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Sample records for bacterial rrna extraction

  1. Direct ribosome isolation from soil to extract bacterial rRNA for community analysis.

    PubMed

    Felske, A; Engelen, B; Nübel, U; Backhaus, H

    1996-11-01

    A simple method that combines an adapted ribosome isolation method and a common RNA extraction step has been developed for selective recovery of intact rRNA from natural microbial communities in soil. After mechanical cell lysis, ribosomes are separated by centrifugation steps, avoiding massive humic acid contamination and RNA degradation. The protocol accommodates the complex composition of soils by blocking adsorbing surfaces and humic acids with polyvinylpyrrolidone and bovine serum albumin. A usual RNA extraction step yields rRNA accessible for hybridization or reverse transcription-PCR. Hybridization with specific oligonucleotide probes was used for group-specific yield comparison. By using a probe hybridizing to the 16S rRNA of the bacterial kingdom, total bacterial rRNA yield was estimated to be in the range of 0.2 microgram per g for different soils. Group-specific probes did not indicate a selectivity of the isolation procedure and differentiated the compositions of different soil microbial communities. The sequence diversity of the isolated RNA population was also revealed by temperature gradient gel electrophoresis of reverse transcription-PCR amplification products by using a region of the 16S rRNA as a target. The pattern obtained by this analysis differed from a similar one resulting from the separation of amplification products of community DNA preparations. This different view of the community composition is attributable to the correlation of ribosome numbers to the metabolic activity of bacteria in the habitat under observation. PMID:8900007

  2. The Influence of DNA Extraction Procedure and Primer Set on the Bacterial Community Analysis by Pyrosequencing of Barcoded 16S rRNA Gene Amplicons

    PubMed Central

    Starke, Ingo C.; Vahjen, Wilfried; Pieper, Robert; Zentek, Jürgen

    2014-01-01

    In this study, the effect of different DNA extraction procedures and primer sets on pyrosequencing results regarding the composition of bacterial communities in the ileum of piglets was investigated. Ileal chyme from piglets fed a diet containing different amounts of zinc oxide was used to evaluate a pyrosequencing study with barcoded 16S rRNA PCR products. Two DNA extraction methods (bead beating versus silica gel columns) and two primer sets targeting variable regions of bacterial 16S rRNA genes (8f-534r versus 968f-1401r) were considered. The SEED viewer software of the MG-RAST server was used for automated sequence analysis. A total of 5.2 × 105 sequences were used for analysis after processing for read length (150 bp), minimum sequence occurrence (5), and exclusion of eukaryotic and unclassified/uncultured sequences. DNA extraction procedures and primer sets differed significantly in total sequence yield. The distribution of bacterial order and main bacterial genera was influenced significantly by both parameters. However, this study has shown that the results of pyrosequencing studies using barcoded PCR amplicons of bacterial 16S rRNA genes depend on DNA extraction and primer choice, as well as on the manner of downstream sequence analysis. PMID:25120931

  3. Effects of DNA extraction and universal primers on 16S rRNA gene-based DGGE analysis of a bacterial community from fish farming water

    NASA Astrophysics Data System (ADS)

    Luo, Peng; Hu, Chaoqun; Zhang, Lüping; Ren, Chunhua; Shen, Qi

    2007-07-01

    Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis. In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples. The results indicate that the number of discernable bands in the DGGE fingerprint differed with the primer pairs used; the bands produced by 63f/518r, 341f/926r and 933f/1387r primer pairs were obviously fewer than those by 968f/1401r. Also, we found that each DNA extraction method resulted in different community profiles, reflected by the number and intensity of bands in the DGGE fingerprint. Furthermore, the main bands (theoretically representing dominant bacteria) differed with the extraction methods applied. It is therefore believed that the effects of universal primers and DNA extraction should be given more attention and carefully chosen before performing an investigation into a new environment with DGGE.

  4. The Variability of the 16S rRNA Gene in Bacterial Genomes and Its Consequences for Bacterial Community Analyses

    PubMed Central

    V?trovsk, Tom; Baldrian, Petr

    2013-01-01

    16S ribosomal RNA currently represents the most important target of study in bacterial ecology. Its use for the description of bacterial diversity is, however, limited by the presence of variable copy numbers in bacterial genomes and sequence variation within closely related taxa or within a genome. Here we use the information from sequenced bacterial genomes to explore the variability of 16S rRNA sequences and copy numbers at various taxonomic levels and apply it to estimate bacterial genome and DNA abundances. In total, 7,081 16S rRNA sequences were in silico extracted from 1,690 available bacterial genomes (115 per genome). While there are several phyla containing low 16S rRNA copy numbers, in certain taxa, e.g., the Firmicutes and Gammaproteobacteria, the variation is large. Genome sizes are more conserved at all tested taxonomic levels than 16S rRNA copy numbers. Only a minority of bacterial genomes harbors identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers. While certain taxa harbor dissimilar 16S rRNA genes, others contain sequences common to multiple species. Sequence identity clusters (often termed operational taxonomic units) thus provide an imperfect representation of bacterial taxa of a certain phylogenetic rank. We have demonstrated that the information on 16S rRNA copy numbers and genome sizes of genome-sequenced bacteria may be used as an estimate for the closest related taxon in an environmental dataset to calculate alternative estimates of the relative abundance of individual bacterial taxa in environmental samples. Using an example from forest soil, this procedure would increase the abundance estimates of Acidobacteria and decrease these of Firmicutes. Using the currently available information, alternative estimates of bacterial community composition may be obtained in this way if the variation of 16S rRNA copy numbers among bacteria is considered. PMID:23460914

  5. Evaluating bias of illumina-based bacterial 16S rRNA gene profiles.

    PubMed

    Kennedy, Katherine; Hall, Michael W; Lynch, Michael D J; Moreno-Hagelsieb, Gabriel; Neufeld, Josh D

    2014-09-01

    Massively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing. PMID:25002428

  6. 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 517596?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

  7. Infective Arthritis: Bacterial 23S rRNA Gene Sequencing as a Supplementary Diagnostic Method.

    PubMed

    Moser, Claus; Andresen, Keld; Kjerulf, Anne; Salamon, Suheil; Kemp, Michael; Christensen, Jens Jørgen

    2008-01-01

    Consecutively collected synovial fluids were examined for presence of bacterial DNA (a 700-bp fragment of the bacterial 23S rRNA gene) followed by DNA sequencing of amplicons, and by conventional bacteriological methods. One or more microorganisms were identified in 22 of the 227 synovial fluids (9,7%) originating from 17 patients. Sixteen of the patients had clinical signs of arthritis. For 11 patients molecular and conventional bacterial examinations were in agreement. Staphylococcus aureus, Streptococcus dysgalactiae subspecies equisimilis and Streptococcus pneumoniae, were detected in synovial fluids from 6, 2 and 2 patients, respectively. In 3 patients only 23S rRNA analysis was positive; 2 synovial fluids contained S. dysgalactiae subspecies equisimilis and 1 S. pneumoniae). The present study indicates a significant contribution by PCR with subsequent DNA sequencing of the 23S rRNA gene analysis in recognizing and identification of microorganisms from synovial fluids. PMID:19088916

  8. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

  9. Nave Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy?

    PubMed Central

    Wang, Qiong; Garrity, George M.; Tiedje, James M.; Cole, James R.

    2007-01-01

    The Ribosomal Database Project (RDP) Classifier, a nave Bayesian classifier, can rapidly and accurately classify bacterial 16S rRNA sequences into the new higher-order taxonomy proposed in Bergey's Taxonomic Outline of the Prokaryotes (2nd ed., release 5.0, Springer-Verlag, New York, NY, 2004). It provides taxonomic assignments from domain to genus, with confidence estimates for each assignment. The majority of classifications (98%) were of high estimated confidence (?95%) and high accuracy (98%). In addition to being tested with the corpus of 5,014 type strain sequences from Bergey's outline, the RDP Classifier was tested with a corpus of 23,095 rRNA sequences as assigned by the NCBI into their alternative higher-order taxonomy. The results from leave-one-out testing on both corpora show that the overall accuracies at all levels of confidence for near-full-length and 400-base segments were 89% or above down to the genus level, and the majority of the classification errors appear to be due to anomalies in the current taxonomies. For shorter rRNA segments, such as those that might be generated by pyrosequencing, the error rate varied greatly over the length of the 16S rRNA gene, with segments around the V2 and V4 variable regions giving the lowest error rates. The RDP Classifier is suitable both for the analysis of single rRNA sequences and for the analysis of libraries of thousands of sequences. Another related tool, RDP Library Compare, was developed to facilitate microbial-community comparison based on 16S rRNA gene sequence libraries. It combines the RDP Classifier with a statistical test to flag taxa differentially represented between samples. The RDP Classifier and RDP Library Compare are available online at http://rdp.cme.msu.edu/. PMID:17586664

  10. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  11. 16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

    EPA Science Inventory

    We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

  12. Direct Amplification of rRNA Genes in Diagnosis of Bacterial Infections

    PubMed Central

    Rantakokko-Jalava, Kaisu; Nikkari, Simo; Jalava, Jari; Eerola, Erkki; Skurnik, Mikael; Meurman, Olli; Ruuskanen, Olli; Alanen, Anna; Kotilainen, Esa; Toivanen, Paavo; Kotilainen, Pirkko

    2000-01-01

    A broad-range bacterial PCR targeting rRNA genes (rDNAs) was used to directly analyze 536 clinical samples obtained from 459 hospitalized patients during a 4-year study period. The molecular diagnosis based on DNA sequencing of the PCR product was compared to that obtained by bacterial culture. The bacteriological diagnosis was concordant for 447 (83%) specimens. Broad-range rDNA PCR was the only method that yielded an etiologic diagnosis for 11 (2.4%) of 459 patients. Compared to culture and clinical assessment, the sensitivity of the PCR method combined with sequencing was 74.2%, and the specificity was between 98.7 and 99.6%. At present, the described molecular approach proved superior to bacterial culture in two clinical situations: infections caused by bacteria with unusual growth requirements and specimens taken during antimicrobial treatment of the patient. PMID:10618059

  13. Systematic 16S rRNA Gene Sequencing of Atypical Clinical Isolates Identified 27 New Bacterial Species Associated with Humans

    PubMed Central

    Drancourt, M.; Berger, P.; Raoult, D.

    2004-01-01

    Clinical microorganisms isolated during a 5-year study in our hospital that could not be identified by conventional criteria were studied by 16S rRNA gene sequence analysis. Each isolate yielded a ?1,400-bp sequence containing <5 ambiguities which was compared with the GenBank 16S rRNA gene library; 1,404 such isolates were tested, and 120 were considered unique (27 isolates) or rare (?10 cases reported in the literature) human pathogens. Eleven new species, Actinobaculum massiliae, Candidatus Actinobaculum timonae, Paenibacillus sanguinis, Candidatus Bacteroides massiliae, Chryseobacterium massiliae, Candidatus Chryseobacterium timonae, Paenibacillus massiliensis, Candidatus Peptostreptococcus massiliae, Candidatus Prevotella massiliensis, Rhodobacter massiliensis, and Candidatus Veillonella atypica were identified. Sixteen species were obtained from humans for the first time. Our results show the important role that 16S rRNA gene sequence-based bacterial identification currently plays in recognizing unusual and emerging bacterial diseases. PMID:15131188

  14. Biological activity of bacterial surface components: bacterial extracts and defined bacterial cell wall components as immunomodulators.

    PubMed

    Bessler, W G; Kleine, B; Martinez Alonso, C; Biesert, L; Strecker, M; Wiesmller, K H; Metzger, J; Jung, G

    1990-01-01

    Bacterial extracts obtained from pathogenic strains occurring in lung infections (Broncho Vaxom) or urogenital infections (Urovaxom) as well as defined surface components of Gram-negative bacteria purified from bacteria or obtained by chemical synthesis were tested for their immunomodulatory properties in a murine system. The bacterial extracts were able to act as immunogens inducing an antigen-specific response. Both the bacterial extracts and the purified bacterial cell wall components constituted polyclonal activators of murine splenic B cells, as demonstrated by proliferation assays measuring the incorporation of [3H]thymidine into DNA. They were also able to act as immunoadjuvants increasing the SRBC and the BSA-TNP specific immune response, and could induce tumor cytotoxicity in bone marrow-derived macrophages. The results show that bacterial extracts and defined bacterial surface components constitute immunogens as well as immunomodulators in vitro and in vivo. PMID:2117181

  15. Phylogeny of the Main Bacterial 16S rRNA Sequences in Drentse A Grassland Soils (The Netherlands)

    PubMed Central

    Felske, Andreas; Wolterink, Arthur; Van Lis, Robert; Akkermans, Antoon D. L.

    1998-01-01

    The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing. Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria. After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR. The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA. Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced. The relationships of the sequences to those of cultured organisms of known phylogeny were determined. Most of the amplicons originated from organisms closely related to Bacillus species. Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for Firmicutes with low G+C content counted for about 50% of all bacterial rRNA. The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from Firmicutes. The most active bacteria apparently were Bacillus species, from which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely related to known Firmicutes with a high G+C content. Other sequences were related to Proteobacteria, mainly the alpha subclass. PMID:9501427

  16. Comparative metagenomic and rRNA microbial diversity characterization using Archaeal and Bacterial synthetic communities

    PubMed Central

    Shakya, Migun; Quince, Christopher; Campbell, James H.; Yang, Zamin K.; Schadt, Christopher W.; Podar, Mircea

    2013-01-01

    Summary Next generation sequencing has dramatically changed the landscape of microbial ecology, large-scale and in-depth diversity studies being now widely accessible. However, determining the accuracy of taxonomic and quantitative inferences and comparing results obtained with different approaches are complicated by incongruence of experimental and computational data types and also by lack of knowledge of the true ecological diversity. Here we used highly diverse bacterial and archaeal synthetic communities assembled from pure genomic DNAs to compare inferences from metagenomic and SSU rRNA amplicon sequencing. Both Illumina and 454 metagenomic data outperformed amplicon sequencing in quantifying the community composition, but the outcome was dependent on analysis parameters and platform. New approaches in processing and classifying amplicons can reconstruct the taxonomic composition of the community with high reproducibility within primer sets, but all tested primers sets lead to significant taxon-specific biases. Controlled synthetic communities assembled to broadly mimic the phylogenetic richness in target environments can provide important validation for fine-tuning experimental and computational parameters used to characterize natural communities. PMID:23387867

  17. Development of a 9600-clone procedure for oligonucleotide fingerprinting of rRNA genes: utilization to identify soil bacterial rRNA genes that correlate in abundance with the development of avocado root rot.

    PubMed

    Bent, Elizabeth; Yin, Bei; Figueroa, Andres; Ye, Jingxiao; Fu, Qi; Liu, Zheng; McDonald, Virginia; Jeske, Daniel; Jiang, Tao; Borneman, James

    2006-10-01

    Oligonucleotide fingerprinting of rRNA genes (OFRG) is an array-based method that generates microbial community profiles through analysis of rRNA gene clone libraries. The original OFRG method allowed 1536 clones to be analyzed per experiment. This report describes a procedure for analyzing 9600 clones per experiment, including a new probe set for bacterial analysis, and improved data processing and statistical analysis tools. The software tools are available at the OFRG website (). Use of the 9600-clone procedure was demonstrated by examining the bacterial rRNA gene compositions of soils subjected to various temperature treatments. These treatments produced a series of soils with a range of abilities to suppress avocado root rot, enabling the identification of bacterial rRNA genes that correlate in abundance with root rot suppressiveness. OFRG analysis of these soils produced 8876 bacterial rRNA gene fingerprints grouped into 5123 clusters, or operational taxonomic units (OTUs). Eleven OTUs exhibited a positive correlation between the number of clones and the percentage of healthy roots. An in silico analysis was performed to examine the relationship between the number of rRNA genes analyzed and the number of correlates (rRNA gene-avocado root rot symptoms) identified. As the number of clones decreased, fewer correlates were identified. To further increase the throughput of the OFRG method, use of a glass slide-fluorescent probe microarray format was also explored. PMID:16712989

  18. Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples.

    PubMed

    Zhen, Huajun; Krumins, Valdis; Fennell, Donna E; Mainelis, Gediminas

    2015-10-01

    Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between cellular rRNA abundance and bacterial activity. PMID:26241659

  19. An alternative strategy for bacterial ribosome synthesis: Bacillus subtilis rRNA transcription regulation

    PubMed Central

    Krásný, Libor; Gourse, Richard L

    2004-01-01

    As an approach to the study of rRNA synthesis in Gram-positive bacteria, we characterized the regulation of the Bacillus subtilis rrnB and rrnO rRNA promoters. We conclude that B. subtilis and Escherichia coli use different strategies to control rRNA synthesis. In contrast to E. coli, it appears that the initiating NTP for transcription from B. subtilis rRNA promoters is GTP, promoter strength is determined primarily by the core promoter (−10/−35 region), and changes in promoter activity always correlate with changes in the intracellular GTP concentration. rRNA promoters in B. subtilis appear to be regulated by changes in the initiating NTP pools, but in some growth transitions, changes in rRNA promoter activity are also dependent on relA, which codes for ppGpp synthetase. In contrast to the situation for E. coli where ppGpp decreases rRNA promoter activity by directly inhibiting RNA polymerase, it appears that ppGpp may not inhibit B. subtilis RNA polymerase directly. Rather, increases in the ppGpp concentration might reduce the available GTP pools, thereby modulating rRNA promoter activity indirectly. PMID:15496987

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

    PubMed

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

    2009-08-01

    Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples. PMID:19763412

  1. Analysis of Bacterial Communities in the Rhizosphere of Chrysanthemum via Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA as Well as DNA Fragments Coding for 16S rRNA

    PubMed Central

    Duineveld, Bernadette M.; Kowalchuk, George A.; Keijzer, Anneke; van Elsas, Jan Dirk; van Veen, Johannes A.

    2001-01-01

    The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal DNA (rDNA) and 16S rRNA, respectively, and the products were subjected to denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands were excised and sequenced to gain insight into the identities of predominantly present (PCR) and predominantly active (RT-PCR) bacterial populations. The majority of DGGE band sequences were related to bacterial genera previously associated with the rhizosphere, such as Pseudomonas, Comamonas, Variovorax, and Acetobacter, or typical of root-free soil environments, such as Bacillus and Arthrobacter. The PCR-DGGE patterns observed for bulk soil were somewhat more complex than those obtained from rhizosphere samples, and the latter contained a subset of the bands present in bulk soil. DGGE analysis of RT-PCR products detected a subset of bands visible in the rDNA-based analysis, indicating that some dominantly detected bacterial populations did not have high levels of metabolic activity. The sequences detected by the RT-PCR approach were, however, derived from a wide taxonomic range, suggesting that activity in the rhizosphere was not determined at broad taxonomic levels but rather was a strain- or species-specific phenomenon. Comparative analysis of DGGE profiles grouped all DNA-derived root tip samples together in a cluster, and within this cluster the root tip samples from young plants formed a separate subcluster. Comparison of rRNA-derived bacterial profiles showed no grouping of root tip samples versus root base samples. Rather, all profiles derived from 2-week-old plant rhizosphere soils grouped together regardless of location along the root. PMID:11133442

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

    PubMed

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

    2015-03-01

    The rates at which wastewater treatment plant (WWTP) microbial communities biotransform specific substrates can differ by orders of magnitude among WWTP communities. Differences in taxonomic compositions among WWTP communities may predict differences in the rates of some types of biotransformations. In this work, we present a novel framework for establishing predictive relationships between specific bacterial 16S rRNA sequence abundances and biotransformation rates. We selected ten WWTPs with substantial variation in their environmental and operational metrics and measured the in situ ammonia biotransformation rate constants in nine of them. We isolated total RNA from samples from each WWTP and analyzed 16S rRNA sequence reads. We then developed multivariate models between the measured abundances of specific bacterial 16S rRNA sequence reads and the ammonia biotransformation rate constants. We constructed model scenarios that systematically explored the effects of model regularization, model linearity and non-linearity, and aggregation of 16S rRNA sequences into operational taxonomic units (OTUs) as a function of sequence dissimilarity threshold (SDT). A large percentage (greater than 80%) of model scenarios resulted in well-performing and significant models at intermediate SDTs of 0.13-0.14 and 0.26. The 16S rRNA sequences consistently selected into the well-performing and significant models at those SDTs were classified as Nitrosomonas and Nitrospira groups. We then extend the framework by applying it to the biotransformation rate constants of ten micropollutants measured in batch reactors seeded with the ten WWTP communities. We identified phylogenetic groups that were robustly selected into all well-performing and significant models constructed with biotransformation rates of isoproturon, propachlor, ranitidine, and venlafaxine. These phylogenetic groups can be used as predictive biomarkers of WWTP microbial community activity towards these specific micropollutants. This work is an important step towards developing tools to predict biotransformation rates in WWTPs based on taxonomic composition. PMID:25594727

  3. Bacterial diversity of a Carolina Bay as determined by 16S rRNA gene analysis: Confirmation of novel taxa

    SciTech Connect

    Wise, M.G.; Shimkets, L.J.; McArthur, J.V.

    1997-04-01

    Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhabit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow Bay, located on the Savannah River Site, near Aiken, S.C. Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T. Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system. Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project`s taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria. One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed. Two clones did not consistently cluster with specific groups and may be chimeric sequences. None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species. In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. 50 refs., 7 figs., 1 tab.

  4. Comparison of Bacterial Extracellular Polymer Extraction Methods

    PubMed Central

    Brown, Melanie J.; Lester, John N.

    1980-01-01

    Five different bacterial extracellular polymer extraction methods and a combination of two of these methods were compared on cultures of activated sludge, synthetic activated sludge, and Klebsiella aerogenes. High-speed centrifugation was the most effective extraction method for the K. aerogenes culture, based on the comparatively small amount of cell disruption and the relatively high extracellular polymer yield. Steaming treatment was the most effective extraction method for the activated sludges, since it released a significant quantity of extracellular polymers from the flocs and caused less cellular disruption than ethylenediaminetetraacetic acid and sodium hydroxide treatments. Sodium hydroxide treatment caused extensive disruption in all cultures. Ultrasonication released low concentrations of extracellular polymers from all cultures. However, it caused no significant cell disruption and therefore may be useful as a preliminary treatment in conjunction with another extraction method. PMID:16345600

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

    DOE PAGESBeta

    None

    2014-12-01

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

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

    SciTech Connect

    2014-12-01

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

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

    PubMed Central

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-01-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

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

    PubMed

    Rubin, Benjamin E R; Sanders, Jon G; Hampton-Marcell, Jarrad; Owens, Sarah M; Gilbert, Jack A; Moreau, Corrie S

    2014-12-01

    The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied. PMID:25257543

  9. Application of 16S rRNA metagenomics to analyze bacterial communities at a respiratory care centre in Taiwan.

    PubMed

    Tang, Chuan Yi; Yiu, Siu-Ming; Kuo, Han-Yueh; Tan, Te-Sheng; Liao, Ki-Hok; Liu, Chih-Chin; Hon, Wing-Kai; Liou, Ming-Li

    2015-03-01

    In this study, we applied a 16S ribosomal RNA (rRNA) metagenomics approach to survey inanimate hospital environments (IHEs) in a respiratory care center (RCC). A total of 16 samples, including 9 from medical devices and 7 from workstations, were analyzed. Besides, clinical isolates were retrospectively analyzed during the sampling period in the RCC. A high amount of microbial diversity was detected, with an average of 1,836 phylotypes per sample. In addition to Acinetobacter, more than 60 % of the bacterial communities present among the top 25 abundant genera were dominated by skin-associated bacteria. Differences in bacterial profiles were restricted to individual samples. Furthermore, compliance with hand hygiene guidelines may be unsatisfactory among hospital staff according to a principal coordinate analysis that indicated clustering of bacterial communities between devices and workstations for most of the sampling sites. Compared to the high incidence of clinical isolates in the RCC, only Staphylococcus and Acinetobacter were highly abundant in the IHEs. Despite Acinetobacter was the most abundant genus present in IHEs of the RCC, potential pathogens, e.g., Acinetobacter baumannii, might remain susceptible to carbapenem. This study is the first in Taiwan to demonstrate a high diversity of human-associated bacteria in the RCC via 16S rRNA metagenomics, which allows for new assessment of potential health risks in RCCs, aids in the evaluation of existing sanitation protocols, and furthers our understanding of the development of healthcare-associated infections. PMID:25359480

  10. A simple one-step PCR walking method and its application of bacterial rRNA for sequencing identification.

    PubMed

    Zhang, Hongfa; You, Chunping; Ren, Jing; Xu, Dan; Han, Mei; Liao, Wenyan

    2014-04-01

    There are many PCR walking methods applied currently, and they all have examples of successful application in organisms which are more complex than bacteria. However, to a certain extent, it will be more convenient for researchers if the complicated operation and poor specificity for bacteria can be improved. Here, we introduced an improved one-step PCR walking method of bacteria. Using a specific primer of the known sequence together with a universal semirandom primer, the unknown sequence adjacent to a known sequence can be obtained easily by just one ordinary round PCR. The products can be gel purified and directly sequenced. Specific primers were designed according to the gene sequence of bacterial rRNA, and the variable and adjacent gene sequences were obtained by this method. The sequence analysis of the product showed that it can improve the resolution of bacterial identification to the species level. PMID:24322403

  11. 16S rRNA gene-based metagenomic analysis identifies a novel bacterial co-prevalence pattern in dental caries

    PubMed Central

    Jagathrakshakan, Sri Nisha; Sethumadhava, Raghavendra Jayesh; Mehta, Dhaval Tushar; Ramanathan, Arvind

    2015-01-01

    Objective: To identify the prevalence of acidogenic and nonacidogenic bacteria in patients with polycaries lesions, and to ascertain caries specific bacterial prevalence in relation to noncaries controls. Materials and Methods: Total genomic DNA extracted from saliva of three adults and four children from the same family were subjected to 16S rRNA gene sequencing analysis on a next generation sequencer, the PGS-Ion Torrent. Those bacterial genera with read counts > 1000 were considered as significant in each of the subject and used to associate the occurrence with caries. Results and Conclusion: Sequencing analysis indicated a higher prevalence of Streptococcus, Rothia, Granulicatella, Gemella, Actinomyces, Selenomonas, Haemophilus and Veillonella in the caries group relative to controls. While higher prevalence of Streptococcus, Rothia and Granulicatella were observed in all caries samples, the prevalence of others was observable in 2957% of samples. Interestingly, Rothia and Selenomonas, which are known to occur within anaerobic environments of dentinal caries and subgingival plaque biofilms, were seen in the saliva of these caries patients. Taken together, the study has identified for the first time a unique co-prevalence pattern of bacteria in caries patients that may be explored as distinct caries specific bacterial signature to predict cariogenesis in high-risk primary and mixed dentition age groups. PMID:25713496

  12. Bacterial rRNA Genes Associated with Soil Suppressiveness against the Plant-Parasitic Nematode Heterodera schachtii

    PubMed Central

    Yin, Bei; Valinsky, Lea; Gao, Xuebiao; Becker, J. Ole; Borneman, James

    2003-01-01

    The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii. Since H. schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H. schachtii cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with H. schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, ?-Proteobacteria, ?-Proteobacteria, and ?-Proteobacteria) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H. schachtii suppressiveness. When these three groups were examined with specific PCR analyses performed on H. schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured ?-proteobacterial clones was consistently associated with the highly suppressive treatments. A quantitative PCR analysis confirmed the association of this Rhizobium-like rDNA group with the H. schachtii suppressiveness. PMID:12620845

  13. Resistance to ketolide antibiotics by coordinated expression of rRNA methyltransferases in a bacterial producer of natural ketolides.

    PubMed

    Almutairi, Mashal M; Park, Sung Ryeol; Rose, Simon; Hansen, Douglas A; Vzquez-Laslop, Nora; Douthwaite, Stephen; Sherman, David H; Mankin, Alexander S

    2015-10-20

    Ketolides are promising new antimicrobials effective against a broad range of Gram-positive pathogens, in part because of the low propensity of these drugs to trigger the expression of resistance genes. A natural ketolide pikromycin and a related compound methymycin are produced by Streptomyces venezuelae strain ATCC 15439. The producer avoids the inhibitory effects of its own antibiotics by expressing two paralogous rRNA methylase genes pikR1 and pikR2 with seemingly redundant functions. We show here that the PikR1 and PikR2 enzymes mono- and dimethylate, respectively, the N6 amino group in 23S rRNA nucleotide A2058. PikR1 monomethylase is constitutively expressed; it confers low resistance at low fitness cost and is required for ketolide-induced activation of pikR2 to attain high-level resistance. The regulatory mechanism controlling pikR2 expression has been evolutionary optimized for preferential activation by ketolide antibiotics. The resistance genes and the induction mechanism remain fully functional when transferred to heterologous bacterial hosts. The anticipated wide use of ketolide antibiotics could promote horizontal transfer of these highly efficient resistance genes to pathogens. Taken together, these findings emphasized the need for surveillance of pikR1/pikR2-based bacterial resistance and the preemptive development of drugs that can remain effective against the ketolide-specific resistance mechanism. PMID:26438831

  14. 16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia

    PubMed Central

    Xia, Li-Ping; Bian, Long-Yan; Xu, Min; Liu, Ying; Tang, Ai-Ling; Ye, Wen-Qin

    2015-01-01

    Ventilator-associated pneumonia (VAP) is an acquired respiratory tract infection following tracheal intubation. The most common hospital-acquired infection among patients with acute respiratory failure, VAP is associated with a mortality rate of 20-30%. The standard bacterial culture method for identifying the etiology of VAP is not specific, timely, or accurate in identifying the bacterial pathogens. This study used 16S rRNA gene metagenomic sequencing to identify and quantify the pathogenic bacteria in lower respiratory tract and oropharyngeal samples of 55 VAP patients. Sequencing of the 16S rRNA gene has served as a valuable tool in bacterial identification, particularly when other biochemical, molecular, or phenotypic identification techniques fail. In this study, 16S rRNA gene sequencing was performed in parallel with the standard bacterial culture method to identify and quantify bacteria present in the collected patient samples. Sequence analysis showed the colonization of multidrug-resistant strains in VAP secretions. Further, this method identified Prevotella, Proteus, Aquabacter, and Sphingomonas bacterial genera that were not detected by the standard bacterial culture method. Seven categories of bacteria, Streptococcus, Neisseria, Corynebacterium, Acinetobacter, Staphylococcus, Pseudomonas and Klebsiella, were detectable by both 16S rRNA gene sequencing and standard bacterial culture methods. Further, 16S rRNA gene sequencing had a significantly higher sensitivity in detecting Streptococcus and Pseudomonas when compared to standard bacterial culture. Together, these data present 16S rRNA gene sequencing as a novel VAP diagnosis tool that will further enable pathogen-specific treatment of VAP.

  15. 16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia.

    PubMed

    Xia, Li-Ping; Bian, Long-Yan; Xu, Min; Liu, Ying; Tang, Ai-Ling; Ye, Wen-Qin

    2015-01-01

    Ventilator-associated pneumonia (VAP) is an acquired respiratory tract infection following tracheal intubation. The most common hospital-acquired infection among patients with acute respiratory failure, VAP is associated with a mortality rate of 20-30%. The standard bacterial culture method for identifying the etiology of VAP is not specific, timely, or accurate in identifying the bacterial pathogens. This study used 16S rRNA gene metagenomic sequencing to identify and quantify the pathogenic bacteria in lower respiratory tract and oropharyngeal samples of 55 VAP patients. Sequencing of the 16S rRNA gene has served as a valuable tool in bacterial identification, particularly when other biochemical, molecular, or phenotypic identification techniques fail. In this study, 16S rRNA gene sequencing was performed in parallel with the standard bacterial culture method to identify and quantify bacteria present in the collected patient samples. Sequence analysis showed the colonization of multidrug-resistant strains in VAP secretions. Further, this method identified Prevotella, Proteus, Aquabacter, and Sphingomonas bacterial genera that were not detected by the standard bacterial culture method. Seven categories of bacteria, Streptococcus, Neisseria, Corynebacterium, Acinetobacter, Staphylococcus, Pseudomonas and Klebsiella, were detectable by both 16S rRNA gene sequencing and standard bacterial culture methods. Further, 16S rRNA gene sequencing had a significantly higher sensitivity in detecting Streptococcus and Pseudomonas when compared to standard bacterial culture. Together, these data present 16S rRNA gene sequencing as a novel VAP diagnosis tool that will further enable pathogen-specific treatment of VAP. PMID:26770469

  16. The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing

    PubMed Central

    Kennedy, Nicholas A.; Walker, Alan W.; Berry, Susan H.; Duncan, Sylvia H.; Farquarson, Freda M.; Louis, Petra; Thomson, John M.; Ahmad, T; Anderson, CA; Barrett, JC; Drummond, H; Edwards, C; Hart, A; Hawkey, C; Henderson, P; Khan, M; Lamb, CA; Lee, JC; Mansfield, JC; Mathew, CG; Mowat, C; Newman, WG; Prescott, NJ; Simmons, A; Simpson, P; Taylor, K; Taylor, K; Wilson, DC; Satsangi, Jack; Flint, Harry J.; Parkhill, Julian

    2014-01-01

    Introduction Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Methods Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. Results DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). Conclusion This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples in a study are prepared with the same method, and the need for caution when cross-comparing studies that use different methods. PMID:24586470

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

    PubMed Central

    Peng, Mu; Zi, Xiaoxue; Wang, Qiuyu

    2015-01-01

    Soil bacteria play a major role in ecological and biodegradable function processes in oil-contaminated soils. Here, we assessed the bacterial diversity and changes therein in oil-contaminated soils exposed to different periods of oil pollution using 454 pyrosequencing of 16S rRNA genes. No less than 24,953 valid reads and 6246 operational taxonomic units (OTUs) were obtained from all five studied samples. OTU richness was relatively higher in contaminated soils than clean samples. Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Proteobacteria were the dominant phyla among all the soil samples. The heatmap plot depicted the relative percentage of each bacterial family within each sample and clustered five samples into two groups. For the samples, bacteria in the soils varied at different periods of oil exposure. The oil pollution exerted strong selective pressure to propagate many potentially petroleum degrading bacteria. Redundancy analysis (RDA) indicated that organic matter was the highest determinant factor for explaining the variations in community compositions. This suggests that compared to clean soils, oil-polluted soils support more diverse bacterial communities and soil bacterial community shifts were mainly controlled by organic matter and exposure time. These results provide some useful information for bioremediation of petroleum contaminated soil in the future. PMID:26404329

  18. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    SciTech Connect

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.

