liao, Li; Wang, Chun-Sheng; Ma, Wei-Lin; Meng, Fan-Xu; Wu, Min; Xu, Xue-Wei

2013-09-01

Deep-sea polymetallic nodules, rich in metals such as Fe, Mn, and Ni, are potential resources for future exploitation. Early culturing and microscopy studies suggest that polymetallic nodules are at least partially biogenic. To understand the microbial communities in this environment, we compared microbial community composition and diversity inside nodules and in the surrounding sediments. Three sampling sites in the Pacific Ocean containing polymetallic nodules were used for culture-independent investigations of microbial diversity. A total of 1013 near full-length bacterial 16S rRNA gene sequences and 640 archaeal 16S rRNA gene sequences with ~650 bp from nodules and the surrounding sediments were analyzed. Bacteria showed higher diversity than archaea. Interestingly, sediments contained more diverse bacterial communities than nodules, while the opposite was detected for archaea. Bacterial communities tend to be mostly unique to sediments or nodules, with only 13.3% of sequences shared. The most abundant bacterial groups detected only in nodules were Pseudoalteromonas and Alteromonas, which were predicted to play a role in building matrix outside cells to induce or control mineralization. However, archaeal communities were mostly shared between sediments and nodules, including the most abundant OTU containing 290 sequences from marine group I Thaumarchaeota. PcoA analysis indicated that microhabitat (i.e., nodule or sediment) seemed to be a major factor influencing microbial community composition, rather than sampling locations or distances between locations.

Comparison of pectin-degrading fungal communities in temperate forests using glycosyl hydrolase family 28 pectinase primers targeting Ascomycete fungi

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

Gacura, Matthew D.; Sprockett, Daniel D.; Heidenreich, Bess

Here, fungi have developed a wide assortment of enzymes to break down pectin, a prevalent polymer in plant cell walls that is important in plant defense and structure. One enzyme family used to degrade pectin is the glycosyl hydrolase family 28 (GH28). In this studywe developed primers for the amplification of GH28 coding genes from a database of 293 GH28 sequences from40 fungal genomes. The primerswere used to successfully amplify GH28 pectinases from all Ascomycota cultures tested, but only three out of seven Basidiomycota cultures. In addition, we further tested the primers in PCRs on metagenomic DNA extracted from senescedmore » tree leaves from different forest ecosystems, followed by cloning and sequencing. Taxonomic specificity for Ascomycota GH28 genes was tested by comparing GH28 composition in leaves to internal transcribed spacer (ITS) amplicon composition using pyrosequencing. All sequences obtained from GH28 primers were classified as Ascomycota; in contrast, ITS sequences indicated that fungal communitieswere up to 39% Basidiomycetes. Analysis of leaf samples indicated that both forest stand and ecosystemtype were important in structuring fungal communities. However, site played the prominent role in explaining GH28 composition, whereas ecosystem type was more important for ITS composition, indicating possible genetic drift between populations of fungi. Overall, these primers will have utility in understanding relationships between fungal community composition and ecosystem processes, as well as detection of potentially pathogenic Ascomycetes.« less

Comparison of pectin-degrading fungal communities in temperate forests using glycosyl hydrolase family 28 pectinase primers targeting Ascomycete fungi

DOE PAGES

Gacura, Matthew D.; Sprockett, Daniel D.; Heidenreich, Bess; ...

2016-02-17

Here, fungi have developed a wide assortment of enzymes to break down pectin, a prevalent polymer in plant cell walls that is important in plant defense and structure. One enzyme family used to degrade pectin is the glycosyl hydrolase family 28 (GH28). In this studywe developed primers for the amplification of GH28 coding genes from a database of 293 GH28 sequences from40 fungal genomes. The primerswere used to successfully amplify GH28 pectinases from all Ascomycota cultures tested, but only three out of seven Basidiomycota cultures. In addition, we further tested the primers in PCRs on metagenomic DNA extracted from senescedmore » tree leaves from different forest ecosystems, followed by cloning and sequencing. Taxonomic specificity for Ascomycota GH28 genes was tested by comparing GH28 composition in leaves to internal transcribed spacer (ITS) amplicon composition using pyrosequencing. All sequences obtained from GH28 primers were classified as Ascomycota; in contrast, ITS sequences indicated that fungal communitieswere up to 39% Basidiomycetes. Analysis of leaf samples indicated that both forest stand and ecosystemtype were important in structuring fungal communities. However, site played the prominent role in explaining GH28 composition, whereas ecosystem type was more important for ITS composition, indicating possible genetic drift between populations of fungi. Overall, these primers will have utility in understanding relationships between fungal community composition and ecosystem processes, as well as detection of potentially pathogenic Ascomycetes.« less

Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

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

Watanabe, Masahito; Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571; Umeyama, Kazuhiro

2010-11-05

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor themore » exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.« less

Comparison of methods for identifying causative bacterial microorganisms in presumed acute endophthalmitis: conventional culture, blood culture, and PCR.

PubMed

Pongsachareonnont, Pear; Honglertnapakul, Worawalun; Chatsuwan, Tanittha

2017-02-21

Identification of bacterial pathogens in endophthalmitis is important to inform antibiotic selection and treatment decisions. Hemoculture bottles and polymerase chain reaction (PCR) analysis have been proposed to offer good detection sensitivity. This study compared the sensitivity and accuracy of a blood culture system, a PCR approach, and conventional culture methods for identification of causative bacteria in cases of acute endophthalmitis. Twenty-nine patients with a diagnosis of presumed acute bacterial endophthalmitis who underwent vitreous specimen collection at King Chulalongkorn Memorial Hospital were enrolled in this study. Forty-one specimens were collected. Each specimen was divided into three parts, and each part was analyzed using one of three microbial identification techniques: conventional plate culture, blood culture, and polymerase chain reaction and sequencing. The results of the three methods were then compared. Bacteria were identified in 15 of the 41 specimens (36.5%). Five (12.2%) specimens were positive by conventional culture methods, 11 (26.8%) were positive by hemoculture, and 11 (26.8%) were positive by PCR. Cohen's kappa analysis revealed p-values for conventional methods vs. hemoculture, conventional methods vs. PCR, and hemoculture vs. PCR of 0.057, 0.33, and 0.009, respectively. Higher detection rates of Enterococcus faecalis were observed for hemoculture and PCR than for conventional methods. Blood culture bottles and PCR detection may facilitate bacterial identification in cases of presumed acute endophthalmitis. These techniques should be used in addition to conventional plate culture methods because they provide a greater degree of sensitivity than conventional plate culture alone for the detection of specific microorganisms such as E. faecalis. Thai Clinical Trial Register No. TCTR20110000024 .

ACVP-05: Virus Genetic Analysis from Cell-Free Plasma, Virally Infected Cells or Tissues and Cultured Supernatant Via Single Genome Amplification and Direct Sequencing | Frederick National Laboratory for Cancer Research

Cancer.gov

The Viral Evolution Core within the AIDS and Cancer Virus Program will extract viral RNA/DNA from cell-free or cell-associated samples. Complementary (cDNA) will be generated as needed, and cDNA or DNA will be diluted to a single copy prior to nested

Establishment and characterization of a testicular Sertoli cell line from olive flounder Paralichthys olivaceus

NASA Astrophysics Data System (ADS)

Peng, Limin; Zheng, Yuan; You, Feng; Wu, Zhihao; Zou, Yuxia; Zhang, Peijun

2016-09-01

The culture of Sertoli cells has become an indispensable resource in studying spermatogenesis. A new Sertoli cell line (POSC) that consisted predominantly of fibroblast-like cells was derived from the testis of the olive flounder Paralichthys olivaceus and sub-cultured for 48 passages. Analysis of the mtDNA COI gene partial sequence confirmed that the cell line was from P. olivaceus. Cells were optimally maintained at 25°C in DMEM/F12 medium supplemented with fetal bovine serum, basic fibroblast growth factor, and epidermal growth factor. The growth curve of POSC showed a typical "S" shape. Chromosome analysis revealed that the cell line possessed the normal P. olivaceus diploid karyotype of 2n=48t. POSC expressed dmrt1 but not vasa, which was detected using RT-PCR and sequencing. Immunocytochemistry revealed that the cells exhibited the testicular Sertoli cell marker FasL. Therefore, POSC appeared to consist of testicular Sertoli cells. Bright fluorescent signals were observed after the cells were transfected with pEGFP-N3 plasmid, with the transfection efficiency reaching 10%. This research not only offers an ideal model for further gene expression and regulation studies on P. olivaceus, but also serves as valuable material in studying fish spermatogenesis, Sertoli cell-germ cell interactions, and the mechanism of growth and development of testis.

Spatial and temporal single-cell volume estimation by a fluorescence imaging technique with application to astrocytes in primary culture

NASA Astrophysics Data System (ADS)

Khatibi, Siamak; Allansson, Louise; Gustavsson, Tomas; Blomstrand, Fredrik; Hansson, Elisabeth; Olsson, Torsten

1999-05-01

Cell volume changes are often associated with important physiological and pathological processes in the cell. These changes may be the means by which the cell interacts with its surrounding. Astroglial cells change their volume and shape under several circumstances that affect the central nervous system. Following an incidence of brain damage, such as a stroke or a traumatic brain injury, one of the first events seen is swelling of the astroglial cells. In order to study this and other similar phenomena, it is desirable to develop technical instrumentation and analysis methods capable of detecting and characterizing dynamic cell shape changes in a quantitative and robust way. We have developed a technique to monitor and to quantify the spatial and temporal volume changes in a single cell in primary culture. The technique is based on two- and three-dimensional fluorescence imaging. The temporal information is obtained from a sequence of microscope images, which are analyzed in real time. The spatial data is collected in a sequence of images from the microscope, which is automatically focused up and down through the specimen. The analysis of spatial data is performed off-line and consists of photobleaching compensation, focus restoration, filtering, segmentation and spatial volume estimation.

Structural characterisation of a water-soluble polysaccharide from tissue-cultured Dendrobium huoshanense C.Z. Tang et S.J. Cheng.

PubMed

Si, Hua-Yang; Chen, Nai-Fu; Chen, Nai-Dong; Huang, Cheng; Li, Jun; Wang, Hui

2018-02-01

A water-soluble polysaccharide TC-DHPA4 with a molecular weight of 8.0 × 10 5  Da was isolated from tissue-cultured Dendrobium huoshanense by anion exchange and gel permeation chromatography. Monosaccharide analysis revealed that the homogeneous polysaccharide was made up of rhamnose, arabinose, mannose, glucose, galactose and glucuronic acid with a molar ratio of 1.28:1:1.67:4.71:10.43:1.42. The sugar residue sequence analysis based on the GC-MS files and NMR spectra indicated that the backbone of TC-DHPA4 consisted of the repeated units:→6)-β-Galp-(1→6)-β-Galp-(1→4)-β-GlcpA-(1→6)-β-Glcp-(1→6)-β-Glcp-(→. The sugar residue sequences β-Glcp-(1→)-α-Rhap-(1→3)-β-Galp-(1→, β-Glcp-(1→4)-α-Rhap-(1→3)-β-Galp-(1→, β-Galp-(1→6)-β-Manp-(1→3)-β-Galp-(1→, and α-l-Araf-(1→2)-β-Manp-(1→3)-β-Galp-(1→ were identified as the branches attached to the C-3 position of (1→6)-linked galactose in the backbone.

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India.

PubMed

Anand, T Prem; Bhat, Abdul Wajid; Shouche, Yogesh S; Roy, Upal; Siddharth, Jay; Sarma, Siddhartha P

2006-01-01

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.

Draft Genome Sequences of Pseudomonas fluorescens BS2 and Pusillimonas noertemannii BS8, Soil Bacteria That Cooperate To Degrade the Poly-γ-d-Glutamic Acid Anthrax Capsule.

PubMed

Stabler, Richard A; Negus, David; Pain, Arnab; Taylor, Peter W

2013-01-01

A mixed culture of Pseudomonas fluorescens BS2 and Pusillimonas noertemannii BS8 degraded poly-γ-d-glutamic acid; when the 2 strains were cultured separately, no hydrolytic activity was apparent. Here we report the draft genome sequences of both soil isolates.

Comparative transcriptome analysis of microsclerotia development in Nomuraea rileyi

PubMed Central

2013-01-01

Background Nomuraea rileyi is used as an environmental-friendly biopesticide. However, mass production and commercialization of this organism are limited due to its fastidious growth and sporulation requirements. When cultured in amended medium, we found that N. rileyi could produce microsclerotia bodies, replacing conidiophores as the infectious agent. However, little is known about the genes involved in microsclerotia development. In the present study, the transcriptomes were analyzed using next-generation sequencing technology to find the genes involved in microsclerotia development. Results A total of 4.69 Gb of clean nucleotides comprising 32,061 sequences was obtained, and 20,919 sequences were annotated (about 65%). Among the annotated sequences, only 5928 were annotated with 34 gene ontology (GO) functional categories, and 12,778 sequences were mapped to 165 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we assessed the transcriptomic differences between cultures grown in minimal and amended medium. In total, 4808 sequences were found to be differentially expressed; 719 differentially expressed unigenes were assigned to 25 GO classes and 1888 differentially expressed unigenes were assigned to 161 KEGG pathways, including 25 enrichment pathways. Subsequently, we examined the up-regulation or uniquely expressed genes following amended medium treatment, which were also expressed on the enrichment pathway, and found that most of them participated in mediating oxidative stress homeostasis. To elucidate the role of oxidative stress in microsclerotia development, we analyzed the diversification of unigenes using quantitative reverse transcription-PCR (RT-qPCR). Conclusion Our findings suggest that oxidative stress occurs during microsclerotia development, along with a broad metabolic activity change. Our data provide the most comprehensive sequence resource available for the study of N. rileyi. We believe that the transcriptome datasets will serve as an important public information platform to accelerate studies on N. rileyi microsclerotia. PMID:23777366

Life cycle as a stable trait in the evaluation of diversity of Nostoc from biofilms in rivers.

PubMed

Mateo, Pilar; Perona, Elvira; Berrendero, Esther; Leganés, Francisco; Martín, Marta; Golubić, Stjepko

2011-05-01

The diversity within the genus Nostoc is still controversial and more studies are needed to clarify its heterogeneity. Macroscopic species have been extensively studied and discussed; however, the microscopic forms of the genus, especially those from running waters, are poorly known and likely represented by many more species than currently described. Nostoc isolates from biofilms of two Spanish calcareous rivers were characterized comparing the morphology and life cycle in two culture media with different levels of nutrients and also comparing the 16S rRNA gene sequences. The results showed that trichome shape and cellular dimensions varied considerably depending on the culture media used, whereas the characteristics expressed in the course of the life cycle remained stable for each strain independent of the culture conditions. Molecular phylogenetic analysis confirmed the distinction between the studied strains established on morphological grounds. A balanced approach to the evaluation of diversity of Nostoc in the service of autecological studies requires both genotypic information and the evaluation of stable traits. The results of this study show that 16S rRNA gene sequence similarity serves as an important criterion for characterizing Nostoc strains and is consistent with stable attributes, such as the life cycle. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Origins and genetic features of the Okhotsk people, revealed by ancient mitochondrial DNA analysis.

PubMed

Sato, Takehiro; Amano, Tetsuya; Ono, Hiroko; Ishida, Hajime; Kodera, Haruto; Matsumura, Hirofumi; Yoneda, Minoru; Masuda, Ryuichi

2007-01-01

In order to investigate the phylogenetic status of the Okhotsk people that were distributed in northern and eastern Hokkaido as well as southern Sakhalin during the fifth to the thirteenth centuries, DNA was carefully extracted from human bone and tooth remains excavated from archaeological sites. The hypervariable region 1 sequences of the mitochondrial DNA (mtDNA) control region were successfully amplified and 16 mtDNA haplotypes were identified from 37 individuals of the Okhotsk people. Of the 16 haplotypes found, 6 were unique to the Okhotsk people, whereas the other 10 were shared by northeastern Asian people that are currently distributed around Sakhalin and downstream of the Amur River. The phylogenetic relationships inferred from mtDNA sequences showed that the Okhotsk people were more closely related to the Nivkhi and Ulchi people among populations of northeastern Asia. In addition, the Okhotsk people had a relatively closer genetic affinity with the Ainu people of Hokkaido, and were likely intermediates of gene flow from the northeastern Asian people to the Ainu people. These findings support the hypothesis that the Okhotsk culture joined the Satsumon culture (direct descendants of the Jomon people) resulting in the Ainu culture, as suggested by previous archaeological and anthropological studies.

Tandem mass spectrometry for the detection of plant pathogenic fungi and the effects of database composition on protein inferences.

PubMed

Padliya, Neerav D; Garrett, Wesley M; Campbell, Kimberly B; Tabb, David L; Cooper, Bret

2007-11-01

LC-MS/MS has demonstrated potential for detecting plant pathogens. Unlike PCR or ELISA, LC-MS/MS does not require pathogen-specific reagents for the detection of pathogen-specific proteins and peptides. However, the MS/MS approach we and others have explored does require a protein sequence reference database and database-search software to interpret tandem mass spectra. To evaluate the limitations of database composition on pathogen identification, we analyzed proteins from cultured Ustilago maydis, Phytophthora sojae, Fusarium graminearum, and Rhizoctonia solani by LC-MS/MS. When the search database did not contain sequences for a target pathogen, or contained sequences to related pathogens, target pathogen spectra were reliably matched to protein sequences from nontarget organisms, giving an illusion that proteins from nontarget organisms were identified. Our analysis demonstrates that when database-search software is used as part of the identification process, a paradox exists whereby additional sequences needed to detect a wide variety of possible organisms may lead to more cross-species protein matches and misidentification of pathogens.

Characterization of an isoproturon mineralizing bacterial culture enriched from a French agricultural soil.

PubMed

Hussain, Sabir; Sørensen, Sebastian R; Devers-Lamrani, Marion; El-Sebai, Talaat; Martin-Laurent, Fabrice

2009-11-01

The phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized by a bacterial culture isolated from an agricultural soil regularly exposed to IPU. Molecular analysis of the bacterial culture by DNA fingerprinting, cloning and sequencing of the 16S rRNA genes revealed that it consisted of six different members among whom the dominant was related to Sphingomonas sp. Six bacterial strains belonging to genera Ancylobacter, Pseudomonas, Stenotrophomonas, Methylobacterium, Variovorax and Agrobacterium were isolated from the IPU-degrading culture. None of these were able to degrade IPU in pure culture and only the intact culture sustained the ability to mineralize IPU. The composition of the culture appeared stable suggesting that yet unknown interactions are involved in the IPU mineralization. IPU degradation involved the transitory accumulation of three known IPU metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropylaniline and their further degradation. Thus, it indicates a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain. This culture did not degrade other structurally related phenylurea herbicides. The degrading activity of the bacterial culture was deeply influenced by the pH, being completely inhibited at pH 5.5 and optimal at pH 7.5.

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

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

[Big Data Revolution or Data Hubris? : On the Data Positivism of Molecular Biology].

PubMed

Gramelsberger, Gabriele

2017-12-01

Genome data, the core of the 2008 proclaimed big data revolution in biology, are automatically generated and analyzed. The transition from the manual laboratory practice of electrophoresis sequencing to automated DNA-sequencing machines and software-based analysis programs was completed between 1982 and 1992. This transition facilitated the first data deluge, which was considerably increased by the second and third generation of DNA-sequencers during the 2000s. However, the strategies for evaluating sequence data were also transformed along with this transition. The paper explores both the computational strategies of automation, as well as the data evaluation culture connected with it, in order to provide a complete picture of the complexity of today's data generation and its intrinsic data positivism. This paper is thereby guided by the question, whether this data positivism is the basis of the big data revolution of molecular biology announced today, or it marks the beginning of its data hubris.

Application of rDNA-PCR amplification and DGGE fingerprinting for detection of microbial diversity in a Malaysian crude oil.

PubMed

Liew, Pauline Woanying; Jong, Bor Chyan

2008-05-01

Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.

Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

PubMed

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2005-03-01

Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes

PubMed Central

Pereiro, Patricia; Balseiro, Pablo; Romero, Alejandro; Dios, Sonia; Forn-Cuni, Gabriel; Fuste, Berta; Planas, Josep V.; Beltran, Sergi; Novoa, Beatriz; Figueras, Antonio

2012-01-01

Background Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations. Methodology/Principal Findings Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways. Conclusions/Significance To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms. PMID:22629298

Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.

PubMed

Kamoun, Jannet; Schué, Mathieu; Messaoud, Wala; Baignol, Justine; Point, Vanessa; Mateos-Diaz, Eduardo; Mansuelle, Pascal; Gargouri, Youssef; Parsiegla, Goetz; Cavalier, Jean-François; Carrière, Frédéric; Aloulou, Ahmed

2015-02-01

Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica. Copyright © 2014 Elsevier B.V. All rights reserved.

Pyrosequencing the Canine Faecal Microbiota: Breadth and Depth of Biodiversity

PubMed Central

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

2013-01-01

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

PCR amplification of 16S rDNA from lyophilized cell cultures facilitates studies in molecular systematics

NASA Technical Reports Server (NTRS)

Wisotzkey, J. D.; Jurtshuk, P. Jr; Fox, G. E.

1990-01-01

The sequence of the major portion of a Bacillus cycloheptanicus strain SCH(T) 16S rRNA gene is reported. This sequence suggests that B. cycloheptanicus is genetically quite distinct from traditional Bacillus strains (e.g., B. subtilis) and may be properly regarded as belonging to a different genus. The sequence was determined from DNA that was produced by direct amplification of ribosomal DNA from a lyophilized cell pellet with straightforward polymerase chain reaction (PCR) procedures. By obviating the need to revive cell cultures from the lyophile pellet, this approach facilitates rapid 16S rDNA sequencing and thereby advances studies in molecular systematics.

Bacillus odysseyi isolate

NASA Technical Reports Server (NTRS)

La Duc, Myron Thomas (Inventor); Venkateswaran, Kasthuri (Inventor)

2007-01-01

The present invention relates to discovery and isolation of a biologically pure culture of a Bacillus odysseyi isolate with high adherence and sterilization resistant properties. B. odysseyi is a round spore forming Bacillus species that produces an exosporium. This novel species has been characterized on the basis of phenotypic traits, 16S rDNA sequence analysis and DNA-DNA hybridization. According to the results of these analyses, this strain belongs to the genus Bacillus and the type strain is 34hs-1.sup.T (=ATCC PTA-4993.sup.T=NRRL B-30641.sup.T=NBRC 100172.sup.T). The GenBank accession number for the 16S rDNA sequence of strain 34hs-1.sup.T is AF526913.

Sequence Memory Constraints Give Rise to Language-Like Structure through Iterated Learning

PubMed Central

Cornish, Hannah; Dale, Rick; Kirby, Simon; Christiansen, Morten H.

2017-01-01

Human language is composed of sequences of reusable elements. The origins of the sequential structure of language is a hotly debated topic in evolutionary linguistics. In this paper, we show that sets of sequences with language-like statistical properties can emerge from a process of cultural evolution under pressure from chunk-based memory constraints. We employ a novel experimental task that is non-linguistic and non-communicative in nature, in which participants are trained on and later asked to recall a set of sequences one-by-one. Recalled sequences from one participant become training data for the next participant. In this way, we simulate cultural evolution in the laboratory. Our results show a cumulative increase in structure, and by comparing this structure to data from existing linguistic corpora, we demonstrate a close parallel between the sets of sequences that emerge in our experiment and those seen in natural language. PMID:28118370

Sequence Memory Constraints Give Rise to Language-Like Structure through Iterated Learning.

PubMed

Cornish, Hannah; Dale, Rick; Kirby, Simon; Christiansen, Morten H

2017-01-01

Human language is composed of sequences of reusable elements. The origins of the sequential structure of language is a hotly debated topic in evolutionary linguistics. In this paper, we show that sets of sequences with language-like statistical properties can emerge from a process of cultural evolution under pressure from chunk-based memory constraints. We employ a novel experimental task that is non-linguistic and non-communicative in nature, in which participants are trained on and later asked to recall a set of sequences one-by-one. Recalled sequences from one participant become training data for the next participant. In this way, we simulate cultural evolution in the laboratory. Our results show a cumulative increase in structure, and by comparing this structure to data from existing linguistic corpora, we demonstrate a close parallel between the sets of sequences that emerge in our experiment and those seen in natural language.

Interaction of Extreme Halophilic Archaea With the Evaporites of the Solar Salterns Guerrero Negro Baja California, Mexico

NASA Astrophysics Data System (ADS)

Tamez, P.; Lopez-Cortés, A.

2008-12-01

Hypersaline environments have been significant reservoirs for the long-term evolution of specifically adapted microorganisms. Characterized to have higher salt concentrations (up to 35 g/L), they are worldwide distributed and have a commercial significance. Exportadora de Sal, Guerrero Negro, Mexico has a multipond salterns system designed to harvest common salt (NaCl) from sea water. To achieve this purpose, sea water is pumped through a set of shallow ponds where water evaporates and salts concentrate. Sequential precipitation of CaCO3, CaSO4 2H2O and NaCl occurs in a mineral formations call it evaporites. In the interior of those gypsum-encrusted and halite-encrusted minerals, communities of extremely salt-loving archaea prosper. Previous studies have showed the influence of Haloarchaeal cells in the formation of larger fluid inclusions than crystals formed in sterile salt solutions. S-layer envelopes and cells of Haloarcula strain SP8807 contributed to the nucleation of new crystals of NaCl. Given the significance of the scope in phylogenetic archaeal diversity research, this study had a polyphasic approach. SEM micrographs from a 21- 31% (w/v) gradient salt multipond system evaporites, gave an insight profile of the extreme halophilic archaeal communities thriving in the surface of the gypsum and halite evaporites. Halite crystals were form after 21 days of incubation in solid medium with archaeal cells. Both culture and non-culture dependent methods, Nested-PCR-DGGE analysis and sequencing of 16S rDNA amplified fragment genes from environmental samples and isolated strains were used for this purpose. We isolate three strains from Pond 9 (21.07% total salt concentration) and one strain from Cristallizer 20 (25.15% total salt concentration). 16S rDNA signaling gave 99% of similarity with Halogeometricum borinquense, sequence AF002984, two other strains were 99% of similarity with Halobacterium salinarum, sequence AJ496185 these strains shown different colony morphology. Other strain match, with 99% of similarity, with three sequences Haloarcula: Haloarcula japonica (EF645686), Haloarcula hispanica (DQ089681) and Haloarcula marismortui (X61689), differences among them were only 3 base pairs. We suggest that strain, C205030908, could correspond to Haloarcula californiae previously reported by Javor et al, (1982), from which there is not sequence known. Nested-PCR-DGGE analysis showed a pattern of 11 bands, meaning the archaeal community is more complex than we could detect by culture approach.

[Occurrence of Giardia species and genotypes in humans and animals in Wielkopolska region, Poland].

PubMed

Solarczyk, Piotr

2009-01-01

Giardia is the most common intestinal protozoan parasite found in humans and animals worldwide. Although it has been known for three hundred years, the nomenclature, taxonomy, host specificity, and pathogenicity of Giardia still arouse numerous controversies and ambiguities. Giardia is classified into six species, that are characterised by various ranges of hosts. The most dubious species is G. intestinalis, which includes a dozen or so genotypes, and only two of them (genotype A and B) have wide ranges of hosts, including humans. Moreover, in some genotype assemblages of G. intestinalis certain subgenotypes were distinguished and it was proven that in the same host species various subgenotypes of this parasite may occur. Bearing in mind the significant genetic heterogeneity of G. intestinalis and the fact that various genotypes and subgenotypes of this parasite are characterised by the broad or narrow host specificity, the data concerning the frequency of giardiosis occurrence are insufficient. It is necessary to use molecular biology techniques in order to define the genotype and/or the subgenotype of G. intestinalis that are found in humans and in certain animal species. Furthermore, since more and more pieces of evidence connected with a possibility of the sexual recombination of Giardia are gathered, it is unknown if genotypes and subgenotypes of this parasite are stable in time. The aim of this thesis was to define the frequency of Giardia occurrence in humans and animals in Wielkopolska region, to identify species and genotypes of Giardia that occur in humans and animals, as well as to obtain an axenic culture of the chosen isolates of Giardia from animals and to compare the sequence of the beta-giardin gene fragment obtained from the DNA isolated from cysts and trophozoites in order to check if the axenisation of G. intestinalis leads to the selection of genotypes or if Giardia genotypes are stable in time. Altogether, 2183 faecal samples were examined for the presence of Giardia cysts; 447 faecal samples were taken from 232 persons coming from 5 cities situated in Wielkopolska, and 1736 faecal samples were obtained from 123 animal species, including: 266 faecal samples from 113 species of animals kept in the Zoological Garden in Poznań, 1286 samples from 4 species of breeding animals, 118 samples from dogs, and 66 samples from 5 species of wild animals. Faecal samples were taken from animals coming from 25 places in Wielkopolska. Moreover, seven isolates of G. intestinalis were used in the studies, which were obtained from various species of hosts and kept in an axenic in vitro culture. Microscopic, molecular and bio-informative methods were used in the studies. From each faecal sample fresh smears were made in a 0.6% solution of physiological salt and in Lugol's solution, as well as a permanent smear stained with trichrome was made. Moreover, the following molecular techniques were implemented in the studies: DNA extraction and purification, the PCR technique (two molecular markers), electrophoresis and visualisation of PCR products, and sequencing. A fragment of the beta-giardin gene was used as a molecular marker in order to define the genotype and subgenotype of Giardia. Only in the case of genotyping of two Giardia isolates obtained from Peromyscus eremicus another molecular marker (SSU rRNA)was additionally used. Some widely available computer programmes (Chromas, CAP 3, BioEdit, BLASTn, MEGA version 4.0) were utilised in the analysis of the sequence of the beta-giardin gene fragment and in the phylogenetic analysis. The culture of Giardia trophozoites was established to compare the sequence of the partial beta-giardin gene from cysts and trophozoites. Concentration and purification of Giardia cysts in the saccharose gradient, and the excystation technique were applied in the studies to obtaining an axenic in vitro culture. In this study, Giardia cysts were found in 12 faecal samples obtained from 3 persons and 5 animal species. Giardia cysts were found only in faecal samples from humans living in Poznań and the samples obtained from animals coming from Poznań and around Puszczykowo. The highest frequency of infection was stated in domestic animals (2.5%) and in animals kept in the Zoological Garden (2.0%), whereas a slightly lower frequency was noticed in wild animals (1.5%) and in humans (1.3%). No Giardia cysts were found in the faecal samples collected from breeding animals. Two new species of Giardia hosts were identified, namely Rhinella marina and Peromyscus eremicus; however, due to a minimal amount of faecal samples supplied for the study it was impossible to define the species and genotype of this parasite. PCR products (the partial of beta-giardin gene) were obtained in seven faecal samples out of the ten studied, including three samples from people and four faecal samples derived from three animal species (i.e. dog, tamandua, red deer). Moreover, molecular characterization of seven Giardia isolates from three persons and four animal species (red-bellied monkey, silver marmoset, Thomson's gazelle, and sheep) kept in an axenic in vitro culture was performed. Based on the beta-giardin sequence fragment analysis, four assemblages of G. intestinalis genotypes were identified (A, B, C and D). In humans, A and B G. intestinalis genotypes and three subgenotypes, including a cosmopolitan subgenotype A2 and two new subgenotypes A and B were detected. Furthermore, four G. intestinalis genotypes were found in animals, including three genotypes which are non-infectious to humans, namely: genotypes C and D in dogs and a cervids-specific genotype A in red deer (Cervus elaphus), which indicate that these animals do not constitute the source of infection to humans. On the other hand, in a tamandua from the Zoological Garden in Poznań a new subgenotype B of G. intestinalis was identified, which due to a close relationship with Giardia isolates obtained from humans is potentially infectious to man. In none of the studied faecal samples a mixed infection of Giardia was found. To date, nine sequences of the partial beta-giardin gene have been deposited in the National Center for Biotechnology Information (NCBI), including two sequences of Giardia isolates obtained from humans (GenBank accession numbers FJ009207, FJ009208), three sequences of isolate obtained from red deer (GenBank accession numbers EU621373, EU626198, EU216429), two sequences of both Giardia isolates obtained from dogs (GenBank accession numbers FJ009205, FJ009206), and the single sequences obtained from tamandua (GenBank accession number FJ009209) and from Thomson's gazelle (GenBank accession number EU626199). According to the literature, an axenic in vitro culture of G. intestinalis was obtained from a red deer for the first time. Based on the analysis of the sequence of the beta-giardin gene fragment obtained from the DNA isolated from cysts and trophozoites it was proven that the red deer was infected with a single population of Giardia and that during the axenisation of the culture no mutation in the DNA of the parasite's trophozoites took place. Probably the time distance that the DNA was isolated from the trophozoites kept in the culture was too short to cause the mutation. This suggestion is confirmed by the results of the genotyping of seven G. intestinalis isolates obtained from various host species and kept in an axenic in vitro culture for at least a number of years. Based on the molecular characteristics it was stated that all the studied isolates from the axenic culture were identical and belonged to the same assemblage B. The comparision with the sequences from GenBank database revealed that all mentioned isolates were 99% similar to the sequence of Giardia Nij5 isolate obtained from a person from the Netherlands and characterised as genotype B1. Due to the sameness of the molecular marker sequences it seems improbable that the identical G. intestinalis genotype occurred in various time periods (the largest difference was 14 years) in humans and in a number of animal species in diverse areas of Wielkopolska region. Quite opposite, the long-term keeping of these isolates in the homogenous conditions of an axenic in vitro culture leads to the selection of a genotype or proves the instability of genotypes of this parasite. Long-term studies need to be conducted in order to verify these hypothesis. Their results will have a key meaning in explaining the genetic structure of the Giardia population and in understanding the molecular epidemiology of giardiosis.