  19. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens

    DOE PAGESBeta

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.; Heimesaat, Markus M.

    2015-02-06

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n =more » 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.« less

  20. Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

    PubMed Central

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L.; Lynch, Susan V.

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Nave Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

  1. Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens.

    PubMed

    Srinivasan, Ramya; Karaoz, Ulas; Volegova, Marina; MacKichan, Joanna; Kato-Maeda, Midori; Miller, Steve; Nadarajan, Rohan; Brodie, Eoin L; Lynch, Susan V

    2015-01-01

    According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Nave Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci. PMID:25658760

  2. Description of Drinking Water Bacterial Communities Using 16S rRNA Gene Sequence Analyses

    EPA Science Inventory

    Descriptions of bacterial communities inhabiting water distribution systems (WDS) have mainly been accomplished using culture-based approaches. Due to the inherent selective nature of culture-based approaches, the majority of bacteria inhabiting WDS remain uncharacterized. The go...

  3. Soil bacterial diversity screening using single 16S rRNA gene V regions coupled with multi-million read generating sequencing technologies.

    PubMed

    Vasileiadis, Sotirios; Puglisi, Edoardo; Arena, Maria; Cappa, Fabrizio; Cocconcelli, Pier S; Trevisan, Marco

    2012-01-01

    The novel multi-million read generating sequencing technologies are very promising for resolving the immense soil 16S rRNA gene bacterial diversity. Yet they have a limited maximum sequence length screening ability, restricting studies in screening DNA stretches of single 16S rRNA gene hypervariable (V) regions. The aim of the present study was to assess the effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. Using an in silico approach, the performance of each V region was compared with the complete 16S rRNA gene stretch. We assessed related properties of the soil derived bacterial sequence collection of the Ribosomal Database Project (RDP) database and concomitantly performed simulations based on published datasets. Results indicate that overall the most prominent V region for soil bacterial diversity studies was V3, even though it was outperformed in some of the tests. Despite its high performance during most tests, V4 was less conserved along flanking sites, thus reducing its ability for bacterial diversity coverage. V5 performed well in the non-redundant RDP database based analysis. However V5 did not resemble the full-length 16S rRNA gene sequence results as well as V3 and V4 did when the natural sequence frequency and occurrence approximation was considered in the virtual experiment. Although, the highly conserved flanking sequence regions of V6 provide the ability to amplify partial 16S rRNA gene sequences from very diverse owners, it was demonstrated that V6 was the least informative compared to the rest examined V regions. Our results indicate that environment specific database exploration and theoretical assessment of the experimental approach are strongly suggested in 16S rRNA gene based bacterial diversity studies. PMID:22880076

  4. Bacterial diversity assessment of pristine mangrove microbial community from Dhulibhashani, Sundarbans using 16S rRNA gene tag sequencing

    PubMed Central

    Basak, Pijush; Pramanik, Arnab; Sengupta, Sohan; Nag, Sudip; Bhattacharyya, Anish; Roy, Debojyoti; Pattanayak, Rudradip; Ghosh, Abhrajyoti; Chattopadhyay, Dhrubajyoti; Bhattacharyya, Maitree

    2015-01-01

    The global knowledge of microbial diversity and function in Sundarbans ecosystem is still scarce, despite global advancement in understanding the microbial diversity. In the present study, we have analyzed the diversity and distribution of bacteria in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. Metagenome is comprised of 1,53,926 sequences with 108.8 Mbp data and with 55 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA245459. Bacterial community metagenome sequences were analyzed by MG-RAST software representing the presence of 56,547 species belonging to 44 different phyla. The taxonomic analysis revealed the dominance of phyla Proteobacteria within our dataset. Further taxonomic analysis revealed abundance of Bacteroidetes, Acidobactreia, Firmicutes, Actinobacteria, Nitrospirae, Cyanobacteria, Planctomycetes and Fusobacteria group as the predominant bacterial assemblages in this largely pristine mangrove habitat. The distribution of different community datasets obtained from four sediment samples originated from one sampling station at two different depths providing better understanding of the sediment bacterial diversity and its relationship to the ecosystem dynamics of this pristine mangrove sediment of Dhulibhashani in, Sundarbans. PMID:26981367

  5. Bacterial diversity assessment of pristine mangrove microbial community from Dhulibhashani, Sundarbans using 16S rRNA gene tag sequencing.

    PubMed

    Basak, Pijush; Pramanik, Arnab; Sengupta, Sohan; Nag, Sudip; Bhattacharyya, Anish; Roy, Debojyoti; Pattanayak, Rudradip; Ghosh, Abhrajyoti; Chattopadhyay, Dhrubajyoti; Bhattacharyya, Maitree

    2016-03-01

    The global knowledge of microbial diversity and function in Sundarbans ecosystem is still scarce, despite global advancement in understanding the microbial diversity. In the present study, we have analyzed the diversity and distribution of bacteria in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. Metagenome is comprised of 1,53,926 sequences with 108.8 Mbp data and with 55 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA245459. Bacterial community metagenome sequences were analyzed by MG-RAST software representing the presence of 56,547 species belonging to 44 different phyla. The taxonomic analysis revealed the dominance of phyla Proteobacteria within our dataset. Further taxonomic analysis revealed abundance of Bacteroidetes, Acidobactreia, Firmicutes, Actinobacteria, Nitrospirae, Cyanobacteria, Planctomycetes and Fusobacteria group as the predominant bacterial assemblages in this largely pristine mangrove habitat. The distribution of different community datasets obtained from four sediment samples originated from one sampling station at two different depths providing better understanding of the sediment bacterial diversity and its relationship to the ecosystem dynamics of this pristine mangrove sediment of Dhulibhashani in, Sundarbans. PMID:26981367

  6. Bacterial community analysis of a gas-phase biotrickling filter for biogas mimics desulfurization through the rRNA approach.

    PubMed

    Maestre, Juan P; Rovira, R; Alvarez-Hornos, F J; Fortuny, M; Lafuente, J; Gamisans, X; Gabriel, D

    2010-08-01

    The bacterial composition of a lab-scale biotrickling filter (BTF) treating high loads of H(2)S was investigated by the rRNA approach. Two 16S rRNA gene clone libraries were established 42 and 189 d after reactor startup, while fluorescent in-situ hybridization (FISH) with DNA probes was performed throughout 260d of reactor operation. Diversity, community structure and metamorphosis were studied from reactor startup to fully-established pseudo-steady state operation at near neutral pH and at an inlet H(2)S concentration of 2000 ppmv (load of 55.6g H(2)S m(-3)h(-1)). In addition, FISH was used for assessing the spatial distribution of sulfur-oxidizing bacteria (SOB) along the length of the reactor under pseudo-steady state operation. A major shift in the diversity of the community was observed with the operating time, from a well-diverse community at startup to pseudo-steady state operation with a majority of retrieved sequences affiliated to SOB of the sulfur cycle including Thiothrix spp., Thiobacillus spp., and Sulfurimonas denitrificans. Although aerobic species were predominant along the BTF, a vertical stratification was encountered, in which facultative anaerobes had a major relative abundance in the inlet part of the BTF, where the sulfide to oxygen ratio was higher. The observed changes were related to the trophic properties of the community, the DO concentration, the accumulation of elemental sulfur and the operation at neutral pH. PMID:20554311

  7. Simultaneous detection of microorganisms in soil suspension based on PCR amplification of bacterial 16S rRNA fragments.

    PubMed

    McGregor, D P; Forster, S; Steven, J; Adair, J; Leary, S E; Leslie, D L; Harris, W J; Titball, R W

    1996-09-01

    The effect of buffer composition on simultaneous PCR amplification of 16S rRNA gene fragments of five bacterial species was examined using a number of different buffer systems. Tris-based PCR buffers at final concentrations of 10 mM proved unreliable. However, when the final concentration of Tris was increased to 75 mM, all five samples were routinely detected. The use of other buffers, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO) and 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO), resulted in PCR amplification of five products even at low final concentrations (10 mM). The presence of certain proteins in the amplification reaction could overcome an inhibitory effect seen when soil suspension was present in the reaction, as might occur when testing field samples for the presence of bacteria. Bovine serum albumin was found to be the most effective additive tested in overcoming inhibition. PMID:8879586

  8. Phylotype-level 16S rRNA analysis reveals new bacterial indicators of health state in acute murine colitis

    PubMed Central

    Berry, David; Schwab, Clarissa; Milinovich, Gabriel; Reichert, Jochen; Ben Mahfoudh, Karim; Decker, Thomas; Engel, Marion; Hai, Brigitte; Hainzl, Eva; Heider, Susanne; Kenner, Lukas; Müller, Mathias; Rauch, Isabella; Strobl, Birgit; Wagner, Michael; Schleper, Christa; Urich, Tim; Loy, Alexander

    2012-01-01

    Human inflammatory bowel disease and experimental colitis models in mice are associated with shifts in intestinal microbiota composition, but it is unclear at what taxonomic/phylogenetic level such microbiota dynamics can be indicative for health or disease. Here, we report that dextran sodium sulfate (DSS)-induced colitis is accompanied by major shifts in the composition and function of the intestinal microbiota of STAT1−/− and wild-type mice, as determined by 454 pyrosequencing of bacterial 16S rRNA (gene) amplicons, metatranscriptomics and quantitative fluorescence in situ hybridization of selected phylotypes. The bacterial families Ruminococcaceae, Bacteroidaceae, Enterobacteriaceae, Deferribacteraceae and Verrucomicrobiaceae increased in relative abundance in DSS-treated mice. Comparative 16S rRNA sequence analysis at maximum possible phylogenetic resolution identified several indicator phylotypes for DSS treatment, including the putative mucin degraders Akkermansia and Mucispirillum. The analysis additionally revealed strongly contrasting abundance changes among phylotypes of the same family, particularly within the Lachnospiraceae. These extensive phylotype-level dynamics were hidden when reads were grouped at higher taxonomic levels. Metatranscriptomic analysis provided insights into functional shifts in the murine intestinal microbiota, with increased transcription of genes associated with regulation and cell signaling, carbohydrate metabolism and respiration and decreased transcription of flagellin genes during inflammation. These findings (i) establish the first in-depth inventory of the mouse gut microbiota and its metatranscriptome in the DSS colitis model, (ii) reveal that family-level microbial community analyses are insufficient to reveal important colitis-associated microbiota shifts and (iii) support a scenario of shifting intra-family structure and function in the phylotype-rich and phylogenetically diverse Lachnospiraceae in DSS-treated mice. PMID:22572638

  9. Adhesion molecules in antibacterial defenses: effects of bacterial extracts.

    PubMed

    Marchant, A; Duchow, J; Goldman, M

    1992-01-01

    Adhesion of polymorphonuclear leukocytes (PMN) to vascular endothelium is one of the first events in their response against local bacterial infection. Different adhesion molecules sequentially mediate PMN adherence to endothelium and extravasation into inflamed tissues. We show that bacterial extracts OM-85 BV and OM-89 increase the expression of adhesion molecules at the surface of PMN and we suggest that this upregulation could be linked to the beneficial effect of bacterial extracts in the prevention of respiratory tract infections. PMID:1439236

  10. Response of a Soil Bacterial Community to Grassland Succession as Monitored by 16S rRNA Levels of the Predominant Ribotypes

    PubMed Central

    Felske, Andreas; Wolterink, Arthur; Van Lis, Robert; De Vos, Willem M.; Akkermans, Antoon D. L.

    2000-01-01

    The composition of predominant soil bacteria during grassland succession was investigated in the Dutch Drentse A area. Five meadows, taken out of agricultural production at different time points, and one currently fertilized plot represented different stages of grassland succession. Since fertilization and agricultural production were stopped, the six plots showed a constant decline in the levels of nutrients and vegetation changes. The activity of the predominant bacteria was monitored by direct ribosome isolation from soil and temperature gradient gel electrophoresis of reverse transcription (RT)-PCR products generated from bacterial 16S rRNA. The amounts of 16S rRNA of 20 predominant ribosome types per gram of soil were monitored via multiple competitive RT-PCR in six plots at different succession stages. These ribosome types mainly represented Bacillus and members of the Acidobacterium cluster and the ? subclass of the class Proteobacteria. The 20 16S rRNA molecules monitored represented approximately half of all bacterial soil rRNA which was estimated by dot blot hybridizations of soil rRNA with the Bacteria probe EUB338. The grasslands showed highly reproducible and specific shifts of bacterial ribosome type composition. The total bacterial ribosome level increased during the first years after agricultural production and fertilization stopped. This correlated with the collapse of the dominant Lolium perenne population and an increased rate of mineralization of organic matter. The results indicate that there is a true correlation between the total activity of the bacterial community in soil and the amount of bacterial ribosomes. PMID:10966420

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

    PubMed

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

    2014-03-01

    Crop rotation is a practice harmonized with the sustainable rice production. Nevertheless, the implications of this empirical practice are not well characterized, mainly in relation to the bacterial community composition and structure. In this study, the bacterial communities of two adjacent paddy fields in the 3rd and 4th year of the crop rotation cycle and of a nonseeded subplot were characterized before rice seeding and after harvesting, using 454-pyrosequencing of the 16S rRNA gene. Although the phyla Acidobacteria, Proteobacteria, Chloroflexi, Actinobacteria and Bacteroidetes predominated in all the samples, there were variations in relative abundance of these groups. Samples from the 3rd and 4th years of the crop rotation differed on the higher abundance of groups of presumable aerobic bacteria and of presumable anaerobic and acidobacterial groups, respectively. Members of the phylum Nitrospira were more abundant after rice harvest than in the previously sampled period. Rice cropping was positively correlated with the abundance of members of the orders Acidobacteriales and 'Solibacterales' and negatively with lineages such as Chloroflexi 'Ellin6529'. Studies like this contribute to understand variations occurring in the microbial communities in soils under sustainable rice production, based on real-world data. PMID:24245591

  12. 23S rRNA as an a-Maz-ing new bacterial toxin target

    PubMed Central

    Schifano, Jason M; Woychik, Nancy A

    2014-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is a bacterium with the unique ability to persist for years or decades as a latent infection. This latent state, during which bacteria have a markedly altered physiology and are thought to be dormant, is crucial for the bacteria to survive the stressful environments it encounters in the human host. Importantly, M. tuberculosis cells in the dormant state are generally refractory to antibiotics, most of which target cellular processes occurring in actively replicating bacteria. The molecular switches that enable M. tuberculosis to slow or stop its replication and become dormant remain unknown. However, the slow growth and dormant state that are hallmarks of latent tuberculosis infection have striking parallels to the quasi-dormant state of Escherichia coli cells caused by the toxin components of chromosomal toxin-antitoxin (TA) modules. An unusually large number of TA modules in M. tuberculosis, including nine in the mazEF family, may contribute to initiating this latent state or to adapting to stress conditions in the host. Toward filling the gap in our understanding of the physiological role of TA modules in M. tuberculosis, we are interested in identifying their molecular mechanisms to better understand how toxins impart growth control. Our recent publication1uncovered a novel function of a MazF toxin in M. tuberculosis that had not been associated with any other MazF ortholog. This toxin, MazF-mt6, can disrupt protein synthesis by cleavage of 23S rRNA at a single location in an evolutionarily conserved five-base sequence in the ribosome active center. PMID:24525465

  13. Variability in abundance of the Bacterial and Archaeal 16S rRNA and amoA genes in water columns of northern South China Sea

    NASA Astrophysics Data System (ADS)

    Liu, H.; Yang, C.; Chen, S.; Xie, W.; Wang, P.; Zhang, C. L.

    2014-12-01

    Recent advances in marine microbial ecology have shown that ammonia-oxidizing Archaea (AOA) are more abundant than ammonia-oxidizing bacteria (AOB), although total Bacteria are more abundant than total Archaea in marine environments. This study aimed to examine the spatial distribution and abundance of planktonic archaeal and bacterial 16S rRNA- and amoA genes in the northern South China Sea. Water samples were collected at different depths at six stations (maximum depth ranging from 1800 m to 3200 m)with four stations (B2, B3, B6, B7) located along a transect from the northeastern continental slope to the Bashi Strait and the other two (D3, D5) located southwest of this transect. Quantitative PCR of the 16S rRNA- and amoA genes was used to estimate the abundances of total Archaea, total Bacteria, and AOA and AOB, respectively. At the B series stations, the abundance of bacterial 16S rRNA gene was twofold to 36fold higher than that of the archaeal 16S rRNA gene while fivefold lower to sixfold higher at the two D stations, with both genes showing peak values slightly below sea surface (5-75 m depths) at all stations. The archaeal amoA gene had similar variations with the archaeal 16S rRNA gene, but was 1-4 orders of magnitude lower than the archaeal 16S rRNA gene at all stations. Bacterial amoA gene was below the detection at all stations. Our results also show the difference in depth profiles among these stations, which may be caused by the difference in water movement between these regions. The non-detection of bacterial amoA gene indicates that ammonia-oxidizing Archaea are the dominant group of microorganisms in nitrification of the South China Sea, which is consistent with observations in other oceans.

  14. Extraction of Bacterial RNA from Soil: Challenges and Solutions

    PubMed Central

    Wang, Yong; Hayatsu, Masahito; Fujii, Takeshi

    2012-01-01

    Detection of bacterial gene expression in soil emerged in the early 1990s and provided information on bacterial responses in their original soil environments. As a key procedure in the detection, extraction of bacterial RNA from soil has attracted much interest, and many methods of soil RNA extraction have been reported in the past 20 years. In addition to various RT-PCR-based technologies, new technologies for gene expression analysis, such as microarrays and high-throughput sequencing technologies, have recently been applied to examine bacterial gene expression in soil. These technologies are driving improvements in RNA extraction protocols. In this mini-review, progress in the extraction of bacterial RNA from soil is summarized with emphasis on the major difficulties in the development of methodologies and corresponding strategies to overcome them. PMID:22791042

  15. Phylogenetic analysis of 16S rRNA gene sequences reveals rumen bacterial diversity in Yaks (Bos grunniens).

    PubMed

    Yang, L Y; Chen, J; Cheng, X L; Xi, D M; Yang, S L; Deng, W D; Mao, H M

    2010-01-01

    Six matured male Yaks (Bos grunniens) with a mean live weight of 450 +/- 23 kg (mean +/- SD), were housed indoors in metabolism cages and fed pelleted lucerne (Medicago sativum). After an adjustment period of 24 days of feeding the diet, samples of rumen content were obtained for analysis of the bacteria in the liquor. The diversity of rumen bacteria was investigated by constructing a 16S rRNA gene clone library using the general bacterial primers F27 and R1492. A total of 130 clones, comprising nearly full length sequences (approx. 1.5 kb) were sequenced and submitted to BLAST and phylogenetic analysis. Using the criterion that similarity of 97% or greater with the sequences of cultivated bacteria, 16 clones were identified as Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Ruminococcus flavefaciens, Succiniclasticum ruminis, Selenomonas ruminantium and Prevotella ruminicola, respectively. A further 10 clones shared similarity ranging from 90 to 97% with cultivated bacteria but the similarity in sequences for the remaining 104 clones were less than 90% of those of cultivated bacteria. Using a phylogenetic analysis it was found that the majority of the clones identified (63.8%) were located in the Low G + C Subdivision, with most of the remainder (35.4% of clones) located in the Cytophaga-Flexibacter-Bacteroides phylum and one clone (0.8%) was identified as a Proteobacteria. It was apparent that Yaks have a large and diverse range of bacteria in the rumen content which differ from those of cattle and other ruminants. PMID:19757178

  16. 16S rRNA phylogenetic analysis of the bacterial endosymbionts associated with cytoplasmic incompatibility in insects.

    PubMed Central

    O'Neill, S L; Giordano, R; Colbert, A M; Karr, T L; Robertson, H M

    1992-01-01

    Bacterial endosymbionts of insects have long been implicated in the phenomenon of cytoplasmic incompatibility, in which certain crosses between symbiont-infected individuals lead to embryonic death or sex ratio distortion. The taxonomic position of these bacteria has, however, not been known with any certainty. Similarly, the relatedness of the bacteria infecting various insect hosts has been unclear. The inability to grow these bacteria on defined cell-free medium has been the major factor underlying these uncertainties. We circumvented this problem by selective PCR amplification and subsequent sequencing of the symbiont 16S rRNA genes directly from infected insect tissue. Maximum parsimony analysis of these sequences indicates that the symbionts belong in the alpha-subdivision of the Proteobacteria, where they are most closely related to the Rickettsia and their relatives. They are all closely related to each other and are assigned to the type species Wolbachia pipientis. Lack of congruence between the phylogeny of the symbionts and their insect hosts suggest that horizontal transfer of symbionts between insect species may occur. Comparison of the sequences for W. pipientis and for Wolbachia persica, an endosymbiont of ticks, shows that the genus Wolbachia is polyphyletic. A PCR assay based on 16S primers was designed for the detection of W. pipientis in insect tissue, and initial screening of insects indicates that cytoplasmic incompatibility may be a more general phenomenon in insects than is currently recognized. Images PMID:1557375

  17. The GA motif: an RNA element common to bacterial antitermination systems, rRNA, and eukaryotic RNAs.

    PubMed

    Winkler, W C; Grundy, F J; Murphy, B A; Henkin, T M

    2001-08-01

    Two different transcription termination control mechanisms, the T box and S box systems, are used to regulate transcription of many bacterial aminoacyl-tRNA synthetase, amino acid biosynthesis, and amino acid transport genes. Both of these regulatory mechanisms involve an untranslated mRNA leader region capable of adopting alternate structural conformations that result in transcription termination or transcription elongation into the downstream region. Comparative analyses revealed a small RNA secondary structural element, designated the GA motif, that is highly conserved in both T box and S box leader sequences. The motif consists of two short helices separated by an asymmetric internal loop, with highly conserved GA dinucleotide sequences on either side of the internal loop. Site-directed mutagenesis of this motif in model T and S box leader sequences indicated that it is essential for transcriptional regulation in both systems. This motif is similar to the binding site of yeast ribosomal protein L30, the Snu13p binding sites found in U4 snRNA and box C/D snoRNAs, and two elements in 23S rRNA. PMID:11497434

  18. Diversity of endophytic bacteria in Malaysian plants as revealed by 16S rRNA encoding gene sequence based method of bacterial identification?

    PubMed Central

    Loh, Chye Ying; Tan, Yin Yin; Rohani, Rahim; Weber, Jean-Frdric F.; Bhore, Subhash Janardhan

    2013-01-01

    Bacterial endophytes do have several potential applications in pharmacy, medicine and agricultural biotech industry. The main objective of this study was to understand types of bacterial endophytes associated with dicotyledonous (dicot) and monocotyledonous (monocot) plant species. Isolation of the endophytic bacteria was performed using surface-sterilized various tissue samples, and identification of the endophytic bacterial isolates (EBIs) was completed using 16S rRNA encoding gene sequence similarity based method. In total, 996 EBIs were isolated and identified from 1055 samples of 31 monocot and 65 dicot plant species from Peninsular Malaysia. The 996 EBIs represented 71 different types of bacterial species. Twelve (12) out of 71 species are reported as endophytes for the first time. We conclude that diverse types of bacterial endophytes are associated with dicot and monocot plants, and could be useful in pharmacy, medicine and agricultural biotechnology for various potential applications. PMID:24396249

  19. Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences. [Calyptogena magnifica; Bathymodiolus thermophilus; Lucinoma annulata; Lucinoma aequizonata; Codakia orbicularis

    SciTech Connect

    Distel, D.L.; Lane, D.J.; Olsen, G.J.; Giovannoni, S.J.; Pace, B.; Pace, N.R.; Stahl, D.A.; Felbeck, H.

    1988-06-01

    The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis. Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.

  20. Selective Extraction of Bacterial DNA from the Surfaces of Macroalgae▿

    PubMed Central

    Burke, Catherine; Kjelleberg, Staffan; Thomas, Torsten

    2009-01-01

    A novel method has been developed for the selective extraction of DNA from surface-associated bacterial communities from the two model marine benthic algae Ulva australis and Delisea pulchra. The extracted DNA had no detectable contamination with host DNA, was recovered in high yield and quality, and was representative of the bacterial community on the algal surfaces. The DNA is suitable for a variety of subsequent applications, including the construction of large-insert clone libraries and metagenomic sequencing. PMID:18978081

  1. Sponge-associated actinobacterial diversity: validation of the methods of actinobacterial DNA extraction and optimization of 16S rRNA gene amplification.

    PubMed

    Yang, Qi; Franco, Christopher M M; Zhang, Wei

    2015-10-01

    Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity. PMID:26245685

  2. Bacterial Community Shift in Treated Periodontitis Patients Revealed by Ion Torrent 16S rRNA Gene Amplicon Sequencing

    PubMed Central

    Jnemann, Sebastian; Prior, Karola; Szczepanowski, Rafael; Harks, Inga; Ehmke, Benjamin; Goesmann, Alexander; Stoye, Jens; Harmsen, Dag

    2012-01-01

    Periodontitis, one of the most common diseases in the world, is caused by a mixture of pathogenic bacteria and inflammatory host responses and often treated by antimicrobials as an adjunct to scaling and root planing (SRP). Our study aims to elucidate explorative and descriptive temporal shifts in bacterial communities between patients treated by SRP alone versus SRP plus antibiotics. This is the first metagenomic study using an Ion Torrent Personal Genome Machine (PGM). Eight subgingival plaque samples from four patients with chronic periodontitis, taken before and two months after intervention were analyzed. Amplicons from the V6 hypervariable region of the 16S rRNA gene were generated and sequenced each on a 314 chip. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance), described by community metrics, and taxonomically classified. Reads ranging from 599,933 to 650,416 per sample were clustered into 1,648 to 2,659 non-singleton OTUs, respectively. Increased diversity (Shannon and Simpson) in all samples after therapy was observed regardless of the treatment type whereas richness (ACE) showed no correlation. Taxonomic analysis revealed different microbial shifts between both therapy approaches at all taxonomic levels. Most remarkably, the genera Porphyromonas, Tannerella, Treponema, and Filifactor all harboring periodontal pathogenic species were removed almost only in the group treated with SPR and antibiotics. For the species T. forsythia and P. gingivalis results were corroborated by real-time PCR analysis. In the future, hypothesis free metagenomic analysis could be the key in understanding polymicrobial diseases and be used for therapy monitoring. Therefore, as read length continues to increase and cost to decrease, rapid benchtop sequencers like the PGM might finally be used in routine diagnostic. PMID:22870235

  3. Bacterial diversity among small-subunit rRNA gene clones and cellular isolates from the same seawater sample

    SciTech Connect

    Suzuki, M.T.; Rappe, M.S.; Haimberger, Z.W.

    1997-03-01

    Numerous investigations applying the cloning and sequencing of rRNA genes (rDNAs) to the study of marine bacterioplankton diversity have shown that the sequences of genes cloned directly from environmental DNA do not correspond to the genes of cultured marine taxa. These results have been interpreted as support for the hypothesis that the most abundant heterotrophic marine bacterioplankton species are not readily culturable by commonly used methods. However, an alternative explanation is that marine bacterioplankton can be easily cultured but are not well represented in sequence databases. To further examine this question, we compared the small-subunit (SSU) rDNAs of 127 cellular clones isolated from a water sample collected off the Oregon coast to 58 bacterial SSU rDNAs cloned from environmental DNAs from the same water sample. The results revealed little overlap between partial SSU rDNA sequences from the cellular clones and the environmental clone library. An exception was the SSU rDNA sequence recovered from a cellular clone belonging to the Pseudomonas subgroup of the {gamma} subclass of the class Proteobacteria, which was related to a single gene cloned directly from the same water sample (OCS181) (similarity, 94.6%). In addition, partial SSU rDNA sequences from three of the cultured strains matched a novel rDNA clone related to the {gamma} subclass of the Proteobacteria found previously in an environmental clone library from marine aggregates (AGG53) (similarity, 94.3 to 99.6%). Our results support the hypothesis that many of the most abundant bacterioplankton species are not readily culturable by standard methods but also show that heterotrophic bacterioplankton that are culturable on media with high organic contents include many strains for which SSU rDNA sequences are not available in sequence databases. 34 refs., 4 figs., 3 tabs.

  4. Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

    PubMed Central

    Siibak, Triinu; Remme, Jaanus

    2010-01-01

    Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly. PMID:20719918

  5. Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags.

    PubMed

    Peng, Xin; Yu, Ke-Qiang; Deng, Guan-Hua; Jiang, Yun-Xia; Wang, Yu; Zhang, Guo-Xia; Zhou, Hong-Wei

    2013-12-01

    Low cost and high throughput capacity are major advantages of using next generation sequencing (NGS) techniques to determine metagenomic 16S rRNA tag sequences. These methods have significantly changed our view of microorganisms in the fields of human health and environmental science. However, DNA extraction using commercial kits has shortcomings of high cost and time constraint. In the present study, we evaluated the determination of fecal microbiomes using a direct boiling method compared with 5 different commercial extraction methods, e.g., Qiagen and MO BIO kits. Principal coordinate analysis (PCoA) using UniFrac distances and clustering showed that direct boiling of a wide range of feces concentrations gave a similar pattern of bacterial communities as those obtained from most of the commercial kits, with the exception of the MO BIO method. Fecal concentration by boiling method affected the estimation of ?-diversity indices, otherwise results were generally comparable between boiling and commercial methods. The operational taxonomic units (OTUs) determined through direct boiling showed highly consistent frequencies with those determined through most of the commercial methods. Even those for the MO BIO kit were also obtained by the direct boiling method with high confidence. The present study suggested that direct boiling could be used to determine the fecal microbiome and using this method would significantly reduce the cost and improve the efficiency of the sample preparation for studying gut microbiome diversity. PMID:23899773

  6. Ultradeep 16S rRNA Sequencing Analysis of Geographically Similar but Diverse Unexplored Marine Samples Reveal Varied Bacterial Community Composition

    PubMed Central

    Karutha Pandian, Shunmugiah

    2013-01-01

    Background Bacterial community composition in the marine environment differs from one geographical location to another. Reports that delineate the bacterial diversity of different marine samples from geographically similar location are limited. The present study aims to understand whether the bacterial community compositions from different marine samples harbour similar bacterial diversity since these are geographically related to each other. Methods and Principal Findings In the present study, 16S rRNA deep sequencing analysis targeting V3 region was performed using Illumina bar coded sequencing. A total of 22.44 million paired end reads were obtained from the metagenomic DNA of Marine sediment, Rhizosphere sediment, Seawater and the epibacterial DNA of Seaweed and Seagrass. Diversity index analysis revealed that Marine sediment has the highest bacterial diversity and the least bacterial diversity was observed in Rhizosphere sediment. Proteobacteria, Actinobacteria and Bacteroidetes were the dominant taxa present in all the marine samples. Nearly 6271% of rare species were identified in all the samples and most of these rare species were unique to a particular sample. Further taxonomic assignment at the phylum and genus level revealed that the bacterial community compositions differ among the samples. Conclusion This is the first report that supports the fact that, bacterial community composition is specific for specific samples irrespective of its similar geographical location. Existence of specific bacterial community for each sample may drive overall difference in bacterial structural composition of each sample. Further studies like whole metagenomic sequencing will throw more insights to the key stone players and its interconnecting metabolic pathways. In addition, this is one of the very few reports that depicts the unexplored bacterial diversity of marine samples (Marine sediment, Rhizosphere sediment, Seawater) and the host associated marine samples (Seaweed and Seagrass) at higher depths from uncharacterised coastal region of Palk Bay, India using next generation sequencing technology. PMID:24167548

  7. Accurate transcription of homologous 5S rRNA and tRNA genes and splicing of tRNA in vitro by soluble extracts of Neurospora.

    PubMed Central

    Tyler, B M; Giles, N H

    1984-01-01

    We have developed soluble extracts from Neurospora crassa capable of accurately and efficiently transcribing homologous 5S rRNA and tRNA genes. The extracts also appear to quantitatively end-process and splice the primary tRNA transcripts. Although the extracts could not transcribe a heterologous (yeast) 5S rRNA gene, they did transcribe a yeast tRNALeu gene and slowly process the transcripts. In addition, we have developed a novel strategy for rapidly sequencing uniformly labelled RNAs using base-specific ribonucleases. We have used this procedure to verify the identity of the in vitro transcripts and processing products. Images PMID:6235482

  8. Isolation, characterization, and identification of bacterial contaminants in semifinal gelatin extracts.

    PubMed

    De Clerck, E; Vanhoutte, T; Hebb, T; Geerinck, J; Devos, J; De Vos, P

    2004-06-01

    Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA gene sequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications. PMID:15184171

  9. Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts

    PubMed Central

    De Clerck, E.; Vanhoutte, T.; Hebb, T.; Geerinck, J.; Devos, J.; De Vos, P.