The genus Bipolaris

PubMed Central

Manamgoda, D.S.; Rossman, A.Y.; Castlebury, L.A.; Crous, P.W.; Madrid, H.; Chukeatirote, E.; Hyde, K.D.

2014-01-01

The genus Bipolaris includes important plant pathogens with worldwide distribution. Species recognition in the genus has been uncertain due to the lack of molecular data from ex-type cultures as well as overlapping morphological characteristics. In this study, we revise the genus Bipolaris based on DNA sequence data derived from living cultures of fresh isolates, available ex-type cultures from worldwide collections and observation of type and additional specimens. Combined analyses of ITS, GPDH and TEF gene sequences were used to reconstruct the molecular phylogeny of the genus Bipolaris for species with living cultures. The GPDH gene is determined to be the best single marker for species of Bipolaris. Generic boundaries between Bipolaris and Curvularia are revised and presented in an updated combined ITS and GPDH phylogenetic tree. We accept 47 species in the genus Bipolaris and clarify the taxonomy, host associations, geographic distributions and species’ synonymies. Modern descriptions and illustrations are provided for 38 species in the genus with notes provided for the other taxa when recent descriptions are available. Bipolaris cynodontis, B. oryzae, B. victoriae, B. yamadae and B. zeicola are epi- or neotypified and a lectotype is designated for B. stenospila. Excluded and doubtful species are listed with notes on taxonomy and phylogeny. Seven new combinations are introduced in the genus Curvularia to accomodate the species of Bipolaris transferred based on the phylogenetic analysis. A taxonomic key is provided for the morphological identification of species within the genus. PMID:25492990

Determination of Spoilage Microbiota of Pacific White Shrimp During Ambient and Cold Storage Using Next-Generation Sequencing and Culture-Dependent Method.

PubMed

Yang, Sheng-Ping; Xie, Jing; Qian, Yun-Fang

2017-05-01

This study was conducted to determine the initial and spoilage microbiota of Pacific white shrimp during ambient and cold storage using next-generation sequencing (NGS) and a culture-dependent method. The quality changes were also evaluated by sensory analysis and total volatile basic nitrogen (TVB-N) values. After 1 d of storage, the psychrotrophic bacteria were only 5.97 log CFU/g, accounting for 1.1% of the mesophilic bacteria counts (7.94 log CFU/g). The psychrotrophic bacteria counts exceeded the counts of mesophilic bacteria for shrimp stored at 4 °C after 6 d of storage, indicating that psychrotrophic bacteria became predominant. The NGS was used to identify the bacterial species in samples stored at 25 and 4 °C. The results showed that the dominant microorganisms were Vibrio at 25 °C, and Acinetobacter, Psychrobacter, and Shewanella at 4 °C. By the culture-dependent method based on 16S rRNA gene and VITEK®2 CompactA system, it showed that the dominant microorganisms were Proteus spp. at 25 °C, and Shewanella putrefaciens, Acinetobacter johnsonii, and Aeromonas sobria at 4 °C. In conclusion, differences in results of microbiota analyzed by culture dependent and independent approaches were observed. The combination of both methodologies may provide more comprehensive information about the dominant spoilage microbiota in Pacific white shrimp during ambient and cold storage. © 2017 Institute of Food Technologists®.

Trading genes along the silk road: mtDNA sequences and the origin of central Asian populations.

PubMed Central

Comas, D; Calafell, F; Mateu, E; Pérez-Lezaun, A; Bosch, E; Martínez-Arias, R; Clarimon, J; Facchini, F; Fiori, G; Luiselli, D; Pettener, D; Bertranpetit, J

1998-01-01

Central Asia is a vast region at the crossroads of different habitats, cultures, and trade routes. Little is known about the genetics and the history of the population of this region. We present the analysis of mtDNA control-region sequences in samples of the Kazakh, the Uighurs, the lowland Kirghiz, and the highland Kirghiz, which we have used to address both the population history of the region and the possible selective pressures that high altitude has on mtDNA genes. Central Asian mtDNA sequences present features intermediate between European and eastern Asian sequences, in several parameters-such as the frequencies of certain nucleotides, the levels of nucleotide diversity, mean pairwise differences, and genetic distances. Several hypotheses could explain the intermediate position of central Asia between Europe and eastern Asia, but the most plausible would involve extensive levels of admixture between Europeans and eastern Asians in central Asia, possibly enhanced during the Silk Road trade and clearly after the eastern and western Eurasian human groups had diverged. Lowland and highland Kirghiz mtDNA sequences are very similar, and the analysis of molecular variance has revealed that the fraction of mitochondrial genetic variance due to altitude is not significantly different from zero. Thus, it seems unlikely that altitude has exerted a major selective pressure on mitochondrial genes in central Asian populations. PMID:9837835

Functional Genomics Analysis of Singapore Grouper Iridovirus: Complete Sequence Determination and Proteomic Analysis

PubMed Central

Song, Wen Jun; Qin, Qi Wei; Qiu, Jin; Huang, Can Hua; Wang, Fan; Hew, Choy Leong

2004-01-01

Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products. PMID:15507645

A long natural-antisense RNA is accumulated in the conidia of Aspergillus oryzae.

PubMed

Tsujii, Masaru; Okuda, Satoshi; Ishi, Kazutomo; Madokoro, Kana; Takeuchi, Michio; Yamagata, Youhei

2016-01-01

Analysis of expressed sequence tag libraries from various culture conditions revealed the existence of conidia-specific transcripts assembled to putative conidiation-specific reductase gene (csrA) in Aspergillus oryzae. However, the all transcripts were transcribed with opposite direction to the gene csrA. The sequence analysis of the transcript revealed that the RNA overlapped mRNA of csrA with 3'-end, and did not code protein longer than 60 amino acid residues. We designated the transcript Conidia Specific Long Natural-antisense RNA (CSLNR). The real-time PCR analysis demonstrated that the CSLNR is conidia-specific transcript, which cannot be transcribed in the absence of brlA, and the amount of CSLNR was much more than that of the transcript from csrA in conidia. Furthermore, the csrA deletion, also lacking coding region of CSLNR in A. oryzae reduced the number of conidia. Overexpression of CsrA demonstrated the inhibition of growth and conidiation, while CSLNR did not affect conidiation.

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis.

PubMed Central

Nübel, U; Engelen, B; Felske, A; Snaidr, J; Wieshuber, A; Amann, R I; Ludwig, W; Backhaus, H

1996-01-01

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts. PMID:8824607

Assembling the Marine Metagenome, One Cell at a Time

PubMed Central

Woyke, Tanja; Xie, Gary; Copeland, Alex; González, José M.; Han, Cliff; Kiss, Hajnalka; Saw, Jimmy H.; Senin, Pavel; Yang, Chi; Chatterji, Sourav; Cheng, Jan-Fang; Eisen, Jonathan A.; Sieracki, Michael E.; Stepanauskas, Ramunas

2009-01-01

The difficulty associated with the cultivation of most microorganisms and the complexity of natural microbial assemblages, such as marine plankton or human microbiome, hinder genome reconstruction of representative taxa using cultivation or metagenomic approaches. Here we used an alternative, single cell sequencing approach to obtain high-quality genome assemblies of two uncultured, numerically significant marine microorganisms. We employed fluorescence-activated cell sorting and multiple displacement amplification to obtain hundreds of micrograms of genomic DNA from individual, uncultured cells of two marine flavobacteria from the Gulf of Maine that were phylogenetically distant from existing cultured strains. Shotgun sequencing and genome finishing yielded 1.9 Mbp in 17 contigs and 1.5 Mbp in 21 contigs for the two flavobacteria, with estimated genome recoveries of about 91% and 78%, respectively. Only 0.24% of the assembling sequences were contaminants and were removed from further analysis using rigorous quality control. In contrast to all cultured strains of marine flavobacteria, the two single cell genomes were excellent Global Ocean Sampling (GOS) metagenome fragment recruiters, demonstrating their numerical significance in the ocean. The geographic distribution of GOS recruits along the Northwest Atlantic coast coincided with ocean surface currents. Metabolic reconstruction indicated diverse potential energy sources, including biopolymer degradation, proteorhodopsin photometabolism, and hydrogen oxidation. Compared to cultured relatives, the two uncultured flavobacteria have small genome sizes, few non-coding nucleotides, and few paralogous genes, suggesting adaptations to narrow ecological niches. These features may have contributed to the abundance of the two taxa in specific regions of the ocean, and may have hindered their cultivation. We demonstrate the power of single cell DNA sequencing to generate reference genomes of uncultured taxa from a complex microbial community of marine bacterioplankton. A combination of single cell genomics and metagenomics enabled us to analyze the genome content, metabolic adaptations, and biogeography of these taxa. PMID:19390573

Parasitemia in an immunocompetent horse experimentally challenged with Sarcocystis neurona sporocysts.

PubMed

Rossano, M G; Schott, H C; Murphy, A J; Kaneene, J B; Sellon, D C; Hines, M T; Hochstatter, T; Bell, J A; Mansfield, L S

2005-01-04

Equine protozoal myeloencephalitis (EPM) is a serious neurological disease of horses in Americans. Most cases are attributed to infection of the central nervous system with Sarcocystis neurona. Parasitemia has not been demonstrated in immunocompetent horses, but has been documented in one immunocompromised foal. The objective of this study was to isolate viable S. neurona from the blood of immunocompetent horses. Horses used in this study received orally administered S. neurona sporocysts (strain SN 37-R) daily for 112 days at the following doses: 100/day for 28 days, followed by 500/day for 28 days, followed by 1000/day for 56 days. On day 98 of the study, six yearling colts were selected for attempted culture of S. neurona from blood, two testing positive, two testing suspect and two testing negative for antibodies against S. neurona on day 84 of the study. Two 10 ml tubes with EDTA were filled from each horse by jugular venipuncture and the plasma fraction rich in mononuclear cells was pipetted onto confluent equine dermal cell cultures. The cultures were monitored weekly for parasite growth for 12 weeks. Merozoites grown from cultures were harvested and tested using S. neurona-specific PCR with RFLP to confirm species identity. PCR products were sequenced and compared to known strains of S. neurona. After 38 days of in vitro incubation, one cell culture from a horse testing positive for antibodies against S. neurona was positive for parasite growth while the five remaining cultures remained negative for parasite growth for all 12 weeks. The Sarcocystis isolate recovered from cell culture was confirmed to be S. neurona by PCR with RFLP. Gene sequence analysis revealed that the isolate was identical to the challenge strain SN-37R and differed from two known strains UCD1 and MIH1. To our knowledge this is the first report of parasitemia with S. neurona in an immunocompetent horse.

Isolation and characterization of a novel Rhabdovirus from a wild boar (Sus scrofa) in Japan.

PubMed

Sakai, Kouji; Hagiwara, Katsuro; Omatsu, Tsutomu; Hamasaki, Chinami; Kuwata, Ryusei; Shimoda, Hiroshi; Suzuki, Kazuo; Endoh, Daiji; Nagata, Noriyo; Nagai, Makoto; Katayama, Yukie; Oba, Mami; Kurane, Ichiro; Saijo, Masayuki; Morikawa, Shigeru; Mizutani, Tetsuya; Maeda, Ken

2015-09-30

A novel rhabdovirus was isolated from the serum of a healthy Japanese wild boar (Sus scrofa leucomystax) and identified using the rapid determination system for viral nucleic acid sequences (RDV), next-generation sequencing, and electron microscopy. The virus was tentatively named wild boar rhabdovirus 1 (WBRV1). Phylogenetic analysis of the entire genome sequence indicated that WBRV1 is closely related to Tupaia rhabdovirus (TRV), which was isolated from cultured cells of hepatocellular carcinoma tissue of tree shrew. TRV has not been assigned to any genus of Rhabdoviridae till date. Analysis of the L gene indicated that WBRV1 belongs to the genus Vesiculovirus. These observations suggest that both TRV and WBRV1 belong to a new genus of Rhabdoviridae. Next-generation genome sequencing of WBRV1 revealed 5 open reading frames of 1329, 765, 627, 1629, and 6336 bases in length. The WBRV1 gene sequences are similar to those of other rhabdoviruses. Epizootiological analysis of a population of wild boars in Wakayama prefecture in Japan indicated that 6.5% were positive for the WBRV1 gene and 52% were positive for WBRV1-neutralizing antibodies. Furthermore, such viral neutralizing antibodies were found in domestic pigs in another prefecture. WBRV1 was inoculated intranasally and intraperitoneally into SCID and BALB/c mice and viral RNA was detected in SCID mice, suggesting that WBRV1 can replicate in immunocompromised mice. These results indicate this novel virus is endemic in wild animals and livestock in Japan. Copyright © 2015 Elsevier B.V. All rights reserved.

Draft Genome Sequence of Staphylococcus carnosus subsp. utilis LTH 7013, Isolated from South Tyrolean Ham.

PubMed

Müller, Anne; Huptas, Christopher; Wenning, Mareike; Schmidt, Herbert; Weiss, Agnes

2015-05-14

Staphylococcus carnosus is used as a starter culture in meat fermentation, where it contributes to color formation and produces aromatic compounds. Here, we report the first draft genome sequence of an S. carnosus subsp. utilis strain, LTH 7013, isolated from South Tyrolean ham, with potential application as a starter culture. Copyright © 2015 Müller et al.

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 treatment and morbidity/mortality outcomes. PMID:28739600

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 treatment and morbidity/mortality outcomes. Copyright © 2017 Owen et al.

Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager Yeast

PubMed Central

Walther, Andrea; Hesselbart, Ana; Wendland, Jürgen

2014-01-01

Lager yeast beer production was revolutionized by the introduction of pure culture strains. The first established lager yeast strain is known as the bottom fermenting Saccharomyces carlsbergensis, which was originally termed Unterhefe No. 1 by Emil Chr. Hansen and has been used in production in since 1883. S. carlsbergensis belongs to group I/Saaz-type lager yeast strains and is better adapted to cold growth conditions than group II/Frohberg-type lager yeasts, e.g., the Weihenstephan strain WS34/70. Here, we sequenced S. carlsbergensis using next generation sequencing technologies. Lager yeasts are descendants from hybrids formed between a S. cerevisiae parent and a parent similar to S. eubayanus. Accordingly, the S. carlsbergensis 19.5-Mb genome is substantially larger than the 12-Mb S. cerevisiae genome. Based on the sequence scaffolds, synteny to the S. cerevisae genome, and by using directed polymerase chain reaction for gap closure, we generated a chromosomal map of S. carlsbergensis consisting of 29 unique chromosomes. We present evidence for genome and chromosome evolution within S. carlsbergensis via chromosome loss and loss of heterozygosity specifically of parts derived from the S. cerevisiae parent. Based on our sequence data and via fluorescence-activated cell-sorting analysis, we determined the ploidy of S. carlsbergensis. This inferred that this strain is basically triploid with a diploid S. eubayanus and haploid S. cerevisiae genome content. In contrast the Weihenstephan strain, which we resequenced, is essentially tetraploid composed of two diploid S. cerevisiae and S. eubayanus genomes. Based on conserved translocations between the parental genomes in S. carlsbergensis and the Weihenstephan strain we propose a joint evolutionary ancestry for lager yeast strains. PMID:24578374

Diversity of Pea-Associated F. proliferatum and F. verticillioides Populations Revealed by FUM1 Sequence Analysis and Fumonisin Biosynthesis

PubMed Central

Waśkiewicz, Agnieszka; Stępień, Łukasz; Wilman, Karolina; Kachlicki, Piotr

2013-01-01

Fusarium proliferatum and F. verticillioides are considered as minor pathogens of pea (Pisum sativum L.). Both species can survive in seed material without visible disease symptoms, but still contaminating it with fumonisins. Two populations of pea-derived F. proliferatum and F. verticillioides strains were subjected to FUM1 sequence divergence analysis, forming a distinct group when compared to the collection strains originating from different host species. Furthermore, the mycotoxigenic abilities of those strains were evaluated on the basis of in planta and in vitro fumonisin biosynthesis. No differences were observed in fumonisin B (FB) levels measured in pea seeds (maximum level reached 1.5 μg g−1); however, in rice cultures, the majority of F. proliferatum genotypes produced higher amounts of FB1–FB3 than F. verticillioides strains. PMID:23470545

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, combined with the increasing availability of portable, random-access sequencing technology, offer the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI. PMID:28490492

Got black swimming dots in your cell culture? Identification of Achromobacter as a novel cell culture contaminant

PubMed Central

Gray, Jennifer Sue; Birmingham, Janette Marie; Fenton, Jenifer Imig

2009-01-01

ARTICLE SUMMARY Cell culture model systems are utilized for their ease of use, relative inexpensiveness, and potentially limitless sample size. Reliable results cannot be obtained, however, when cultures contain contamination. This report discusses the observation and identification of mobile black specks observed in multiple cell lines. Cultures of the contamination were grown, and DNA was purified from isolated colonies. The 16S rDNA gene was PCR amplified using primers that will amplify the gene from many genera, and then sequenced. Sequencing results matched the members of the genus Achromobacter, bacteria common in the environment. Achromobacter species have been shown to be resistant to multiple antibiotics. Attempts to decontaminate the eukaryotic cell culture used multiple antibiotics at different concentrations. The contaminating Achromobacter was eventually eliminated, without permanently harming the eukaryotic cells, using a combination of the antibiotics ciprofloxacin and piperacillin. PMID:19926304

Rapid, comprehensive, and affordable mycobacterial diagnosis with whole-genome sequencing: a prospective study

PubMed Central

Pankhurst, Louise J; del Ojo Elias, Carlos; Votintseva, Antonina A; Walker, Timothy M; Cole, Kevin; Davies, Jim; Fermont, Jilles M; Gascoyne-Binzi, Deborah M; Kohl, Thomas A; Kong, Clare; Lemaitre, Nadine; Niemann, Stefan; Paul, John; Rogers, Thomas R; Roycroft, Emma; Smith, E Grace; Supply, Philip; Tang, Patrick; Wilcox, Mark H; Wordsworth, Sarah; Wyllie, David; Xu, Li; Crook, Derrick W

2016-01-01

Summary Background Slow and cumbersome laboratory diagnostics for Mycobacterium tuberculosis complex (MTBC) risk delayed treatment and poor patient outcomes. Whole-genome sequencing (WGS) could potentially provide a rapid and comprehensive diagnostic solution. In this prospective study, we compare real-time WGS with routine MTBC diagnostic workflows. Methods We compared sequencing mycobacteria from all newly positive liquid cultures with routine laboratory diagnostic workflows across eight laboratories in Europe and North America for diagnostic accuracy, processing times, and cost between Sept 6, 2013, and April 14, 2014. We sequenced specimens once using local Illumina MiSeq platforms and processed data centrally using a semi-automated bioinformatics pipeline. We identified species or complex using gene presence or absence, predicted drug susceptibilities from resistance-conferring mutations identified from reference-mapped MTBC genomes, and calculated genetic distance to previously sequenced UK MTBC isolates to detect outbreaks. WGS data processing and analysis was done by staff masked to routine reference laboratory and clinical results. We also did a microcosting analysis to assess the financial viability of WGS-based diagnostics. Findings Compared with routine results, WGS predicted species with 93% (95% CI 90–96; 322 of 345 specimens; 356 mycobacteria specimens submitted) accuracy and drug susceptibility also with 93% (91–95; 628 of 672 specimens; 168 MTBC specimens identified) accuracy, with one sequencing attempt. WGS linked 15 (16% [95% CI 10–26]) of 91 UK patients to an outbreak. WGS diagnosed a case of multidrug-resistant tuberculosis before routine diagnosis was completed and discovered a new multidrug-resistant tuberculosis cluster. Full WGS diagnostics could be generated in a median of 9 days (IQR 6–10), a median of 21 days (IQR 14–32) faster than final reference laboratory reports were produced (median of 31 days [IQR 21–44]), at a cost of £481 per culture-positive specimen, whereas routine diagnosis costs £518, equating to a WGS-based diagnosis cost that is 7% cheaper annually than are present diagnostic workflows. Interpretation We have shown that WGS has a scalable, rapid turnaround, and is a financially feasible method for full MTBC diagnostics. Continued improvements to mycobacterial processing, bioinformatics, and analysis will improve the accuracy, speed, and scope of WGS-based diagnosis. Funding National Institute for Health Research, Department of Health, Wellcome Trust, British Colombia Centre for Disease Control Foundation for Population and Public Health, Department of Clinical Microbiology, Trinity College Dublin. PMID:26669893

Culturing Heterotrophic Protists from the Baltic Sea: Mostly the "Usual Suspects" but a Few Novelties as Well.

PubMed

Weber, Felix; Mylnikov, Alexander P; Jürgens, Klaus; Wylezich, Claudia

2017-03-01

The study of cultured strains has a long tradition in protistological research and has greatly contributed to establishing the morphology, taxonomy, and ecology of many protist species. However, cultivation-independent techniques, based on 18S rRNA gene sequences, have demonstrated that natural protistan assemblages mainly consist of hitherto uncultured protist lineages. This mismatch impedes the linkage of environmental diversity data with the biological features of cultured strains. Thus, novel taxa need to be obtained in culture to close this knowledge gap. In this study, traditional cultivation techniques were applied to samples from coastal surface waters and from deep oxygen-depleted waters of the Baltic Sea. Based on 18S rRNA gene sequencing, 126 monoclonal cultures of heterotrophic protists were identified. The majority of the isolated strains were affiliated with already cultured and described taxa, mainly chrysophytes and bodonids. This was likely due to "culturing bias" but also to the eutrophic nature of the Baltic Sea. Nonetheless, ~ 12% of the isolates in our culture collection showed highly divergent 18S rRNA gene sequences compared to those of known organisms and thus may represent novel taxa, either at the species level or at the genus level. Moreover, we also obtained evidence that some of the isolated taxa are ecologically relevant, under certain conditions, in the Baltic Sea. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

Phylogenetic and functional diversity within toluene-degrading, sulphate-reducing consortia enriched from a contaminated aquifer.

PubMed

Kuppardt, Anke; Kleinsteuber, Sabine; Vogt, Carsten; Lüders, Tillmann; Harms, Hauke; Chatzinotas, Antonis

2014-08-01

Three toluene-degrading microbial consortia were enriched under sulphate-reducing conditions from different zones of a benzene, toluene, ethylbenzene and xylenes (BTEX) plume of two connected contaminated aquifers. Two cultures were obtained from a weakly contaminated zone of the lower aquifer, while one culture originated from the highly contaminated upper aquifer. We hypothesised that the different habitat characteristics are reflected by distinct degrader populations. Degradation of toluene with concomitant production of sulphide was demonstrated in laboratory microcosms and the enrichment cultures were phylogenetically characterised. The benzylsuccinate synthase alpha-subunit (bssA) marker gene, encoding the enzyme initiating anaerobic toluene degradation, was targeted to characterise the catabolic diversity within the enrichment cultures. It was shown that the hydrogeochemical parameters in the different zones of the plume determined the microbial composition of the enrichment cultures. Both enrichment cultures from the weakly contaminated zone were of a very similar composition, dominated by Deltaproteobacteria with the Desulfobulbaceae (a Desulfopila-related phylotype) as key players. Two different bssA sequence types were found, which were both affiliated to genes from sulphate-reducing Deltaproteobacteria. In contrast, the enrichment culture from the highly contaminated zone was dominated by Clostridia with a Desulfosporosinus-related phylotype as presumed key player. A distinct bssA sequence type with high similarity to other recently detected sequences from clostridial toluene degraders was dominant in this culture. This work contributes to our understanding of the niche partitioning between degrader populations in distinct compartments of BTEX-contaminated aquifers.

FTA liver impressions as DNA template for detecting and genotyping fowl adenovirus.

PubMed

Moscoso, Hugo; Bruzual, Jose J; Sellers, Holly; Hofacre, Charles L

2007-03-01

The feasibility of using liver impressions on Flinders Technology Associates (FTA filter paper for the collection, inactivation, and molecular analysis of fowl adenovirus (FAV) was evaluated. FAV I European Union (EU) serotype 1 spotted on FTA was shown to be inactivated using specific-pathogen-free (SPF) primary chicken embryo liver cell culture as indicated by absence of cytopathic effect. Sensitivity of the polymerase chain reaction (PCR) test using tenfold dilutions of allantoic fluid from 100 to 10-4 for the detection of adenovirus serotype 1 on FTA cards was determined to be 0.0005 mean tissue culture infectious dose per FTA spot. The stability of the DNA from liver impressions on the FTA was found to be 198 days when stored at -20 degrees C. In a trial, inclusion body hepatitis (IBH) was experimentally reproduced in SPF chickens inoculated with FAV I EU serogroup 1, 4, 8, or 11, which presented weakness, pallor, depression, dehydration, and mortality within 6 days after inoculation. PCR performed on FTA liver impressions from the inoculated birds was able to detect all four viruses, and the nucleotide sequence analysis of the amplified PCR products (1219 bp of the hexone gene) revealed the expected serotypes. In addition to the trial, 55 clinical samples were analyzed from liver impressions on FTA cards, and FAV was detected in 11 of 55 (20%). Sequencing analysis showed that the viruses were EU serotypes 4, 5, 9, and 10. The results demonstrate that FTA filter paper inactivates the FAV I and maintains the DNA template for molecular analysis.

Isolation of Separate Ureaplasma Species From Endotracheal Secretions of Twin Patients.

PubMed

Beeton, Michael L; Maxwell, Nicola C; Chalker, Victoria J; Brown, Rebecca J; Aboklaish, Ali F; Spiller, O Brad

2016-08-01

Isolation of Ureaplasma spp. from preterm neonates and the association with development of bronchopulmonary dysplasia has been previously investigated. However, few studies have contrasted the nature of infection in twins. In this article, we report that dizygotic twins (1 girl, 1 boy) born at 24 weeks gestation both yielded culturable Ureaplasma from endotracheal secretions. The samples were part of a serial blind collection cohort of ventilated premature neonates, and analysis of repeat cultures showed stable, separate infections over a period of 17 and 21 days, respectively. Immunoblot and probe-specific quantitative polymerase chain reaction analysis determined that Twin 1 was solely infected with Ureaplasma parvum (specifically, serovar 6 by gene sequencing), whereas Twin 2 was solely infected with Ureaplasma urealyticum (specifically, genotype A- serovars 2, 5, and 8 by gene sequencing). Immunoblot analysis found that the major surface antigen (multiple-banded antigen) altered relative mass for both strains during the course of infection. Quantitative polymerase chain reaction analysis of extracted endotracheal aspirates confirmed no evidence of mixed infection for either twin. Failure of sentinel ventilated preterm infants on the same ward to acquire Ureaplasma infection after the first week of birth suggests no cot-to-cot transfer of Ureaplasma infection occurred. This study demonstrated not only a contrasting clinical outcome for a set of twins infected with 2 separate species of Ureaplasma, but also the first real-time demonstration of multiple-banded antigen alteration and evolution of Ureaplasma over the course of a clinical infection. Copyright © 2016 by the American Academy of Pediatrics.

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

PubMed

Spaethling, Jennifer M; Na, Young-Ji; Lee, Jaehee; Ulyanova, Alexandra V; Baltuch, Gordon H; Bell, Thomas J; Brem, Steven; Chen, H Isaac; Dueck, Hannah; Fisher, Stephen A; Garcia, Marcela P; Khaladkar, Mugdha; Kung, David K; Lucas, Timothy H; O'Rourke, Donald M; Stefanik, Derek; Wang, Jinhui; Wolf, John A; Bartfai, Tamas; Grady, M Sean; Sul, Jai-Yoon; Kim, Junhyong; Eberwine, James H

2017-01-17

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles.

PubMed

Gotoh, Aina; Nara, Misaki; Sugiyama, Yuta; Sakanaka, Mikiyasu; Yachi, Hiroyuki; Kitakata, Aya; Nakagawa, Akira; Minami, Hiromichi; Okuda, Shujiro; Katoh, Toshihiko; Katayama, Takane; Kurihara, Shin

2017-10-01

Recently, a "human gut microbial gene catalogue," which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the "human gut microbial gene catalogue" were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.

Single cell genomics of uncultured marine alveolates shows paraphyly of basal dinoflagellates.

PubMed

Strassert, Jürgen F H; Karnkowska, Anna; Hehenberger, Elisabeth; Del Campo, Javier; Kolisko, Martin; Okamoto, Noriko; Burki, Fabien; Janouškovec, Jan; Poirier, Camille; Leonard, Guy; Hallam, Steven J; Richards, Thomas A; Worden, Alexandra Z; Santoro, Alyson E; Keeling, Patrick J

2018-01-01

Marine alveolates (MALVs) are diverse and widespread early-branching dinoflagellates, but most knowledge of the group comes from a few cultured species that are generally not abundant in natural samples, or from diversity analyses of PCR-based environmental SSU rRNA gene sequences. To more broadly examine MALV genomes, we generated single cell genome sequences from seven individually isolated cells. Genes expected of heterotrophic eukaryotes were found, with interesting exceptions like presence of proteorhodopsin and vacuolar H + -pyrophosphatase. Phylogenetic analysis of concatenated SSU and LSU rRNA gene sequences provided strong support for the paraphyly of MALV lineages. Dinoflagellate viral nucleoproteins were found only in MALV groups that branched as sister to dinokaryotes. Our findings indicate that multiple independent origins of several characteristics early in dinoflagellate evolution, such as a parasitic life style, underlie the environmental diversity of MALVs, and suggest they have more varied trophic modes than previously thought.

Interdisciplinary Perspectives on Culture, Conflict, and Negotiation

DTIC Science & Technology

2009-08-28

negotiator cognition: Judgement accuracy and negotiation processes in individualistic and collectivistic cultures ", Organizational Behavior and Human...2004, Adair, Okumura, and Brett, 2001). Communication sequences are also affected by culture . Negotiators from collectivistic cultures use more... individualistic cultures (Adail and Brett, 2005; Adair, Okumura, and Brett, 2001). Research in DB/psychology has increasingly examined situational factors that

The Culturally Intelligent Negotiator: The Impact of Cultural Intelligence (CQ) on Negotiation Sequences and Outcomes

ERIC Educational Resources Information Center

Imai, Lynn; Gelfand, Michele J.

2010-01-01

Although scholars and practitioners have repeatedly touted the importance of negotiating effectively across cultures, paradoxically, little research has addressed what predicts intercultural negotiation effectiveness. In this research, we examined the impact of cultural intelligence (CQ) on intercultural negotiation processes and outcomes,…

Application of Metagenomic Sequencing to Food Safety: Detection of Shiga Toxin-Producing Escherichia coli on Fresh Bagged Spinach

PubMed Central

Leonard, Susan R.; Mammel, Mark K.; Lacher, David W.

2015-01-01

Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection. PMID:26386062

A Foray into Fungal Ecology: Understanding Fungi and Their Functions Across Ecosystems

NASA Astrophysics Data System (ADS)

Francis, N.; Dunkirk, N. C.; Peay, K.

2015-12-01

Despite their incredible diversity and importance to terrestrial ecosystems, fungi are not included in a standard high school science curriculum. This past summer, however, my work for the Stanford EARTH High School Internship program introduced me to fungal ecology through experiments involving culturing, genomics and root dissections. The two fungal experiments I worked on had very different foci, both searching for answers to broad ecological questions of fungal function and physiology. The first, a symbiosis experiment, sought to determine if the partners of the nutrient exchange between pine trees and their fungal symbionts could choose one another. The second experiment, a dung fungal succession project, compared the genetic sequencing results of fungal extractions from dung versus fungal cultures from dung. My part in the symbiosis experiment involved dissection, weighing and encapsulation of root tissue samples characterized based on the root thickness and presence of ectomycorrhizal fungi. The dung fungi succession project required that I not only learn how to culture various genera of dung fungi but also learn how to extract DNA and RNA for sequencing from the fungal tissue. Although I primarily worked with dung fungi cultures and thereby learned about their unique physiologies, I also learned about the different types of genetic sequencing since the project compared sequences of cultured fungi versus Next Generation sequencing of all fungi present within a dung pellet. Through working on distinct fungal projects that reassess how information about fungi is known within the field of fungal ecology, I learned not only about the two experiments I worked on but also many past related experiments and inquiries through reading scientific papers. Thanks to my foray into fungal research, I now know not only the broader significance of fungi in ecological research but also how to design and conduct ecological experiments.

Diversity of virus-host systems in hypersaline Lake Retba, Senegal.

PubMed

Sime-Ngando, Télesphore; Lucas, Soizick; Robin, Agnès; Tucker, Kimberly Pause; Colombet, Jonathan; Bettarel, Yvan; Desmond, Elie; Gribaldo, Simonetta; Forterre, Patrick; Breitbart, Mya; Prangishvili, David

2011-08-01

Remarkable morphological diversity of virus-like particles was observed by transmission electron microscopy in a hypersaline water sample from Lake Retba, Senegal. The majority of particles morphologically resembled hyperthermophilic archaeal DNA viruses isolated from extreme geothermal environments. Some hypersaline viral morphotypes have not been previously observed in nature, and less than 1% of observed particles had a head-and-tail morphology, which is typical for bacterial DNA viruses. Culture-independent analysis of the microbial diversity in the sample suggested the dominance of extremely halophilic archaea. Few of the 16S sequences corresponded to known archeal genera (Haloquadratum, Halorubrum and Natronomonas), whereas the majority represented novel archaeal clades. Three sequences corresponded to a new basal lineage of the haloarchaea. Bacteria belonged to four major phyla, consistent with the known diversity in saline environments. Metagenomic sequencing of DNA from the purified virus-like particles revealed very few similarities to the NCBI non-redundant database at either the nucleotide or amino acid level. Some of the identifiable virus sequences were most similar to previously described haloarchaeal viruses, but no sequence similarities were found to archaeal viruses from extreme geothermal environments. A large proportion of the sequences had similarity to previously sequenced viral metagenomes from solar salterns. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

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.

Identification, Purification and Characterization of Laterosporulin, a Novel Bacteriocin Produced by Brevibacillus sp. Strain GI-9

PubMed Central

Singh, Pradip Kumar; Chittpurna; Ashish; Sharma, Vikas; Patil, Prabhu B.; Korpole, Suresh

2012-01-01

Background Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology. Methodology/Findings The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database. Conclusions We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity. PMID:22403615

Identification, purification and characterization of laterosporulin, a novel bacteriocin produced by Brevibacillus sp. strain GI-9.

PubMed

Singh, Pradip Kumar; Chittpurna; Ashish; Sharma, Vikas; Patil, Prabhu B; Korpole, Suresh

2012-01-01

Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology. The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database. We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.

1,4-Benzoquinone reductase from Phanerochaete chrysosporium: cDNA cloning and regulation of expression

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

Akileswaran, L.; Brock, B.J.; Cereghino, J.L.

1999-02-01

A cDNA clone encoding a quinone reductase (QR) from the white rot basidiomycete Phanerochaete chrysosporium was isolated and sequenced. The cDNA consisted of 1,007 nucleotides and a poly(A) tail and encoded a deduced protein containing 271 amino acids. The experimentally determined eight-amino-acid N-germinal sequence of the purified QR protein from P. chrysosporium matched amino acids 72 to 79 of the predicted translation product of the cDNA. The M{sub r} of the predicted translation product, beginning with Pro-72, was essentially identical to the experimentally determined M{sub r} of one monomer of the QR dimer, and this finding suggested that QR ismore » synthesized as a proenzyme. The results of in vitro transcription-translation experiments suggested that QR is synthesized as a proenzyme with a 71-amino-acid leader sequence. This leader sequence contains two potential KEX2 cleavage sites and numerous potential cleavage sites for dipeptidyl aminopeptidase. The QR activity in cultures of P. chrysosporium increased following the addition of 2-dimethoxybenzoquinone, vanillic acid, or several other aromatic compounds. An immunoblot analysis indicated that induction resulted in an increase in the amount of QR protein, and a Northern blot analysis indicated that this regulation occurs at the level of the qr mRNA.« less

[Genetic characterization analysis on the first imported measles virus of genotype D8 in Chinese mainland].

PubMed

Sun, Xiao-Dong; Li, Chong-Shan; Tang, Xian; Li, Zhi; Zhang, Yan; Tang, Wei; Wang, Jing; Wang, Hui-Ling; Yang, Yan-Ji; Li, Jia; Yuan, Zheng-An; Xu, Wen-Bo

2013-11-01

This study analyzed the genetic characterization on first imported measles virus of genotype D8 in Chinese mainland. Serums were collected from the suspicious MV patients to detect IgM antibody in ELISA. Throat swabs were cultured in Vero/SLAM cell line to get measles virus isolates. Part of the nucleotide sequence of the 3' terminus of nucleoprotein (N) gene of these isolates were amplified by RT-PCR, and the amplicons were directly sequenced. The phylogenetic analysis was based on the nucleotide sequence about 456 base pairs of the 3' terminus of nucleoprotein (N) gene. Results showed that it reported 1 105 suspicious measles cases in shanghai, 2012, including 590 confirmed cases and 2 clinical case. The reported morbidity was 2.52 per one hundred thousand. 247 measles viruses were isolated from 984 throat swabs specimen. Most of them belonged to sub-genotype H1a except Shanghai12-239 was genotype D8. The homology of nucleotide and amino acid sequences were 97.8% and 98.6% respectively between Shanghai12-239 and WHO reference strain (Manchester. UNK30.94(D8)AF280803). Those were 89.6%-94.5% and 88.7%-95.3% between Shanghai12-239 and WHO reference strains of other genotypes.

Genetic and Antigenic Typing of Seasonal Influenza Virus Breakthrough Cases from a 2008-2009 Vaccine Efficacy Trial

PubMed Central

Durviaux, Serge; Treanor, John; Beran, Jiri; Duval, Xavier; Esen, Meral; Feldman, Gregory; Frey, Sharon E.; Launay, Odile; Leroux-Roels, Geert; McElhaney, Janet E.; Nowakowski, Andrzej; Ruiz-Palacios, Guillermo M.; van Essen, Gerrit A.; Oostvogels, Lidia; Devaster, Jeanne-Marie

2014-01-01

Estimations of the effectiveness of vaccines against seasonal influenza virus are guided by comparisons of the antigenicities between influenza virus isolates from clinical breakthrough cases with strains included in a vaccine. This study examined whether the prediction of antigenicity using a sequence analysis of the hemagglutinin (HA) gene-encoded HA1 domain is a simpler alternative to using the conventional hemagglutination inhibition (HI) assay, which requires influenza virus culturing. Specimens were taken from breakthrough cases that occurred in a trivalent influenza virus vaccine efficacy trial involving >43,000 participants during the 2008-2009 season. A total of 498 influenza viruses were successfully subtyped as A(H3N2) (380 viruses), A(H1N1) (29 viruses), B(Yamagata) (23 viruses), and B(Victoria) (66 viruses) from 603 PCR- or culture-confirmed specimens. Unlike the B strains, most A(H3N2) (377 viruses) and all A(H1N1) viruses were classified as homologous to the respective vaccine strains based on their HA1 domain nucleic acid sequence. HI titers relative to the respective vaccine strains and PCR subtyping were determined for 48% (182/380) of A(H3N2) and 86% (25/29) of A(H1N1) viruses. Eighty-four percent of the A(H3N2) and A(H1N1) viruses classified as homologous by sequence were matched to the respective vaccine strains by HI testing. However, these homologous A(H3N2) and A(H1N1) viruses displayed a wide range of relative HI titers. Therefore, although PCR is a sensitive diagnostic method for confirming influenza virus cases, HA1 sequence analysis appeared to be of limited value in accurately predicting antigenicity; hence, it may be inappropriate to classify clinical specimens as homologous or heterologous to the vaccine strain for estimating vaccine efficacy in a prospective clinical trial. PMID:24371255

Malassezia furfur in infantile seborrheic dermatitis.

PubMed

Wananukul, Siriwan; Chindamporn, Ariya; Yumyourn, Poomjit; Payungporn, Sunchai; Samathi, Chanchuree; Poovorawan, Yong

2005-01-01

Our objective was to study both incidence and various strains of Malassezia in infantile seborrheic dermatitis (ISD). Sixty infants between 2 weeks and 2 years old with clinical diagnosis of ISD at the Department of Pediatrics, King Chulalongkorn Memorial Hospital from May 2002 to April 2003 were recruited. Malassezia spp. were isolated from cultured skin samples of the patients, genomic DNA was extracted and the ITS1 rDNA region was amplified. The PCR product was examined by agarose gel electrophoresis and DNA sequences were determined. The ITS1 sequences were also subjected to phylogenetic analysis and species identification. ISD is most commonly found in infants below the age of 2 months (64%), followed by those between 2 and 4 months (28%) old. Cultures yielded yeast-like colonies in 15 specimens. PCR yielded 200-bp products (Candida) in 3 patients and 300-bp products (Malassezia furfur) in 12 patients (18%). Sugar fermentation using API 20C aux performed on the three 200-bp PCR products yielded Candida species. M. furfur was the only Malassezia recovered from skin scrapings of children with ISD.

Culture-Independent Identification of Periodontitis-Associated Porphyromonas and Tannerella Populations by Targeted Molecular Analysis

PubMed Central

de Lillo, A.; Booth, V.; Kyriacou, L.; Weightman, A. J.; Wade, W. G.

2004-01-01

Periodontitis is the commonest bacterial disease of humans and is the major cause of adult tooth loss. About half of the oral microflora is unculturable; and 16S rRNA PCR, cloning, and sequencing techniques have demonstrated the high level of species richness of the oral microflora. In the present study, a PCR primer set specific for the genera Porphyromonas and Tannerella was designed and used to analyze the bacterial populations in subgingival plaque samples from inflamed shallow and deep sites in subjects with periodontitis and shallow sites in age- and sex-matched controls. A total of 308 clones were sequenced and found to belong to one of six Porphyromonas or Tannerella species or phylotypes, one of which, Porphyromonas P3, was novel. Tannerella forsythensis was found in significantly higher proportions in patients than in controls. Porphyromonas catoniae and Tannerella phylotype BU063 appeared to be associated with shallow sites. Targeted culture-independent molecular ecology studies have a valuable role to play in the identification of bacterial targets for further investigations of the pathogenesis of bacterial infections. PMID:15583276

Isolation and characterization of two crude oil-degrading fungi strains from Rumaila oil field, Iraq.

PubMed

Al-Hawash, Adnan B; Alkooranee, Jawadayn T; Abbood, Hayder A; Zhang, Jialong; Sun, Jin; Zhang, Xiaoyu; Ma, Fuying

2018-03-01

Among four crude oil-degrading fungi strains that were isolated from a petroleum-polluted area in the Rumaila oil field, two fungi strains showed high activity in aliphatic hydrocarbon degradation. ITS sequencing and analysis of morphological and biochemical characteristics identified these strains as Penicillium sp. RMA1 and RMA2. Gravimetric and gas chromatography analysis of the crude oil remaining in the culture medium after 14 days of incubation at 30 °C showed that RMA1 and RMA2 degraded the crude oil by 57% and 55%, respectively. These strains reduced surface tension when cultured on crude oil (1% v/v) and exhibited a cell surface hydrophobicity of more than 70%. These results suggested that RMA1 and RMA2 performed effective crude oil-degrading activity and crude oil emulsification. In conclusion, these fungal strains can be used in bioremediation process and oil pollution reduction in aquatic ecosystems.

Yeast species diversity in apple juice for cider production evidenced by culture-based method.

PubMed

Lorenzini, Marilinda; Simonato, Barbara; Zapparoli, Giacomo

2018-05-07

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.

Phylogeography of infectious haematopoietic necrosis virus in North America

USGS Publications Warehouse

Kurath, Gael; Garver, Kyle A.; Troyer, Ryan M.; Emmenegger, Eveline J.; Einer-Jensen, Katja; Anderson, Eric D.

2003-01-01

Infectious hematopoietic necrosis virus (IHNV) is a rhabdoviral pathogen that infects wild and cultured salmonid fish throughout the Pacific Northwest of North America. IHNV causes severe epidemics in young fish and can cause disease or occur asymptomatically in adults. In a broad survey of 323 IHNV field isolates, sequence analysis of a 303 nucleotide variable region within the glycoprotein gene revealed a maximum nucleotide diversity of 8.6 %, indicating low genetic diversity overall for this virus. Phylogenetic analysis revealed three major virus genogroups, designated U, M and L, which varied in topography and geographical range. Intragenogroup genetic diversity measures indicated that the M genogroup had three- to fourfold more diversity than the other genogroups and suggested relatively rapid evolution of the M genogroup and stasis within the U genogroup. We speculate that factors influencing IHNV evolution may have included ocean migration ranges of their salmonid host populations and anthropogenic effects associated with fish culture.

DNA Sequencing in Cultural Heritage.

PubMed

Vai, Stefania; Lari, Martina; Caramelli, David

2016-02-01

During the last three decades, DNA analysis on degraded samples revealed itself as an important research tool in anthropology, archaeozoology, molecular evolution, and population genetics. Application on topics such as determination of species origin of prehistoric and historic objects, individual identification of famous personalities, characterization of particular samples important for historical, archeological, or evolutionary reconstructions, confers to the paleogenetics an important role also for the enhancement of cultural heritage. A really fast improvement in methodologies in recent years led to a revolution that permitted recovering even complete genomes from highly degraded samples with the possibility to go back in time 400,000 years for samples from temperate regions and 700,000 years for permafrozen remains and to analyze even more recent material that has been subjected to hard biochemical treatments. Here we propose a review on the different methodological approaches used so far for the molecular analysis of degraded samples and their application on some case studies.

Loktanella spp. Gb03 as an algicidal bacterium, isolated from the culture of Dinoflagellate Gambierdiscus belizeanus.

PubMed

Bloh, Anmar Hameed; Usup, Gires; Ahmad, Asmat

2016-02-01

Bacteria associated with harmful algal blooms can play a crucial role in regulating algal blooms in the environment. This study aimed at isolating and identifying algicidal bacteria in Dinoflagellate culture and to determine the optimum growth requirement of the algicidal bacteria, Loktanella sp. Gb-03. The Dinoflagellate culture used in this study was supplied by Professor Gires Usup's Laboratory, School of Environmental and Natural Resources Sciences, Faculty of Science and Technology, University Kebangsaan Malaysia, Malaysia. The culture was used for the isolation of Loktanella sp., using biochemical tests, API 20 ONE kits. The fatty acid content of the isolates and the algicidal activity were further evaluated, and the phenotype was determined through the phylogenetic tree. Gram-negative, non-motile, non-spore-forming, short rod-shaped, aerobic bacteria (Gb01, Gb02, Gb03, Gb04, Gb05, and Gb06) were isolated from the Dinoflagellate culture. The colonies were pink in color, convex with a smooth surface and entire edge. The optimum growth temperature for the Loktanella sp. Gb03 isolate was determined to be 30°C, in 1% of NaCl and pH7. Phylogenetic analysis based on 16S rRNA gene sequences showed that the bacterium belonged to the genus Loktanella of the class Alphaproteobacteria and formed a tight cluster with the type strain of Loktanella pyoseonensis (97.0% sequence similarity). On the basis of phenotypic, phylogenetic data and genetic distinctiveness, strain Gb-03, were placed in the genus Loktanella as the type strain of species. Moreover, it has algicidal activity against seven toxic Dinoflagellate. The algicidal property of the isolated Loktanella is vital, especially where biological control is needed to mitigate algal bloom or targeted Dinoflagellates.