    2004-01-01

    Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications. PMID:15184171

  10. Bacterial community composition during two consecutive NE Monsoon periods in the Arabian Sea studied by denaturing gradient gel electrophoresis (DGGE) of rRNA genes

    NASA Astrophysics Data System (ADS)

    Riemann, Lasse; Steward, Grieg F.; Fandino, Laura B.; Campbell, Lisa; Landry, Michael R.; Azam, Farooq

    1999-08-01

    Horizontal and vertical variations in bacterial community composition were examined in samples collected during two Joint Global Ocean Flux Study (JGOFS) Arabian Sea cruises in 1995. The cruises, 11 months apart, took place during two consecutive NE Monsoon periods (January and December). Bacteria were harvested by filtration from samples collected in the mixed layer, mid-water, and deep sea at stations across the study area. Total bacterial community genomic DNA was analyzed by PCR amplification of 16S rRNA gene fragments, followed by denaturing gradient gel electrophoresis (DGGE). In total, 20 DGGE bands reflecting unique or varying phylotypes were excised, cloned and sequenced. Amplicons were dominated by bacterial groups commonly found in oceanic waters (e.g., the SAR11 cluster of ?-Proteobacteria and cyanobacteria), but surprisingly none of the sequenced amplicons were related to ?-Proteobacteria or to members of the Cytophaga-Flavobacter-Bacteroides phylum. Amplicons related to magnetotactic bacteria were found for the first time in pelagic oceanic waters. The DGGE banding patterns revealed a dominance of ?15 distinguishable amplicons in all samples. In the mixed layer the bacterial community was dominated by the same ?15 phylotypes at all stations, but unique phylotypes were found with increasing depth. Except for cyanobacteria, comparison of the bacterial community composition in surface waters from January and December 1995 showed only minor differences, despite significant differences in environmental parameters. These data suggest a horizontal homogeneity and some degree of seasonal predictability of bacterial community composition in the Arabian Sea.

  11. Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

    PubMed Central

    Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

    2001-01-01

    The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

  12. Comparison between MICROCARDFISH and 16S rRNA gene clone libraries to assess the active versus total bacterial community in the coastal Arctic

    PubMed Central

    De Corte, Daniele; Sintes, Eva; Yokokawa, Taichi; Herndl, Gerhard J

    2013-01-01

    We collected surface- and deep-water samples (maximum depth 300 m) during the springsummer transition in the coastal Arctic along a transect in the Kongsfjorden (Ny-lesund, Spitsbergen, Norway) to determine the structure of the active versus total marine bacterioplankton community using different approaches. Catalysed reporter depositionfluorescence in situ hybridization combined with microautoradiography (MICROCARDFISH) was used to determine the abundance and activity of different bacterial groups. The bacterial communities were dominated by members of Alphaproteobacteria followed by Bacteroidetes, whereas Gammaproteobacteria were present at low abundance but exhibited a high percentage of active cells taking up leucine. The clone libraries of 16S rRNA genes (16S rDNA) and 16S rRNA from two different depths were used to decipher the bacterial community structure. Independently of the type of clone libraries analysed (16S rDNA- or 16S rRNA-based), four major and four minor taxonomic groups were detected. The bacterioplankton community was mainly dominated at both the DNA and the RNA levels by Alphaproteobacteria followed by Gammaproteobacteria. The Rhodobacteriaceae were the most abundant members of the Alphaproteobacteria in both DNA and RNA clone libraries, followed by the SAR11 clade, which was only detectable at the 16S rDNA level. Moreover, there was a general agreement between the results obtained with both techniques, although some specific phylogenetic groups, such as SAR11 and Roseobacter, deviated substantially from this relation. These discrepancies are most likely linked to different physiological states among members of the bacterioplankton community. Combined, MICROCARDFISH and DNA and RNA clone libraries, however, allowed for accurately quantifying different bacterial groups and their activity as well as a detailed phylogenetic insight into the fractions of present versus metabolically active bacterial groups. PMID:23565124

  13. How much can we learn about the function of bacterial rRNA modification by mining large-scale experimental datasets?

    PubMed Central

    Sergiev, Petr V.; Golovina, Anna Y.; Sergeeva, Olga V.; Osterman, Ilya A.; Nesterchuk, Mikhail V.; Bogdanov, Alexey A.; Dontsova, Olga A.

    2012-01-01

    Modification of ribosomal RNA is ubiquitous among living organisms. Its functional role is well established for only a limited number of modified nucleotides. There are examples of rRNA modification involvement in the gene expression regulation in the cell. There is a need for large data set analysis in the search for potential functional partners for rRNA modification. In this study, we extracted phylogenetic profile, genome neighbourhood, co-expression and phenotype profile and co-purification data regarding Escherichia coli rRNA modification enzymes from public databases. Results were visualized as graphs using Cytoscape and analysed. Majority linked genes/proteins belong to translation apparatus. Among co-purification partners of rRNA modification enzymes are several candidates for experimental validation. Phylogenetic profiling revealed links of pseudouridine synthetases with RF2, RsmH with translation factors IF2, RF1 and LepA and RlmM with RdgC. Genome neighbourhood connections revealed several putative functionally linked genes, e.g. rlmH with genes coding for cell wall biosynthetic proteins and others. Comparative analysis of expression profiles (Gene Expression Omnibus) revealed two main associations, a group of genes expressed during fast growth and association of rrmJ with heat shock genes. This study might be used as a roadmap for further experimental verification of predicted functional interactions. PMID:22411911

  14. Immunostimulatory activity of the bacterial extract OM-8.

    PubMed

    Huber, M; Ayoub, M; Pfannes, S D; Mittenbhler, K; Weis, K; Bessler, W G; Baier, W

    2000-03-27

    The bacterial extract OM-89 used for the prevention and treatment of recurrent urinary tract infections constitutes an effective immunostimulant in vitro and in vivo. Here we demonstrate that OM-89 shows mitogenic properties towards murine spleen cell cultures from LPS responder and non-responder mice. In macrophages the extract induces the translocation of NF-kappaB into the cell nucleus and RNI (radical nitrogen intermediates) release, which could be attributed to single fractions of the extract. Our findings on the in vitro immunostimulatory effect of OM-89, as well as its immunogenic and adjuvant properties, are of importance for understanding its therapeutic efficacy as demonstrated in clinical studies. PMID:10756163

  15. Different bacterial communities in heat and gamma irradiation treated replant disease soils revealed by 16S rRNA gene analysis – contribution to improved aboveground apple plant growth?

    PubMed Central

    Yim, Bunlong; Winkelmann, Traud; Ding, Guo-Chun; Smalla, Kornelia

    2015-01-01

    Replant disease (RD) severely affects apple production in propagation tree nurseries and in fruit orchards worldwide. This study aimed to investigate the effects of soil disinfection treatments on plant growth and health in a biotest in two different RD soil types under greenhouse conditions and to link the plant growth status with the bacterial community composition at the time of plant sampling. In the biotest performed we observed that the aboveground growth of apple rootstock M26 plants after 8 weeks was improved in the two RD soils either treated at 50°C or with gamma irradiation compared to the untreated RD soils. Total community DNA was extracted from soil loosely adhering to the roots and quantitative real-time PCR revealed no pronounced differences in 16S rRNA gene copy numbers. 16S rRNA gene-based bacterial community analysis by denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing revealed significant differences in the bacterial community composition even after 8 weeks of plant growth. In both soils, the treatments affected different phyla but only the relative abundance of Acidobacteria was reduced by both treatments. The genera Streptomyces, Bacillus, Paenibacillus, and Sphingomonas had a higher relative abundance in both heat treated soils, whereas the relative abundance of Mucilaginibacter, Devosia, and Rhodanobacter was increased in the gamma-irradiated soils and only the genus Phenylobacterium was increased in both treatments. The increased abundance of genera with potentially beneficial bacteria, i.e., potential degraders of phenolic compounds might have contributed to the improved plant growth in both treatments. PMID:26635733

  16. Biological activity of bacterial cell-wall components: immunogenicity of the bacterial extract OM-89.

    PubMed

    Sedelmeier, E A; Bessler, W G

    1995-02-01

    The bacterial extract OM-89 is used for the treatment of recurrent infections of the urinary tract. It consists of immunostimulating components derived from 18 Escherichia coli bacterial strains and constitutes a non-specific leukocyte activator. We here could demonstrate for the first time that, in a long-term immunization protocol, OM-89 acts as an effective immunogen in mice. After multiple parenteral applications of the extract, OM-89-specific antisera were obtained; the maximum serum antibody content was found after 6-9 immunizations. The antibodies induced were mainly of the IgG isotype, and a weak increase of IgM was observed. The sera also bound to the bacterial strains used for the preparation of the extract. Moreover, we could show that defined bacterial cell-wall components like protein I (porin), lipopeptide and murein were recognized. Our findings on the immunogenicity of OM-89 might partially explain the therapeutic effect of the extract as shown in clinical studies. PMID:7768669

  17. Cloning of 16S rRNA genes amplified from normal and disturbed vaginal microflora suggests a strong association between Atopobium vaginae, Gardnerella vaginalis and bacterial vaginosis

    PubMed Central

    Verhelst, Rita; Verstraelen, Hans; Claeys, Geert; Verschraegen, Gerda; Delanghe, Joris; Van Simaey, Leen; De Ganck, Catharine; Temmerman, Marleen; Vaneechoutte, Mario

    2004-01-01

    Background The pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the normal and disturbed vaginal microflora. Results A total of 150 vaginal swab samples from healthy women (115 pregnant and 35 non-pregnant) were categorized on the basis of Gram stain of direct smear as grade I (n = 112), grade II (n = 26), grade III (n = 9) or grade IV (n = 3). The composition of the vaginal microbial community of eight of these vaginal swabs (three grade I, two grade II and three grade III), all from non-pregnant women, were studied by culture and by cloning of the 16S rRNA genes obtained after direct amplification. Forty-six cultured isolates were identified by tDNA-PCR, 854 cloned 16S rRNA gene fragments were analysed of which 156 by sequencing, yielding a total of 38 species, including 9 presumptively novel species with at least five species that have not been isolated previously from vaginal samples. Interestingly, cloning revealed that Atopobium vaginae was abundant in four out of the five non-grade I specimens. Finally, species specific PCR for A. vaginae and Gardnerella vaginalis pointed to a statistically significant co-occurrence of both species in the bacterial vaginosis samples. Conclusions Although historically the literature regarding bacterial vaginosis has largely focused on G. vaginalis in particular, several findings of this study – like the abundance of A. vaginae in disturbed vaginal microflora and the presence of several novel species – indicate that much is to be learned about the composition of the vaginal microflora and its relation to the etiology of BV. PMID:15102329

  18. Quantifying Microbial Utilization of Petroleum Hydrocarbons in Salt Marsh Sediments by Using the 13C Content of Bacterial rRNA?

    PubMed Central

    Pearson, Ann; Kraunz, Kimberly S.; Sessions, Alex L.; Dekas, Anne E.; Leavitt, William D.; Edwards, Katrina J.

    2008-01-01

    Natural remediation of oil spills is catalyzed by complex microbial consortia. Here we took a whole-community approach to investigate bacterial incorporation of petroleum hydrocarbons from a simulated oil spill. We utilized the natural difference in carbon isotopic abundance between a salt marsh ecosystem supported by the 13C-enriched C4 grass Spartina alterniflora and 13C-depleted petroleum to monitor changes in the 13C content of biomass. Magnetic bead capture methods for selective recovery of bacterial RNA were used to monitor the 13C content of bacterial biomass during a 2-week experiment. The data show that by the end of the experiment, up to 26% of bacterial biomass was derived from consumption of the freshly spilled oil. The results contrast with the inertness of a nearby relict spill, which occurred in 1969 in West Falmouth, MA. Sequences of 16S rRNA genes from our experimental samples also were consistent with previous reports suggesting the importance of Gamma- and Deltaproteobacteria and Firmicutes in the remineralization of hydrocarbons. The magnetic bead capture approach makes it possible to quantify uptake of petroleum hydrocarbons by microbes in situ. Although employed here at the domain level, RNA capture procedures can be highly specific. The same strategy could be used with genus-level specificity, something which is not currently possible using the 13C content of biomarker lipids. PMID:18083852

  19. Bacterial taxa associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR amplification and TTGE fingerprinting.

    PubMed

    Valiente Moro, Claire; Thioulouse, Jean; Chauve, Claude; Normand, Philippe; Zenner, Lionel

    2009-01-01

    Dermanyssus gallinae (Arthropoda, Mesostigmata) is suspected to be involved in the transmission of a wide variety of pathogens, but nothing is known about its associated non-pathogenic bacterial community. To address this question, we examined the composition of bacterial communities in D. gallinae collected from standard poultry farms in Brittany, France. Genetic fingerprints of bacterial communities were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis. Most of the sequences belonged to the Proteobacteria and Firmicute phyla, with a majority of sequences corresponding to the Enterobacteriales order and the Staphylococcus genus. By using statistical analysis, we showed differences in biodiversity between poultry farms. We also determined the major phylotypes that compose the characteristic microbiota associated with D. gallinae. Saprophytes, opportunistic pathogens and pathogenic agents such as Pasteurella multocida, Erysipelothrix rhusiopathiae and sequences close to the genus Aerococcus were identified. Endosymbionts such as Schineria sp., Spiroplasma sp. Anistosticta, "Candidatus Cardinium hertigii" and Rickettsiella sp. were also present in the subdominant bacterial community. Identification of potential targets within the symbiont community may be considered in the future as a means of ectoparasite control. PMID:19027065

  20. Bacterial Population Changes in a Membrane Bioreactor for Graywater Treatment Monitored by Denaturing Gradient Gel Electrophoretic Analysis of 16S rRNA Gene Fragments

    PubMed Central

    Stamper, David M.; Walch, Marianne; Jacobs, Rachel N.

    2003-01-01

    The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD5), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time. PMID:12571004

  1. Bacterial Community Composition in the Gut Content and Ambient Sediment of Sea Cucumber Apostichopus japonicus Revealed by 16S rRNA Gene Pyrosequencing

    PubMed Central

    Gao, Fei; Li, Fenghui; Tan, Jie; Yan, Jingping; Sun, Huiling

    2014-01-01

    The composition of the bacterial communities in the contents of the foregut and hindgut of the sea cucumber Apostichopus japonicus and in the ambient surface sediment was surveyed by 16S rRNA gene 454-pyrosequencing. A total of 188,623 optimized reads and 15,527 operational taxonomic units (OTUs) were obtained from the ten gut contents samples and four surface sediment samples. The sequences in the sediments, foregut contents, and hindgut contents were assigned to 38.0±4.7, 31.2±6.2 and 27.8±6.5 phyla, respectively. The bacterial richness and Shannon diversity index were both higher in the ambient sediments than in the gut contents. Proteobacteria was the predominant phylum in both the gut contents and sediment samples. The predominant classes in the foregut, hindgut, and ambient sediment were Holophagae and Gammaproteobacteria, Deltaproteobacteria and Gammaproteobacteria, and Gammaproteobacteria and Deltaproteobacteria, respectively. The potential probiotics, including sequences related to Bacillus, lactic acid bacteria (Lactobacillus, Lactococcus, and Streptococcus) and Pseudomonas were detected in the gut of A. japonicus. Principle component analysis and heatmap figure showed that the foregut, hindgut, and ambient sediment respectively harbored different characteristic bacterial communities. Selective feeding of A. japonicus may be the primary source of the different bacterial communities between the foregut contents and ambient sediments. PMID:24967593

  2. Bacterial community composition in the gut content and ambient sediment of sea cucumber Apostichopus japonicus revealed by 16S rRNA gene pyrosequencing.

    PubMed

    Gao, Fei; Li, Fenghui; Tan, Jie; Yan, Jingping; Sun, Huiling

    2014-01-01

    The composition of the bacterial communities in the contents of the foregut and hindgut of the sea cucumber Apostichopus japonicus and in the ambient surface sediment was surveyed by 16S rRNA gene 454-pyrosequencing. A total of 188,623 optimized reads and 15,527 operational taxonomic units (OTUs) were obtained from the ten gut contents samples and four surface sediment samples. The sequences in the sediments, foregut contents, and hindgut contents were assigned to 38.0±4.7, 31.2±6.2 and 27.8±6.5 phyla, respectively. The bacterial richness and Shannon diversity index were both higher in the ambient sediments than in the gut contents. Proteobacteria was the predominant phylum in both the gut contents and sediment samples. The predominant classes in the foregut, hindgut, and ambient sediment were Holophagae and Gammaproteobacteria, Deltaproteobacteria and Gammaproteobacteria, and Gammaproteobacteria and Deltaproteobacteria, respectively. The potential probiotics, including sequences related to Bacillus, lactic acid bacteria (Lactobacillus, Lactococcus, and Streptococcus) and Pseudomonas were detected in the gut of A. japonicus. Principle component analysis and heatmap figure showed that the foregut, hindgut, and ambient sediment respectively harbored different characteristic bacterial communities. Selective feeding of A. japonicus may be the primary source of the different bacterial communities between the foregut contents and ambient sediments. PMID:24967593

  3. Development of Bacterial Contamination during Production of Yeast Extracts

    PubMed Central

    Barrette, Julie; Champagne, Claude P.; Goulet, Jacques

    1999-01-01

    Bakers yeast suspensions having bacterial populations of 106 and 108 CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH. PMID:10388734

  4. Horizon-Specific Bacterial Community Composition of German Grassland Soils, as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes ?

    PubMed Central

    Will, Christiane; Thrmer, Andrea; Wollherr, Antje; Nacke, Heiko; Herold, Nadine; Schrumpf, Marion; Gutknecht, Jessica; Wubet, Tesfaye; Buscot, Franois; Daniel, Rolf

    2010-01-01

    The diversity of bacteria in soil is enormous, and soil bacterial communities can vary greatly in structure. Here, we employed a pyrosequencing-based analysis of the V2-V3 16S rRNA gene region to characterize the overall and horizon-specific (A and B horizons) bacterial community compositions in nine grassland soils, which covered three different land use types. The entire data set comprised 752,838 sequences, 600,544 of which could be classified below the domain level. The average number of sequences per horizon was 41,824. The dominant taxonomic groups present in all samples and horizons were the Acidobacteria, Betaproteobacteria, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Deltaproteobacteria, Chloroflexi, Firmicutes, and Bacteroidetes. Despite these overarching dominant taxa, the abundance, diversity, and composition of bacterial communities were horizon specific. In almost all cases, the estimated bacterial diversity (H?) was higher in the A horizons than in the corresponding B horizons. In addition, the H? was positively correlated with the organic carbon content, the total nitrogen content, and the C-to-N ratio, which decreased with soil depth. It appeared that lower land use intensity results in higher bacterial diversity. The majority of sequences affiliated with the Actinobacteria, Bacteroidetes, Cyanobacteria, Fibrobacteres, Firmicutes, Spirochaetes, Verrucomicrobia, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria were derived from A horizons, whereas the majority of the sequences related to Acidobacteria, Chloroflexi, Gemmatimonadetes, Nitrospira, TM7, and WS3 originated from B horizons. The distribution of some bacterial phylogenetic groups and subgroups in the different horizons correlated with soil properties such as organic carbon content, total nitrogen content, or microbial biomass. PMID:20729324

  5. Bacterial diversity of soil in the vicinity of Pindari glacier, Himalayan mountain ranges, India, using culturable bacteria and soil 16S rRNA gene clones.

    PubMed

    Shivaji, S; Pratibha, M S; Sailaja, B; Hara Kishore, K; Singh, Ashish K; Begum, Z; Anarasi, Uttam; Prabagaran, S R; Reddy, G S N; Srinivas, T N R

    2011-01-01

    Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected near Pindari glacier, Himalayas. The three libraries yielded a total of 703 clones. Actinobacteria, Firmicutes and Proteobacteria were common to the three libraries. In addition to the above P1L and P8L shared the phyla Acidobacteria, Bacteroidetes, Gemmatimonadetes and Planctomycetes. Phyla Chlamydiae, Chlorobi, Chloroflexi, Dictyoglomi, Fibrobacteres, Nitrospirae, Verrucomicrobia, candidate division SPAM and candidate TM7s TM7a phylum were present only in P1L. Rarefaction analysis indicated that the bacterial diversity in P4S and P8S soil samples was representative of the sample. Principal component analysis (PCA) revealed that P1S and P8S were different from P4S soil sample. PCA also indicated that arsenic content, pH, Cr and altitude influence the observed differences in the percentage of specific OTUs in the three 16S rRNA gene clone libraries. The observed bacterial diversity was similar to that observed for other Himalayan and non-polar cold habitats. A total of 40 strains of bacteria were isolated from the above three soil samples and based on the morphology 20 bacterial strains were selected for further characterization. The 20 bacteria belonged to 12 different genera. All the isolates were psychro-, halo- and alkalitolerant. Amylase and urease activities were detected in majority of the strains but lipase and protease activities were not detected. Long chain, saturated, unsaturated and branched fatty acids were predominant in the psychrotolerant bacteria. PMID:21061031

  6. Matrix-Assisted Laser Desorption IonizationTime of Flight Mass Spectrometry as an Alternative to 16S rRNA Gene Sequencing for Identification of Difficult-To-Identify Bacterial Strains?

    PubMed Central

    Bizzini, A.; Jaton, K.; Romo, D.; Bille, J.; Prod'hom, G.; Greub, G.

    2011-01-01

    Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionizationtime of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing. PMID:21106794

  7. Sampling of intestinal microbiota and targeted amplification of bacterial 16S rRNA genes for microbial ecologic analysis

    PubMed Central

    Tong, Maomeng; Jacobs, Jonathan P.; McHardy, Ian H.; Braun, Jonathan

    2015-01-01

    Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Appropriate collection and pre-processing of biospecimens from humans or mice is necessary for accurate analysis of microbial composition and functional state. Methods to sample intestinal luminal and mucosal microbiota from humans and mice, and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using this protocol can be used for downstream quantitative analysis of microbial ecology. PMID:25367129

  8. Lactic acid bacterial extract as a biogenic mineral growth modifier

    NASA Astrophysics Data System (ADS)

    Borah, Ballav M.; Singh, Atul K.; Ramesh, Aiyagari; Das, Gopal

    2009-04-01

    The formation of minerals and mechanisms by which bacteria could control their formation in natural habitats is now of current interest for material scientists to have an insight of the mechanism of in vivo mineralization, as well as to seek industrial and technological applications. Crystalline uniform structures of calcium and barium minerals formed micron-sized building blocks when synthesized in the presence of an organic matrix consisting of secreted protein extracts from three different lactic acid bacteria (LAB) viz.: Lactobacillus plantarum MTCC 1325, Lactobacillus acidophilus NRRL B4495 and Pediococcus acidilactici CFR K7. LABs are not known to form organic matrix in biological materialization processes. The influence of these bacterial extracts on the crystallization behavior was investigated in details to test the basic coordination behavior of the acidic protein. In this report, varied architecture of the mineral crystals obtained in presence of high molecular weight protein extracts of three different LAB strains has been discussed. The role of native form of high molecular weight bacterial protein extracts in the generation of nucleation centers for crystal growth was clearly established. A model for the formation of organic matrix-cation complex and the subsequent events leading to crystal growth is proposed.

  9. Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes?

    PubMed Central

    Beier, Sara; Witzel, Karl-Paul; Marxsen, Jrgen

    2008-01-01

    The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions. PMID:18024682

  10. Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis.

    PubMed

    Chim Chan, On; Casper, Peter; Sha, Li Qing; Feng, Zhi Li; Fu, Yun; Yang, Xiao Dong; Ulrich, Andreas; Zou, Xiao Ming

    2008-06-01

    Bacterial community structure is influenced by vegetation, climate and soil chemical properties. To evaluate these influences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from high-plateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced influences than climate and soil chemical properties. PMID:18430004

  11. Conserved Bacterial RNase YbeY Plays Key Roles in 70S Ribosome Quality Control and 16S rRNA Maturation

    PubMed Central

    Jacob, Asha Ivy; Khrer, Caroline; Davies, Bryan William; RajBhandary, Uttam Lal; Walker, Graham Charles

    2012-01-01

    Quality control of ribosomes is critical for cellular function since protein mistranslation leads to severe physiological consequences. We report the first evidence of a ribosome quality control system in bacteria that operates at the level of 70S to remove defective ribosomes. YbeY, a previously unidentified endoribonuclease, and the exonuclease RNase R act together by a process mediated specifically by the 30S ribosomal subunit, to degrade defective 70S ribosomes but not properly matured 70S ribosomes or individual subunits. Furthermore, there is essentially no fully matured 16S rRNA in a ?ybeY mutant at 45C, making YbeY the first endoribonuclease to be implicated in the critically important processing of the 16S rRNA 3' terminus. These key roles in ribosome quality control and maturation indicate why YbeY is a member of the minimal bacterial gene set and suggest that it could be a potential target for antibacterial drugs. PMID:23273979

  12. Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench).

    PubMed

    Maropola, Mapula Kgomotso Annah; Ramond, Jean-Baptiste; Trindade, Marla

    2015-05-01

    Culture-independent studies rely on the quantity and quality of the extracted environmental metagenomic DNA (mDNA). To fully access the plant tissue microbiome, the extracted plant mDNA should allow optimal PCR applications and the genetic content must be representative of the total microbial diversity. In this study, we evaluated the endophytic bacterial diversity retrieved using different mDNA extraction procedures. Metagenomic DNA from sorghum (Sorghum bicolor L. Moench) stem and root tissues were extracted using two classical DNA extraction protocols (CTAB- and SDS-based) and five commercial kits. The mDNA yields and quality as well as the reproducibility were compared. 16S rRNA gene terminal restriction fragment length polymorphism (t-RFLP) was used to assess the impact on endophytic bacterial community structures observed. Generally, the classical protocols obtained high mDNA yields from sorghum tissues; however, they were less reproducible than the commercial kits. Commercial kits retrieved higher quality mDNA, but with lower endophytic bacterial diversities compared to classical protocols. The SDS-based protocol enabled access to the highest sorghum endophytic diversities. Therefore, "SDS-extracted" sorghum root and stem microbiome diversities were analysed via 454 pyrosequencing, and this revealed that the two tissues harbour significantly different endophytic communities. Nevertheless, both communities are dominated by agriculturally important genera such as Microbacterium, Agrobacterium, Sphingobacterium, Herbaspirillum, Erwinia, Pseudomonas and Stenotrophomonas; which have previously been shown to play a role in plant growth promotion. This study shows that DNA extraction protocols introduce biases in culture-independent studies of environmental microbial communities by influencing the mDNA quality, which impacts the microbial diversity analyses and evaluation. Using the broad-spectrum SDS-based DNA extraction protocol allows the recovery of the most diverse endophytic communities associated with sorghum tissues and, as such, establishes a reliable basis for future study of endophytic communities. PMID:25775938

  13. Prevalence of a novel division-level bacterial lineage in Lake Dhanmondi, Dhaka, Bangladesh, as revealed by deep sequencing of 16S rRNA gene amplicons.

    PubMed

    Azmuda, Nafisa; Rahman, Mohammed Ziaur; Madsen, Marit Steine; Khan, Sirajul Islam; Birkeland, Nils-Kre

    2012-10-01

    A culture-independent study of the bacterial diversity in Lake Dhanmondi, located in the central region of Dhaka city, Bangladesh, was carried out using deep sequence analysis of 16S rRNA gene PCR amplicons. The results revealed the presence of a group of bacteria, termed LD11, phylogenetically unrelated to any previously cultivated bacteria at the phylum level. LD11 sequences comprised about 1.7% of the total sequence reads after quality assessment. LD11 appears to constitute a novel division with a deep evolutionary lineage apparently branching between the Chloroflexi and Thermi-Deinococci phyla. Sequence similarity with molecular data from freshwater environments indicates that LD11 represents a widespread and novel clade of freshwater bacteria for which no cultivated representatives are yet available. PMID:22706799

  14. Bacterial Community Composition of South China Sea Sediments through Pyrosequencing-Based Analysis of 16S rRNA Genes

    PubMed Central

    Zhu, Daochen; Tanabe, Shoko-Hosoi; Yang, Chong; Zhang, Weimin; Sun, Jianzhong

    2013-01-01

    Background Subseafloor sediments accumulate large amounts of organic and inorganic materials that contain a highly diverse microbial ecosystem. The aim of this study was to survey the bacterial community of subseafloor sediments from the South China Sea. Methodology/Principal Findings Pyrosequencing of over 265,000 amplicons of the V3 hypervariable region of the 16S ribosomal RNA gene was performed on 16 sediment samples collected from multiple locations in the northern region of the South China Sea from depths ranging from 35 to 4000 m. A total of 9,726 operational taxonomic units (OTUs; between 695 and 2819 unique OTUs per sample) at 97% sequence similarity level were generated. In total, 40 bacterial phyla including 22 formally described phyla and 18 candidate phyla, with Proteobacteria, Firmicutes, Planctomycetes, Actinobacteria and Chloroflexi being most diverse, were identified. The most abundant phylotype, accounting for 42.6% of all sequences, belonged to Gammaproteobacteria, which possessed absolute predominance in the samples analyzed. Among the 18 candidate phyla, 12 were found for the first time in the South China Sea. Conclusions This study provided a novel insight into the composition of bacterial communities of the South China Sea subseafloor. Furthermore, abundances and community similarity analysis showed that the compositions of the bacterial communities are very similar at phylum level at different depths from 35-4000 m. PMID:24205246

  15. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples.

    PubMed

    Rothrock, Michael J; Hiett, Kelli L; Gamble, John; Caudill, Andrew C; Cicconi-Hogan, Kellie M; Caporaso, J Gregory

    2014-01-01

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples. PMID:25548939

  16. Rapid 16S rRNA Next-Generation Sequencing of Polymicrobial Clinical Samples for Diagnosis of Complex Bacterial Infections

    PubMed Central

    Salipante, Stephen J.; Sengupta, Dhruba J.; Rosenthal, Christopher; Costa, Gina; Spangler, Jessica; Sims, Elizabeth H.; Jacobs, Michael A.; Miller, Samuel I.; Hoogestraat, Daniel R.; Cookson, Brad T.; McCoy, Connor; Matsen, Frederick A.; Shendure, Jay; Lee, Clarence C.; Harkins, Timothy T.; Hoffman, Noah G.

    2013-01-01

    Classifying individual bacterial species comprising complex, polymicrobial patient specimens remains a challenge for culture-based and molecular microbiology techniques in common clinical use. We therefore adapted practices from metagenomics research to rapidly catalog the bacterial composition of clinical specimens directly from patients, without need for prior culture. We have combined a semiconductor deep sequencing protocol that produces reads spanning 16S ribosomal RNA gene variable regions 1 and 2 (∼360 bp) with a de-noising pipeline that significantly improves the fraction of error-free sequences. The resulting sequences can be used to perform accurate genus- or species-level taxonomic assignment. We explore the microbial composition of challenging, heterogeneous clinical specimens by deep sequencing, culture-based strain typing, and Sanger sequencing of bulk PCR product. We report that deep sequencing can catalog bacterial species in mixed specimens from which usable data cannot be obtained by conventional clinical methods. Deep sequencing a collection of sputum samples from cystic fibrosis (CF) patients reveals well-described CF pathogens in specimens where they were not detected by standard clinical culture methods, especially for low-prevalence or fastidious bacteria. We also found that sputa submitted for CF diagnostic workup can be divided into a limited number of groups based on the phylogenetic composition of the airway microbiota, suggesting that metagenomic profiling may prove useful as a clinical diagnostic strategy in the future. The described method is sufficiently rapid (theoretically compatible with same-day turnaround times) and inexpensive for routine clinical use. PMID:23734239

  17. Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and pyrosequencing for the characterization of the dentine caries-associated microbiome

    PubMed Central

    Schulze-Schweifing, Kathrin; Banerjee, Avijit; Wade, William G.

    2014-01-01

    Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas, and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture. PMID:25429361

  18. Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: A tool for rapid bacterial identification

    PubMed Central

    von Wintzingerode, Friedrich; Bcker, Sebastian; Schltelburg, Cord; Chiu, Norman H. L.; Storm, Niels; Jurinke, Christian; Cantor, Charles R.; Gbel, Ulf B.; van den Boom, Dirk

    2002-01-01

    A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria. PMID:11983869

  19. [Dominant phylotypes in the 16S rRNA gene clone libraries from bacterial mats of the Uzon caldera (Kamchatka, Russia) hydrothermal springs].