Loktanella spp. Gb03 as an algicidal bacterium, isolated from the culture of Dinoflagellate Gambierdiscus belizeanus

PubMed Central

Bloh, Anmar Hameed; Usup, Gires; Ahmad, Asmat

2016-01-01

Aim: Bacteria associated with harmful algal blooms can play a crucial role in regulating algal blooms in the environment. This study aimed at isolating and identifying algicidal bacteria in Dinoflagellate culture and to determine the optimum growth requirement of the algicidal bacteria, Loktanella sp. Gb-03. Materials and Methods: The Dinoflagellate culture used in this study was supplied by Professor Gires Usup's Laboratory, School of Environmental and Natural Resources Sciences, Faculty of Science and Technology, University Kebangsaan Malaysia, Malaysia. The culture was used for the isolation of Loktanella sp., using biochemical tests, API 20 ONE kits. The fatty acid content of the isolates and the algicidal activity were further evaluated, and the phenotype was determined through the phylogenetic tree. Results: Gram-negative, non-motile, non-spore-forming, short rod-shaped, aerobic bacteria (Gb01, Gb02, Gb03, Gb04, Gb05, and Gb06) were isolated from the Dinoflagellate culture. The colonies were pink in color, convex with a smooth surface and entire edge. The optimum growth temperature for the Loktanella sp. Gb03 isolate was determined to be 30°C, in 1% of NaCl and pH7. Phylogenetic analysis based on 16S rRNA gene sequences showed that the bacterium belonged to the genus Loktanella of the class Alphaproteobacteria and formed a tight cluster with the type strain of Loktanella pyoseonensis (97.0% sequence similarity). Conclusion: On the basis of phenotypic, phylogenetic data and genetic distinctiveness, strain Gb-03, were placed in the genus Loktanella as the type strain of species. Moreover, it has algicidal activity against seven toxic Dinoflagellate. The algicidal property of the isolated Loktanella is vital, especially where biological control is needed to mitigate algal bloom or targeted Dinoflagellates. PMID:27051199

Genome-wide transcriptome and expression profile analysis of Phalaenopsis during explant browning.

PubMed

Xu, Chuanjun; Zeng, Biyu; Huang, Junmei; Huang, Wen; Liu, Yumei

2015-01-01

Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning.

Genome-Wide Transcriptome and Expression Profile Analysis of Phalaenopsis during Explant Browning

PubMed Central

Xu, Chuanjun; Zeng, Biyu; Huang, Junmei; Huang, Wen; Liu, Yumei

2015-01-01

Background Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. Methodology/Principal Findings We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Conclusions/Significance Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning. PMID:25874455

Genome Sequence of the Novel Marine Member of the Gammaproteobacteria Strain HTCC5015▿

PubMed Central

Thrash, J. Cameron; Stingl, Ulrich; Cho, Jang-Cheon; Ferriera, Steve; Johnson, Justin; Vergin, Kevin L.; Giovannoni, Stephen J.

2010-01-01

HTCC5015 is a novel, highly divergent marine member of the Gammaproteobacteria, currently without a cultured representative with greater than 89% 16S rRNA gene identity to itself. The organism was isolated from water collected from Hydrostation S south of Bermuda using high-throughput dilution-to-extinction culturing techniques. Here we present the genome sequence of the unique Gammaproteobacterium strain HTCC5015. PMID:20472792

Legionella confirmation in cooling tower water. Comparison of culture, real-time PCR and next generation sequencing.

PubMed

Farhat, Maha; Shaheed, Raja A; Al-Ali, Haider H; Al-Ghamdi, Abdullah S; Al-Hamaqi, Ghadeer M; Maan, Hawraa S; Al-Mahfoodh, Zainab A; Al-Seba, Hussain Z

2018-02-01

To investigate the presence of Legionella spp in cooling tower water. Legionella proliferation in cooling tower water has serious public health implications as it can be transmitted to humans via aerosols and cause Legionnaires' disease. Samples of cooling tower water were collected from King Fahd Hospital of the University (KFHU) (Imam Abdulrahman Bin Faisal University, 2015/2016). The water samples were analyzed by a standard Legionella culture method, real-time polymerase chain reaction (RT-PCR), and 16S rRNA next-generation sequencing. In addition, the bacterial community composition was evaluated. All samples were negative by conventional Legionella culture. In contrast, all water samples yielded positive results by real-time PCR (105 to 106 GU/L). The results of 16S rRNA next generation sequencing showed high similarity and reproducibility among the water samples. The majority of sequences were Alpha-, Beta-, and Gamma-proteobacteria, and Legionella was the predominant genus. The hydrogen-oxidizing gram-negative bacterium Hydrogenophaga was present at high abundance, indicating high metabolic activity. Sphingopyxis, which is known for its resistance to antimicrobials and as a pioneer in biofilm formation, was also detected. Our findings indicate that monitoring of Legionella in cooling tower water would be enhanced by use of both conventional culturing and molecular methods.

Francisella marina sp. nov., etiologic agent of systemic disease in cultured spotted rose snapper (Lutjanus guttatus) in Central America.

PubMed

Soto, Esteban; Griffin, Matt J; Morales, Juan Alberto; Barquero Calvo, Elías; de Alexandre Sebastião, Fernanda; Lopez Porras, Adrián; Víquez-Rodríguez, Xindy; Reichley, Stephen R; Rosser, Thomas G; Ware, Cynthia; Byrne, Barbara A; García, Julio C; LaFrentz, Benjamin R; Camus, Alvin C

2018-06-18

Historically, piscine francisellosis in various warm, temperate and coldwater fish hosts has been attributed to Francisella noatunensis From 2015-2016, an undescribed Francisella sp. was recovered during mortality events in cultured spotted rose snapper ( Lutjanus guttatus ) off the Pacific coast of Central America. Despite high mortality and emaciation, limited gross findings were observed in affected fish. Histological examination revealed multifocal granulomatous lesions, with the presence of numerous small, pleomorphic coccobacilli, predominantly in the peritoneum, spleen, kidneys, liver, pancreas, heart, and intestine. Sequencing of an ∼1400 bp fragment of the 16S rRNA gene demonstrated these isolates to be most similar (99.9% identity) to Francisella sp. isolate TX077308 cultured from sea water in the Gulf of Mexico, while sharing <99% similarity to other Fransicella spp. Biochemical analysis, multi-locus sequence comparisons of select housekeeping-genes, repetitive extragenic palindromic PCR fingerprinting, matrix-assisted laser-desorption ionization-time of flight mass spectrometry, and fatty acid methyl esters analysis revealed marked differences between these isolates and other described members of the genus. Koch's postulates were fulfilled by experimental intracoelomic injection and immersion trials using Nile ( Oreochromis niloticus ) and blue tilapia ( O. aureus ). Based on observed phenotypic and genotypic differences from recognized Francisella spp., the name Francisella marina sp. nov. (NRRL # B-65518) is proposed to accommodate these novel strains. Importance Finfish aquaculture is the fastest growing global food production sector. Infectious disease, particularly emergent pathogens, pose a significant threat to established and nascent aquaculture industries worldwide. Herein, we characterize a novel pathogen isolated from mortality events in cultured spotted rose snapper in Central America. The bacteria recovered from these outbreaks were genetically and phenotypically dissimilar from other known Francisella spp. from fish, representing a previously unrecognized member of the genus Francisella , for which the name Francisella marina sp. nov. is proposed. Copyright © 2018 American Society for Microbiology.

The Fungal Frontier: A Comparative Analysis of Methods Used in the Study of the Human Gut Mycobiome.

PubMed

Huseyin, Chloe E; Rubio, Raul Cabrera; O'Sullivan, Orla; Cotter, Paul D; Scanlan, Pauline D

2017-01-01

The human gut is host to a diverse range of fungal species, collectively referred to as the gut "mycobiome". The gut mycobiome is emerging as an area of considerable research interest due to the potential roles of these fungi in human health and disease. However, there is no consensus as to what the best or most suitable methodologies available are with respect to characterizing the human gut mycobiome. The aim of this study is to provide a comparative analysis of several previously published mycobiome-specific culture-dependent and -independent methodologies, including choice of culture media, incubation conditions (aerobic versus anaerobic), DNA extraction method, primer set and freezing of fecal samples to assess their relative merits and suitability for gut mycobiome analysis. There was no significant effect of media type or aeration on culture-dependent results. However, freezing was found to have a significant effect on fungal viability, with significantly lower fungal numbers recovered from frozen samples. DNA extraction method had a significant effect on DNA yield and quality. However, freezing and extraction method did not have any impact on either α or β diversity. There was also considerable variation in the ability of different fungal-specific primer sets to generate PCR products for subsequent sequence analysis. Through this investigation two DNA extraction methods and one primer set was identified which facilitated the analysis of the mycobiome for all samples in this study. Ultimately, a diverse range of fungal species were recovered using both approaches, with Candida and Saccharomyces identified as the most common fungal species recovered using culture-dependent and culture-independent methods, respectively. As has been apparent from ecological surveys of the bacterial fraction of the gut microbiota, the use of different methodologies can also impact on our understanding of gut mycobiome composition and therefore requires careful consideration. Future research into the gut mycobiome needs to adopt a common strategy to minimize potentially confounding effects of methodological choice and to facilitate comparative analysis of datasets.

Terrestrial microbes in martian and chondritic meteorites

NASA Astrophysics Data System (ADS)

Airieau, S.; Piceno, Y.; Andersen, G.

2007-08-01

Good extraterrestrial analogs for microbiology are SNC meteorites as Mars analogs, and chondrites as early planet analogs. Chondrites and SNCs are used to trace processes in the early solar system and on Mars. Yet, questions about terrestrial contamination and its effects on the isotopic, chemical and mineral characteristics often arise. A wide biodiversity was found in 21 chondrites of groups CR, CV, CK, CO from ANSMET, CI and CM Falls, and 8 SNCs. Studies documented the alteration of meteorites by weathering and biology [1]-[6], and during aqueous extraction for oxygen isotopic analysis [7], visible biofilms grew in the meteorite solutions in days. To assess biological isotopic and chemical impacts, cultures were incubated 11 months and analyzed by PCR. The sequences for 2 isolates from EET 87770 and Leoville were of a good quality with long sequence reads. In EET 87770, the closest matches were in the genus Microbacterium. Soil and plant isolates were close relatives by sequence comparison. Bacillus, a common soil bacterial genus, grew in a Leoville culture. All SNCs exhibited biological activity measured independently by LAL but only 1 colony was successfully cultured from grains of the SNC Los Angeles. Isotopic analyses of samples with various amounts of microbial contamination could help quantified isotopic impact of microbes on protoplanetary chemistry in these rocks. References: [1] Gounelle, M.& Zolensky M. (2001) LPS XXXII, Abstract #999. [2] Fries, M. et al. (2005) Meteoritical Society Meeting 68, Abstract # 5201. [3] Burckle, L. H. & Delaney, J. S (1999) Meteoritics & Planet. Sci., 32, 475. [4] Whitby, C. et al. (2000) LPS XXXI, Abstract #1732. [5] Tyra M. et al., (2007) Geochim. Cosmochim. Acta, 71, 782 [6] Toporski, J. & Steele A., (2007) Astrobiology, 7, 389 [7]Airieau, S. et al (2005) Geochim. Cosmochim. Acta, 69, 4166.

Virological investigation of hand, foot, and mouth disease in a tertiary care center in South India.

PubMed

Vijayaraghavan, Pavithra M; Chandy, Sara; Selvaraj, Kavitha; Pulimood, Susanne; Abraham, Asha M

2012-07-01

Hand, foot, and mouth disease (HFMD) remains a common problem in India, yet its etiology is largely unknown as diagnosis is based on clinical characteristics. There are very few laboratory-based molecular studies on HFMD outbreaks. The aim of this study was to characterize HFMD-related isolates by molecular techniques. Between 2005 and 2008, during two documented HFMD outbreaks, 30 suspected HFMD cases presented at the Outpatient Unit of the Department of Dermatology, Christian Medical College (CMC), Vellore. Seventy-eight clinical specimens (swabs from throat, mouth, rectum, anus, buttocks, tongue, forearm, sole, and foot) were received from these patients at the Department of Clinical Virology, CMC, for routine diagnosis of hand, foot, and mouth disease. Samples from these patients were cultured in Vero and rhabdomyosarcoma (RD) cell lines. Isolates producing enterovirus-like cytopathogenic effect (CPE) in cell culture were identified by a nested reverse transcription-based polymerase chain reaction (RT-PCR) and sequenced. The nucleotide sequences were analyzed using the BioEdit sequence program. Homology searches were performed using the Basic Local Alignment Search Tool (BLAST) algorithm. The statistical analysis was performed using Epi Info version 6.04b and Microsoft Excel 2002 (Microsoft Office XP). Of the 30 suspected HFMD cases, only 17 (57%) were laboratory confirmed and Coxsackievirus A16 (CVA16) was identified as the etiological agent in all these cases. Coxsackievirus A16 (CVA16) was identified as the virus that caused the HFMD outbreaks in Vellore between 2005 and 2008. Early confirmation of HFMD helps to initiate control measures to interrupt virus transmission. In the laboratory, classical diagnostic methods, culture and serological tests are being replaced by molecular techniques. Routine surveillance systems will help understand the epidemiology of HFMD in India.

Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil.

PubMed

Cavalcanti, Sarah Desirée Barbosa; Vidal, Mônica Scarpelli Martinelli; Sousa, Maria da Glória Teixeira de; Del Negro, Gilda Maria Barbaro

2013-01-01

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.

PubMed

Quick, Joshua; Grubaugh, Nathan D; Pullan, Steven T; Claro, Ingra M; Smith, Andrew D; Gangavarapu, Karthik; Oliveira, Glenn; Robles-Sikisaka, Refugio; Rogers, Thomas F; Beutler, Nathan A; Burton, Dennis R; Lewis-Ximenez, Lia Laura; de Jesus, Jaqueline Goes; Giovanetti, Marta; Hill, Sarah C; Black, Allison; Bedford, Trevor; Carroll, Miles W; Nunes, Marcio; Alcantara, Luiz Carlos; Sabino, Ester C; Baylis, Sally A; Faria, Nuno R; Loose, Matthew; Simpson, Jared T; Pybus, Oliver G; Andersen, Kristian G; Loman, Nicholas J

2017-06-01

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.

Use of Fe(III) as an electron acceptor to recover previously uncultured hyperthermophiles: isolation and characterization of Geothermobacterium ferrireducens gen. nov., sp. nov.

PubMed

Kashefi, Kazem; Holmes, Dawn E; Reysenbach, Anna-Louise; Lovley, Derek R

2002-04-01

It has recently been recognized that the ability to use Fe(III) as a terminal electron acceptor is a highly conserved characteristic in hyperthermophilic microorganisms. This suggests that it may be possible to recover as-yet-uncultured hyperthermophiles in pure culture if Fe(III) is used as an electron acceptor. As part of a study of the microbial diversity of the Obsidian Pool area in Yellowstone National Park, Wyo., hot sediment samples were used as the inoculum for enrichment cultures in media containing hydrogen as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. A pure culture was recovered on solidified, Fe(III) oxide medium. The isolate, designated FW-1a, is a hyperthermophilic anaerobe that grows exclusively by coupling hydrogen oxidation to the reduction of poorly crystalline Fe(III) oxide. Organic carbon is not required for growth. Magnetite is the end product of Fe(III) oxide reduction under the culture conditions evaluated. The cells are rod shaped, about 0.5 microm by 1.0 to 1.2 microm, and motile and have a single flagellum. Strain FW-1a grows at circumneutral pH, at freshwater salinities, and at temperatures of between 65 and 100 degrees C with an optimum of 85 to 90 degrees C. To our knowledge this is the highest temperature optimum of any organism in the Bacteria. Analysis of the 16S ribosomal DNA (rDNA) sequence of strain FW-1a places it within the Bacteria, most closely related to abundant but uncultured microorganisms whose 16S rDNA sequences have been previously recovered from Obsidian Pool and a terrestrial hot spring in Iceland. While previous studies inferred that the uncultured microorganisms with these 16S rDNA sequences were sulfate-reducing organisms, the physiology of the strain FW-1a, which does not reduce sulfate, indicates that these organisms are just as likely to be Fe(III) reducers. These results further demonstrate that Fe(III) may be helpful for recovering as-yet-uncultured microorganisms from hydrothermal environments and illustrate that caution must be used in inferring the physiological characteristics of at least some thermophilic microorganisms solely from 16S rDNA sequences. Based on both its 16S rDNA sequence and physiological characteristics, strain FW-1a represents a new genus among the Bacteria. The name Geothermobacterium ferrireducens gen. nov., sp. nov., is proposed (ATCC BAA-426).