    PubMed

    Akimov, V N; Podosokorskaia, O A; Shliapnikov, M G; Gal'chenko, V F

    2013-01-01

    In situ analysis of the 16S rRNA genes form bacterial mats of five hydrothermal springs (36-58 degrees C) in the Uzon caldera (Kamchatka, Russia) was carried out using clone libraries. Eight clone libraries contained 18 dominant phylotypes (over 4-5%). In most clone libraries, the phylotype of the green sulfur bacterium Chlorobaculum sp. was among the dominant ones. The phylotypes of the green nonsulfur bacteria Chloroflexus and Roseiflexus and of purple nonsulfur bacteria Rhodoblastus, Rhodopseudomonas, and Rhodoferax were also among the dominant ones. Cyanobacteria were represented by one dominant phylotype in a single spring. Among nonphototrophic bacteria, the dominant phylotypes belonged to Sulfyrihydrogenibium sp., Geothrixsp., Acidobacterium sp., Meiothermus sp., Thiomonas sp., Thiofaba sp., and Spirochaeta sp. Three phylotypes were not identified at the genus level. Most genera of phototrophic and nonphototrophic organisms corresponding to the phylotypes from Uzon hydrotherms have been previously revealed in the hydrotherms of volcanically active regions of America, Asia, and Europe. These results indicate predominance of bacterial mats carrying out anaerobic photosynthesis in the hydrotherms of the Uzon caldera. PMID:25509409

  20. Abundance and Activity of 16S rRNA, AmoA and NifH Bacterial Genes During Assisted Phytostabilization of Mine Tailings.

    PubMed

    Nelson, Karis N; Neilson, Julia W; Root, Robert A; Chorover, Jon; Maier, Raina M

    2015-01-01

    Mine tailings in semiarid regions are highly susceptible to erosion and are sources of dust pollution and potential avenues of human exposure to toxic metals. One constraint to revegetation of tailings by phytostabilization is the absence of microbial communities critical for biogeochemical cycling of plant nutrients. The objective of this study was to evaluate specific genes as in situ indicators of biological soil response during phytoremediation. The abundance and activity of 16S rRNA, nifH, and amoA were monitored during a nine month phytostabilization study using buffalo grass and quailbush grown in compost-amended, metalliferous tailings. The compost amendment provided a greater than 5-log increase in bacterial abundance, and survival of this compost-inoculum was more stable in planted treatments. Despite increased abundance, the activity of the introduced community was low, and significant increases were not detected until six and nine months in quailbush, and unplanted compost and buffalo grass treatments, respectively. In addition, increased abundances of nitrogen-fixation (nifH) and ammonia-oxidizing (amoA) genes were observed in rhizospheres of buffalo grass and quailbush, respectively. Thus, plant establishment facilitated the short term stabilization of introduced bacterial biomass and supported the growth of two key nitrogen-cycling populations in compost-amended tailings. PMID:25495940

  1. Bacterial cellulose production from the litchi extract by Gluconacetobacter xylinus.

    PubMed

    Yang, Xiao-Yan; Huang, Chao; Guo, Hai-Jun; Xiong, Lian; Luo, Jun; Wang, Bo; Lin, Xiao-Qing; Chen, Xue-Fang; Chen, Xin-De

    2016-01-01

    Although litchi has both nutrient and edible value, the extremely short preservation time limited its further market promotion. To explore processed litchi products with longer preservation time, litchi extract was selected as an alternative feedstock for production of bacterial cellulose (BC). After 2 weeks of static fermentation, 2.53g/L of the BC membrane was obtained. The trace elements including magnesium (Mg) and sodium (Na) in the litchi extract were partly absorbed in the BC membrane, but no potassium (K) element was detected in it, curiously. Scanning electron microscope (SEM) photographs exhibited an ultrafine network nanostructure for the BC produced in the litchi extract. Analysis of the fourier-transform infrared spectroscopy (FTIR) confirmed the pellicles to be a cellulosic material. Interestingly, X-ray diffraction (XRD) results showed the BC membrane obtained from litchi extract had higher crystallinity of 94.0% than that from HS medium. Overall, the work showed the potential of producing high value-added polymer from litchi resources. PMID:25181328

  2. Binding thermodynamics of paromomycin, neomycin, neomycin-dinucleotide and -diPNA conjugates to bacterial and human rRNA.

    PubMed

    Alguacil, Javier; Robles, Jordi; Ràfols, Clara; Bosch, Elisabeth

    2016-04-01

    Isothermal titration calorimetry (ITC) is a powerful technique able to evaluate the energetics of target-drug binding within the context of drug discovery. In this work, the interactions of RNAs reproducing bacterial and human ribosomal A-site, with two well-known antibiotic aminoglycosides, Paromomycin and Neomycin, as well as several Neomycin-dinucleotide and -diPNA conjugates, have been evaluated by ITC and the corresponding thermodynamic quantities determined. The comparison of the thermodynamic data of aminoglycosides and their chemical analogues allowed to select Neomycin-diPNA conjugates as the best candidates for antimicrobial activity. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26530431

  3. Evaluation of bacterial communities by bacteriome analysis targeting 16S rRNA genes and quantitative analysis of ammonia monooxygenase gene in different types of compost.

    PubMed

    Kitamura, Rika; Ishii, Kazuo; Maeda, Isamu; Kozaki, Toshinori; Iwabuchi, Kazunori; Saito, Takahiro

    2016-01-01

    Biofiltration technology based on microbial degradation and assimilation is used for the removal of malodorous compounds, such as ammonia. Microbes that degrade malodorous and/or organic substances are involved in composting and are retained after composting; therefore, mature composts can serve as an ideal candidate for a biofilter medium. In this study, we focused on different types of raw compost materials, as these are important factors determining the bacterial community profile and the chemical component of the compost. Therefore, bacterial community profiles, the abundance of the bacterial ammonia monooxygenase gene (amoA), and the quantities of chemical components were analyzed in composts produced from either food waste or cattle manure. The community profiles with the lowest beta diversity were obtained from single type of cattle manure compost. However, cattle manure composts showed greater alpha diversity, contained higher amounts of various rRNA gene fragments than those of food waste composts and contained the amoA gene by relative quantification, and Proteobacteria were abundantly found and nitrifying bacteria were detected in it. Nitrifying bacteria are responsible for ammonia oxidation and mainly belong to the Proteobacteria or Nitrospira phyla. The quantities of chemical components, such as salt, phosphorus, and nitrogen, differed between the cattle manure and food waste composts, indicating that the raw materials provided different fermentation environments that were crucial for the formation of different community profiles. The results also suggest that cattle manure might be a more suitable raw material for the production of composts to be used in the biofiltration of ammonia. PMID:26111599

  4. Bacterial communities in thermophilic H2-producing reactors investigated using 16S rRNA 454 pyrosequencing.

    PubMed

    Ratti, Regiane Priscila; Delforno, Tiago Palladino; Okada, Dagoberto Yukio; Varesche, Maria Bernadete Amncio

    2015-04-01

    In this study, the composition and diversity of the bacterial community in thermophilic H2-producing reactors fed with glucose were investigated using pyrosequencing. The H2-producing experiments in batch were conducted using 0.5 and 2.0 g l(-1) glucose at 550 C. Under the two conditions, the H2 production and yield were 1.3 and 1.6 mol H2 mol glucose(-1), respectively. Acetic, butyric, iso-butyric, lactic and propionic acids were detected in the two reactors. The increase in substrate concentration favored a high H2 yield. In this reactor, a predominance of acetic and iso-butyric acids, 27.7% and 40%, were measured, respectively. By means of pyrosequencing, a total of 323 and 247 operational taxonomic units were obtained, with a predominance of the phylum Firmicutes (68.73-67.61%) for reactors with 0.5 and 2.0 g l(-1) glucose, respectively. Approximately 40.55% and 62.34% of sequences were affiliated with Thermoanaerobacterium and Thermohydrogenium, microorganisms that produce H2 under thermophilic conditions. PMID:25801966

  5. Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities

    PubMed Central

    Hanshew, Alissa S.; Mason, Charles J.; Raffa, Kenneth F.; Currie, Cameron R.

    2014-01-01

    Chloroplast sequence contamination in 16S ribosomal RNA gene (16S) analyses can be particularly problematic when sampling microbial communities in plants and folivorous arthropods. We previously encountered high levels of plastid contamination in herbivorous insect samples when we used the predominant 454 pyrosequencing 16S methodologies described in the literature. 799F, a primer previously found to exclude chloroplast sequences, was modified to enhance its efficacy, and we describe, in detail, our methodology throughout amplicon pyrosequencing. Thirteen versions of 799F were assessed for the exclusion of chloroplast sequences from our samples. We found that a shift in the mismatch between 799F and chloroplast 16S resulted in significant reduction of chloroplast reads. Our results also indicate that amplifying sequences from environmental samples in a two-step PCR process, with the addition of the multiplex identifiers and 454 adapters in a second round of PCR, further improved primer specificity. Primers that included 3′ phosphorothioate bonds, which were designed to block primer degradation, did not amplify consistently across samples. The different forward primers do not appear to bias the bacterial communities detected. We provide a methodological framework for reducing chloroplast reads in high-throughput sequencing data sets that can be applied to a number of environmental samples and sequencing techniques. PMID:23968645

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

    PubMed

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

    2015-04-01

    The bacterial diversity involved in food fermentations is one of the most important factors shaping the final characteristics of traditional foods. Knowledge about this diversity can be greatly improved by the application of high-throughput sequencing technologies (HTS) coupled to the PCR amplification of the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were carried out in order to be compared with HTS results. A total of 722,196 high quality sequences were obtained after trimming, paired-reads assembly and quality screening of raw reads obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable regions V3 and V4; manual curation of 16S database allowed a correct taxonomical classification at the species for 99.5% of these reads. Results confirmed the presence of main bacterial species involved in the fermentation of salami as assessed by PCR-DGGE, but with a greater extent of resolution and quantitative assessments that are not possible by the mere analyses of gel banding patterns. Thirty-two different Staphylococcus and 33 Lactobacillus species where identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the observed diversity. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating that HTS has the potential for fine scale differentiation of local fermented foods. PMID:25475305

  7. Deep 16S rRNA pyrosequencing reveals a bacterial community associated with Banana Fusarium Wilt disease suppression induced by bio-organic fertilizer application.

    PubMed

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

    2014-01-01

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

  8. Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.

    PubMed

    Lindsay, Brianna; Pop, Mihai; Antonio, Martin; Walker, Alan W; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Li, Shan; Magder, Laurence S; Paulson, Joseph N; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E; Hannis, James; Manalili, Sheri; Deleon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J; Hossain, M Anowar; Breiman, Robert F; Morris, J Glenn; McDaniel, Timothy K; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O Colin; Nataro, James P

    2013-10-01

    Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

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

    PubMed Central

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

    2014-01-01

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

  10. Identification of Bacterial Populations in Dairy Wastewaters by Use of 16S rRNA Gene Sequences and Other Genetic Markers

    PubMed Central

    McGarvey, Jeffery A.; Miller, William G.; Sanchez, Susan; Stanker, Larry

    2004-01-01

    Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms. Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system. Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California. Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water. The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria). The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences. Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences. Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment. PMID:15240310

  11. Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection

    PubMed Central

    Pop, Mihai; Antonio, Martin; Walker, Alan W.; Mai, Volker; Ahmed, Dilruba; Oundo, Joseph; Tamboura, Boubou; Panchalingam, Sandra; Levine, Myron M.; Kotloff, Karen; Li, Shan; Magder, Laurence S.; Paulson, Joseph N.; Liu, Bo; Ikumapayi, Usman; Ebruke, Chinelo; Dione, Michel; Adeyemi, Mitchell; Rance, Richard; Stares, Mark D.; Ukhanova, Maria; Barnes, Bret; Lewis, Ian; Ahmed, Firoz; Alam, Meer Taifur; Amin, Ruhul; Siddiqui, Sabbir; Ochieng, John B.; Ouma, Emmanuel; Juma, Jane; Mailu, Eunice; Omore, Richard; O'Reilly, Ciara E.; Hannis, James; Manalili, Sheri; DeLeon, Jonna; Yasuda, Irene; Blyn, Lawrence; Ranken, Raymond; Li, Feng; Housley, Roberta; Ecker, David J.; Hossain, M. Anowar; Breiman, Robert F.; Morris, J. Glenn; McDaniel, Timothy K.; Parkhill, Julian; Saha, Debasish; Sampath, Rangarajan; Stine, O. Colin; Nataro, James P.

    2013-01-01

    Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens. PMID:23884998

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

    PubMed Central

    Bolhuis, Henk; Stal, Lucas J

    2011-01-01

    Coastal microbial mats are small-scale and largely closed ecosystems in which a plethora of different functional groups of microorganisms are responsible for the biogeochemical cycling of the elements. Coastal microbial mats play an important role in coastal protection and morphodynamics through stabilization of the sediments and by initiating the development of salt-marshes. Little is known about the bacterial and especially archaeal diversity and how it contributes to the ecological functioning of coastal microbial mats. Here, we analyzed three different types of coastal microbial mats that are located along a tidal gradient and can be characterized as marine (ST2), brackish (ST3) and freshwater (ST3) systems. The mats were sampled during three different seasons and subjected to massive parallel tag sequencing of the V6 region of the 16S rRNA genes of Bacteria and Archaea. Sequence analysis revealed that the mats are among the most diverse marine ecosystems studied so far and consist of several novel taxonomic levels ranging from classes to species. The diversity between the different mat types was far more pronounced than the changes between the different seasons at one location. The archaeal community for these mats have not been studied before and revealed a strong reaction on a short period of draught during summer resulting in a massive increase in halobacterial sequences, whereas the bacterial community was barely affected. We concluded that the community composition and the microbial diversity were intrinsic of the mat type and depend on the location along the tidal gradient indicating a relation with salinity. PMID:21544102

  13. Multiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay.

    PubMed

    Pingle, Maneesh R; Granger, Kathleen; Feinberg, Philip; Shatsky, Rebecca; Sterling, Bram; Rundell, Mark; Spitzer, Eric; Larone, Davise; Golightly, Linnie; Barany, Francis

    2007-06-01

    We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity. PMID:17428930

  14. Phylogenetic Analysis of Bacterial Communities Associated with Leaves of the Seagrass Halophila stipulacea by a Culture-Independent Small-Subunit rRNA Gene Approach.

    PubMed

    Weidner; Arnold; Stackebrandt; Phler

    2000-01-01

    The phylogenetic diversity of the bacterial community associated with leaves of the marine plant Halophila stipulacea in the northern Gulf of Elat was examined by 16S rRNA gene (rDNA) sequence analyses of a clone library. For 59 clones corresponding to 51 ARDRA (amplified rDNA restriction analysis) groups, the sequence of approximately 1 kb was determined, and the fraction of the corresponding ARDRA groups of the leaf library was calculated. The class Proteobacteria was represented by 62.6% of the clone sequences. Most sequences originated from members of the gamma-subclass (27.3%), affiliated with members of the genera Pseudomonas, Vibrio, Marinomonas, Oceanospirillum, and other marine groups. Affiliation to the alpha-subclass was determined for 24.2% of the sequences. They were related to the genera Hyphomonas, Roseobacter, Ruegeria, and Rhizobiaceae. Several alpha-proteobacterial sequences were distantly related to known sequences. Only 4% of the clone sequences were related to beta-Proteobacteria. Additionally, 7.1% of the sequences possibly belonged to the class Proteobacteria, but branched deeply from known subclasses. Several sequences were affiliated to members of the orders Verrucomicrobiales and Planctomycetales, the Holophaga/Acidobacterium phylum, and chloroplasts of marine diatoms.

  15. Multiplexed Identification of Blood-Borne Bacterial Pathogens by Use of a Novel 16S rRNA Gene PCR-Ligase Detection Reaction-Capillary Electrophoresis Assay?

    PubMed Central

    Pingle, Maneesh R.; Granger, Kathleen; Feinberg, Philip; Shatsky, Rebecca; Sterling, Bram; Rundell, Mark; Spitzer, Eric; Larone, Davise; Golightly, Linnie; Barany, Francis

    2007-01-01

    We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 blood-borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by CE. Each organism generates a specific pattern of ligation products, which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested, with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR-LDR-CE assay can successfully identify, in a multiplexed fashion, a panel of 20 blood-borne pathogens with high sensitivity and specificity. PMID:17428930

  16. Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

    PubMed Central

    Terrat, Sebastien; Plassart, Pierre; Bourgeois, Emilie; Ferreira, Stphanie; Dequiedt, Samuel; Adele-Dit-De-Renseville, Nathalie; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Ranjard, Lionel

    2015-01-01

    This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard. PMID:25195809

  17. Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition.

    PubMed

    Terrat, Sebastien; Plassart, Pierre; Bourgeois, Emilie; Ferreira, Stphanie; Dequiedt, Samuel; Adele-Dit-De-Renseville, Nathalie; Lemanceau, Philippe; Bispo, Antonio; Chabbi, Abad; Maron, Pierre-Alain; Ranjard, Lionel

    2015-01-01

    This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO-11063, GnS-GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico-chemical characteristics and land use. A meta-barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS-GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO-11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO-11063 standard. PMID:25195809

  18. Synthesis of bacterial cellulose using hot water extracted wood sugars.

    PubMed

    Erbas Kiziltas, Esra; Kiziltas, Alper; Gardner, Douglas J

    2015-06-25

    Bacterial cellulose (BC), a type of nanopolymer produced by Acetobacter xylinum is a nanostructured material with unique properties and wide applicability. However, a standard medium used for the cultivation of BC, the Hestrin-Schramm medium, is expensive and prevents wide scale extension of BC applications. In this research, a relatively low-cost culture media was successfully developed from wood hot water extracts for the Acetobacter xylinus 23769 strain. Hot water extract (HWE) is a residual material originating from pulp mills and lignocellulosic biorefineries and consists of mainly monomeric sugars, organic acids and organics. The effects of different pH (5, 6, 7 and 8) and temperatures (26, 28 and 30°C) were also examined in this research. There were no significant differences in the crystallinity and the recorded Iα fraction of cellulose produced between Hestrin-Schramm and the HWE medium. The maximum production of 0.15g/l of BC was obtained at a pH of 8 and temperature of 28°C. Glucose and xylose in the HWE were the main nutrient sources utilized in all BC cultivations based on high-pressure liquid chromatography (HPLC) results. HWE was shown to be a suitable carbon source for BC production, and a process was established for BC production from lignocellulosic feedstocks without using any modification of the HWE. HWE is an abundant and relatively inexpensive forest by-product. Using HWE for BC production could reduce burdens on the environment and also, achieve the goal of large scale BC production at low cost without using added culture nutrients. PMID:25839803

  19. Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen suppressive soil

    SciTech Connect

    Hjort, K.; Bergstrom, M.; Adesina, M.F.; Jansson, J.K.; Smalla, K.; Sjoling, S.

    2009-09-01

    Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal-restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal-restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF{sup 103} of the isolate, Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

  20. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRNA Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  1. Changes in the Composition of Drinking Water Bacterial Clone Libraries Introduced by Using Two Different 16S rRna Gene PCR Primers

    EPA Science Inventory

    Sequence analysis of 16S rRNA gene clone libraries is a popular tool used to describe the composition of natural microbial communities. Commonly, clone libraries are developed by direct cloning of 16S rRNA gene PCR products. Different primers are often employed in the initial amp...

  2. Profiling the Succession of Bacterial Communities throughout the Life Stages of a Higher Termite Nasutitermes arborum (Termitidae, Nasutitermitinae) Using 16S rRNA Gene Pyrosequencing

    PubMed Central

    Diouf, Michel; Roy, Virginie; Mora, Philippe; Frechault, Sophie; Lefebvre, Thomas; Hervé, Vincent; Rouland-Lefèvre, Corinne; Miambi, Edouard

    2015-01-01

    Previous surveys of the gut microbiota of termites have been limited to the worker caste. Termite gut microbiota has been well documented over the last decades and consists mainly of lineages specific to the gut microbiome which are maintained across generations. Despite this intimate relationship, little is known of how symbionts are transmitted to each generation of the host, especially in higher termites where proctodeal feeding has never been reported. The bacterial succession across life stages of the wood-feeding higher termite Nasutitermes arborum was characterized by 16S rRNA gene deep sequencing. The microbial community in the eggs, mainly affiliated to Proteobacteria and Actinobacteria, was markedly different from the communities in the following developmental stages. In the first instar and last instar larvae and worker caste termites, Proteobacteria and Actinobacteria were less abundant than Firmicutes, Bacteroidetes, Spirochaetes, Fibrobacteres and the candidate phylum TG3 from the last instar larvae. Most of the representatives of these phyla (except Firmicutes) were identified as termite-gut specific lineages, although their relative abundances differed. The most salient difference between last instar larvae and worker caste termites was the very high proportion of Spirochaetes, most of which were affiliated to the Treponema Ic, Ia and If subclusters, in workers. The results suggest that termite symbionts are not transmitted from mother to offspring but become established by a gradual process allowing the offspring to have access to the bulk of the microbiota prior to the emergence of workers, and, therefore, presumably through social exchanges with nursing workers. PMID:26444989

  3. Structural motifs of the bacterial ribosomal proteins S20, S18 and S16 that contact rRNA present in the eukaryotic ribosomal proteins S25, S26 and S27A, respectively

    PubMed Central

    Malygin, Alexey A.; Karpova, Galina G.

    2010-01-01

    The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively. PMID:20034956

  4. Simple and inexpensive DNA extraction protocol for studying the bacterial composition of sludges used in microbial fuel cells.

    PubMed

    Canto-Canch, B; Tzec-Sim, M; Vzquez-Lora, J I; Espadas-lvarez, H; Ch-Manzanero, B H; Rojas-Herrera, R; Valdez-Ojeda, R; Alzate-Gaviria, L

    2013-01-01

    Bacteria oxidize organic matter and nutrients to produce electric energy in microbial fuel cells (MFC) - a technology of increasing importance because of its sustainability. To improve the performance of MFCs, it is necessary not only to gain a better understanding of MFC engineering designs, but also to improve the understanding of the composition of the microbial communities in MFCs. Fast and efficient DNA extraction protocols that are suitable for extracting diverse bacterial genomes are necessary to identify the bacterial diversity present in MFCs and to further monitor the dynamic changes of microbial communities. This study focused on testing different direct cell lysis protocols to extract DNA from a microbial sludge harvested from an MFC. The protocol that achieved the best results was based on a previous study, but was modified by eliminating a chaotropic salt and the special columns used for nucleic acid purification. The efficiency of this less expensive and more straightforward protocol was confirmed by PCR amplification of the 16S rRNA gene and denaturing gradient gel electrophoresis analysis, which confirmed the extraction of multiple genomes. The sequences of 10 clones revealed the presence of phyla, Proteobacteria, Firmicutes and Actinobacteria, comprising both Gram-negative and Gram-positive bacteria. Some of these bacteria were identified at the genus level, e.g., Clostridium, Pseudoxanthomonas, Tistrella, and Enterobacter; these genera have been described in active sludges from wastewater treatment, supporting the congruency of our results. Therefore, this protocol is a useful tool for analysis of the bacteria responsible for energy production in MFCs. PMID:23408415

  5. Investigation of antibacterial mechanism and identification of bacterial protein targets mediated by antibacterial medicinal plant extracts.

    PubMed

    Yong, Ann-Li; Ooh, Keng-Fei; Ong, Hean-Chooi; Chai, Tsun-Thai; Wong, Fai-Chu

    2015-11-01

    In this paper, we investigated the antibacterial mechanism and potential therapeutic targets of three antibacterial medicinal plants. Upon treatment with the plant extracts, bacterial proteins were extracted and resolved using denaturing gel electrophoresis. Differentially-expressed bacterial proteins were excised from the gels and subjected to sequence analysis by MALDI TOF-TOF mass spectrometry. From our study, seven differentially expressed bacterial proteins (triacylglycerol lipase, N-acetylmuramoyl-L-alanine amidase, flagellin, outer membrane protein A, stringent starvation protein A, 30S ribosomal protein s1 and 60 kDa chaperonin) were identified. Additionally, scanning electron microscope study indicated morphological damages induced on bacterial cell surfaces. To the best of our knowledge, this represents the first time these bacterial proteins are being reported, following treatments with the antibacterial plant extracts. Further studies in this direction could lead to the detailed understanding of their inhibition mechanism and discovery of target-specific antibacterial agents. PMID:25976788

  6. Immunostimulation by bacterial components: II. Efficacy studies and meta-analysis of the bacterial extract OM-89.

    PubMed

    Huber, M; Krauter, K; Winkelmann, G; Bauer, H W; Rahlfs, V W; Lauener, P A; Blessmann, G S; Bessler, W G

    2000-12-01

    The bacterial extract OM-89 (Uro-Vaxom) consisting of immunostimulating components derived from 18 Escherichia coli strains is used for the treatment of recurrent urinary tract infections. We investigated in the mouse the immunogenicity of the bacterial extract after oral administration. After repeated administration of OM-89, a specific serum IgG and IgA response against a number of bacterial strains was obtained. Supernatants of cell cultures prepared from the urogenital tract of immunized mice also contained increased levels of strain specific IgG and IgA. We could show a bias towards a Th1 type immune response as indicated by increased IgG2a levels in sera, and increased IFNgamma levels in supernatants of spleen cells. These findings may contribute to an understanding of the therapeutic effect of Uro-Vaxom: the metaanalysis of several clinical studies confirmed that Uro-Vaxom constitutes an effective prophylaxis for urinary tract infections. PMID:11137617

  7. Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications.

    PubMed

    Kajiura, Lauren N; Stewart, Scott D; Dresios, John; Uyehara, Catherine F T

    2015-12-01

    Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics. PMID:26543438

  8. Simultaneous Extraction of Viral and Bacterial Nucleic Acids for Molecular Diagnostic Applications

    PubMed Central

    Kajiura, Lauren N.; Stewart, Scott D.; Dresios, John; Uyehara, Catherine F. T.

    2015-01-01

    Molecular detection of microbial pathogens in clinical samples requires the application of efficient sample lysis protocols and subsequent extraction and isolation of their nucleic acids. Here, we describe a simple and time-efficient method for simultaneous extraction of genomic DNA from gram-positive and -negative bacteria, as well as RNA from viral agents present in a sample. This method compared well with existing bacterial- and viral-specialized extraction protocols, worked reliably on clinical samples, and was not pathogen specific. This method may be used to extract DNA and RNA concurrently from viral and bacterial pathogens present in a sample and effectively detect coinfections in routine clinical diagnostics. PMID:26543438

  9. Bacterial consortium for copper extraction from sulphide ore consisting mainly of chalcopyrite

    PubMed Central

    Romo, E.; Weinacker, D.F.; Zepeda, A.B.; Figueroa, C.A.; Chavez-Crooker, P.; Farias, J.G.

    2013-01-01

    The mining industry is looking forward for bacterial consortia for economic extraction of copper from low-grade ores. The main objective was to determine an optimal bacterial consortium from several bacterial strains to obtain copper from the leach of chalcopyrite. The major native bacterial species involved in the bioleaching of sulphide ore (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans and Leptospirillum ferriphilum) were isolated and the assays were performed with individual bacteria and in combination with At. thiooxidans. In conclusion, it was found that the consortium integrated by At. ferrooxidans and At. thiooxidans removed 70% of copper in 35 days from the selected ore, showing significant differences with the other consortia, which removed only 35% of copper in 35 days. To validate the assays was done an escalation in columns, where the bacterial consortium achieved a higher percentage of copper extraction regarding to control. PMID:24294251

  10. Biological activity of bacterial surface components. Immunogenicity and immunomodulatory properties of a bacterial extract from Escherichia coli.

    PubMed

    Bessler, W G; Beck, P; Konetznick, U; Loleit, M; Sedelmeier, E; Hoffmann, P; Strecker, M; Stcklin, S

    1991-03-01

    The immunogenic and immunomodulatory properties of a lysed fraction from selected E. coli strains (OM-89, Uro-Vaxom) were determined in vivo and in vitro. It could be demonstrated that OM-89 constitutes an active immunogen in mice. Maximum OM-89-specific antibody titers were obtained after 4-5 i.p. immunizations; the titers could be further enhanced by the simultaneous injection of lipopeptide adjuvants. It was shown by ELISA that the antibodies obtained bound to the bacterial strains used for the preparation of the OM-89 extract. Immunogenicity was observed both after intraperitoneal and oral application of the extract. Besides being active as an immunogen. OM-89 was able to act in vitro as a polyclonal lymphocyte activator, as determined in splenocyte cultures of different inbred murine strains, and in cultures of human peripheral blood lymphocytes. Our results show that the B lymphocyte stimulating activity of the bacterial extract OM-89 was comparable to that of lipopeptide adjuvants. In conclusion, the bacterial extract both an active immunogen in vivo, and a polyclonal B cell activator in vitro. These findings may be of importance for the understanding of the therapeutic effect of OM-89. PMID:1867666

  11. Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library.

    PubMed

    Garcia-Armisen, Tamara; Papalexandratou, Zoi; Hendryckx, Hugo; Camu, Nicholas; Vrancken, Gino; De Vuyst, Luc; Cornelis, Pierre

    2010-08-01

    Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation. PMID:20559826

  12. Soil bacterial community structure in five tropical forests in Malaysia and one temperate forest in Japan revealed by pyrosequencing analyses of 16S rRNA gene sequence variation.

    PubMed

    Miyashita, Naohiko T; Iwanaga, Hiroko; Charles, Suliana; Diway, Bibian; Sabang, John; Chong, Lucy

    2013-01-01

    Bacterial community structure was investigated in five tropical rainforests in Sarawak, Malaysia and one temperate forest in Kyoto, Japan. A hierarchical sampling approach was employed, in which soil samples were collected from five sampling-sites within each forest. Pyrosequencing was performed to analyze a total of 493,790 16S rRNA amplicons. Despite differences in aboveground conditions, the composition of bacterial groups was similar across all sampling-sites and forests, with Acidobacteria, Proteobacteria, Verrucomicrobia, Planctomycetes and Bacteroidetes accounting for 90% of all Phyla detected. At higher taxonomic levels, the same taxa were predominant, although there was significant heterogeneity in relative abundance of specific taxa across sampling-sites within one forest or across different forests. In all forests, the level of bacterial diversity, estimated using the Chao1 index, was on the order of 1,000, suggesting that tropical rainforests did not necessarily have a large soil bacterial diversity. The average number of reads per species (OTUs) per sampling-site was 8.0, and more than 40-50% of species were singletons, indicating that most bacterial species occurred infrequently and that few bacterial species achieved high predominance. Approximately 30% of species were specific to one sampling-site within a forest, and 40-60% of species were uniquely detected in one of the six forests studied here. Only 0.2% of species were detected in all forests, while on average 32.1% of species were detected in all sampling-sites within a forest. The results suggested that bacterial communities adapted to specific micro- and macro-environments, but macro-environmental diversity made a larger contribution to total bacterial diversity in forest soil. PMID:23832301

  13. Wild Mushroom Extracts as Inhibitors of Bacterial Biofilm Formation

    PubMed Central

    Alves, Maria José; Ferreira, Isabel C. F. R.; Lourenço, Inês; Costa, Eduardo; Martins, Anabela; Pintado, Manuela

    2014-01-01

    Microorganisms can colonize a wide variety of medical devices, putting patients in risk for local and systemic infectious complications, including local-site infections, catheter-related bloodstream infections, and endocarditis. These microorganisms are able to grow adhered to almost every surface, forming architecturally complex communities termed biofilms. The use of natural products has been extremely successful in the discovery of new medicine, and mushrooms could be a source of natural antimicrobials. The present study reports the capacity of wild mushroom extracts to inhibit in vitro biofilm formation by multi-resistant bacteria. Four Gram-negative bacteria biofilm producers (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii) isolated from urine were used to verify the activity of Russula delica, Fistulina hepatica, Mycena rosea, Leucopaxilus giganteus, and Lepista nuda extracts. The results obtained showed that all tested mushroom extracts presented some extent of inhibition of biofilm production. Pseudomonas aeruginosa was the microorganism with the highest capacity of biofilm production, being also the most susceptible to the extracts inhibition capacity (equal or higher than 50%). Among the five tested extracts against E. coli, Leucopaxillus giganteus (47.8%) and Mycenas rosea (44.8%) presented the highest inhibition of biofilm formation. The extracts exhibiting the highest inhibitory effect upon P. mirabilis biofilm formation were Sarcodon imbricatus (45.4%) and Russula delica (53.1%). Acinetobacter baumannii was the microorganism with the lowest susceptibility to mushroom extracts inhibitory effect on biofilm production (highest inhibition—almost 29%, by Russula delica extract). This is a pioneer study since, as far as we know, there are no reports on the inhibition of biofilm production by the studied mushroom extracts and in particular against multi-resistant clinical isolates; nevertheless, other studies are required to elucidate the mechanism of action. PMID:25438017

  14. Comparison of Methods for Extraction of Bacterial Adenine Nucleotides Determined by Firefly Assay

    PubMed Central

    Lundin, Arne; Thore, Anders

    1975-01-01

    Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view. PMID:812422

  15. Phylogenetic relationships of chemoautotrophic bacterial symbionts of Solemya velum say (Mollusca: Bivalvia) determined by 16S rRNA gene sequence analysis.