FARME DB: a functional antibiotic resistance element database

PubMed Central

Wallace, James C.; Port, Jesse A.; Smith, Marissa N.; Faustman, Elaine M.

2017-01-01

Antibiotic resistance (AR) is a major global public health threat but few resources exist that catalog AR genes outside of a clinical context. Current AR sequence databases are assembled almost exclusively from genomic sequences derived from clinical bacterial isolates and thus do not include many microbial sequences derived from environmental samples that confer resistance in functional metagenomic studies. These environmental metagenomic sequences often show little or no similarity to AR sequences from clinical isolates using standard classification criteria. In addition, existing AR databases provide no information about flanking sequences containing regulatory or mobile genetic elements. To help address this issue, we created an annotated database of DNA and protein sequences derived exclusively from environmental metagenomic sequences showing AR in laboratory experiments. Our Functional Antibiotic Resistant Metagenomic Element (FARME) database is a compilation of publically available DNA sequences and predicted protein sequences conferring AR as well as regulatory elements, mobile genetic elements and predicted proteins flanking antibiotic resistant genes. FARME is the first database to focus on functional metagenomic AR gene elements and provides a resource to better understand AR in the 99% of bacteria which cannot be cultured and the relationship between environmental AR sequences and antibiotic resistant genes derived from cultured isolates. Database URL: http://staff.washington.edu/jwallace/farme PMID:28077567

Sequence-based analysis of the microbial composition of water kefir from multiple sources.

PubMed

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

2013-11-01

Water kefir is a water-sucrose-based beverage, fermented by a symbiosis of bacteria and yeast to produce a final product that is lightly carbonated, acidic and that has a low alcohol percentage. The microorganisms present in water kefir are introduced via water kefir grains, which consist of a polysaccharide matrix in which the microorganisms are embedded. We aimed to provide a comprehensive sequencing-based analysis of the bacterial population of water kefir beverages and grains, while providing an initial insight into the corresponding fungal population. To facilitate this objective, four water kefirs were sourced from the UK, Canada and the United States. Culture-independent, high-throughput, sequencing-based analyses revealed that the bacterial fraction of each water kefir and grain was dominated by Zymomonas, an ethanol-producing bacterium, which has not previously been detected at such a scale. The other genera detected were representatives of the lactic acid bacteria and acetic acid bacteria. Our analysis of the fungal component established that it was comprised of the genera Dekkera, Hanseniaspora, Saccharomyces, Zygosaccharomyces, Torulaspora and Lachancea. This information will assist in the ultimate identification of the microorganisms responsible for the potentially health-promoting attributes of these beverages. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge.

PubMed

Brulle, Franck; Jeffroy, Fanny; Madec, Stéphanie; Nicolas, Jean-Louis; Paillard, Christine

2012-10-01

The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams. Copyright © 2012 Elsevier Ltd. All rights reserved.

Chromosomal Diversity in Lactococcus lactis and the Origin of Dairy Starter Cultures

PubMed Central

Kelly, William J.; Ward, Lawrence J. H.; Leahy, Sinead C.

2010-01-01

A large collection of Lactococcus lactis strains, including wild-type isolates and dairy starter cultures, were screened on the basis of their phenotype and the macrorestriction patterns produced from pulsed-field gel electrophoresis (PFGE) analysis of SmaI digests of genomic DNA. Three groups of dairy starter cultures, used for different purposes in the dairy industry, and a fourth group made up of strains isolated from the environment were selected for analysis of their chromosomal diversity using the endonuclease I-CeuI. Chromosome architecture was largely conserved with each strain having six copies of the rRNA genes, and the chromosome size of individual strains ranged between 2,240 and 2,688 kb. The origin of L. lactis strains showed the greatest correlation with chromosome size, and dairy strains, particularly those with the cremoris phenotype, had smaller chromosomes than wild-type strains. Overall, this study, coupled with analysis of the sequenced L. lactis genomes, provides evidence that defined strain dairy starter cultures have arisen from plant L. lactis strains. Adaptation of these strains to the dairy environment has involved loss of functions resulting in smaller chromosomes and acquisition of genes (usually plasmid associated) that facilitate growth in milk. We conclude that dairy starter cultures generally and the industrially used cremoris and diacetylactis phenotype strains in particular comprise a specialized group of L. lactis strains that have been selected to become an essential component of industrial processes and have evolved accordingly, so that they are no longer fit to survive outside the dairy environment. PMID:20847124

Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep.

PubMed

Hecht, Jochen; Kuhl, Heiner; Haas, Stefan A; Bauer, Sebastian; Poustka, Albert J; Lienau, Jasmin; Schell, Hanna; Stiege, Asita C; Seitz, Volkhard; Reinhardt, Richard; Duda, Georg N; Mundlos, Stefan; Robinson, Peter N

2006-07-05

The sheep is an important model animal for testing novel fracture treatments and other medical applications. Despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and DNA sequences are available for only a small number of sheep genes. In this work we have sequenced over 47 thousand expressed sequence tags (ESTs) from libraries developed from healing bone in a sheep model of fracture healing. These ESTs were clustered with the previously available 10 thousand sheep ESTs to a total of 19087 contigs with an average length of 603 nucleotides. We used the newly identified sequences to develop RT-PCR assays for 78 sheep genes and measured differential expression during the course of fracture healing between days 7 and 42 postfracture. All genes showed significant shifts at one or more time points. 23 of the genes were differentially expressed between postfracture days 7 and 10, which could reflect an important role for these genes for the initiation of osteogenesis. The sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for Ovis aries, with partial or complete sequences being made available for over 5,800 previously unsequenced sheep genes.

Analysis of simian immunodeficiency virus sequence variation in tissues of rhesus macaques with simian AIDS.

PubMed Central

Kodama, T; Mori, K; Kawahara, T; Ringler, D J; Desrosiers, R C

1993-01-01

One rhesus macaque displayed severe encephalomyelitis and another displayed severe enterocolitis following infection with molecularly cloned simian immunodeficiency virus (SIV) strain SIVmac239. Little or no free anti-SIV antibody developed in these two macaques, and they died relatively quickly (4 to 6 months) after infection. Manifestation of the tissue-specific disease in these macaques was associated with the emergence of variants with high replicative capacity for macrophages and primary infection of tissue macrophages. The nature of sequence variation in the central region (vif, vpr, and vpx), the env gene, and the nef long terminal repeat (LTR) region in brain, colon, and other tissues was examined to see whether specific genetic changes were associated with SIV replication in brain or gut. Sequence analysis revealed strong conservation of the intergenic central region, nef, and the LTR. However, analysis of env sequences in these two macaques and one other revealed significant, interesting patterns of sequence variation. (i) Changes in env that were found previously to contribute to the replicative ability of SIVmac for macrophages in culture were present in the tissues of these animals. (ii) The greatest variability was located in the regions between V1 and V2 and from "V3" through C3 in gp120, which are different in location from the variable regions observed previously in animals with strong antibody responses and long-term persistent infection. (iii) The predominant sequence change of D-->N at position 385 in C3 is most surprising, since this change in both SIV and human immunodeficiency virus type 1 has been associated with dramatically diminished affinity for CD4 and replication in vitro. (iv) The nature of sequence changes at some positions (146, 178, 345, 385, and "V3") suggests that viral replication in brain and gut may be facilitated by specific sequence changes in env in addition to those that impart a general ability to replicate well in macrophages. These results demonstrate that complex selective pressures, including immune responses and varying cell and tissue specificity, can influence the nature of sequence changes in env. Images PMID:8411355

Microbial forensics: fiber optic microarray subtyping of Bacillus anthracis

NASA Astrophysics Data System (ADS)

Shepard, Jason R. E.

2009-05-01

The past decade has seen increased development and subsequent adoption of rapid molecular techniques involving DNA analysis for detection of pathogenic microorganisms, also termed microbial forensics. The continued accumulation of microbial sequence information in genomic databases now better positions the field of high-throughput DNA analysis to proceed in a more manageable fashion. The potential to build off of these databases exists as technology continues to develop, which will enable more rapid, cost effective analyses. This wealth of genetic information, along with new technologies, has the potential to better address some of the current problems and solve the key issues involved in DNA analysis of pathogenic microorganisms. To this end, a high density fiber optic microarray has been employed, housing numerous DNA sequences simultaneously for detection of various pathogenic microorganisms, including Bacillus anthracis, among others. Each organism is analyzed with multiple sequences and can be sub-typed against other closely related organisms. For public health labs, real-time PCR methods have been developed as an initial preliminary screen, but culture and growth are still considered the gold standard. Technologies employing higher throughput than these standard methods are better suited to capitalize on the limitless potential garnered from the sequence information. Microarray analyses are one such format positioned to exploit this potential, and our array platform is reusable, allowing repetitive tests on a single array, providing an increase in throughput and decrease in cost, along with a certainty of detection, down to the individual strain level.

Bifidobacterium pseudolongum has characteristics of a keystone species in bifidobacterial blooms in the ceca of rats fed Hi-Maize starch.

PubMed

Centanni, Manuela; Lawley, Blair; Butts, Christine A; Roy, Nicole; Lee, Julian; Kelly, William J; Tannock, Gerald W

2018-05-25

Starches resistant to mammalian digestion are present in foods and pass to the large bowel where they may be degraded and fermented by the microbiota. Increases in relative abundances of bifidobacteria (blooms) have been reported in rats whose diet was supplemented with Hi-Maize resistant starch. We determined that the bifidobacterial species present in the rat cecum under these circumstances mostly belonged to Bifidobacterium animalis However, cultures of B. animalis isolated from the rats failed to degrade Hi-Maize starch to any extent. In contrast, Bifidobacterium pseudolongum also detected in the rat microbiota had high starch-degrading ability. Transcriptional comparisons showed increased expression of a Type 1 pullulanase, alpha amylase, and 'glycogen debranching enzyme' by B. pseudolongum when cultured in medium containing Hi-Maize starch. Maltose was released into the culture medium and B. animalis cultures had shorter doubling times in maltose medium compared to B. pseudolongum Thus B. pseudolongum, which was present at a consistently low abundance in the microbiota, but which has extensive enzymic capacity to degrade resistant starch, showed the attributes of a keystone species associated with the bifidobacterial bloom. IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the rat gut) using DNA-based observations and in vitro experimentation. The microbial community (microbiota, microbiome) of the large bowel of animals, including humans, has been studied extensively by use of high throughput DNA sequencing methods and advanced bioinformatics analysis. These studies reveal the compositions and genetic capacities of microbiotas, but not the intricacies of how microbial communities function. Our work, combining DNA sequence analysis and laboratory experiments with cultured strains of bacteria, revealed that increased abundance of bifidobacteria in the rat gut, induced by feeding indigestible starch, involved a species that cannot itself degrade the starch ( Bifidobacterium animalis ) but cohabits with a species that can ( Bifidobacterium pseudolongum ). This latter species has the characteristics of a keystone species in the community because it had low abundance but high ability to perform a critical function (hydrolysis of resistant starch). Copyright © 2018 American Society for Microbiology.

Comparison of pectin-degrading fungal communities in temperate forests using glycosyl hydrolase family 28 pectinase primers targeting Ascomycete fungi.

PubMed

Gacura, Matthew D; Sprockett, Daniel D; Heidenreich, Bess; Blackwood, Christopher B

2016-04-01

Fungi have developed a wide assortment of enzymes to break down pectin, a prevalent polymer in plant cell walls that is important in plant defense and structure. One enzyme family used to degrade pectin is the glycosyl hydrolase family 28 (GH28). In this study we developed primers for the amplification of GH28 coding genes from a database of 293 GH28 sequences from 40 fungal genomes. The primers were used to successfully amplify GH28 pectinases from all Ascomycota cultures tested, but only three out of seven Basidiomycota cultures. In addition, we further tested the primers in PCRs on metagenomic DNA extracted from senesced tree leaves from different forest ecosystems, followed by cloning and sequencing. Taxonomic specificity for Ascomycota GH28 genes was tested by comparing GH28 composition in leaves to internal transcribed spacer (ITS) amplicon composition using pyrosequencing. All sequences obtained from GH28 primers were classified as Ascomycota; in contrast, ITS sequences indicated that fungal communities were up to 39% Basidiomycetes. Analysis of leaf samples indicated that both forest stand and ecosystem type were important in structuring fungal communities. However, site played the prominent role in explaining GH28 composition, whereas ecosystem type was more important for ITS composition, indicating possible genetic drift between populations of fungi. Overall, these primers will have utility in understanding relationships between fungal community composition and ecosystem processes, as well as detection of potentially pathogenic Ascomycetes. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L.

PubMed

Toubart, P; Desiderio, A; Salvi, G; Cervone, F; Daroda, L; De Lorenzo, G

1992-05-01

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGIP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone et al., 1987). The amino acid sequences of the N-terminus and one internal tryptic peptide of the PGIP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758-bp DNA as a hybridization probe, a lambda clone containing the PGIP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGIP gene, contained on a subcloned 3.3 kb SalI-SalI DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)+ RNAs, isolated from various tissues of bean seedlings or from suspension-cultured bean cells, were also performed using the cloned PCR-generated DNA as a probe. A 1.2 kb transcript was detected in suspension-cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers.(ABSTRACT TRUNCATED AT 250 WORDS)

Composition of Micro-eukaryotes on the Skin of the Cascades Frog (Rana cascadae) and Patterns of Correlation between Skin Microbes and Batrachochytrium dendrobatidis.

PubMed

Kueneman, Jordan G; Weiss, Sophie; McKenzie, Valerie J

2017-01-01

Global amphibian decline linked to fungal pathogens has galvanized research on applied amphibian conservation. Skin-associated bacterial communities of amphibians have been shown to mediate fungal skin infections and the development of probiotic treatments with antifungal bacteria has become an emergent area of research. While exploring the role of protective bacteria has been a primary focus for amphibian conservation, we aim to expand and study the other microbes present in amphibian skin communities including fungi and other micro-eukaryotes. Here, we characterize skin-associated bacteria and micro-eukaryotic diversity found across life stages of Cascades frog ( Rana cascadae ) and their associated aquatic environments using culture independent 16S and 18S rRNA marker-gene sequencing. Individuals of various life stages of Cascades frogs were sampled from a population located in the Trinity Alps in Northern California during an epidemic of the chytrid fungus, Batrachochytrium dendrobatidis . We filtered the bacterial sequences against a published database of bacteria known to inhibit B. dendrobatidis in co-culture to estimate the proportion of the skin bacterial community that is likely to provide defense against B. dendrobatidis . Tadpoles had a significantly higher proportion of B. dendrobatidis -inhibitory bacterial sequence matches relative to subadult and adult Cascades frogs. We applied a network analysis to examine patterns of correlation between bacterial taxa and B. dendrobatidis , as well as micro-eukaryotic taxa and B. dendrobatidis . Combined with the published database of bacteria known to inhibit B. dendrobatidis , we used the network analysis to identify bacteria that negatively correlated with B. dendrobatidis and thus could be good probiotic candidates in the Cascades frog system.

A meta-barcoding approach to assess and compare the storage temperature-dependent bacterial diversity of gilt-head sea bream (Sparus aurata) originating from fish farms from two geographically distinct areas of Greece.

PubMed

Parlapani, Foteini F; Michailidou, Sofia; Pasentsis, Konstantinos; Argiriou, Anagnostis; Krey, Grigorios; Boziaris, Ioannis S

2018-08-02

Bacterial diversity of whole gilt-head sea bream (Sparus aurata L. 1758) originating from Ionian and Aegean Sea aquaculture farms and stored at 0 (ice), 4 and 8 °C was determined by 16S rRNA gene amplicon sequencing method using the Illumina's MiSeq platform. The composition of Aerobic Plate Counts (APC) was also monitored by 16S rRNA gene sequencing. The rejection time point of sea bream from either area, as determined by sensory evaluation, was about 14, 6 and 3 days at 0, 4 and 8 °C, respectively. APC was approximately 4.5 log cfu/g at day 0 and ranged from 7.5 to 8.5 log cfu/g at sensory rejection. Culture-depended analysis showed that Pseudomonas and Shewanella were the most abundant microorganisms grown on plates for both seas. Moreover, culture-independent analysis of DNA extracted directly from fish flesh showed that sea bream originating from different geographical areas exhibited different bacterial diversity. Pseudomonas and Psychrobacter were the dominant microorganisms of chill-stored fish from Ionian (apart from 8 °C, where Carnobacterium dominated) and Aegean Sea, respectively. In addition, small changes of storage temperature greatly affected bacterial microbiota of stored fish. Various bacterial species, not detected by conventional microbiological methods, were also revealed through 16S amplicon sequencing. In conclusion, the use of NGS approach is a promising methodology for assessing bacterial diversity of sea bream originating from different geographical areas and stored at various temperatures. Copyright © 2018 Elsevier B.V. All rights reserved.

Bacteria and Genes Involved in Arsenic Speciation in Sediment Impacted by Long-Term Gold Mining

PubMed Central

Costa, Patrícia S.; Scholte, Larissa L. S.; Reis, Mariana P.; Chaves, Anderson V.; Oliveira, Pollyanna L.; Itabayana, Luiza B.; Suhadolnik, Maria Luiza S.; Barbosa, Francisco A. R.; Chartone-Souza, Edmar; Nascimento, Andréa M. A.

2014-01-01

The bacterial community and genes involved in geobiocycling of arsenic (As) from sediment impacted by long-term gold mining were characterized through culture-based analysis of As-transforming bacteria and metagenomic studies of the arsC, arrA, and aioA genes. Sediment was collected from the historically gold mining impacted Mina stream, located in one of the world’s largest mining regions known as the “Iron Quadrangle”. A total of 123 As-resistant bacteria were recovered from the enrichment cultures, which were phenotypically and genotypically characterized for As-transformation. A diverse As-resistant bacteria community was found through phylogenetic analyses of the 16S rRNA gene. Bacterial isolates were affiliated with Proteobacteria, Firmicutes, and Actinobacteria and were represented by 20 genera. Most were AsV-reducing (72%), whereas AsIII-oxidizing accounted for 20%. Bacteria harboring the arsC gene predominated (85%), followed by aioA (20%) and arrA (7%). Additionally, we identified two novel As-transforming genera, Thermomonas and Pannonibacter. Metagenomic analysis of arsC, aioA, and arrA sequences confirmed the presence of these genes, with arrA sequences being more closely related to uncultured organisms. Evolutionary analyses revealed high genetic similarity between some arsC and aioA sequences obtained from isolates and clone libraries, suggesting that those isolates may represent environmentally important bacteria acting in As speciation. In addition, our findings show that the diversity of arrA genes is wider than earlier described, once none arrA-OTUs were affiliated with known reference strains. Therefore, the molecular diversity of arrA genes is far from being fully explored deserving further attention. PMID:24755825

Expression and characterization of constitutive heat shock protein 70.1 (HSPA-1A) gene in in vitro produced and in vivo-derived buffalo (Bubalus bubalis) embryos.

PubMed

Sharma, G T; Nath, A; Prasad, S; Singhal, S; Singh, N; Gade, N E; Dubey, P K; Saikumar, G

2012-12-01

Cells are blessed with a group of stress protector molecules known as heat shock proteins (HSPs), amongst them HSP70, encoded by HSPA-1A gene, is most abundant and highly conserved protein. Variety of stresses hampers the developmental competence of embryos under in vivo and in vitro conditions. Present work was designed to study the quantitative expression of HSPA-1A mRNA in immature oocytes (IMO), matured oocytes (MO), in vitro produced (IVP) and in vivo-derived (IVD) buffalo embryos to assess the level of stress to which embryos are exposed under in vivo and in vitro culture conditions. Further, HSPA-1A gene sequence was analysed to determine its homology with other mammalian sequences. The mRNA expression analysis was carried out on 72 oocytes (40 IMO; 32 MO), 76 IVP and 55 IVD buffalo embryos. Expression of HSPA-1A was found in oocytes and throughout the developmental stages of embryos examined irrespective of the embryo source; however, higher (p < 0.05) expression was observed in 8-16 cell, morula and blastocyst stages of IVP embryos as compared to IVD embryos. Phylogenetic analysis of bubaline HSPA-1A revealed that it shares 91-98% identity with other mammalian sequences. It can be concluded that higher level of HSPA-1A mRNA in IVP embryos in comparison with in vivo-derived embryos is an indicator of cellular stress in IVP system. This study suggests need for further optimization of in vitro culture system in which HSPA-1A gene could be used as a stress biomarker during pre-implantation development. © 2012 Blackwell Verlag GmbH.

Development of a systematic feedback isolation approach for targeted strains from mixed culture systems.

PubMed

Poudel, Pramod; Tashiro, Yukihiro; Miyamoto, Hirokuni; Miyamoto, Hisashi; Okugawa, Yuki; Sakai, Kenji

2017-01-01

Elucidation of functions of bacteria in a mixed culture system (MCS) such as composting, activated sludge system is difficult, since the system is complicating with many unisolated bacteria. Here, we developed a systematic feedback isolation strategy for the isolation and rapid screening of multiple targeted strains from MCS. Six major strains (Corynebacterium sphenisci, Bacillus thermocloacae, Bacillus thermoamylovorans, Bacillus smithii, Bacillus humi, and Bacillus coagulans), which are detected by denaturing gradient gel electrophoresis (DGGE) analysis in our previous study on MCS for l-lactic acid production, were targeted for isolation. Based on information of suitable cultivation conditions (e.g., media, pH, temperature) from the literature, feedback isolation was performed to form 136 colonies. The following direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was optimised as the second screening to narrow down 20 candidate colonies from similar spectra patterns with six closest type strains. This step could distinguish bacteria at the species level with distance similarity scores ≥0.55 corresponding to 16S rRNA gene sequence similarity ≥98.2%, suggesting that this is an effective technique to minimize isolates close to targeted type strains. Analysis of 16S rRNA gene sequences indicated that two targeted strains and one strain related to the target had successfully been isolated, showing high similarities (99.5-100%) with the sequences from the DGGE bands, and that the other candidates were affiliated with three strains that were closely related to the target species. This study proposes a new method for systematic feedback isolation that may be useful for isolating targeted strains from MCS for further investigation. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

The Still Bay and Howiesons Poort at Sibudu and Blombos: Understanding Middle Stone Age Technologies.

PubMed

Soriano, Sylvain; Villa, Paola; Delagnes, Anne; Degano, Ilaria; Pollarolo, Luca; Lucejko, Jeannette J; Henshilwood, Christopher; Wadley, Lyn

2015-01-01

The classification of archaeological assemblages in the Middle Stone Age of South Africa in terms of diversity and temporal continuity has significant implications with respect to recent cultural evolutionary models which propose either gradual accumulation or discontinuous, episodic processes for the emergence and diffusion of cultural traits. We present the results of a systematic technological and typological analysis of the Still Bay assemblages from Sibudu and Blombos. A similar approach is used in the analysis of the Howiesons Poort (HP) assemblages from Sibudu seen in comparison with broadly contemporaneous assemblages from Rose Cottage and Klasies River Cave 1A. Using our own and published data from other sites we report on the diversity between stone artifact assemblages and discuss to what extent they can be grouped into homogeneous lithic sets. The gradual evolution of debitage techniques within the Howiesons Poort sequence with a progressive abandonment of the HP technological style argues against the saltational model for its disappearance while the technological differences between the Sibudu and Blombos Still Bay artifacts considerably weaken an interpretation of similarities between the assemblages and their grouping into the same cultural unit. Limited sampling of a fragmented record may explain why simple models of cultural evolution do not seem to apply to a complex reality.

The Still Bay and Howiesons Poort at Sibudu and Blombos: Understanding Middle Stone Age Technologies

PubMed Central

Soriano, Sylvain; Villa, Paola; Delagnes, Anne; Degano, Ilaria; Pollarolo, Luca; Lucejko, Jeannette J.; Henshilwood, Christopher; Wadley, Lyn

2015-01-01

The classification of archaeological assemblages in the Middle Stone Age of South Africa in terms of diversity and temporal continuity has significant implications with respect to recent cultural evolutionary models which propose either gradual accumulation or discontinuous, episodic processes for the emergence and diffusion of cultural traits. We present the results of a systematic technological and typological analysis of the Still Bay assemblages from Sibudu and Blombos. A similar approach is used in the analysis of the Howiesons Poort (HP) assemblages from Sibudu seen in comparison with broadly contemporaneous assemblages from Rose Cottage and Klasies River Cave 1A. Using our own and published data from other sites we report on the diversity between stone artifact assemblages and discuss to what extent they can be grouped into homogeneous lithic sets. The gradual evolution of debitage techniques within the Howiesons Poort sequence with a progressive abandonment of the HP technological style argues against the saltational model for its disappearance while the technological differences between the Sibudu and Blombos Still Bay artifacts considerably weaken an interpretation of similarities between the assemblages and their grouping into the same cultural unit. Limited sampling of a fragmented record may explain why simple models of cultural evolution do not seem to apply to a complex reality. PMID:26161665

Microbial degradation of high impact polystyrene (HIPS), an e-plastic with decabromodiphenyl oxide and antimony trioxide.

PubMed

Sekhar, Vini C; Nampoothiri, K Madhavan; Mohan, Arya J; Nair, Nimisha R; Bhaskar, Thallada; Pandey, Ashok

2016-11-15

Accumulation of electronic waste has increased catastrophically and out of that various plastic resins constitute one of the leading thrown out materials in the electronic machinery. Enrichment medium, containing high impact polystyrene (HIPS) with decabromodiphenyl oxide and antimony trioxide as sole carbon source, was used to isolate microbial cultures. The viability of these cultures in the e-plastic containing mineral medium was further confirmed by triphenyl tetrazolium chloride (TTC) reduction test. Four cultures were identified by 16S rRNA sequencing as Enterobacter sp., Citrobacter sedlakii, Alcaligenes sp. and Brevundimonas diminuta. Biodegradation experiments were carried out in flask level and gelatin supplementation (0.1% w/v) along with HIPS had increased the degradation rate to a maximum of 12.4% (w/w) within 30days. This is the first report for this kind of material. The comparison of FTIR, NMR, and TGA analysis of original and degraded e-plastic films revealed structural changes under microbial treatment. Polystyrene degradation intermediates in the culture supernatant were also detected using HPLC analysis. The gravity of biodegradation was validated by morphological changes under scanning electron microscope. All isolates displayed depolymerase activity to substantiate enzymatic degradation of e-plastic. Copyright © 2016 Elsevier B.V. All rights reserved.

Osteogenic differentiation of 3D cultured mesenchymal stem cells induced by bioactive peptides.

PubMed

Lukasova, Vera; Buzgo, Matej; Sovkova, Vera; Dankova, Jana; Rampichova, Michala; Amler, Evzen

2017-08-01

Bioactive peptides derived from receptor binding motifs of native proteins are a potent source of bioactive molecules that can induce signalling pathways. These peptides could substitute for osteogenesis promoting supplements. The work presented here compares three kinds of bioactive peptides derived from collagen III, bone morphogenetic protein 7 (BMP-7) and BMP-2 with their potential osteogenic activity on the model of porcine mesenchymal stem cells (pMSCs). pMSCs were cultured on electrospun polycaprolactone nanofibrous scaffolds with different concentrations of the bioactive peptides without addition of any osteogenic supplement. Analysis of pMSCs cultures included measurement of the metabolic activity and proliferation, immunofluorescence staining and also qPCR. Results showed no detrimental effect of the bioactive peptides to cultured pMSCs. Based on qPCR analysis, the bioactive peptides are specific for osteogenic differentiation with no detectable expression of collagen II. Our results further indicate that peptide derived from BMP-2 protein promoted the expression of mRNA for osteocalcin (OCN) and collagen I significantly compared to control groups and also supported deposition of OCN as observed by immunostaining method. The data suggest that bioactive peptide with an amino acid sequence of KIPKASSVPTELSAISTLYL derived from BMP-2 protein was the most potent for triggering osteogenic differentiation of pMSCs. © 2017 John Wiley & Sons Ltd.

Differences between culture & non-culture confirmed invasive meningococci with a focus on factor H-binding protein distribution.

PubMed

Clark, Stephen A; Lekshmi, Aiswarya; Lucidarme, Jay; Hao, Li; Tsao, How; Lee-Jones, Lisa; Jansen, Kathrin U; Newbold, Lynne S; Anderson, Annaliesa S; Borrow, Ray

2016-07-01

To compare the distribution of capsular groups and factor H-binding protein (fHBP) variants among meningococcal isolates and non-culture clinical specimens and to assess the representativeness of group B isolates amongst group B cases as a whole. A PCR sequencing assay was used to characterise fHBP from non-culture cases confirmed from January 2011 to December 2013. These were compared to genotypic data derived from whole genome analysis of isolates received during the same period. Group W and Y strains were more common among isolates than non-culture strains. The distribution of fHBP variants among group B non-culture cases generally reflected that seen in the corresponding isolates. Nonetheless, the non-culture subset contained a greater proportion of fHBP variant 15/B44, associated with the ST-269 cluster sublineage. Differences in capsular group and fHBP distribution among culture and non-culture cases may be indicative of variation in strain viability, diagnostic practice, disease severity and/or clinical presentation. Future analyses combining clinical case information with laboratory data may help to further explore these differences. Group B isolates provide a good representation of group B disease in E&W and, therefore, can reliably be used in fHBP strain coverage predictions of recently-licensed vaccines. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Design, synthesis and biological evaluation of non-peptide PAR1 thrombin receptor antagonists based on small bifunctional templates: arginine and phenylalanine side chain groups are keys for receptor activity.

PubMed

Androutsou, Maria-Eleni; Saifeddine, Mahmoud; Hollenberg, Morley D; Matsoukas, John; Agelis, George

2010-04-01

In the present study, we report the synthesis and biological evaluation of a series of new non-peptide PAR(1) mimetic receptor antagonists, based on conformational analysis of the S(42)FLLR(46) tethered ligand (TL) sequence of PAR(1). These compounds incorporate the key pharmacophore groups in the TL sequence, guanidyl, amino and phenyl, which are essential for triggering receptor activity. Compounds 5 and 15 (50-100 microM) inhibited both TFLLR-amide (10 microM) and thrombin-mediated (0.5 and 1 U/ml; 5 and 10 microM) calcium signaling in a cultured human HEK cell assay.

Sequence analysis of the lactococcal plasmid pNP40: a mobile replicon for coping with environmental hazards.

PubMed

O'Driscoll, Jonathan; Glynn, Frances; Fitzgerald, Gerald F; van Sinderen, Douwe

2006-09-01

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, possesses a potent complement of bacteriophage resistance systems, which has stimulated its application as a fitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of this plasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocin resistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adaptation and DNA damage repair were identified, further confirming pNP40's contribution to environmental stress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resulting in a "stockpiling" of bacteriophage resistance systems.

Community Structure Comparisons of Hydrothermal Vent Microbial Mats Along the Mariana Arc and Back-arc

NASA Astrophysics Data System (ADS)

Hager, K. W.; Fullerton, H.; Moyer, C. L.

2015-12-01

Hydrothermal vents along the Mariana Arc and back-arc represent a hotspot of microbial diversity that has not yet been fully recognized. The Mariana Arc and back-arc contain hydrothermal vents with varied vent effluent chemistry and temperature, which translates to diverse community composition. We have focused on iron-rich sites where the dominant primary producers are iron oxidizing bacteria. Because microbes from these environments have proven elusive in culturing efforts, we performed culture independent analysis among different microbial communities found at these hydrothermal vents. Terminal-restriction fragment length polymorphism (T-RFLP) and Illumina sequencing of small subunit ribosomal gene amplicons were used to characterize community members and identify samples for shotgun metagenomics. Used in combination, these methods will better elucidate the composition and characteristics of the bacterial communities at these hydrothermal vent systems. The overarching goal of this study is to evaluate and compare taxonomic and metabolic diversity among different communities of microbial mats. We compared communities collected on a fine scale to analyze the bacterial community based on gross mat morphology, geography, and nearby vent effluent chemistry. Taxa richness and evenness are compared with rarefaction curves to visualize diversity. As well as providing a survey of diversity this study also presents a juxtaposition of three methods in which ribosomal small subunit diversity is compared with T-RFLP, next generation amplicon sequencing, and metagenomic shotgun sequencing.