    PubMed Central

    Eisen, J A; Smith, S W; Cavanaugh, C M

    1992-01-01

    The protobranch bivalve Solemya velum Say (Mollusca: Bivalvia) houses chemoautotrophic symbionts intracellularly within its gills. These symbionts were characterized through sequencing of polymerase chain reaction-amplified 16S rRNA coding regions and hybridization of an Escherichia coli gene probe to S. velum genomic DNA restriction fragments. The symbionts appeared to have only one copy of the 16S rRNA gene. The lack of variability in the 16S sequence and hybridization patterns within and between individual S. velum organisms suggested that one species of symbiont is dominant within and specific for this host species. Phylogenetic analysis of the 16S sequences of the symbionts indicates that they lie within the chemoautotrophic cluster of the gamma subdivision of the eubacterial group Proteobacteria. Images PMID:1577710

  16. Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis

    PubMed Central

    Siala, Mariam; Gdoura, Radhouane; Fourati, Hela; Rihl, Markus; Jaulhac, Benoit; Younes, Mohamed; Sibilia, Jean; Baklouti, Sofien; Bargaoui, Naceur; Sellami, Slaheddine; Sghir, Abdelghani; Hammami, Adnane

    2009-01-01

    Introduction Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. Methods We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10. Conclusions Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis. PMID:19570210

  17. Presence of Chlamydiales DNA in samples negative by broad-range bacterial 16S rRNA PCRs: new insights into chlamydial pathogenic role.

    PubMed

    Tagini, F; Jaton, K; Manuel, O; Greub, G

    2016-05-01

    Since routine eubacterial 16S rRNA PCR does not amplify members of the Chlamydiales order, we tested all samples received in our laboratory during a 10 months period using a pan-Chlamydiales real-time PCR. 3 of 107 samples (2.8%) revealed to be positive, suggesting a role of some Chlamydiales in the pathogenesis of chronic bronchial stenosis or bronchial stenosis superinfection and as agents of orthopaedic prosthesis infections. PMID:27014466

  18. Presence of Chlamydiales DNA in samples negative by broad-range bacterial 16S rRNA PCRs: new insights into chlamydial pathogenic role

    PubMed Central

    Tagini, F.; Jaton, K.; Manuel, O.; Greub, G.

    2016-01-01

    Since routine eubacterial 16S rRNA PCR does not amplify members of the Chlamydiales order, we tested all samples received in our laboratory during a 10 months period using a pan-Chlamydiales real-time PCR. 3 of 107 samples (2.8%) revealed to be positive, suggesting a role of some Chlamydiales in the pathogenesis of chronic bronchial stenosis or bronchial stenosis superinfection and as agents of orthopaedic prosthesis infections. PMID:27014466

  19. Enhanced Mucosal Antibody Production and Protection against Respiratory Infections Following an Orally Administered Bacterial Extract.

    PubMed

    Pasquali, Christian; Salami, Olawale; Taneja, Manisha; Gollwitzer, Eva S; Trompette, Aurelien; Pattaroni, Cline; Yadava, Koshika; Bauer, Jacques; Marsland, Benjamin J

    2014-01-01

    Secondary bacterial infections following influenza infection are a pressing problem facing respiratory medicine. Although antibiotic treatment has been highly successful over recent decades, fatalities due to secondary bacterial infections remain one of the leading causes of death associated with influenza. We have assessed whether administration of a bacterial extract alone is sufficient to potentiate immune responses and protect against primary infection with influenza, and secondary infections with either Streptococcus pneumoniae or Klebsiella pneumoniae in mice. We show that oral administration with the bacterial extract, OM-85, leads to a maturation of dendritic cells and B-cells characterized by increases in MHC II, CD86, and CD40, and a reduction in ICOSL. Improved immune responsiveness against influenza virus reduced the threshold of susceptibility to secondary bacterial infections, and thus protected the mice. The protection was associated with enhanced polyclonal B-cell activation and release of antibodies that were effective at neutralizing the virus. Taken together, these data show that oral administration of bacterial extracts provides sufficient mucosal immune stimulation to protect mice against a respiratory tract viral infection and associated sequelae. PMID:25593914

  20. Enhanced Mucosal Antibody Production and Protection against Respiratory Infections Following an Orally Administered Bacterial Extract

    PubMed Central

    Pasquali, Christian; Salami, Olawale; Taneja, Manisha; Gollwitzer, Eva S.; Trompette, Aurelien; Pattaroni, Céline; Yadava, Koshika; Bauer, Jacques; Marsland, Benjamin J.

    2014-01-01

    Secondary bacterial infections following influenza infection are a pressing problem facing respiratory medicine. Although antibiotic treatment has been highly successful over recent decades, fatalities due to secondary bacterial infections remain one of the leading causes of death associated with influenza. We have assessed whether administration of a bacterial extract alone is sufficient to potentiate immune responses and protect against primary infection with influenza, and secondary infections with either Streptococcus pneumoniae or Klebsiella pneumoniae in mice. We show that oral administration with the bacterial extract, OM-85, leads to a maturation of dendritic cells and B-cells characterized by increases in MHC II, CD86, and CD40, and a reduction in ICOSL. Improved immune responsiveness against influenza virus reduced the threshold of susceptibility to secondary bacterial infections, and thus protected the mice. The protection was associated with enhanced polyclonal B-cell activation and release of antibodies that were effective at neutralizing the virus. Taken together, these data show that oral administration of bacterial extracts provides sufficient mucosal immune stimulation to protect mice against a respiratory tract viral infection and associated sequelae. PMID:25593914

  1. Bacterial Primary Colonization and Early Succession on Surfaces in Marine Waters as Determined by Amplified rRNA Gene Restriction Analysis and Sequence Analysis of 16S rRNA Genes

    PubMed Central

    Dang, Hongyue; Lovell, Charles R.

    2000-01-01

    The nearly universal colonization of surfaces in marine waters by bacteria and the formation of biofilms and biofouling communities have important implications for ecological function and industrial processes. However, the dynamics of surface attachment and colonization in situ, particularly during the early stages of biofilm establishment, are not well understood. Experimental surfaces that differed in their degrees of hydrophilicity or hydrophobicity were incubated in a salt marsh estuary tidal creek for 24 or 72 h. The organisms colonizing these surfaces were examined by using a cultivation-independent approach, amplified ribosomal DNA restriction analysis. The goals of this study were to assess the diversity of bacterial colonists involved in early succession on a variety of surfaces and to determine the phylogenetic affiliations of the most common early colonists. Substantial differences in the representation of different cloned ribosomal DNA sequences were found when the 24- and 72-h incubations were compared, indicating that some new organisms were recruited and some other organisms were lost. Phylogenetic analyses of the most common sequences recovered showed that the colonists were related to organisms known to inhabit surfaces or particles in marine systems. A total of 22 of the 26 clones sequenced were affiliated with the Roseobacter subgroup of the ? subdivision of the division Proteobacteria (?-Proteobacteria), and most of these clones were recovered at a high frequency from all surfaces after 24 or 72 h of incubation. Two clones were affiliated with the Alteromonas group of the ?-Proteobacteria and appeared to be involved only in the very early stages of colonization (within the first 24 h). A comparison of the colonization patterns on the test surfaces indicated that the early bacterial community succession rate and/or direction may be influenced by surface physicochemical properties. However, organisms belonging to the Roseobacter subgroup are ubiquitous and rapid colonizers of surfaces in coastal environments. PMID:10653705

  2. Evaluation of a fluorescence-labelled oligonucleotide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears.

    PubMed Central

    Nordentoft, S; Christensen, H; Wegener, H C

    1997-01-01

    A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all of the S. enterica subspecies and S. bongori, an 18-mer oligonucleotide probe was selected. The specificity of the probe was tested by in situ hybridization to bacterial cell smears of pure cultures. Forty-nine of 55 tested Salmonella serovars belonging to subspecies I, II, IIIb, IV, and VI hybridized with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin-embedded tissue from experimentally infected mice or from animals with a history of clinical salmonellosis. In these tissue sections the probe hybridized specifically to Salmonella serovars, allowing for the detection of single bacterial cells. The development of a fluorescence-labelled specific oligonucleotide probe makes the FISH technique a promising tool for the rapid identification of S. enterica in bacterial smears, as well as for the detection of S. enterica in histological tissue sections. PMID:9316923

  3. Evaluation of a fluorescence-labelled oligonucleotide probe targeting 23S rRNA for in situ detection of Salmonella serovars in paraffin-embedded tissue sections and their rapid identification in bacterial smears.

    PubMed

    Nordentoft, S; Christensen, H; Wegener, H C

    1997-10-01

    A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all of the S. enterica subspecies and S. bongori, an 18-mer oligonucleotide probe was selected. The specificity of the probe was tested by in situ hybridization to bacterial cell smears of pure cultures. Forty-nine of 55 tested Salmonella serovars belonging to subspecies I, II, IIIb, IV, and VI hybridized with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin-embedded tissue from experimentally infected mice or from animals with a history of clinical salmonellosis. In these tissue sections the probe hybridized specifically to Salmonella serovars, allowing for the detection of single bacterial cells. The development of a fluorescence-labelled specific oligonucleotide probe makes the FISH technique a promising tool for the rapid identification of S. enterica in bacterial smears, as well as for the detection of S. enterica in histological tissue sections. PMID:9316923

  4. Bacterial diversity analysis of Huanglongbing pathogen-infected citrus, using PhyloChip and 16S rRNA gene clone library sequencing

    SciTech Connect

    Shankar Sagaram, U.; DeAngelis, K.M.; Trivedi, P.; Andersen, G.L.; Lu, S.-E.; Wang, N.

    2009-03-01

    The bacterial diversity associated with citrus leaf midribs was characterized 1 from citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rDNA microarray and 16S rDNA clone library sequencing to determine the microbial community composition of symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria from 15 phyla were present in the citrus leaf midribs while 20 orders from phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs compared to asymptomatic midribs. Candidatus Liberibacter asiaticus (Las) was detected at a very low level in asymptomatic plants, but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis was further verified by sequencing 16S rDNA clone libraries, which indicated the dominance of Las in symptomatic leaves. These data implicate Las as the pathogen responsible for HLB disease. Citrus is the most important commercial fruit crop in Florida. In recent years, citrus Huanglongbing (HLB), also called citrus greening, has severely affected Florida's citrus production and hence has drawn an enormous amount of attention. HLB is one of the most devastating diseases of citrus (6,13), characterized by blotchy mottling with green islands on leaves, as well as stunting, fruit decline, and small, lopsided fruits with poor coloration. The disease tends to be associated with a phloem-limited fastidious {alpha}-proteobacterium given a provisional Candidatus status (Candidatus Liberobacter spp. later changed to Candidatus Liberibacter spp.) in nomenclature (18,25,34). Previous studies indicate that HLB infection causes disorder in the phloem and severely impairs the translocation of assimilates in host plants (5,27,40). Tatineni and colleagues discovered that the HLB bacteria were unevenly distributed in phloem of bark tissue, vascular tissue of the leaf midrib, roots, and different floral and fruit parts (43). Unsuccessful attempts in culturing the pathogen are notably hampering efforts to understand its biology and pathogenesis mechanism. Using a modified Koch's Postulates approach, Jagoueix and colleagues were able to re-infect periwinkle plants from a mixed microbial community harvested from HLB diseased plants (25). Emergence of the disease in otherwise healthy plants led to the conclusion that HLB was associated with Candidatus Liberibacter sp. based on its 16S rDNA sequence (18,25). Currently, three species of the pathogen are recognized from trees with HLB disease based on 16S rDNA sequence: Ca. Liberibacter asiaticus (Las), Ca. Liberibacter africanus (Laf), and Ca. Liberibacter americanus (Lam); Las is the most prevalent species among HLB diseased trees (5,12,18,25,44). Las is naturally transmitted to citrus by the psyllid, Diaphorina citri Kuwayama, and can be artificially transmitted by grafting from citrus to citrus and dodder (Cuscuta campestris) to periwinkle (Catharanthus roseus) or tobacco (Nicotiana tabacum Xanthi) (5). Based on current research regarding the associations of Liberibacter in planta there is not enough evidence to implicate Liberibacter as the definitive causal agent of HLB disease due to its resistance to cultivation in vitro. It is possible that HLB disease may be the result of complex etiology where Liberibacter interacts with other endophytic bacteria. However, there is not enough evidence regarding its association(s) in planta to make this conclusion, nor is it known whether associated microbial communities play a role in expression of pathogenic traits. The main objective of the study was to test the hypothesis that other bacteria besides Ca. Liberibacter spp. are associated with citrus greening disease. The differences between the relative abundance, species richness and phylogenetic diversity of the microbial communities associated with the leaf midribs of HLB symptomatic and asymptomatic citrus trees were investigated using high-density 16S rDNA microarray PhyloChip and 16S rRNA gene clone library methods.

  5. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis.

    PubMed

    Salonen, Anne; Nikkilä, Janne; Jalanka-Tuovinen, Jonna; Immonen, Outi; Rajilić-Stojanović, Mirjana; Kekkonen, Riina A; Palva, Airi; de Vos, Willem M

    2010-05-01

    Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories. PMID:20171997

  6. Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaa and minas cheese production using culture-independent 16s rRNA gene sequence analysis

    PubMed Central

    Lacerda, Inayara C. A.; Gomes, Ftima C. O.; Borelli, Beatriz M.; Faria Jr., Csar L. L.; Franco, Gloria R.; Mouro, Marina M.; Morais, Paula B.; Rosa, Carlos A.

    2011-01-01

    We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaa and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaa samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent. PMID:24031676

  7. Identification of the bacterial community responsible for traditional fermentation during sour cassava starch, cachaa and minas cheese production using culture-independent 16s rRNA gene sequence analysis.

    PubMed

    Lacerda, Inayara C A; Gomes, Ftima C O; Borelli, Beatriz M; Faria, Csar L L; Franco, Gloria R; Mouro, Marina M; Morais, Paula B; Rosa, Carlos A

    2011-04-01

    We used a cultivation-independent, clone library-based 16S rRNA gene sequence analysis to identify bacterial communities present during traditional fermentation in sour cassava starch, cachaa and cheese production in Brazil. Partial 16S rRNA gene clone sequences from sour cassava starch samples collected on day five of the fermentation process indicated that Leuconostoc citreum was the most prevalent species, representing 47.6% of the clones. After 27 days of fermentation, clones (GenBank accession numbers GQ999786 and GQ999788) related to unculturable bacteria were the most prevalent, representing 43.8% of the clones from the bacterial community analyzed. The clone represented by the sequence GQ999786 was the most prevalent at the end of the fermentation period. The majority of clones obtained from cachaa samples during the fermentation of sugar cane juice were from the genus Lactobacillus. Lactobacillus nagelli was the most prevalent at the beginning of the fermentation process, representing 76.9% of the clones analyzed. After 21 days, Lactobacillus harbinensis was the most prevalent species, representing 75% of the total clones. At the end of the fermentation period, Lactobacillus buchneri was the most prevalent species, representing 57.9% of the total clones. In the Minas cheese samples, Lactococcus lactis was the most prevalent species after seven days of ripening. After 60 days of ripening, Streptococcus salivarius was the most prevalent species. Our data show that these three fermentation processes are conducted by a succession of bacterial species, of which lactic acid bacteria are the most prevalent. PMID:24031676

  8. Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions, and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

    PubMed Central

    Cruaud, Perrine; Vigneron, Adrien; Lucchetti-Miganeh, Céline; Ciron, Pierre Emmanuel; Godfroy, Anne

    2014-01-01

    Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities. PMID:24837380

  9. Bacterial characterization of Beijing drinking water by flow cytometry and MiSeq sequencing of the 16S rRNA gene.

    PubMed

    Liu, Tingting; Kong, Weiwen; Chen, Nan; Zhu, Jing; Wang, Jingqi; He, Xiaoqing; Jin, Yi

    2016-02-01

    Flow cytometry (FCM) and 16S rRNA gene sequencing data are commonly used to monitor and characterize microbial differences in drinking water distribution systems. In this study, to assess microbial differences in drinking water distribution systems, 12 water samples from different sources water (groundwater, GW; surface water, SW) were analyzed by FCM, heterotrophic plate count (HPC), and 16S rRNA gene sequencing. FCM intact cell concentrations varied from 2.2 × 10(3) cells/mL to 1.6 × 10(4) cells/mL in the network. Characteristics of each water sample were also observed by FCM fluorescence fingerprint analysis. 16S rRNA gene sequencing showed that Proteobacteria (76.9-42.3%) or Cyanobacteria (42.0-3.1%) was most abundant among samples. Proteobacteria were abundant in samples containing chlorine, indicating resistance to disinfection. Interestingly, Mycobacterium, Corynebacterium, and Pseudomonas, were detected in drinking water distribution systems. There was no evidence that these microorganisms represented a health concern through water consumption by the general population. However, they provided a health risk for special crowd, such as the elderly or infants, patients with burns and immune-compromised people exposed by drinking. The combined use of FCM to detect total bacteria concentrations and sequencing to determine the relative abundance of pathogenic bacteria resulted in the quantitative evaluation of drinking water distribution systems. Knowledge regarding the concentration of opportunistic pathogenic bacteria will be particularly useful for epidemiological studies. PMID:26941936

  10. Antibacterial activity of plant extracts on foodborne bacterial pathogens and food spoilage bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacterial foodborne diseases are caused by consumption of foods contaminated with bacteria and/or their toxins. In this study, we evaluated antibacterial properties of twelve different extracts including turmeric, lemon and different kinds of teas against four major pathogenic foodborne bacteria inc...

  11. Bacterial cell wall components as immunomodulators--II. The bacterial cell wall extract OM-85 BV as unspecific activator, immunogen and adjuvant in mice.

    PubMed

    Bessler, W G; Huber, M; Baier, W

    1997-01-01

    The bacterial extract Broncho-Vaxom used for the prevention and treatment of recurrent respiratory tract infections is an immunomodulator in vitro and in vivo, as determined in a murine model. The extract acts, on the one hand, as macrophage activator and polyclonal B-lymphocyte stimulant. On the other hand, after repeated intraperitoneal or oral immunizations, the extract is immunogenic, inducing serum IgG binding to the bacterial strains used for the preparation of the extract. On bacteria, the sera recognize the cell wall components porin, lipoprotein/lipopeptide and murein. The bacterial extract also exhibits adjuvant properties when applied in mixture with antigens, such as TNP-BSA or an influenza vaccine preparation. The unspecific and the immunospecific stimulatory effect of the extract as well as its adjuvant properties could be of importance for understanding its therapeutic effect. PMID:9637353

  12. Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys

    SciTech Connect

    Walters , William; Hyde, Embriette R.; Berg-Lyons, Donna; Ackermann, Gail; Humphrey, Greg; Parada , Alma; Gilbert, Jack A.; Jansson, Janet K.; Caporaso, Greg; Fuhrman, Jed A.; Apprill, Amy; Knight, Rob

    2015-12-22

    Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of datasets amplified with varied primers requires attention. Here we examine the performance of modified 16S rRNA gene and ITS primers for archaea/bacteria and fungi, respectively, with non-aquatic samples. We moved primer barcodes to the 5’-end, allowing for a range of different 3’ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4-5 of the 16S rRNA gene. We additionally demonstrate that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.

  13. The bioactivity of plant extracts against representative bacterial pathogens of the lower respiratory tract

    PubMed Central

    Bocanegra-García, Virgilio; del Rayo Camacho-Corona, María; Ramírez-Cabrera, Mónica; Rivera, Gildardo; Garza-González, Elvira

    2009-01-01

    Background Lower respiratory tract infections are a major cause of illness and death. Such infections are common in intensive care units (ICU) and their lethality persists despite advances in diagnosis, treatment and prevention. In Mexico, some plants are used in traditional medicine to treat respiratory diseases or ailments such as cough, bronchitis, tuberculosis and other infections. Medical knowledge derived from traditional societies has motivated searches for new bioactive molecules derived from plants that show potent activity against bacterial pathogens. Therefore, the aim of this study was to evaluate the effect of hexanic, chloroformic (CLO), methanolic (MET) and aqueous extracts from various plants used in Mexican traditional medicine on various microorganisms associated with respiratory disease. Methods thirty-five extracts prepared from nine plants used in Mexican traditional medicine for the treatment of respiratory infections were evaluated against 15 control bacterial species and clinical isolates. Results Both chloroformic (CLO) and methanolic (MET) extracts of Larrea tridentata were active against Methicillin-resistant S. aureus, B. subtilis and L. monocytogenes. A MET extract of L. tridentata was also active against S. aureus, S. pneumoniae, S. maltophilia, E. faecalis and H. influenzae and the CLO extract was active against A. baumannii. An Aqueous extract of M. acumitata and a MET extract of N. officinale were active against S. pneumoniae. CLO and MET extracts of L. tridentata were active against clinical isolates of S. aureus, S. pneumoniae and E. faecalis. Conclusion Overall, our results support the potential use of L. tridentata as a source of antibacterial compounds. PMID:19486533

  14. High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

    PubMed Central

    Sergeant, Martin J.; Constantinidou, Chrystala; Cogan, Tristan; Penn, Charles W.; Pallen, Mark J.

    2012-01-01

    The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers. PMID:22666455

  15. Composition and Metabolic Activities of the Bacterial Community in Shrimp Sauce at the Flavor-Forming Stage of Fermentation As Revealed by Metatranscriptome and 16S rRNA Gene Sequencings.

    PubMed

    Duan, Shan; Hu, Xiaoxi; Li, Mengru; Miao, Jianyin; Du, Jinghe; Wu, Rongli

    2016-03-30

    The bacterial community and the metabolic activities involved at the flavor-forming stage during the fermentation of shrimp sauce were investigated using metatranscriptome and 16S rRNA gene sequencings. Results showed that the abundance of Tetragenococcus was 95.1%. Tetragenococcus halophilus was identified in 520 of 588 transcripts annotated in the Nr database. Activation of the citrate cycle and oxidative phosphorylation, along with the absence of lactate dehydrogenase gene expression, in T. halophilus suggests that T. halophilus probably underwent aerobic metabolism during shrimp sauce fermentation. The metabolism of amino acids, production of peptidase, and degradation of limonene and pinene were very active in T. halophilus. Carnobacterium, Pseudomonas, Escherichia, Staphylococcus, Bacillus, and Clostridium were also metabolically active, although present in very small populations. Enterococcus, Abiotrophia, Streptococcus, and Lactobacillus were detected in metatranscriptome sequencing, but not in 16S rRNA gene sequencing. Many minor taxa showed no gene expression, suggesting that they were in dormant status. PMID:26978261

  16. A comparison of five methods for extraction of bacterial DNA from human faecal samples.

    PubMed

    McOrist, Alexandra L; Jackson, Michelle; Bird, Anthony R

    2002-07-01

    The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type. PMID:11997164

  17. Estimation of diversity and community structure through restriction fragment length polymorphism distribution analysis of bacterial 16S rRNA genes from a microbial mat at an active, hydrothermal vent system, Loihi Seamount, Hawaii.

    PubMed Central

    Moyer, C L; Dobbs, F C; Karl, D M

    1994-01-01

    PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete operational taxonomic units (OTUs). The rank order abundance of these putative OTUs was measured, and the two most abundant OTUs accounted for 72.9% of all of the 16S rDNA clones. Among the remaining 27.1% of the 16S rDNA clones, none of the 10 OTUs was represented by more than three individual clones. The cumulative OTU distribution for 48 bacterial 16S rDNA clones demonstrated that the majority of taxa represented in the clone library were detected, a result which we assume to be an estimate of the diversity of bacteria in the native hydrothermal vent habitat. 16S rDNA fingerprinting of individual clones belonging to particular OTUs by using an oligonucleotide probe that binds to a universally conserved region of the 16S rDNA fragments was conducted to confirm OTU specificity and 16S rDNA identity. Images PMID:7512808

  18. Effects of the Bacterial Extract OM-85 on Phagocyte Functions and the Stress Response

    PubMed Central

    Baladi, S.; Kantengwa, S.; Donati, Y. R. A.; Polla, B. S.

    1994-01-01

    The effects of the bacterial extract OM-85 on the respiratory burst, intracellular calcium and the stress response have been investigated in human peripheral blood monocytes from normal donors. Activation of the respiratory burst during bacterial phagocytosis has been previously associated with heat shock/stress proteins synthesis. Whereas OM-85 stimulated superoxide production and increased Ca2+ mobilization, it fared to induce synthesis of classical HSPs. The lack of stress protein induction was observed even in the presence of iron which potentiates both oxidative injury and stress protein induction during bacterial phagocytosis. However OM-85 induced a 7578 kDa protein, which is likely to be a glucose regulated protein (GRP78), and enhanced intracellular expression of interleukin-l? precursor. PMID:18472933

  19. Effects of the Bacterial Extract OM-85 on Phagocyte Functions and the Stress Response.

    PubMed

    Baladi, S; Kantengwa, S; Donati, Y R; Polla, B S

    1994-01-01

    The effects of the bacterial extract OM-85 on the respiratory burst, intracellular calcium and the stress response have been investigated in human peripheral blood monocytes from normal donors. Activation of the respiratory burst during bacterial phagocytosis has been previously associated with heat shock/stress proteins synthesis. Whereas OM-85 stimulated superoxide production and increased Ca(2+) mobilization, it fared to induce synthesis of classical HSPs. The lack of stress protein induction was observed even in the presence of iron which potentiates both oxidative injury and stress protein induction during bacterial phagocytosis. However OM-85 induced a 75-78 kDa protein, which is likely to be a glucose regulated protein (GRP78), and enhanced intracellular expression of interleukin-lbeta precursor. PMID:18472933

  20. Extraction, purification and identification of bacterial signal molecules based on N?acyl homoserine lactones

    PubMed Central

    Wang, Jianhua; Quan, Chunshan; Wang, Xue; Zhao, Pengchao; Fan, Shengdi

    2011-01-01

    Summary Bacteria possess an extraordinary repertoire for intercellular communication and social behaviour. This repertoire for bacterial communication, termed as quorum sensing (QS), depends on specific diffusible signal molecules. There are many different kinds of signal molecules in the bacterial community. Among those signal molecules, N?acyl homoserine lactones (HSLs, in other publications also referred to as AHLs, acy?HSLs etc.) are often employed as QS signal molecules for many Gram?negative bacteria. Due to the specific structure and tiny amount of those HSL signal molecules, the characterization of HSLs has been the subject of extensive investigations in the last decades and has become a paradigm for bacteria intercellular signalling. In this article, different methods, including extraction, purification and characterization of HSLs, are reviewed. The review provides an insight into identification and characterization of new HSLs and other signal molecules for bacterial intercellular communication. PMID:21375695

  1. Comprehensive Meta-analysis of Ontology Annotated 16S rRNA Profiles Identifies Beta Diversity Clusters of Environmental Bacterial Communities.

    PubMed

    Henschel, Andreas; Anwar, Muhammad Zohaib; Manohar, Vimitha

    2015-10-01

    Comprehensive mapping of environmental microbiomes in terms of their compositional features remains a great challenge in understanding the microbial biosphere of the Earth. It bears promise to identify the driving forces behind the observed community patterns and whether community assembly happens deterministically. Advances in Next Generation Sequencing allow large community profiling studies, exceeding sequencing data output of conventional methods in scale by orders of magnitude. However, appropriate collection systems are still in a nascent state. We here present a database of 20,427 diverse environmental 16S rRNA profiles from 2,426 independent studies, which forms the foundation of our meta-analysis. We conducted a sample size adaptive all-against-all beta diversity comparison while also respecting phylogenetic relationships of Operational Taxonomic Units(OTUs). After conventional hierarchical clustering we systematically test for enrichment of Environmental Ontology terms and their abstractions in all possible clusters. This post-hoc algorithm provides a novel formalism that quantifies to what extend compositional and semantic similarity of microbial community samples coincide. We automatically visualize significantly enriched subclusters on a comprehensive dendrogram of microbial communities. As a result we obtain the hitherto most differentiated and comprehensive view on global patterns of microbial community diversity. We observe strong clusterability of microbial communities in ecosystems such as human/mammal-associated, geothermal, fresh water, plant-associated, soils and rhizosphere microbiomes, whereas hypersaline and anthropogenic samples are less homogeneous. Moreover, saline samples appear less cohesive in terms of compositional properties than previously reported. PMID:26458130

  2. Comprehensive Meta-analysis of Ontology Annotated 16S rRNA Profiles Identifies Beta Diversity Clusters of Environmental Bacterial Communities

    PubMed Central

    Henschel, Andreas; Anwar, Muhammad Zohaib; Manohar, Vimitha

    2015-01-01

    Comprehensive mapping of environmental microbiomes in terms of their compositional features remains a great challenge in understanding the microbial biosphere of the Earth. It bears promise to identify the driving forces behind the observed community patterns and whether community assembly happens deterministically. Advances in Next Generation Sequencing allow large community profiling studies, exceeding sequencing data output of conventional methods in scale by orders of magnitude. However, appropriate collection systems are still in a nascent state. We here present a database of 20,427 diverse environmental 16S rRNA profiles from 2,426 independent studies, which forms the foundation of our meta-analysis. We conducted a sample size adaptive all-against-all beta diversity comparison while also respecting phylogenetic relationships of Operational Taxonomic Units(OTUs). After conventional hierarchical clustering we systematically test for enrichment of Environmental Ontology terms and their abstractions in all possible clusters. This post-hoc algorithm provides a novel formalism that quantifies to what extend compositional and semantic similarity of microbial community samples coincide. We automatically visualize significantly enriched subclusters on a comprehensive dendrogram of microbial communities. As a result we obtain the hitherto most differentiated and comprehensive view on global patterns of microbial community diversity. We observe strong clusterability of microbial communities in ecosystems such as human/mammal-associated, geothermal, fresh water, plant-associated, soils and rhizosphere microbiomes, whereas hypersaline and anthropogenic samples are less homogeneous. Moreover, saline samples appear less cohesive in terms of compositional properties than previously reported. PMID:26458130

  3. 16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca.

    PubMed

    Gong, Jianhua; Si, Weiduo; Forster, Robert J; Huang, Ruilin; Yu, Hai; Yin, Yulong; Yang, Chengbo; Han, Yanming

    2007-01-01

    Mucosa-associated microbiota from different regions of the gastrointestinal (GI) tract of adult broilers was studied by analysis of 16S rRNA gene sequences. The microbiota mainly comprised Gram-positive bacteria along the GI tract. Fifty-one operational taxonomic units (OTUs) (from 98 clones) were detected in the ceca, as compared with 13 OTUs (from 49 clones) in the crops, 11 OTUs (from 51 clones) in the gizzard, 14 OTUs (from 52 clones) in the duodenum, 12 OTUs (from 50 clones) in the jejunum and nine OTUs (from 50 clones) in the ileum. Ceca were dominantly occupied by clostridia-related sequences (40%) with other abundant sequences being related to Faecalibacterium prausnitzii (14%), Escherichia coli (11%), lactobacilli (7%) and Ruminococcus (6%). Lactobacilli were predominant in the upper GI tract and had the highest diversity in the crop. Both Lactobacillus aviarius and Lactobacillus salivarius were the predominant species among lactobacilli. Candidatus division Arthromitus was also abundant in the jejunum and ileum. PMID:17233749

  4. The bacterial extract OM-85 BV protects mice against influenza and Salmonella infection.

    PubMed

    Bessler, Wolfgang G; Vor dem Esche, Ulrich; Masihi, Noel

    2010-09-01

    The bacterial extract OM-85 BV has been shown to provide protection against recurrent respiratory infections. We here investigated its efficacy against viral and bacterial infections in murine models. We first evaluated the role of OM-85 BV protecting from an A/PR/8/34 (H1N1) influenza virus infection. In a group treated with 1.75 mg/mouse OM-85 BV all animals survived, compared to 70% in the untreated control group and a group treated with a lower dosage. In addition, the appearance of clinical signs was delayed, their intensity was decreased, and they disappeared faster; also a marked increase in the influenza hemagglutination inhibition antibody level was observed. Since bacterial infections often superimpose viral lung infections, we also investigated on the protection of mice from a Salmonella typhimurium infection after the oral administration of OM-85 BV. Here, 100% of the OM-85 BV treated animals survived compared to 58% of the untreated control group. The mechanism of protection was further investigated: OM-85 BV acts, on the one hand, as an immunogen: the repeated administration of OM-85 BV induced a marked increase in serum antibody levels recognizing pathogenic bacterial strains. On the other hand, the extract acts as a stimulator of the nonspecific macrophage, monocyte, dendritic cell, and granulocyte response. Our findings demonstrate the antimicrobial activity of OM-85 BV against infections, as also has been shown in clinical studies. PMID:20601184

  5. 16S rRNA pyrosequencing-based investigation of the bacterial community in nukadoko, a pickling bed of fermented rice bran.

    PubMed

    Sakamoto, Naoshige; Tanaka, Shigemitsu; Sonomoto, Kenji; Nakayama, Jiro

    2011-01-01

    Nukadoko is a naturally fermented rice bran mash traditionally used for pickling vegetables in Japan; its refreshment and fermentation cycles sometimes continue for many years. Here, we investigated the structure and dynamics of the bacterial community in nukadoko by conducting pyrosequencing and quantitative polymerase chain reaction (PCR) analyses of 16S ribosomal RNA genes (rDNA). Of the 16 different samples studied, 13 showed Lactobacillus-dominated microbiota, suggesting that aged nukadoko samples tend to realize a niche, favorable Lactobacillus species. The lactic acid bacterial community of each of the 16 samples was classified into 3 types according to the presence or absence of 2 predominant species, Lactobacillus namurensis and Lactobacillus acetotolerans. The dynamics of the bacterial community during fermentation and the subsequent ripening process were examined using a laboratory model of nukadoko inoculated with an aged nukadoko sample (inoculated model). Lb. namurensis grew rapidly in the first 2 days, accompanied with a rapid decrease in pH and an increase in lactate levels, while Lb. acetotolerans grew with a longer doubling time and slow acidification during the 20 days after inoculation. On the other hand, spontaneous fermentation of the nukadoko model prepared from fresh rice bran without the nukadoko inoculation (inoculant-free model), showed the growth of some non-Lactobacillus species such as staphylococci and bacilli within the first 10 days; thereafter, Lb. namurensis was dominant, while Lb. acetotolerans was not detected during the 20-day experimental period. These results suggest that the naturally established Lactobacillus community in aged nukadoko is effectively involved in the biocontrol of the microbial community of nukadoko during the refreshment and fermentation cycles. PMID:21084126

  6. Interactions of antibiotics and methanolic crude extracts of Afzelia Africana (Smith.) against drug resistance bacterial isolates.

    PubMed

    Aiyegoro, Olayinka; Adewusi, Adekanmi; Oyedemi, Sunday; Akinpelu, David; Okoh, Anthony

    2011-01-01

    Infection due to multidrug resistance pathogens is difficult to manage due to bacterial virulence factors and because of a relatively limited choice of antimicrobial agents. Thus, it is imperative to discover fresh antimicrobials or new practices that are effective for the treatment of infectious diseases caused by drug-resistant microorganisms. The objective of this experiment is to investigate for synergistic outcomes when crude methanolic extract of the stem bark of Afzelia africana and antibiotics were combined against a panel of antibiotic resistant bacterial strains that have been implicated in infections. Standard microbiological protocols were used to determine the minimum inhibitory concentrations (MICs) of the extract and antibiotics, as well as to investigate the effect of combinations of the methanolic extract of A. africana stem bark and selected antibiotics using the time-kill assay method. The extract of Afzelia africana exhibited antibacterial activities against both Gram-negative and Gram-positive bacteria made up of environmental and standard strains at a screening concentration of 5 mg/mL. The MICs of the crude extracts and the antibiotics varied between 1 ?g/mL and 5.0 mg/mL. Overall, synergistic response constituted about 63.79% of all manner of combinations of extract and antibiotics against all test organisms; antagonism was not detected among the 176 tests carried out. The extract from A. africana stem bark showed potentials of synergy in combination with antibiotics against strains of pathogenic bacteria. The detection of synergy between the extract and antibiotics demonstrates the potential of this plant as a source of antibiotic resistance modulating compounds. PMID:21845091

  7. Assessing bacterial diversity in a seawater-processing wastewater treatment plant by 454-pyrosequencing of the 16S rRNA and amoA genes

    PubMed Central

    Sánchez, Olga; Ferrera, Isabel; González, Jose M; Mas, Jordi

    2013-01-01

    Summary The bacterial community composition of activated sludge from a wastewater treatment plant (Almería, Spain) with the particularity of using seawater was investigated by applying 454-pyrosequencing. The results showed that Deinococcus-Thermus, Proteobacteria, Chloroflexi and Bacteroidetes were the most abundant retrieved sequences, while other groups, such as Actinobacteria, Chlorobi, Deferribacteres, Firmicutes, Planctomycetes, Spirochaetes and Verrumicrobia were reported at lower proportions. Rarefaction analysis showed that very likely the diversity is higher than what could be described despite most of the unknown microorganisms probably correspond to rare diversity. Furthermore, the majority of taxa could not be classified at the genus level and likely represent novel members of these groups. Additionally, the nitrifiers in the sludge were characterized by pyrosequencing the amoA gene. In contrast, the nitrifying bacterial community, dominated by the genera Nitrosomonas, showed a low diversity and rarefaction curves exhibited saturation. These results suggest that only a few populations of low abundant but specialized bacteria are responsible for removal of ammonia in these saline wastewater systems. PMID:23574645

  8. Seasonal change in bacterial flora and biomass in mountain snow from the Tateyama Mountains, Japan, analyzed by 16S rRNA gene sequencing and real-time PCR.

    PubMed

    Segawa, Takahiro; Miyamoto, Koji; Ushida, Kazunari; Agata, Kiyokazu; Okada, Norihiro; Kohshima, Shiro

    2005-01-01

    The bacterial flora and biomass in mountain snow from the Tateyama Mountains, Toyama Prefecture, Japan, one of the heaviest snowfall regions in the world, were analyzed by amplified ribosomal DNA restriction analysis followed by 16S rRNA gene sequencing and DNA quantification by real-time PCR. Samples of surface snow collected in various months during the melting season contained a psychrophilic bacterium, Cryobacterium psychrophilum, and two psychrotrophic bacteria, Variovorax paradoxus and Janthinobacterium lividum. Bacterial colonies that developed in an in situ meltwater medium at 4 degrees C were revealed to be V. paradoxus. The biomasses of C. psychrophilum, J. lividum, and V. paradoxus, as estimated by real-time PCR, showed large increases during the melting season from March to October (2.0 x 10(5)-fold, 1.5 x 10(5)-fold, and 1.0 x 10(4)-fold increases, respectively), suggesting their rapid growth in the surface snow. The biomasses of C. psychrophilum and J. lividum increased significantly from March to April, reached a maximum in August, and dropped at the end of the melting season. In contrast, the biomass of V. paradoxus did not increase as rapidly during the early melting season but continued to increase from June until October. The differences in development observed among these bacterial species suggest that their growth was promoted by different nutrients and/or environmental conditions in the snow. Since these three types of bacteria have also been reported to be present in a glacier in Antarctica and a Greenland ice core, they seem to be specialized members of the snow biota that are distributed in snow and ice environments in various parts of the world. PMID:15640179

  9. CHARACTERIZATION OF BACTERIAL BIOMASS IN MARINE SEDIMENTS BENEATH THE ROSS ICE SHEET, ANTARCTICA BY PHOSPHOLIPIDS ANALYSIS AND 16S RRNA GENE SEQUENCING

    NASA Astrophysics Data System (ADS)

    Carr, S. A.; Glossner, A. W.; Dunbar, R. B.; Vogel, S. W.; Brandes, J.; Sahl, J. W.; Pepe-Ranney, C.; Spear, J. R.; Naish, T.; Powell, R. D.; Mandernack, K. W.

    2009-12-01

    As concerns regarding climate change increase, so does the importance of understanding the biogeochemical cycling of elements such as carbon. In the marine sediments of the Ross Sea, Antarctica, the in situ microbial community plays a significant role in the decomposition, mineralization and recycling of both organic and inorganic carbon. In this study, viable biomass for the top 155 cm below seafloor of sediment cores in the Ross Sea were estimated based on microbial phospholipid concentrations and Acridine Orange direct cell counts (AODC). Results for the biomass estimates suggest that both methods are able to accurately estimate viable biomass. Structural and isotopic analyses of phospholipid fatty acids (PLFAs) and phospholipid ether lipids (PELs), as well as isotopic analyses of carbon sources within sediment porewaters were used to identify changes in microbial metabolic pathways. The ?13C values of dissolved inorganic carbon (DIC) in porewaters ranged from -2.52 to -3.72 while corresponding ?13C values for sedimentary organic carbon (OC) varied from -26.25 to -23.12 in the surface and 155cm porewaters, respectively. The ?13C values of PLFAs are slightly lighter than the ?13C values of the organic carbon, ranging between -29 to -35 throughout the sediment core. 16S ribosomal RNA gene sequencing was preformed to classify the microbial species present at various depths. 16S sequences revealed that members of this microbial community include ?, ?, ?, and ? proteobacteria, acitobacteria, acidobacteria, and flavobacteria, all of which have been previously sequenced from other Antarctic continental shelf sediments. Archaea represent 1 to 3% of the microbial community which is similar to comparable studies. Amongst the sequenced organisms, many have been reported to utilize organic carbon sources such as amino acids, oligosaccharides, and lactose. These heterotropic organisms compliment the constant lipid isotope values and suggest that heterotrophic organisms dominate these sediments, with the implication that primary productivity is derived from above. Integrating structural analyses and ?13C values of phospholipids, porewater chemistry, ?13CDIC and ?13CDIC values with 16S rRNA gene sequences provides a more comprehensive understanding of the biogeochemical influences of microbial carbon cycling that occur beneath marine sediments of Antarctica and elsewhere.

  10. Immunomodulating effects of OM-89, a bacterial extract from Escherichia coli, in murine and human leukocytes.

    PubMed

    Bessler, Wolfgang G; Puce, Karola; vor dem Esche, Ulrich; Kirschning, Carsten; Huber, Maria

    2009-01-01

    OM-89 (Uro-Vaxom) is a bacterial extract prepared from 18 uropathogenic Escherichia coli strains used for the prevention and treatment of recurrent infections of the urinary tract. The immunomodulating effects of the bacterial extract were investigated in a mouse model. After a single oral administration of OM-89, leukocyte activation was demonstrated ex vivo in blood and liver cells using a chemiluminescence assay. An increase of the production of tumor necrosis factor-alpha (TNF-alpha) in supernatants of peritoneal cells was also observed. After repeated oral administration of OM-89, increased serum immunoglobulin G responses against several E. coli strains were found. Also, adjuvant properties of the extract using ovalbumin as an antigen could be demonstrated. In line with these findings in the mouse system, preliminary in vitro data obtained in the human system showed an increase in TNF-alpha and interleukin-6 production after stimulation of monocyte derived dendritic cells with OM-89. The activation of immune cells is likely to be mediated via Toll like receptors (TLRs); thus, the binding of components of the extract to TLR-4 and marginally to TLR-2 could be shown. PMID:20066966

  11. Crude bacterial extracts of two new Streptomyces sp. isolates as bio-colorants for textile dyeing.

    PubMed

    Kramar, Ana; Ilic-Tomic, Tatjana; Petkovic, Milos; Radulović, Niko; Kostic, Mirjana; Jocic, Dragan; Nikodinovic-Runic, Jasmina

    2014-08-01

    Renewed demand for incorporation of natural dyes (bio-colorants) in textile industry could be met through biotechnological production of bacterial pigments. Two new Streptomyces strains (NP2 and NP4) were isolated for the remarkable ability to produce diffusible deep blue and deep red pigment into fermentation medium. Crude mycelial extracts of both strains were used as bio-colorants in conventional textile dyeing procedures avoiding downstream purification procedures. The yields of bio-colorants obtained in this way were 62 and 84 mg per g of mycelia for Streptomyces sp. NP2 and Streptomyces sp. NP4, respectively. Through nuclear magnetic resonance analysis of crude extracts before and after dyeing procedures, it was shown that both extracts contained prodigiosin-like family of compounds that exhibited different dyeing capabilities towards different textile fibers. Polyamide and acrylic fibers were colored to the deepest shade, polyester and triacetate fibers to a noticeable, but much lower shade depth, while cotton and cellulosic fibers stained weakly. These results confirmed that crude bacterial extracts had the characteristics similar to those of ionic and disperse dyes, which was consistent with the identified polypyrrolic prodigiosin-like structures. PMID:24671299

  12. Comparison of seven methods for extraction of bacterial DNA from fecal and cecal samples of mice.

    PubMed

    Ferrand, Janina; Patron, Kevin; Legrand-Frossi, Christine; Frippiat, Jean-Pol; Merlin, Christophe; Alauzet, Corentine; Lozniewski, Alain

    2014-10-01

    Analysis of bacterial DNA from fecal samples of mice is commonly performed in experimental studies. Although DNA extraction is a critical step in various molecular approaches, the efficiency of methods that may be used for DNA extraction from mice fecal samples has never been evaluated. We compared the efficiencies of six widely used commercial kits (MasterPure Gram Positive DNA Purification Kit, QIAamp DNA Stool Mini Kit; NucliSENS easyMAG, ZR Fecal DNA MiniPrep, FastDNA SPIN Kit for Feces and FastDNA SPIN Kit for Soil) and a non-commercial method for DNA isolation from mice feces and cecal contents. DNA quantity and quality were assessed by fluorometry, spectrophotometry, gel electrophoresis and qPCR. Cell lysis efficiencies were evaluated by qPCR targeting three relevant bacteria in spiked specimens. For both feces and intestinal contents, the most efficient extraction method was the FastDNA SPIN Kit for Soil. PMID:25093756

  13. Comparison of different DNA-extraction techniques to investigate the bacterial community of marine copepods

    NASA Astrophysics Data System (ADS)

    Brandt, Petra; Gerdts, Gunnar; Boersma, Maarten; Wiltshire, Karen H.; Wichels, Antje

    2010-12-01

    Marine zooplanktic organisms, such as copepods, are usually associated with large numbers of bacteria. Some of these bacteria live attached to copepods exoskeleton, while others prevail in their intestine and faecal pellets. Until now, general conclusions concerning the identity of these bacteria are problematic since the majority of previous studies focused on cultivable bacteria only. Hence, to date little is known on whether copepod genera or species harbour distinct bacterial populations and about the nature of this association. To shed more light on these copepod/bacteria consortia, the focus of this study was the development and evaluation of a suitable approach to extract bacterial DNA from different North Sea copepod genera. Furthermore, the bacterial DNA was analysed by PCR-DGGE and subsequent sequencing of excised bands. The result of this work was an appropriate extraction method for batches of ten to one copepod specimens and offered first insights as to which bacteria are attached to the copepods Acartia sp . and Temora sp . from Helgoland Roads (German Bight) and a laboratory-grown Acartia tonsa culture. It revealed the prevalence of Alphaproteobacteria.

  14. Pyrosequencing-based profiling of archaeal and bacterial 16S rRNA genes identifies a novel archaeon associated with black band disease in corals.

    PubMed

    Sato, Yui; Willis, Bette L; Bourne, David G

    2013-11-01

    Black band disease (BBD) is a microbial consortium that creates anoxic, sulfide-rich microenvironments and kills underlying coral tissues as it rapidly migrates across colonies. Although bacterial communities associated with BBD have been studied extensively, the presence and roles of archaea are unexplored. Using amplicon-pyrosequencing of 16S ribosomal RNA genes, we investigated the community structure of both archaea and bacteria within microbial lesions of BBD and the less-virulent precursor stage, 'cyanobacterial patches' (CP), affecting the coral Montipora hispida. We detected characteristic shifts in microbial communities during the development of BBD from CP, reflecting microenvironmental changes within lesions. Archaeal profiles in CP suggested a diverse assemblage affiliated with the Thaumarchaeota and Euryarchaeota, similar to communities described for oxic marine environments. In contrast, a novel ribotype, distantly affiliated to the Euryarchaeota, dominated up to 94% of archaeal sequences retrieved from BBD. The physiological characteristics of this dominant archaeal ribotype are unknown because of the novelty of its 16S ribosomal RNA gene sequences; however, their prominent associations with BBD lesions suggest the ability to thrive in the organic- and sulfide-rich anoxic microenvironment characteristic of BBD lesions. Discovery of this novel archaeal ribotype provides new insights into the microbial ecology and aetiology of BBD. PMID:24112537

  15. Analysis of bacterial community structure in Saba-Narezushi (Narezushi of Mackerel) by 16S rRNA gene clone library.

    PubMed

    Matsui, Hiroki; Tsuchiya, Rie; Isobe, Yuka; Narita, Miyo

    2013-08-01

    Narezushi, a derivation of sushi, is a traditional Japanese food made by fermenting salted fish meat and cooked rice together. In this study, the microbial diversity of saba-narezushi (narezushi of mackerel, Scomber japonicus) was analyzed by the 16S ribosomal RNA gene clone library method. Chemical composition was also analyzed to compare with different kinds of narezushi. The chemical composition of the narezushi was similar to those obtained from samma-narezushi. Ninety-four clones were randomly selected and DNA sequences of cloned fragments (approx. 890bp) were analyzed. The DNA sequences obtained were phylogenetically analyzed. The expected operational taxonomy units (OTUs) by Chao1 estimates and Shannon-Wiener index (H') at 97% identity threshold were 48 and 1.822, respectively. The sequence similarity of the cloned fragment was equal to or higher than 98% of the sequence of cultivated bacterial species in the public database. Most of the clones (85%) belonged to lactic acid bacteria (LAB). Lactobacillus curvatus was the most abundant species followed by Lactococcus piscium and Leuconostoc gasicomitatum, suggesting that these bacteria play important roles in the fermentation of saba-narezushi. PMID:24425983

  16. Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol.

    PubMed

    Milani, Christian; Hevia, Arancha; Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martn, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  17. Assessing the Fecal Microbiota: An Optimized Ion Torrent 16S rRNA Gene-Based Analysis Protocol

    PubMed Central

    Foroni, Elena; Duranti, Sabrina; Turroni, Francesca; Lugli, Gabriele Andrea; Sanchez, Borja; Martín, Rebeca; Gueimonde, Miguel; van Sinderen, Douwe; Margolles, Abelardo; Ventura, Marco

    2013-01-01

    Assessing the distribution of 16S rRNA gene sequences within a biological sample represents the current state-of-the-art for determination of human gut microbiota composition. Advances in dissecting the microbial biodiversity of this ecosystem have very much been dependent on the development of novel high-throughput DNA sequencing technologies, like the Ion Torrent. However, the precise representation of this bacterial community may be affected by the protocols used for DNA extraction as well as by the PCR primers employed in the amplification reaction. Here, we describe an optimized protocol for 16S rRNA gene-based profiling of the fecal microbiota. PMID:23869230

  18. RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition

    PubMed Central

    McCarthy, Ann; Chiang, Edna; Schmidt, Marian L.; Denef, Vincent J.

    2015-01-01

    Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples. PMID:25798612

  19. SLiCE: a novel bacterial cell extract-based DNA cloning method

    PubMed Central

    Zhang, Yongwei; Werling, Uwe; Edelmann, Winfried

    2012-01-01

    We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method. PMID:22241772

  20. DNA extraction using modified bacterial magnetic particles in the presence of amino silane compound.

    PubMed

    Yoza, Brandon; Matsumoto, Mitsufumi; Matsunaga, Tadashi

    2002-04-11

    Magnetic particles produced by magnetic bacteria have been used to carry out magnetic separation of DNA. Separation was achieved using magnetite coated with 3-aminopropyltriethoxysilane, N-(trimethoxy-silylpropyl) isothiouronium chloride or 3-[2-(2-aminoethyl)-ethylamino]-propyltrimethoxysilane (AEEA). The DNA binding efficiency increased with the number of amino groups present on the silane compounds and was 14 fold higher than with untreated magnetite. Addition of AEEA to aqueous solutions containing coated magnetite increased efficiency due to co-condensation of DNA. From 10(8) Escherichia coli cells, 7.1 microg of DNA was recovered using 100 microg of magnetite. E. coli DNA extracted with modified bacterial magnetite was suitable for restriction enzyme digestion and polymerase chain reaction (PCR). Ten replicate samples of E. coli cells were extracted using an automated magnetic robot and 9.5 microg of DNA was extracted using 100 microg of modified bacterial magnetite and possessed a 1.94 absorbance ratio (260:280 nm). PMID:11861080

  1. Structure and dynamics of the bacterial communities in fermentation of the traditional Chinese post-fermented pu-erh tea revealed by 16S rRNA gene clone library.

    PubMed

    Zhao, Ming; Xiao, Wei; Ma, Yan; Sun, Tingting; Yuan, Wenxia; Tang, Na; Zhang, Donglian; Wang, Yongxia; Li, Yali; Zhou, Hongjie; Cui, Xiaolong

    2013-10-01

    Microbes are thought to have key roles in the development of the special properties of post-fermented pu-erh tea (pu-erh shucha), a well-known traditional Chinese tea; however, little is known about the bacteria during the fermentation. In this work, the structure and dynamics of the bacterial community involved in the production of pu-erh shucha were investigated using 16S rRNA gene clone libraries constructed from samples collected on days zero (LD-0), 5 (LD-5), 10 (LD-10), 15 (LD-15) and 20 (LD-20) of the fermentation. A total of 747 sequences with individual clone library containing 115-174 sequences and 4-20 unique operational taxonomic units (OTUs) were obtained. These OTUs were grouped into four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and further identified as members of 10 families, such as Alcaligenaceae, Bacillaceae, Enterobacteriaceae, etc. The dominant bacteria were Enterobacteriaceae in the raw material (LD-0) and in the initial stages of fermentation (LD-5 and LD-10), which changed to Bacillaceae at the last stages of fermentation (LD-15 and LD-20) at a temperature of 40-60C. It is interesting that the dominant OTUs in libraries LD-15 and LD-20 were very closely related to Bacillus coagulans, which is a safe thermoduric probiotic. Together the bacterial diversity and dynamics during a fermentation of pu-erh shucha were demonstrated, and a worthy clue for artificial inoculation of B. coagulans to improve the health benefits of pu-erh shucha or produce probiotic pu-erh tea were provided. PMID:23591759

  2. Antibacterial Activity of Salvadora persica L. (Miswak) Extracts against Multidrug Resistant Bacterial Clinical Isolates.

    PubMed

    Al-Ayed, Mohamed Saeed Zayed; Asaad, Ahmed Morad; Qureshi, Mohamed Ansar; Attia, Hany Goda; AlMarrani, Abduljabbar Hadi

    2016-01-01

    Much effort has focused on examining the inhibitory effect of Salvadora persica (miswak) on oral microorganisms, but information concerning its antibacterial activity against other human pathogens, particularly multidrug resistant (MDR) isolates, is scarce. Therefore, this study aimed to assess the in vitro antibacterial activities of Salvadora persica L. extracts against 10 MDR bacterial clinical isolates other than oral pathogens. The antibacterial activity of aqueous and methanol miswak extracts was assessed using the agar dilution and minimum inhibitory concentration (MIC) methods. Overall, the 400 mg/mL of miswak extract was the most effective on all strains. The methanol extract exhibited a stronger antibacterial activity against Gram-negative (3.3-13.6 mm) than Gram-positive (1.8-8.3 mm) bacteria. The lowest MIC value was seen for E. coli (0.39, 1.56 µg/mL), followed by Streptococcus pyogenes (1.56 µg/mL). The highest MIC value (6.25, 12.5 µg/mL) was recorded for methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, and Stenotrophomonas maltophilia. This study demonstrates, for the first time, the moderate to strong antibacterial activity of miswak extracts against all tested MDR-pathogens. Methanol extract appears to be a potent antimicrobial agent that could be considered as complementary and alternative medicine against resistant pathogens. Further studies on a large number of MDR organisms are necessary to investigate and standardize the inhibitory effect of miswak extracts against these emerging pathogens. PMID:26904146

  3. Antibacterial Activity of Salvadora persica L. (Miswak) Extracts against Multidrug Resistant Bacterial Clinical Isolates

    PubMed Central

    Al-Ayed, Mohamed Saeed Zayed; Asaad, Ahmed Morad; Qureshi, Mohamed Ansar; Attia, Hany Goda; AlMarrani, Abduljabbar Hadi

    2016-01-01

    Much effort has focused on examining the inhibitory effect of Salvadora persica (miswak) on oral microorganisms, but information concerning its antibacterial activity against other human pathogens, particularly multidrug resistant (MDR) isolates, is scarce. Therefore, this study aimed to assess the in vitro antibacterial activities of Salvadora persica L. extracts against 10 MDR bacterial clinical isolates other than oral pathogens. The antibacterial activity of aqueous and methanol miswak extracts was assessed using the agar dilution and minimum inhibitory concentration (MIC) methods. Overall, the 400 mg/mL of miswak extract was the most effective on all strains. The methanol extract exhibited a stronger antibacterial activity against Gram-negative (3.3–13.6 mm) than Gram-positive (1.8–8.3 mm) bacteria. The lowest MIC value was seen for E. coli (0.39, 1.56 µg/mL), followed by Streptococcus pyogenes (1.56 µg/mL). The highest MIC value (6.25, 12.5 µg/mL) was recorded for methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii, and Stenotrophomonas maltophilia. This study demonstrates, for the first time, the moderate to strong antibacterial activity of miswak extracts against all tested MDR-pathogens. Methanol extract appears to be a potent antimicrobial agent that could be considered as complementary and alternative medicine against resistant pathogens. Further studies on a large number of MDR organisms are necessary to investigate and standardize the inhibitory effect of miswak extracts against these emerging pathogens. PMID:26904146

  4. Prevention of Bacterial Biofilms Formation on Urinary Catheter by Selected Plant Extracts.

    PubMed

    Adesina, T D; Nwinyi, O C; Olugbuyiro, J A O

    2015-02-01

    In this study, we investigated the feasibility of using Psidium guajava, Mangifera indica and Ocimum gratissimum leaf extracts in preventing Escherichia coli biofilm formation. The plants extractions were done with methanol under cold extraction. The various concentrations 5.0, 10.0 and 20.0 mg mL(-1) were used to coat 63 catheters under mild heat from water bath. Biofilm formation on the catheter was induced using cultures of E. coli. Biofilm formation was evaluated using aerobic plate count and turbidity at 600 nm. From the obtained results, Psidium guajava, Mangifera indica and Ocimum gratissimum delayed the onset of biofilm formation for a week. Ocimum gratissimum coated catheter had the highest inhibitory effect at 5.0, 10.0 and 20.0 mg mL(-1) with bacterial count ranging from 2.2 x 10(5)-7.0 x 10(4) and 5.7 x 10(5)-3.7 x10(5) for 120 and 128 h, respectively. The Psidium guajava coated catheter had the lowest inhibitory effect at 5.0, 10.0 and 20.0 mg mL(-1), with bacterial count ranging between 4.3 x 10(5)-1.9 x 10(3) and 7.7 x 10(5)-3.8 x 10(5) for 120 and 128 h, respectively. Despite the antimicrobial activities, the differences in the activity of these plant extracts were statistically not significant (p < 0.05). PMID:26364356

  5. Electrochemical Biosensor for Rapid and Sensitive Detection of Magnetically Extracted Bacterial Pathogens

    PubMed Central

    Setterington, Emma B.; Alocilja, Evangelyn C.

    2012-01-01

    Biological defense and security applications demand rapid, sensitive detection of bacterial pathogens. This work presents a novel qualitative electrochemical detection technique which is applied to two representative bacterial pathogens, Bacillus cereus (as a surrogate for B. anthracis) and Escherichia coli O157:H7, resulting in detection limits of 40 CFU/mL and 6 CFU/mL, respectively, from pure culture. Cyclic voltammetry is combined with immunomagnetic separation in a rapid method requiring approximately 1 h for presumptive positive/negative results. An immunofunctionalized magnetic/polyaniline core/shell nano-particle (c/sNP) is employed to extract target cells from the sample solution and magnetically position them on a screen-printed carbon electrode (SPCE) sensor. The presence of target cells significantly inhibits current flow between the electrically active c/sNPs and SPCE. This method has the potential to be adapted for a wide variety of target organisms and sample matrices, and to become a fully portable system for routine monitoring or emergency detection of bacterial pathogens. PMID:25585629

  6. Potential applications for Annona squamosa leaf extract in the treatment and prevention of foodborne bacterial disease.

    PubMed

    Dholvitayakhun, Achara; Trachoo, Nathanon; Sakee, Uthai; Cushnie, T P Tim

    2013-03-01

    Foodborne disease is a major public health problem. The present study examined Annona squamosa leaves, which are traditionally used to treat diarrhea and other infections, for their potential to be used in modern food safety or medicine. Active constituents were partially purified by ethanol extraction and column chromatography. MICs of the extract were 62.5 to 125 microg/mL against Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus, and 250 microg/mL against Campylobacter jejuni. In time-kill assays, 500 microg/mL of the extract reduced colony forming unit numbers of C. jejuni almost 10 000-fold within 12 hours. Similar decreases were seen against B. cereus, but over a longer time-frame. LC-MS analysis indicated the presence of reticuline and oxophoebine. Assessment of stability by MIC assay showed activity was heat-labile, with loss of activity greatest following high temperature treatments. Activity was relatively stable at refrigeration temperature. These results indicate A. squamosa has broad-spectrum but heat-labile activity against foodborne bacterial pathogens, and bactericidal activity against B. cereus and C. jejuni. This bactericidal activity is not sufficiently rapid for A. squamosa to be used as a food sanitizer, but the extract could potentially be developed as an additive for refrigerated foods, or a modern treatment for foodborne illness. PMID:23678817

  7. Th1-orientated immunological properties of the bacterial extract OM-85-BV.

    PubMed

    Huber, M; Mossmann, H; Bessler, W G

    2005-05-20

    The bacterial extract OM-85-BV prepared from 21 pathogenic bacterial strains is administered orally to adults and children for the treatment and prevention of recurrent infections of the respiratory tract. We analyzed in vitro and in vivo the immunomodulatory effects of the extract. The lysate acted as a non specific macrophage activator, inducing NO production as well as the translocation of transcription factor NF-kappaB into the nucleus in murine bone marrow-derived macrophages. Besides stimulating unspecifically the immune system, a bacteria-specific humoral immune response was revealed. After oral application, a trend to increase bacteria-specific IgG and IgA in serum was observed. Also a marked increase of bacteria specific IgA in saliva as well as in supernatants of Peyer's patches and mesenteric lymph nodes-derived cell cultures was found. The immunomodulatory properties of the extract were also investigated with respect to shifting the Th1/Th2 bias in an in vivo allergy model. BALB/c mice were orally immunized with OM-85-BV and subsequently sensitized intraperitoneally with the allergen ovalbumin. The group pretreated with OM-85-BV showed a decrease of both total and ovalbumin specific IgE. Accordingly, in spleen cell supernatants the Th1-associated cytokine IFN-gamma was increased, and the Th2-associated cytokine IL-4 was downregulated. Our findings suggest that the immunoprotective effects of OM-85-BV observed in human beings may be correlated to its Th1 augmenting properties. PMID:15946922

  8. Liquid-liquid extraction and liquid chromatography-mass spectrometry detection of curcuminoids from bacterial culture medium.

    PubMed

    Tan, Suryani; Rupasinghe, Thusitha W T; Tull, Dedreia L; Augustin, Mary Ann; Gras, Sally L

    2015-04-15

    Liquid chromatography-mass spectrometry (LC-MS) has been used to detect polyphenolic curcuminoids found in turmeric but studies of metabolism by bacterial and mammalian cells in vitro are compromised by poor recovery from the culture medium. We report a liquid-liquid extraction procedure with ethyl acetate and use LC-MS to quantify extracted curcuminoids. Ethyl acetate allows recoveries of ? 80-86% of curcuminoids from the bacterial growth medium, bacterial cell lysate and combined bacterial cell and growth medium matrices; a clear improvement over acetonitrile where recoveries were ? 25-66%. This optimised method will enable studies of curcuminoid metabolism and may be applicable to other hydrophobic polyphenolic compounds. PMID:25770788

  9. Immunogenicity of an E. coli extract after oral or intraperitoneal administration: induction of antibodies against pathogenic bacterial strains.

    PubMed

    Huber, M; Baier, W; Serr, A; Bessler, W G

    2000-01-01

    For the treatment of recurrent infections of the urinary tract, a bacterial extract (OM-89) consisting of immunostimulating components derived from 18 Escherichia coli strains is orally applied to patients. We investigated in a mouse model the immunogenicity of the bacterial extract after intraperitoneal or oral administration. After repeated administration of the extract, serum IgG and IgA responses against the E. coli strains used for the preparation of OM-89 were obtained. This antisera also recognized a number of bacterial strains isolated from patients with urinary tract and enterohemorrhagic E. coli infections, and bound to a variety of other pathogenic strains. Moreover, the supernatants of cell cultures prepared from the urogenital tract of mice immunized with OM-89 contained increased levels of strain specific and of total IgG and IgA. Our findings may contribute to explain the therapeutic effect of OM-89 demonstrated in clinical studies. PMID:10684989

  10. Induction of apoptosis in cancer cell lines by the Red Sea brine pool bacterial extracts

    PubMed Central

    2013-01-01

    Background Marine microorganisms are considered to be an important source of bioactive molecules against various diseases and have great potential to increase the number of lead molecules in clinical trials. Progress in novel microbial culturing techniques as well as greater accessibility to unique oceanic habitats has placed the marine environment as a new frontier in the field of natural product drug discovery. Methods A total of 24 microbial extracts from deep-sea brine pools in the Red Sea have been evaluated for their anticancer potential against three human cancer cell lines. Downstream analysis of these six most potent extracts was done using various biological assays, such as Caspase-3/7 activity, mitochondrial membrane potential (MMP), PARP-1 cleavage and expression of ?H2Ax, Caspase-8 and -9 using western blotting. Results In general, most of the microbial extracts were found to be cytotoxic against one or more cancer cell lines with cell line specific activities. Out of the 13 most active microbial extracts, six extracts were able to induce significantly higher apoptosis (>70%) in cancer cells. Mechanism level studies revealed that extracts from Chromohalobacter salexigens (P3-86A and P3-86B(2)) followed the sequence of events of apoptotic pathway involving MMP disruption, caspase-3/7 activity, caspase-8 cleavage, PARP-1 cleavage and Phosphatidylserine (PS) exposure, whereas another Chromohalobacter salexigens extract (K30) induced caspase-9 mediated apoptosis. The extracts from Halomonas meridiana (P3-37B), Chromohalobacter israelensis (K18) and Idiomarina loihiensis (P3-37C) were unable to induce any change in MMP in HeLa cancer cells, and thus suggested mitochondria-independent apoptosis induction. However, further detection of a PARP-1 cleavage product, and the observed changes in caspase-8 and -9 suggested the involvement of caspase-mediated apoptotic pathways. Conclusion Altogether, the study offers novel findings regarding the anticancer potential of several halophilic bacterial species inhabiting the Red Sea (at the depth of 15002500m), which constitute valuable candidates for further isolation and characterization of bioactive molecules. PMID:24305113

  11. A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability

    PubMed Central

    Chiba, Akio; Sugimoto, Shinya; Sato, Fumiya; Hori, Seiji; Mizunoe, Yoshimitsu

    2015-01-01

    Biofilm-forming bacteria embedded in polymeric extracellular matrices (ECMs) that consist of polysaccharides, proteins and/or extracellular DNAs (eDNAs) acquire high resistance to antimicrobial agents and host immune systems. To understand molecular mechanisms of biofilm formation and maintenance and to develop therapeutic countermeasures against chronic biofilm-associated infections, reliable methods to isolate ECMs are inevitable. In this study, we refined the ECM extraction method recently reported and evaluated its applicability. Using three Staphylococcus aureus biofilms in which proteins, polysaccharides or eDNAs are major contributors to their integrity, ECMs were extracted using salts and detergents. We found that extraction with 1.5 M sodium chloride (NaCl) could be optimum for not only ECM proteins but also polysaccharides and eDNAs. In addition, long-time incubation was not necessary for efficient ECM isolation. Lithium chloride (LiCl) was comparative to NaCl but is more expensive. In contrast to SDS, NaCl hardly caused leakage of intracellular proteins and did not affect viability of bacterial cells within biofilms. Furthermore, this method is applicable to other bacteria such as Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli and Pseudomonas aeruginosa. Thus, this refined method is very simple, rapid, low cost and non-invasive and could be used for a broad range of applications. PMID:25154775

  12. A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability.

    PubMed

    Chiba, Akio; Sugimoto, Shinya; Sato, Fumiya; Hori, Seiji; Mizunoe, Yoshimitsu

    2015-05-01

    Biofilm-forming bacteria embedded in polymeric extracellular matrices (ECMs) that consist of polysaccharides, proteins and/or extracellular DNAs (eDNAs) acquire high resistance to antimicrobial agents and host immune systems. To understand molecular mechanisms of biofilm formation and maintenance and to develop therapeutic countermeasures against chronic biofilm-associated infections, reliable methods to isolate ECMs are inevitable. In this study, we refined the ECM extraction method recently reported and evaluated its applicability. Using three Staphylococcus aureus biofilms in which proteins, polysaccharides or eDNAs are major contributors to their integrity, ECMs were extracted using salts and detergents. We found that extraction with 1.5 M sodium chloride (NaCl) could be optimum for not only ECM proteins but also polysaccharides and eDNAs. In addition, long-time incubation was not necessary for efficient ECM isolation. Lithium chloride (LiCl) was comparative to NaCl but is more expensive. In contrast to SDS, NaCl hardly caused leakage of intracellular proteins and did not affect viability of bacterial cells within biofilms. Furthermore, this method is applicable to other bacteria such as Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli and Pseudomonas aeruginosa. Thus, this refined method is very simple, rapid, low cost and non-invasive and could be used for a broad range of applications. PMID:25154775

  13. Antibiofilm Activity, Compound Characterization, and Acute Toxicity of Extract from a Novel Bacterial Species of Paenibacillus

    PubMed Central

    Alasil, Saad Musbah; Omar, Rahmat; Yusof, Mohd Yasim

    2014-01-01

    The effectiveness of many antimicrobial agents is currently decreasing; therefore, it is important to search for alternative therapeutics. Our study was carried out to assess the in vitro antibiofilm activity using microtiter plate assay, to characterize the bioactive compounds using Ultra Performance Liquid Chromatography-Diode Array Detection and Liquid Chromatography-Mass Spectrometry and to test the oral acute toxicity on Sprague Dawley rats of extract derived from a novel bacterial species of Paenibacillus strain 139SI. Our results indicate that the crude extract and its three identified compounds exhibit strong antibiofilm activity against a broad range of clinically important pathogens. Three potential compounds were identified including an amino acid antibiotic C8H20N3O4P (MW 253.237), phospholipase A2 inhibitor C21H36O5 (MW 368.512), and an antibacterial agent C14H11N3O2 (MW 253.260). The acute toxicity test indicates that the mortality rate among all rats was low and that the biochemical parameters, hematological profile, and histopathology examination of liver and kidneys showed no significant differences between experimental groups (P > 0.05). Overall, our findings suggest that the extract and its purified compounds derived from novel Paenibacillus sp. are nontoxic exhibiting strong antibiofilm activity against Gram-positive and Gram-negative pathogens that can be useful towards new therapeutic management of biofilm-associated infections. PMID:24790603

  14. Polymeric Cryogel-Based Boronate Affinity Chromatography for Separation of Ribonucleic Acid from Bacterial Extracts.

    PubMed

    Shakya, Akhilesh Kumar; Srivastava, Akshay; Kumar, Ashok

    2015-01-01

    Three-dimensional monolithic columns are preferred stationary phase in column chromatography. Conventional columns based on silica or particles are efficient in bioseparation though associated with limitations of nonspecific interaction and uneven porosity that causes high mass transfer resistance for the movement of big molecules. Cryogels as a monolith column have shown promising application in bioseparation. Cryogels column can be synthesized in the form of a monolith at sub-zero temperature through gelation of pre-synthesized polymers or polymerization of monomers. Cryogels are macroporous and mechanically stable materials. They have open interconnected micron-sized pores with a wide range of porosity (10-200 μm). Current protocol demonstrated the ability of poly(hydroxymethyl methacrylate)-co-vinylphenyl boronic acid p(HEMA-co-VPBA) cryogel matrix for selective separation of RNA from the bacterial crude extract. © 2015 by John Wiley & Sons, Inc. PMID:26623972

  15. Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study used a partial 535 base pair 16S rRNA gene sequence to identify a bacterial isolate. Fatty acid profiles are consistent with the 16S rRNA gene sequence identification of this bacterium. The isolate was obtained from a compost bin in Fort Collins, Colorado, USA. The 16S rRNA gene sequen...

  16. Commercial DNA extraction kits impact observed microbial community composition in permafrost samples.

    PubMed

    Vishnivetskaya, Tatiana A; Layton, Alice C; Lau, Maggie C Y; Chauhan, Archana; Cheng, Karen R; Meyers, Arthur J; Murphy, Jasity R; Rogers, Alexandra W; Saarunya, Geetha S; Williams, Daniel E; Pfiffner, Susan M; Biggerstaff, John P; Stackhouse, Brandon T; Phelps, Tommy J; Whyte, Lyle; Sayler, Gary S; Onstott, Tullis C

    2014-01-01

    The total community genomic DNA (gDNA) from permafrost was extracted using four commercial DNA extraction kits. The gDNAs were compared using quantitative real-time PCR (qPCR) targeting 16S rRNA genes and bacterial diversity analyses obtained via 454 pyrosequencing of the 16S rRNA (V3 region) amplified in single or nested PCR. The FastDNA() SPIN (FDS) Kit provided the highest gDNA yields and 16S rRNA gene concentrations, followed by MoBio PowerSoil() (PS) and MoBio PowerLyzer (PL) kits. The lowest gDNA yields and 16S rRNA gene concentrations were from the Meta-G-Nome (MGN) DNA Isolation Kit. Bacterial phyla identified in all DNA extracts were similar to that found in other soils and were dominated by Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Acidobacteria. Weighted UniFrac and statistical analyses indicated that bacterial community compositions derived from FDS, PS, and PL extracts were similar to each other. However, the bacterial community structure from the MGN extracts differed from other kits exhibiting higher proportions of easily lysed ?- and ?-Proteobacteria and lower proportions of Actinobacteria and Methylocystaceae important in carbon cycling. These results indicate that gDNA yields differ between the extraction kits, but reproducible bacterial community structure analysis may be accomplished using gDNAs from the three bead-beating lysis extraction kits. PMID:24102625

  17. Bacterial removal of quinolizidine alkaloids and other carbon sources from a Lupinus albus aqueous extract.

    PubMed

    Santana, Filomena M C; Pinto, Teresa; Fialho, Arsnio M; S-Correia, Isabel; Empis, Jos M A

    2002-04-10

    Two Gram-negative bacterial strains capable of using lupanine, the predominant quinolizidine alkaloid in Lupinus albus, as a sole carbon source were isolated from soil in which L. albus and L. luteus had been grown [Santana, F. M. et al. J. Ind. Microbiol. 1996, 17, 110-115]. In the present study, we present results suggesting that these isolates are of potential interest for removing lupanine and other quinolizidine alkaloids (QA) from the effluent resulting from the wet processing of Lupinus seeds, at temperatures within the range 20-34 degrees C. Growth in L. albus aqueous extract was diauxic, with a first period of rapid growth leading to the simultaneous consumption of a significant part of the initial concentration of QA (3 g L(-1), being 2 g L(-1) lupanine) and amino acids (1.5 g L(-1)). This period was followed by a second period of slower growth corresponding to the subsequent partial utilization (25%) of the carbohydrates (initial concentration of 20 g L(-1)) together with further removal of QA and amino acids. Despite the differences detected in the susceptibility of the two strains to lupanine toxicity, in particular at supraoptimal temperatures, and in the efficiency of lupanine catabolism, their performance on L. albus extract did not vary significantly. PMID:11929291

  18. Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato

    PubMed Central

    Kwak, A Min; Min, Kyeong Jin; Lee, Sang Yeop

    2015-01-01

    Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding β-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction. PMID:26539048

  19. Water Extract from Spent Mushroom Substrate of Hericium erinaceus Suppresses Bacterial Wilt Disease of Tomato.

    PubMed

    Kwak, A Min; Min, Kyeong Jin; Lee, Sang Yeop; Kang, Hee Wan

    2015-09-01

    Culture filtrates of six different edible mushroom species were screened for antimicrobial activity against tomato wilt bacteria Ralstonia solanacearum B3. Hericium erinaceus, Lentinula edodes (Sanjo 701), Grifola frondosa, and Hypsizygus marmoreus showed antibacterial activity against the bacteria. Water, n-butanol, and ethyl acetate extracts of spent mushroom substrate (SMS) of H. erinaceus exhibited high antibacterial activity against different phytopathogenic bacteria: Pectobacterium carotovorum subsp. carotovorum, Agrobacterium tumefaciens, R. solanacearum, Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, X. axonopodis pv. vesicatoria, X. axonopodis pv. citiri, and X. axonopodis pv. glycine. Quantitative real-time PCR revealed that water extracts of SMS (WESMS) of H. erinaceus induced expressions of plant defense genes encoding β-1,3-glucanase (GluA) and pathogenesis-related protein-1a (PR-1a), associated with systemic acquired resistance. Furthermore, WESMS also suppressed tomato wilt disease caused by R. solanacearum by 85% in seedlings and promoted growth (height, leaf number, and fresh weight of the root and shoot) of tomato plants. These findings suggest the WESMS of H. erinaceus has the potential to suppress bacterial wilt disease of tomato through multiple effects including antibacterial activity, plant growth promotion, and defense gene induction. PMID:26539048

  20. Extraction of copper from an oxidized (lateritic) ore using bacterially catalysed reductive dissolution.

    PubMed

    Nancucheo, Ivan; Grail, Barry M; Hilario, Felipe; du Plessis, Chris; Johnson, D Barrie

    2014-01-01

    An oxidized lateritic ore which contained 0.8 % (by weight) copper was bioleached in pH- and temperature-controlled stirred reactors under acidic reducing conditions using pure and mixed cultures of the acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans. Sulfur was provided as the electron donor for the bacteria, and ferric iron present in goethite (the major ferric iron mineral present in the ore) acted as electron acceptor. Significantly more copper was leached by bacterially catalysed reductive dissolution of the laterite than in aerobic cultures or in sterile anoxic reactors, with up to 78 % of the copper present in the ore being extracted. This included copper that was leached from acid-labile minerals (chiefly copper silicates) and that which was associated with ferric iron minerals in the lateritic ore. In the anaerobic bioreactors, soluble iron in the leach liquors was present as iron (II) and copper as copper (I), but both metals were rapidly oxidized (to iron (III) and copper (II)) when the reactors were aerated. The number of bacteria added to the reactors had a critical role in dictating the rate and yield of copper solubilised from the ore. This work has provided further evidence that reductive bioprocessing, a recently described approach for extracting base metals from oxidized deposits, has the potential to greatly extend the range of metal ores that can be biomined. PMID:24687752

  1. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in th...

  2. The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

    ERIC Educational Resources Information Center

    Falconer, A. C.; Hayes, L. J.

    1986-01-01

    Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

  3. COMPARISON OF RAPID METHODS FOR THE EXTRACTION OF BACTERIAL DNA FROM COLONIC AND CECAL LUMEN CONTENTS OF THE PIG

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increasing use of DNA methodologies to study the microflora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from intestinal samples. Thus, the objective of this study was to determine which extraction methods are most effective for colonic and cecal lumen sampl...

  4. Effect of Punica granatum L. Flower Water Extract on Five Common Oral Bacteria and Bacterial Biofilm Formation on Orthodontic Wire

    PubMed Central

    VAHID DASTJERDI, Elahe; ABDOLAZIMI, Zahra; GHAZANFARIAN, Marzieh; AMDJADI, Parisa; KAMALINEJAD, Mohammad; MAHBOUBI, Arash

    2014-01-01

    Background: Use of herbal extracts and essences as natural antibacterial compounds has become increasingly popular for the control of oral infectious diseases. Therefore, finding natural antimicrobial products with the lowest side effects seems necessary. The present study sought to assess the effect of Punica granatum L. water extract on five oral bacteria and bacterial biofilm formation on orthodontic wire. Methods: Antibacterial property of P. granatum L. water extract was primarily evaluated in brain heart infusion agar medium using well-plate method. The minimum inhibitory concentration and minimum bactericidal concentration were determined by macro-dilution method. The inhibitory effect on orthodontic wire bacterial biofilm formation was evaluated using viable cell count in biofilm medium. At the final phase, samples were fixed and analyzed by Scanning Electron Microscopy. Results: The growth inhibition zone diameter was proportional to the extract concentration. The water extract demonstrated the maximum antibacterial effect on Streptococcus sanguinis ATCC 10556 with a minimum inhibitory concentration of 6.25 mg/ml and maximum bactericidal effect on S. sanguinis ATCC 10556 and S. sobrinus ATCC 27607 with minimum bactericidal concentration of 25 mg/ml. The water extract decreased bacterial biofilm formation by S. sanguinis, S. sobrinus, S. salivarius, S. mutans ATCC 35608 and E. faecalis CIP 55142 by 93.7100%, 40.699.9%, 85.286.5%, 66.484.4% and 35.556.3% respectively. Conclusion: Punica granatum L. water extract had significant antibacterial properties against 5 oral bacteria and prevented orthodontic wire bacterial biofilm formation. However, further investigations are required to generalize these results to the clinical setting. PMID:26171362

  5. Fragmentation of 23S rRNA in Strains of Proteus and Providencia Results from Intervening Sequences in the rrn (rRNA) Genes

    PubMed Central

    Miller, Wayne L.; Pabbaraju, Kanti; Sanderson, Kenneth E.

    2000-01-01

    Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera. Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes. By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation. We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size. Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar. Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred. PMID:10648538

  6. Evaluation of antibacterial activity of crude protein extracts from seeds of six different medical plants against standard bacterial strains

    PubMed Central

    Al Akeel, Raid; Al-Sheikh, Yazeed; Mateen, Ayesha; Syed, Rabbani; Janardhan, K.; Gupta, V.C.

    2013-01-01

    A huge group of natural antimicrobial compounds are active against a large spectrum of bacterial strains causing infectious threat. The present study was conducted to investigate the crude extracts of antimicrobial protein and peptide efficacy from six medicinal plant seeds. Extraction was carried out in Sodium phosphate citrate buffer, and Sodium acetate buffer using different pH. Antimicrobial activities of these plants were determined by the microbiological technique using Agar well diffusion Assay. Extremely strong activity was observed in the seed extracts of Allium ascolinicum extracted in sodium phosphate citrate buffer at pH (5.8) against Proteus vulgaris, Escherichia coli and Staphylococcus aureus with zone of inhibition 17mm, 17mm and 15mm and Rumex vesicarius at pH (7.6), Ammi majus at pH (6.8), Cichorium intybus at pH (7.4) and Cucumis sativus at pH (7.8) also showed better sensitivity against the bacterial strains with zone of inhibition ranges 1610mm and some of the strains were found to be resistant. Antibacterial activity pattern of different plant extracts prepared in sodium acetate buffer pH (6.5), among all the plant seed extracts used Foeniculum vulgare had shown good inhibition in all the bacterial strains used, with zone of inhibition ranges 1112.5mm, The extracts of C. intybus and C. sativus were found to be effective with zone of inhibition 116mm and some of the strains were found to be resistant. Most of the strains found to have shown better sensitivity compared with the standard antibiotic Chloramphenicol (25mcg). Our results showed that the plants used for our study are the richest source for antimicrobial proteins and peptides and they may be used for industrial extraction and isolation of antimicrobial compounds which may find a place in medicine industry as constituents of antibiotics. PMID:24600307

  7. Characteristics and anticancer properties of bacterial cellulose films containing ethanolic extract of mangosteen peel.

    PubMed

    Taokaew, Siriporn; Nunkaew, Natthawut; Siripong, Pongpun; Phisalaphong, Muenduen

    2014-01-01

    Bacterial cellulose (BC) films containing an ethanolic extract of mangosteen peel were prepared and their physical, chemical, and anticancer properties were characterized. The cumulative absorption and release profiles of bioactive compounds in the films were determined based on total phenolic and ?-mangostin content. The BC films were filled with total phenolic compounds expressed as gallic acid equivalent varying from 4.72 to 275.91?mg/cm3 dried film, and ?-mangostin varying from 2.06 to 248.20?mg/cm3 dried film. A Fourier transform infrared spectroscopy evaluation showed that there were weak interactions between the functional groups of the extract and the BC. Decreases in the water absorption capacity and water vapor transmission rate of the modified films were detected. Release studies were performed using Franz diffusion cells. In a non-transdermal system, the release of bioactive compounds from the films depended on concentration, immersion time, and the pH of the dissolution medium. A transdermal diffusion study showed that 59-62% of total phenolic compounds that were initially loaded were released from the films and more than 95% of bioactive compounds released from the films were adsorbed into pig skin. Only very small amount of the bioactive compounds penetrated through pig skin and into phosphate and acetate buffers. In studies of anticancer abilities, the release of 2.0??g/ml ?-mangostin from the BC films could suppress the growth of B16F10 melanoma (approximately 31% survival). With the release of ?-mangostin at greater than 17.4-18.4??g/ml, less than 15 and 5% survival of B16F10 melanoma and MCF-7 breast cancer cells, respectively, was observed. PMID:24802115

  8. METAXA2: improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data.

    PubMed

    Bengtsson-Palme, Johan; Hartmann, Martin; Eriksson, Karl Martin; Pal, Chandan; Thorell, Kaisa; Larsson, Dan Gran Joakim; Nilsson, Rolf Henrik

    2015-11-01

    The ribosomal rRNA genes are widely used as genetic markers for taxonomic identification of microbes. Particularly the small subunit (SSU; 16S/18S) rRNA gene is frequently used for species- or genus-level identification, but also the large subunit (LSU; 23S/28S) rRNA gene is employed in taxonomic assignment. The METAXA software tool is a popular utility for extracting partial rRNA sequences from large sequencing data sets and assigning them to an archaeal, bacterial, nuclear eukaryote, mitochondrial or chloroplast origin. This study describes a comprehensive update to METAXA - METAXA2 - that extends the capabilities of the tool, introducing support for the LSU rRNA gene, a greatly improved classifier allowing classification down to genus or species level, as well as enhanced support for short-read (100 bp) and paired-end sequences, among other changes. The performance of METAXA2 was compared to other commonly used taxonomic classifiers, showing that METAXA2 often outperforms previous methods in terms of making correct predictions while maintaining a low misclassification rate. METAXA2 is freely available from http://microbiology.se/software/metaxa2/. PMID:25732605

  9. Bacterial extract (OM-89) specific and non specific immunomodulation in rheumatoid arthritis patients.

    PubMed

    Toussirot, Eric; Robinet, Eric; Saas, Philippe; Chabod, Jacqueline; Aug, Benot; Cozma, Gabriel; Tiberghien, Pierre; Roudier, Jean; Wendling, Daniel

    2006-06-01

    The Escherichia Coli bacterial extract (OM-89) is used in the treatment of rheumatoid arthritis (RA). We evaluated the immunological changes induced by oral administration of OM-89 in 12 RA patients (polyclonal T cell reactivity to PHA, T cell precursor frequencies specific for OM-89 and Tetanus toxoid (TT), a control antigen and the release of Th1 (IFN-gamma, TNF-alpha), Th2 (IL-4) and T regulatory 1 cell (Tr1) (IL-10) cytokines in the supernatants of PBMC cultures. Stimulation index in response to PHA decreased at month 3 as well as T cell precursor frequencies specific for TT with similar trends for OM-89-specific T cell precursor frequencies. OM-89 induced a strong production of IL-10, a significant decrease in IL-4 production while TNF-alpha and IFN-gamma production tended to decrease during the study. Our results suggest that OM-89 has immunomodulatory properties by inducing changes in PBMC cytokines release suggestive of an induced Tr1 response to OM-89. PMID:16891218

  10. Immunostimulatory properties of the bacterial extract OM-89 in vitro and in vivo.

    PubMed

    Bessler, Wolfgang G; vor dem Esche, Ulrich; Zgaga-Griesz, Andrea; Ataullakhanov, Ravshan

    2010-01-01

    The therapeutic agent OM-89 (Uro-Vaxom) contains lyophilized immunostimulating fractions from 18 Escherichia coil strains. It has been shown to provide protection against recurrent urinary tract infections in humans and against bacterial infections in mice. Here the immunostimulatory properties of OM-89 were investigated by in vitro and in vivo assays. In vitro the activation of murine spleen cells by the AlamarBlue assay was determined. OM-89 was effective in stimulating the metabolism of spleen cells within a concentration range of 0.625-2.5 mg/ml. The activation of murine bone marrow-derived macrophages by OM-89 was shown by the induction of NO production; OM-89 was a most effective stimulant at concentrations around 6 mg/ml. In the human system, the effect of OM-89 was tested in vitro:metabolic activity of peripheral blood lymphocytes (PBL) was stimulated starting at concentrations of approx. 250 microg/ml, and the spontaneous apoptosis of polymorphonuclear neutrophils (PMN) was reduced starting at OM-89 concentrations of approx. 100 microg/ml. Finally, in a mouse model, the in vivo protection of mice against infection with Salmonella typhimurium after the oral administration of OM-89 was tested (100 mg in a volume of 0.5 ml once a day for 10 consecutive days). The extract proved to be effective: 90% of the OM-89-treated animals survived compared to 58% of the untreated control group. PMID:20648922

  11. Bacterial extract OM-85 BV protects mice against experimental chronic rhinosinusitis

    PubMed Central

    Tao, Yanli; Yuan, Tiejun; Li, Xuechang; Yang, Shuqin; Zhang, Fanping; Shi, Li

    2015-01-01

    Objectives: To investigate the therapeutic effects of OM-85 BV as an adjunctive treatment on experimental chronic rhinosinusitis (CRS) in mice. Methodology: Female BALB/c mice aged 8-12 weeks were sensitized and administrated by intranasal Aspergillus fumigatis (AF) three times per week for 1 week, 3 weeks, 2 months and 3 months (n = 10 each time point). The mice were randomly and equally assigned to four groups: normal control group, model group, OM-85-BV plus amoxicillin group, and isolated amoxicillin group. Inflammatory changes were determined by hematoxylin-eosin (HE) staining. The expression levels of suppressor of cytokine signaling (SOCS) 1, SOCS3, tumor necrosis factor (TNF)-?, and interferon (IFN)-? in samples were assessed by using real-time PCR (RT-PCR) and Western blotting. Results: There were significantly inflammatory and structural changes between the model and other groups. Compared to the model group, the mRNA expression levels of SOCS1, SOCS3, TNF-?, and IFN-? were significantly decreased in OM-85-BV plus amoxicillin group and isolated amoxicillin group, along with the protein levels. Conclusion: The bacterial extract OM-85 BV is a low-cost alternatively adjunctive drug to treat CRS with simple oral administration, good safety, and few side effects. PMID:26261565

  12. Activation of natural killer cells and cytokine production in man by bacterial extracts.

    PubMed

    Wybran, J; Libin, M; Schandene, L

    1989-01-01

    Broncho-Vaxon (OM-85 BV) is a bacterial extract of eight bacterias usually involved in the respiratory tract infections. Since Broncho-Vaxom is clinically active in decreasing the incidence of such infections, its immunological effect was investigated, in vitro, using peripheral blood mononuclear cells (PBMC). The experimental data indicate that Broncho-Vaxom can modulate various immune functions. It was shown, using a radioimmunoassay for these cytokines, that Broncho-Vaxom will spontaneously enhance TNF alpha and IL-2 production whereas it has no action on IF gamma production. However, when the PBMC are stimulated with PHA, an increased production for IF gamma, TNF alpha and IL-2 was observed suggesting that, under appropriate conditions, Broncho-Vaxom enhances the production of these cytokines. In other experiments, Broncho-Vaxom was shown to markedly increase the natural killer activity of PBMC. All these results demonstrate that Broncho-Vaxom is an immunomodulator affecting multiple immunological mechanisms including the activation of natural killer cells, of monocytes and of T cells through direct mechanisms or through the cytokine cascade. PMID:2503554

  13. Immunotherapy with an oral bacterial extract (OM-85 BV) for upper respiratory infections.

    PubMed

    Paupe, J

    1991-01-01

    The efficacy of Broncho-Vaxom/Imocur (OM-85 BV), an orally administered lyophilized bacterial extract, for recurrent respiratory and ear, nose and throat (ENT) infections was evaluated in 116 children aged 6 months to 19 years by comparing its activity in 61 children with that of a placebo in 55 children. The study was randomized, double-blind, and comprised a 90-day treatment period followed by a 90-day follow-up period without test drugs. Over the 180 days, 39.5% of patients taking OM-85 BV remained free from infection compared with 16.5% on placebo (p less than 0.01). 44% on OM-85 BV did not need antibiotics compared with 23.5% on placebo (p less than 0.05). These differences were even greater in the subgroup of children aged 6 years and less (34 vs. 3.5% for the absence of infections, p less than 0.01 and 37 vs. 10% for the need of antibiotics, p less than 0.05). Tolerance to OM-85 BV was excellent, and laboratory investigations showed no abnormalities attributable to this product. This work confirms that the immunomodulator OM-85 BV is an effective immunotherapy for recurrent respiratory and ENT infections in children. PMID:1745846

  14. Bacterial extract OM85-BV induces interleukin-12-dependent IFN-gamma production by human CD4+ T cells.

    PubMed

    Byl, B; Libin, M; Grard, M; Clumeck, N; Goldman, M; Mascart-Lemone, F

    1998-10-01

    To obtain insight into the possible mode of action of bacterial extracts used as immunostimulants in Europe, we used the ELISPOT technique to investigate the effects of one of them (OM85-BV, Broncho-Vaxom) on interferon-y (IFN-gamma) production by peripheral blood mononuclear cells (PBMC). We found that (1) OM85-BV stimulates IFN-gamma secretion by PBMC from normal individuals and human immunodeficiency virus (HIV)-infected patients, (2) CD4+ cells represent the major source of IFN-gamma produced in response to OM85-BV, and (3) this effect of OM85-BV involves the induction of interleukin-12 (IL-12) secretion by accessory cells. We conclude that bacterial extracts might enhance antimicrobial defenses by eliciting IL-12-dependent IFN-gamma synthesis by CD4+ T cells. PMID:9809616

  15. Seasonal and Spatial Variability in Lake Michigan Sediment Small-Subunit rRNA Concentrations

    PubMed Central

    MacGregor, Barbara J.; Moser, Duane P.; Baker, Brett J.; Alm, Elizabeth W.; Maurer, Max; Nealson, Kenneth H.; Stahl, David A.

    2001-01-01

    We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4°C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations. PMID:11525985

  16. Anti-bacterial activity and brine shrimp lethality bioassay of methanolic extracts of fourteen different edible vegetables from Bangladesh

    PubMed Central

    Ullah, M. Obayed; Haque, Mahmuda; Urmi, Kaniz Fatima; Zulfiker, Abu Hasanat Md.; Anita, Elichea Synthi; Begum, Momtaj; Hamid, Kaiser

    2013-01-01

    Objective To investigate the antibacterial and cytotoxic activity of fourteen different edible vegetables methanolic extract from Bangladesh. Methods The antibacterial activity was evaluated using disc diffusion assay method against 12 bacteria (both gram positive and gram negative). The plant extracts were also screened for cytotoxic activity using the brine shrimp lethality bioassay method and the lethal concentrations (LC50) were determined at 95% confidence intervals by analyzing the data on a computer loaded with Finney Programme. Results All the vegetable extracts showed low to elevated levels of antibacterial activity against most of the tested strains (zone of inhibition=5-28 mm). The most active extract against all bacterial strains was from Xanthium indicum which showed remarkable antibacterial activity having the diameter of growth inhibition zone ranging from 12 to 28 mm followed by Alternanthera sessilis (zone of inhibition=6-21 mm). All extracts exhibited considerable general toxicity towards brine shrimps. The LC50 value of the tested extracts was within the range of 8.447 to 60.323 g/mL with respect to the positive control (vincristine sulphate) which was 0.91 g/mL. Among all studied extracts, Xanthium indicum displayed the highest cytotoxic effect with LC50 value of 8.447 g/mL. Conclusions The results of the present investigation suggest that most of the studied plants are potentially good source of antibacterial and anticancer agents. PMID:23570009

  17. Identification of the Microbiota in Carious Dentin Lesions Using 16S rRNA Gene Sequencing

    PubMed Central

    Obata, Junko; Takeshita, Toru; Shibata, Yukie; Yamanaka, Wataru; Unemori, Masako; Akamine, Akifumi; Yamashita, Yoshihisa

    2014-01-01

    While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 476 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating. PMID:25083880

  18. Selective Enhancement of Systemic Th1 Immunity in Immunologically Immature Rats with an Orally Administered Bacterial Extract

    PubMed Central

    Bowman, L. M.; Holt, P. G.

    2001-01-01

    Infant rats primed during the first week of life with soluble antigen displayed adult-equivalent levels of T-helper 2 (Th2)-dependent immunological memory development as revealed by production of secondary immunoglobulin G1 (IgG1) antibody responses to subsequent challenge, but in contrast to adults failed to prime for Th1-dependent IgG2b responses. We demonstrate that this Th2 bias in immune function can be redressed by oral administration to neonates of a bacterial extract (Broncho-Vaxom OM-85) comprising lyophilized fractions of several common respiratory tract bacterial pathogens. Animals given OM-85 displayed a selective upregulation in primary and secondary IgG2b responses, accompanied by increased gamma interferon and decreased interleukin-4 production (both antigen specific and polyclonal), and increased capacity for development of Th1-dependent delayed hypersensitivity to the challenge antigen. We hypothesize that the bacterial extract functions via enhancement of the process of postnatal maturation of Th1 function, which is normally driven by stimuli from the gastrointestinal commensal microflora. PMID:11349036

  19. Selective enhancement of systemic Th1 immunity in immunologically immature rats with an orally administered bacterial extract.

    PubMed

    Bowman, L M; Holt, P G

    2001-06-01

    Infant rats primed during the first week of life with soluble antigen displayed adult-equivalent levels of T-helper 2 (Th2)-dependent immunological memory development as revealed by production of secondary immunoglobulin G1 (IgG1) antibody responses to subsequent challenge, but in contrast to adults failed to prime for Th1-dependent IgG2b responses. We demonstrate that this Th2 bias in immune function can be redressed by oral administration to neonates of a bacterial extract (Broncho-Vaxom OM-85) comprising lyophilized fractions of several common respiratory tract bacterial pathogens. Animals given OM-85 displayed a selective upregulation in primary and secondary IgG2b responses, accompanied by increased gamma interferon and decreased interleukin-4 production (both antigen specific and polyclonal), and increased capacity for development of Th1-dependent delayed hypersensitivity to the challenge antigen. We hypothesize that the bacterial extract functions via enhancement of the process of postnatal maturation of Th1 function, which is normally driven by stimuli from the gastrointestinal commensal microflora. PMID:11349036

  20. Yeast Cell Wall Extract Induces Disease Resistance against Bacterial and Fungal Pathogens in Arabidopsis thaliana and Brassica Crop

    PubMed Central

    Narusaka, Mari; Minami, Taichi; Iwabuchi, Chikako; Hamasaki, Takashi; Takasaki, Satoko; Kawamura, Kimito; Narusaka, Yoshihiro

    2015-01-01

    Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods. PMID:25565273

  1. Yeast cell wall extract induces disease resistance against bacterial and fungal pathogens in Arabidopsis thaliana and Brassica crop.

    PubMed

    Narusaka, Mari; Minami, Taichi; Iwabuchi, Chikako; Hamasaki, Takashi; Takasaki, Satoko; Kawamura, Kimito; Narusaka, Yoshihiro

    2015-01-01

    Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods. PMID:25565273

  2. Inhibitory activity of cranberry extract on the bacterial adhesiveness in the urine of women: an ex-vivo study.

    PubMed

    Tempera, G; Corsello, S; Genovese, C; Caruso, F E; Nicolosi, D

    2010-01-01

    Strains of uropathogenic E. coli are responsible for approximately 90% of community-acquired, uncomplicated cystitis, and fimbriae represent the adhesive factors enabling E. coli to be anchored to uroepithelial cells in the first step of the infectious process. Recently, a few studies have shown that a correlation between the consumption of cranberry (Vaccinium macrocarpon) and prevention of UTI is related to the ability of proanthocyanidins to reduce the bacterial adhesion to uroepithelial cells. In this study we evaluate the inhibitory activity of urine of healthy women treated with tablets containing cranberry extract on the adhesiveness of E. coli to uroepithelial human cells. Two groups of 12 female volunteers each, aged between 18 and 65 years, were enrolled, one group with negative history and one group with positive history of recurrent cystitis. Subjects were treated with the active product or placebo in a random, cross-over, double-blinded sequence for one week in each of the two treatment sequences. Urine samples were collected at the beginning and the end of each study period. Tests of bacterial adhesiveness were performed with two strains of E. coli (ATCC 25922 and ATCC 35218) on HT1376 human bladder carcinoma cells. Significant reductions of bacterial adhesiveness were observed in women who received cranberry extract (-50.9%; p less than 0.0001), regardless of their medical history and the treatment period in the cross-over sequence. No changes were observed with placebo (-0.29%; n.s.). This ex-vivo study showed that the assumption of cranberry extract in suitable amounts can have an anti-adhesive activity on uropathogenic E. coli. PMID:20646356

  3. Immunoglobulin isotype isolated from human placental extract does not interfere in complement-mediated bacterial opsonization within the wound milieu

    PubMed Central

    Sharma, Kanika; Bhattacharyya, Debasish

    2015-01-01

    The wound healing potency of an aqueous extract of placenta can be evaluated through the presence of numerous regulatory components. The presence of glycans was detected by thin layer chromatography and fluorophore-assisted carbohydrate electrophoresis. Mass spectrometric analysis revealed the existence of multiple fragments of immunoglobulin G (IgG). IgG was present in the extract at a concentration of 25.2 ± 3.97 μg/ml. IgG possesses anti-complementary activity by diverting the complement activation from target surface. Thus, effect of placental IgG on complement–bacteria interaction was investigated through classical and alternative pathway and the preparation was ascertained to be safe with respect to their interference in the process of bacterial opsonization. PMID:25984442

  4. First experience of a multicenter external quality assessment of molecular 16S rRNA gene detection in bone and joint infections.

    PubMed

    Plouzeau, Chloé; Bémer, Pascale; Valentin, Anne Sophie; Héry-Arnaud, Geneviève; Tandé, Didier; Jolivet-Gougeon, Anne; Vincent, Pascal; Kempf, Marie; Lemarié, Carole; Guinard, Jérôme; Bret, Laurent; Cognée, Anne Sophie; Gibaud, Sophie; Burucoa, Christophe; Corvec, Stéphane

    2015-02-01

    The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI. PMID:25411177

  5. First Experience of a Multicenter External Quality Assessment of Molecular 16S rRNA Gene Detection in Bone and Joint Infections

    PubMed Central

    Bmer, Pascale; Valentin, Anne Sophie; Hry-Arnaud, Genevive; Tand, Didier; Jolivet-Gougeon, Anne; Vincent, Pascal; Kempf, Marie; Lemari, Carole; Guinard, Jrme; Bret, Laurent; Cogne, Anne Sophie; Gibaud, Sophie; Burucoa, Christophe; Corvec, Stphane

    2014-01-01

    The objective of this study was to assess the performance of seven French laboratories for 16S rRNA gene detection by real-time PCR in the diagnosis of bone and joint infection (BJI) to validate a large multicenter study. External quality control (QC) was required owing to the differences in extraction procedures and the molecular equipment used in the different laboratories. Three proficiency sets were organized, including four bacterial DNA extracts and four bead mill-pretreated osteoarticular specimens. Extraction volumes, 16S rRNA gene primers, and sequencing interpretation rules were standardized. In order to assess each laboratory's ability to achieve the best results, scores were assigned, and each QC series was classified as optimal, acceptable, or to be improved. A total of 168 QCs were sent, and 160 responses were analyzed. The expected results were obtained for 93.8%, with the same proportion for extracts (75/80) and clinical specimens (75/80). For the specimens, there was no significant difference between manual and automated extraction. This QC demonstrated the ability to achieve good and homogeneous results using the same 16S rRNA gene PCR with different equipment and validates the possibility of high-quality multicenter studies using molecular diagnosis for BJI. PMID:25411177

  6. The oral administration of bacterial extracts prevents asthma via the recruitment of regulatory T cells to the airways.

    PubMed

    Navarro, S; Cossalter, G; Chiavaroli, C; Kanda, A; Fleury, S; Lazzari, A; Cazareth, J; Sparwasser, T; Dombrowicz, D; Glaichenhaus, N; Julia, V

    2011-01-01

    The prevalence of asthma has steadily increased during the last decade, probably as the result of changes in the environment, including reduced microbial exposure during infancy. Accordingly, experimental studies have shown that deliberate infections with live pathogens prevent the development of allergic airway diseases in mice. Bacterial extracts are currently used in children suffering from repeated upper respiratory tract infections. In the present study, we have investigated whether bacterial extracts, commercially available as Broncho-Vaxom (BV), could prevent allergic airway disease in mice. Oral treatment with BV suppressed airway inflammation through interleukin-10 (IL-10)-dependent and MyD88 (myeloid differentiation primary response gene (88))-dependent mechanisms and induced the conversion of FoxP3 (forkhead box P3)(-) T cells into FoxP3(+) regulatory T cells. Furthermore, CD4(+) T cells purified from the trachea of BV-treated mice conferred protection against airway inflammation when adoptively transferred into sensitized mice. Therefore, treatment with BV could possibly be a safe and efficient strategy to prevent the development of allergic diseases in children. PMID:20811345

  7. Long term effect of methylparathion contamination on soil microbial community diversity estimated by 16S rRNA gene cloning.

    PubMed

    Zhang, Ruifu; Jiang, Jiandong; Gu, Ji-Dong; Li, Shunpeng

    2006-08-01

    The long-term effects of methylparathion contamination on the diversity of soil microbial community was investigated by a culture-independent approach using small subunit ribosomal RNA (SSU rRNA) gene-based cloning. Microbial DNA extracted from both the control soil sample and methylparathion contaminated soil sample was subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences. From the PCR amplification product, clone libraries were constructed for both samples. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and HhaI. A total of 603 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, the phylotype richness, frequency distribution (evenness) of the two clone libraries were compared by using a variety of diversity indices. Phylogenetic analysis of the sequences of the dominant phylotypes revealed that the bacterial communities changed noticeably. In the control soil, the dominant bacterial groups included a member of a novel bacterial division, the bacillus genus, and a member of alpha-proteobacteria, while in methylparathion contaminated soil, the dominant phylotypes were replaced by a member of the flexibactera-cytophaga-bacteroides division and two members of the gamma-proteobacteria subdivision. This is the first report of the long-term effects of methylparathion (one of the major pesticides widely used in developing countries) on soil microbial community diversity and structure by a culture-independent method, and provides the evidences to assess the long-term environmental toxicological effects of methylparathion from the microbial community viewpoint. PMID:16816989

  8. Bacterial extracts for the prevention of acute exacerbations in chronic obstructive pulmonary disease: a point of view.

    PubMed

    Cazzola, Mario; Rogliani, Paola; Curradi, Giacomo

    2008-03-01

    Given the high prevalence of chronic obstructive pulmonary disease (COPD), the impact of exacerbations on quality of life, and the costs incurred, effective ways for the prevention of exacerbations, and for reductions in the severity and duration of COPD symptoms are needed. Bacterial immunostimulation has been advocated as a management strategy in COPD for the purposes of preventing acute exacerbations. In particular, it suggests that the use of oral multicomponent vaccines may reduce the severity and duration of acute episodes. The way in which bacterial extracts may exert their effects is not fully understood although a number of possible specific mechanisms have been suggested. Given the high prevalence of COPD worldwide and the high cost of acute exacerbations, some cost-effectiveness analyses suggest that bacterial immunostimulants may become a key element in the improved control of this condition. Nonetheless, larger and longer clinical trials are needed to investigate efficacy before oral vaccination could be recommended as part of the routine clinical management of COPD, mainly in advanced COPD. It remains also to be investigated whether this protective effect may be additive to the other treatments. In any case, it is well known that for Streptococcus pneumoniae, non-typable Haemophilus influenzae and Moraxella catarrhalis, recurrent infections occur because of strain heterogeneity. Therefore, a single or even multiple strain vaccine with a killed whole cell formulation is possibly not the ideal vaccine. Moreover, the method of inactivation can affect the immunogenicity of essential antigens through denaturation. For this reason, the efficacy of bacterial immunostimulants should not only be assessed but also compared. PMID:18164190

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

    PubMed Central

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

    2014-01-01

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

  10. Illumina amplicon sequencing of 16S rRNA tag reveals bacterial community development in the rhizosphere of apple nurseries at a replant disease site and a new planting site.

    PubMed

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

    2014-01-01

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

  11. Phylogenetic Differences in Attached and Free-Living Bacterial Communities in a Temperate Coastal Lagoon during Summer, Revealed via High-Throughput 16S rRNA Gene Sequencing

    PubMed Central

    Mohit, Vani; Archambault, Philippe; Toupoint, Nicolas

    2014-01-01

    Most of what is known about coastal free-living and attached bacterial diversity is based on open coasts, with high particulate and nutrient riverine supply, terrestrial runoffs, and anthropogenic activities. The Magdalen Islands in the Gulf of St. Lawrence (Canada) are dominated by shallow lagoons with small, relatively pristine catchments and no freshwater input apart from rain. Such conditions provided an opportunity to investigate coastal free-living and attached marine bacterial diversity in the absence of confounding effects of steep freshwater gradients. We found significant differences between the two communities and marked temporal patterns in both. Taxonomic richness and diversity were greater in the attached than in the free-living community, increasing over summer, especially within the least abundant bacterial phyla. The highest number of reads fell within the SAR 11 clade (Pelagibacter, Alphaproteobacteria), which dominated free-living communities. The attached communities had deeper phylum-level diversity than the free-living fraction. Distance-based redundancy analysis indicated that the particulate organic matter (POM) concentration was the main variable separating early and late summer samples with salinity and temperature changes also significantly correlated to bacterial community structure. Our approach using high-throughput sequencing detected differences in free-living versus attached bacteria in the absence of riverine input, in keeping with the concept that marine attached communities are distinct from cooccurring free-living taxa. This diversity likely reflects the diverse microhabitats of available particles, implying that the total bacterial diversity in coastal systems is linked to particle supply and variability, with implications for understanding microbial biodiversity in marine systems. PMID:24463966

  12. EFFECT OF SOLVENT AND EXTRACTION METHODS ON THE BACTERIAL MUTAGENICITY OF SIDESTREAM CIGARETTE SMOKE

    EPA Science Inventory

    The mutagenic activity of sidestream cigarette smoke particles was estimated by testing sidestream cigarette smoke particles which had been collected under controlled burning conditions in the laboratory. Two different extraction methods (Soxhlet and ultrasonic agitation) and 3 d...

  13. Bacterial and fungal DNA extraction from positive blood culture bottles: a manual and an automated protocol.

    PubMed

    Mki, Minna

    2015-01-01

    When adapting a gene amplification-based method in a routine sepsis diagnostics using a blood culture sample as a specimen type, a prerequisite for a successful and sensitive downstream analysis is the efficient DNA extraction step. In recent years, a number of in-house and commercial DNA extraction solutions have become available. Careful evaluation in respect to cell wall disruption of various microbes and subsequent recovery of microbial DNA without putative gene amplification inhibitors should be conducted prior selecting the most feasible DNA extraction solution for the downstream analysis used. Since gene amplification technologies have been developed to be highly sensitive for a broad range of microbial species, it is also important to confirm that the used sample preparation reagents and materials are bioburden-free to avoid any risks for false-positive result reporting or interference of the diagnostic process. Here, one manual and one automated DNA extraction system feasible for blood culture samples are described. PMID:25319779

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

    PubMed Central

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

    2015-01-01

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

  15. Estimating bacterial diversity for ecological studies: methods, metrics, and assumptions.

    PubMed

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

    2015-01-01

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

  16. Inhibition of Bacterial Quorum Sensing by Extracts from Aquatic Fungi: First Report from Marine Endophytes

    PubMed Central

    Martín-Rodríguez, Alberto J.; Reyes, Fernando; Martín, Jesús; Pérez-Yépez, Juan; León-Barrios, Milagros; Couttolenc, Alan; Espinoza, César; Trigos, Ángel; Martín, Víctor S.; Norte, Manuel; Fernández, José J.

    2014-01-01

    In our search for quorum-sensing (QS) disrupting molecules, 75 fungal isolates were recovered from reef organisms (endophytes), saline lakes and mangrove rhizosphere. Their QS inhibitory activity was evaluated in Chromobacterium violaceum CVO26. Four strains of endophytic fungi stood out for their potent activity at concentrations from 500 to 50 μg mL−1. The molecular characterization, based on the internal transcribed spacer (ITS) region sequences (ITS1, 5.8S and ITS2) between the rRNA of 18S and 28S, identified these strains as belonging to four genera: Sarocladium (LAEE06), Fusarium (LAEE13), Epicoccum (LAEE14), and Khuskia (LAEE21). Interestingly, three came from coral species and two of them came from the same organism, the coral Diploria strigosa. Metabolic profiles obtained by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) suggest that a combination of fungal secondary metabolites and fatty acids could be the responsible for the observed activities. The LC-HRMS analysis also revealed the presence of potentially new secondary metabolites. This is, to the best of our knowledge, the first report of QS inhibition by marine endophytic fungi. PMID:25415350

  17. Secondary Metabolites Control the Associated Bacterial Communities of Saprophytic Basidiomycotina Fungi

    PubMed Central

    de Carvalho, Maira Peres; Trck, Patrick; Abraham, Wolf-Rainer

    2015-01-01

    Fungi grow under humid conditions and are, therefore, prone to biofilm infections. A 16S rRNA fingerprint analysis was performed on 49 sporocarps of Basidiomycotina in order to determine whether they are able to control these biofilms. Ninety-five bacterial phylotypes, comprising 4 phyla and 10 families, were identified. While ectomycorrhizal fungi harbored the highest bacterial diversity, saprophytic fungi showed little or no association with bacteria. Seven fungal species were screened for antimicrobial and antibiofilm activities. Biofilm formation and bacterial growth was inhibited by extracts obtained from saprophytic fungi, which confirmed the hypothesis that many fungi modulate biofilm colonization on their sporocarps. PMID:25904019

  18. Bacterial Biodegradation of Extractives and Patterns of Bordered Pit Membrane Attack in Pine Wood

    PubMed Central

    Burnes, Todd A.; Blanchette, Robert A.; Farrell, Roberta L.

    2000-01-01

    Wood extractives, commonly referred to as pitch, cause major problems in the manufacturing of pulp and paper. Treatment of nonsterile southern yellow pine chips for 14 days with Pseudomonas fluorescens, Pseudomonas sp., Xanthomonas campestris, and Serratia marcescens reduced wood extractives by as much as 40%. Control treatments receiving only water lost 11% of extractives due to the growth of naturally occurring microorganisms. Control treatments were visually discolored after the 14-day incubation, whereas bacterium-treated wood chips were free of dark staining. Investigations using P. fluorescens NRRL B21432 showed that all individual resin and fatty acid components of the pine wood extractives were substantially reduced. Micromorphological observations showed that bacteria were able to colonize resin canals, ray parenchyma cells, and tracheids. Tracheid pit membranes within bordered pit chambers were degraded after treatment with P. fluorescens NRRL B21432. P. fluorescens and the other bacteria tested appear to have the potential for biological processing to substantially reduce wood extractives in pine wood chips prior to the paper making process so that problems associated with pitch in pulp mills can be controlled. PMID:11097890

  19. Structure-Activity Relationships of Antimicrobial Gallic Acid Derivatives from Pomegranate and Acacia Fruit Extracts against Potato Bacterial Wilt Pathogen.

    PubMed

    Farag, Mohamed A; Al-Mahdy, Dalia A; Salah El Dine, Riham; Fahmy, Sherifa; Yassin, Aymen; Porzel, Andrea; Brandt, Wolfgang

    2015-06-01

    Bacterial wilts of potato, tomato, pepper, and or eggplant caused by Ralstonia solanacearum are among the most serious plant diseases worldwide. In this study, the issue of developing bactericidal agents from natural sources against R. solanacearum derived from plant extracts was addressed. Extracts prepared from 25 plant species with antiseptic relevance in Egyptian folk medicine were screened for their antimicrobial properties against the potato pathogen R. solancearum by using the disc-zone inhibition assay and microtitre plate dilution method. Plants exhibiting notable antimicrobial activities against the tested pathogen include extracts from Acacia arabica and Punica granatum. Bioactivity-guided fractionation of A. arabica and P. granatum resulted in the isolation of bioactive compounds 3,5-dihydroxy-4-methoxybenzoic acid and gallic acid, in addition to epicatechin. All isolates displayed significant antimicrobial activities against R. solanacearum (MIC values 0.5-9 mg/ml), with 3,5-dihydroxy-4-methoxybenzoic acid being the most effective one with a MIC value of 0.47 mg/ml. We further performed a structure-activity relationship (SAR) study for the inhibition of R. solanacearum growth by ten natural, structurally related benzoic acids. PMID:26080741

  20. Detection and identification of Legionella species in hospital water supplies through Polymerase Chain Reaction (16S rRNA)

    PubMed Central

    2014-01-01

    Legionella spp. are important waterborne pathogens that are normally transmitted through aerosols. The present work was conducted to investigate the presence of Legionella spp. and its common species in hospital water supplies. Considering the limitations of culture method, polymerase chain reaction (PCR) assays were developed to detect the gene 16S rRNA irrespective of the bacterial serotype. Four well-established DNA extraction protocols (freeze & thaw and phenol-chloroform as two manual protocols and two commercial kits) were tested and evaluated to release DNA from bacterial cells. A total of 45 samples were collected from seven distinct hospitals sites during a period of 10months. The PCR assay was used to amplify a 654-bp fragment of the 16S rRNA gene. Legionella were detected in 13 samples (28.9%) by all of the methods applied for DNA extraction. Significant differences were noted in the yield of extracted nucleic acids. Legionella were not detected in any of the samples when DNA extraction by freeze & thaw was used. Excluding this method and comparing manual protocol with commercial kits, Kappa coefficient was calculated as 0.619 with p?extraction with Bioneer kit exhibited a higher sensitivity than classical Qiagen. Showerheads and cold-water taps were the most and least contaminated sources with 55.5 and 9 percent positive samples, respectively. Moreover two positive samples were identified for species by DNA sequencing and submitted to the Gene Bank database with accession Nos. FJ480932 and FJ480933. The results obtained showed that despite the advantages of molecular assays in Legionella tracing in environmental sources, the use of optimised DNA extraction methods is critical. PMID:24860661

  1. Effect of solvent and extraction methods on the bacterial mutagenicity of sidestream cigarette smoke

    SciTech Connect

    Morin, R.S.; Tulis, J.J.; Claxton, L.D.

    1987-01-01

    The mutagenic activity of sidestream cigarette-smoke particles was estimated by testing sidestream cigarette-smoke particles that had been collected under controlled burning conditions in the laboratory. Two different extraction methods (Soxhlet and ultrasonic agitation) and 3 different solvents (dichloromethane, methanol, and acetone) were compared for their efficiencies in the extraction of compounds from sidestream cigarette-smoke particles that are mutagenic in the Ames test. The mutagenic activity of the sidestream smoke particles was estimated to be 15,000-20,000 revertants per cigarette in TA98 with metabolic activation and 12,000-17,000 revertants per cigarette in TA100 without metabolic activation.

  2. Inhibition of bacterial, fungal and plant growth by testa extracts of Citrullus genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Watermelon (Citrullus lanatus var. lanatus (Thunb.) Matsum & Nakai) seed exudates inhibit germination and seedling growth of several plant species and growth of pathogenic fungi and bacteria. This study was conducted to determine if extractable components in testae contribute to the inhibition. T...

  3. An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

    PubMed Central

    Holmes, Ashleigh; Birse, Louise; Jackson, Robert W.; Holden, Nicola J.

    2014-01-01

    Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety. PMID:25018749

  4. Antimicrobial activity of extracts of local cough mixtures on upper respiratory tract bacterial pathogens.

    PubMed

    Adeleye, I A; Opiah, L

    2003-09-01

    The punched-hole and the paper disc diffusion methods were used in screening for the antimicrobial activity of six common ingredients used locally in cough mixtures, against the following bacteria: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus. Enterococcus faecalis, Escherichia coli, Salmonella spp, Salmonella paratyphi, Shigella dysenteria, Shigella sonnei and Candida albicans. The results, evaluated as the diameter of zone of inhibition of microbial growth, showed that lime, garlic onion, onion and honey were active against Staphylococcus aureus, Streptococcus faecalis, Candida albicans, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Salmonella spp and Shigella dysenteriae. Bitter-kola nut extract and palm kernel oil showed no antimicrobial activities against any of the tested organisms. None of the extracts inhibited the growth of Salmonella paratyphi and Shigella sonnei and the most susceptible organisms were Escherichia coli and Salmonella spp. PMID:14649097

  5. Optimization of isolation and cultivation of bacterial endophytes through addition of plant extract to nutrient media

    PubMed Central

    Eevers, N; Gielen, M; Sánchez-López, A; Jaspers, S; White, J C; Vangronsveld, J; Weyens, N

    2015-01-01

    Many endophytes have beneficial effects on plants and can be exploited in biotechnological applications. Studies hypothesize that only 0.001–1% of all plant-associated bacteria are cultivable. Moreover, even after successful isolations, many endophytic bacteria often show reduced regrowth capacity. This research aimed to optimize isolation processes and culturing these bacteria afterwards. We compared several minimal and complex media in a screening. Beside the media themselves, two gelling agents and adding plant extract to media were investigated to enhance the number and diversity of endophytes as well as the growth capacity when regrown after isolation. In this work, 869 medium delivered the highest numbers of cultivable bacteria, as well as the highest diversity. When comparing gelling agents, no differences were observed in the numbers of bacteria. Adding plant extract to the media lead to a slight increase in diversity. However, when adding plant extract to improve the regrowth capacity, sharp increases of viable bacteria occurred in both rich and minimal media. PMID:25997013

  6. Presence of hsp65 in bacterial extracts (OM-89): a possible mediator of orally-induced tolerance?

    PubMed

    Polla, B S; Baladi, S; Fuller, K; Rook, G

    1995-08-16

    Heat shock proteins (HSP) have been implicated in rodent models of autoimmunity, particularly arthritis, and there is suggestive though inconclusive evidence that they may also play a role in human autoimmune disease. The simplest hypothesis is based on molecular mimicry due to the amino-acid sequence homology between mammalian and microbial HSP. Recently OM-89, an extract of several strains of Escherichia coli, has shown some efficacy in the treatment of rheumatoid arthritis (RA) when taken orally. Using species-specific antibodies, we show here that OM-89 contains the 65 kDa HSP (hsp65), while hsp65 was not detected in another bacterial extract containing other microorganisms, including Staphylococcus aureus (OM-85). We suggest that if the human homologue of hsp65 is a relevant target antigen in the human disease, the efficacy of the preparation could be due to induction of oral tolerance or to switching the Th1 response towards Th2. Alternatively, even if the human hsp65 is not a target molecule in RA joints, OM-89 may evoke bystander suppression of joint inflammation via induction of TGF beta-secreting effector cells. These hypotheses should be tested in further studies. PMID:7649235

  7. Development of a bacterial cell enrichment method and its application to the community analysis in soybean stems.

    PubMed

    Ikeda, Seishi; Kaneko, Takakazu; Okubo, Takashi; Rallos, Lynn E E; Eda, Shima; Mitsui, Hisayuki; Sato, Shusei; Nakamura, Yasukazu; Tabata, Satoshi; Minamisawa, Kiwamu

    2009-11-01

    A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans. PMID:19662454

  8. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures.

    PubMed

    Boers, Stefan A; Hays, John P; Jansen, Ruud

    2015-01-01

    16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys). PMID:26373611

  9. Micelle PCR reduces chimera formation in 16S rRNA profiling of complex microbial DNA mixtures

    PubMed Central

    Boers, Stefan A.; Hays, John P.; Jansen, Ruud

    2015-01-01

    16S rRNA gene profiling has revolutionized the field of microbial ecology. Many researchers in various fields have embraced this technology to investigate bacterial compositions of samples derived from many different ecosystems. However, it is important to acknowledge the current limitations and drawbacks of 16S rRNA gene profiling. Although sample handling, DNA extraction methods and the choice of universal 16S rRNA gene PCR primers are well known factors that could seriously affect the final results of microbiota profiling studies, inevitable amplification artifacts, such as chimera formation and PCR competition, are seldom appreciated. Here we report on a novel micelle based amplification strategy, which overcomes these limitations via the clonal amplification of targeted DNA molecules. Our results show that micelle PCR drastically reduces chimera formation by a factor of 38 (1.5% vs. 56.9%) compared with traditional PCR, resulting in improved microbial diversity estimates. In addition, compartmentalization during micelle PCR prevents PCR competition due to unequal amplification rates of different 16S template molecules, generating robust and accurate 16S microbiota profiles required for comparative studies (e.g. longitudinal surveys). PMID:26373611

  10. Effect of a bacterial extract on cellular and humoral immune responses in humans.

    PubMed

    Rosenthal, M

    1986-01-01

    A lyophilized extract from E. coli (OM-89) was studied for its immunomodulating properties and tolerance in humans. Its oral administration to healthy volunteers produced a selective increase in the active T-cell population without changes in other lymphocyte populations. A significant increase in the proliferative response to concanavalin A and phytohemagglutin was recorded, but not to pokeweed mitogen. No significant changes were observed in the serum levels of IgG, IgA and IgM. The clinical and biological tolerance of OM-89 was excellent, without any adverse side-effects or production of circulating immune complexes or of autoantibodies, while the in vitro investigation showed that it is not a mitogen. Thus in healthy subjects OM-89 seems to act mainly on the cell-mediated immune responses. PMID:3531348

  11. Protective Effect of Polygonum orientale L. Extracts against Clavibater michiganense subsp. sepedonicum, the Causal Agent of Bacterial Ring Rot of Potato

    PubMed Central

    Cai, Jin; Xie, Shulian; Feng, Jia; Wang, Feipeng; Xu, Qiufeng

    2013-01-01

    The Polygonum orientale L. extracts were investigated for antibacterial activity against Clavibater michiganense subsp. sepedonicum (Spieckermann & Kotthoff) Davis et al., the causal agent of a serious disease called bacterial ring rot of potato. The results showed that the leaf extracts of P. orientale had significantly (p<0.05) greater antibacterial activity against C. michiganense subsp. sepedonicum than root, stem, flower extracts in vitro. According to the results of single factor experiments and L273(13) orthogonal experiments, optimum extraction conditions were A1B3C1, extraction time 6 h, temperature 80C, solid to liquid ratio 1?10 (g:mL). The highest (p<0.05) antibacterial activity was observed when pH was 5, excluding the effect of control. The extracts were stable under ultraviolet (UV). In vivo analysis revealed that 50 mg/mL of P. orientale leaf extracts was effective in controlling decay. Under field conditions, 50 mg/mL of P. orientale leaf extracts also improved growth parameters (whole plant length, shoot length, root length, plant fresh weight, shoot fresh weight, root fresh weight, dry weight, and number of leaves), in the 2010 and 2011 two growing seasons. Further solvent partition assays showed that the most active compounds were in the petroleum ether fractionation. Transmission electron microscopy (TEM) showed drastic ultrastructural changes caused by petroleum ether fractionation, including bacterial deformation, electron-dense particles, formation of vacuoles and lack of cytoplasmic materials. These results indicated that P. orientale extracts have strong antibacterial activity against C. michiganense subsp. sepedonicum and a promising effect in control of bacterial ring rot of potato disease. PMID:23861908

  12. Automated extraction of typing information for bacterial pathogens from whole genome sequence data: Neisseria meningitidis as an exemplar

    PubMed Central

    Jolley, Keith A.; Maiden, Martin C.J.

    2014-01-01

    Whole genome sequence (WGS) data are becoming a major means of characterising samples of bacterial pathogens. These data have the advantage of providing detailed information on the genotypes and likely phenotypes of aetiological agents, enabling the relationships of samples from potential disease outbreaks to be established precisely. However, the generation of increasing quantities of sequence data does not, in itself, resolve the problems that a wide variety of microbiological typing methods have addressed over the last 100 years or so; indeed, the provision of very high volumes of unstructured data can confuse rather than resolve these issues. Here we review the nascent field of the storage of WGS data for clinical application and show how curated sequence-based typing schemes on websites such as PubMLST.org, accumulated over the past 14 years or so, has generated an infrastructure that can be used to exploit WGS for bacterial typing efficiently. We review the tools that have been implemented within the PubMLST.org website to extract clinically useful, strain characterisation information which can be provided to physicians and public health scientists and officials in a timely, concise and understandable way. These data can be used to inform medical decisions such as how to treat a patient, whether to institute public health action, and what action might be appropriate. The information is compatible both with previous sequence-based typing data and also with data that can be obtained in the absence of WGS data, for example by real-time PCR tests, providing a flexible infrastructure for WGS-based clinical microbiology. PMID:23369391

  13. The majority of in vitro macrophage activation exhibited by extracts of some immune enhancing botanicals is due to bacterial lipoproteins and lipopolysaccharides

    PubMed Central

    Pugh, Nirmal D.; Tamta, Hemlata; Balachandran, Premalatha; Wu, Xiangmei; Howell, J’Lynn; Dayan, Franck E.; Pasco, David S.

    2008-01-01

    We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacterially derived components when tested in in vitro monocyte/macrophage activation systems. In experiments using RAW 264.7 and mouse peritoneal macrophages the majority (85–98%) of the activity within extracts from eight immune enhancing botanicals was eradicated by treatment with agents (lipoprotein lipase and polymyxin B) known to target these two bacterial components. Alfalfa sprouts exhibited the highest activity of those botanicals tested but the appearance of this activity during the germination of surface sterilized seeds was abolished by the presence of antibiotics. These studies indicate that the majority of the in vitro macrophage activating properties in extracts from these botanicals can be attributed to the presence of lipoproteins and lipopolysaccharides derived from bacteria and that bacterial endophytes may be a significant source of these components. PMID:18486914

  14. A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists

    PubMed Central

    Marron, Alan O.; Akam, Michael; Walker, Giselle

    2013-01-01

    Background Cultures of heterotrophic protists often require co-culturing with bacteria to act as a source of nutrition. Such cultures will contain varying levels of intrinsic bacterial contamination that can interfere with molecular research and cause problems with the collection of sufficient material for sequencing. Measuring the levels of bacterial contamination for the purposes of molecular biology research is non-trivial, and can be complicated by the presence of a diverse bacterial flora, or by differences in the relative nucleic acid yield per bacterial or eukaryotic cell. Principal Findings Here we describe a duplex PCR-based assay that can be used to measure the levels of contamination from marine bacteria in a culture of loricate choanoflagellates. By comparison to a standard culture of known target sequence content, the assay can be used to quantify the relative proportions of bacterial and choanoflagellate material in DNA or RNA samples extracted from a culture. We apply the assay to compare methods of purifying choanoflagellate cultures prior to DNA extraction, to determine their effectiveness in reducing bacterial contamination. Together with measurements of the total nucleic acid concentration, the assay can then be used as the basis for determining the absolute amounts of choanoflagellate DNA or RNA present in a sample. Conclusions The assay protocol we describe here is a simple and relatively inexpensive method of measuring contamination levels in nucleic acid samples. This provides a new way to establish quantification and purification protocols for molecular biology and genomics in novel heterotrophic protist species. Guidelines are provided to develop a similar protocol for use with any protistan culture. This assay method is recommended where qPCR equipment is unavailable, where qPCR is not viable because of the nature of the bacterial contamination or starting material, or where prior sequence information is insufficient to develop qPCR protocols. PMID:23593495

  15. Avoiding acidic region streaking in two-dimensional gel electrophoresis: case study with two bacterial whole cell protein extracts.

    PubMed

    Roy, Arnab; Varshney, Umesh; Pal, Debnath

    2014-09-01

    Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost. PMID:25116618

  16. Oral immunization with bacterial extracts for protection against acute bronchitis in elderly institutionalized patients with chronic bronchitis.

    PubMed

    Orcel, B; Delclaux, B; Baud, M; Derenne, J P

    1994-03-01

    Acute bronchitis is a major source of morbidity in elderly patients. The purpose of this study was to assess the preventive effects of oral immunisation with a bacterial extract. Three hundred and fifty four patients with chronic bronchitis, living in institutions for the elderly (aged > 65 yrs), were included in a randomized, placebo-controlled, double-blind study. The purpose of the study was to assess preventive effects of OM-85 BV (an immunostimulating agent consisting of lyophilized fractions of eight of the most common pathogens isolated in respiratory tract infections) against acute lower respiratory tract infections. Two hundred and ninety patients completed the study (143 taking placebo and 147 taking OM-85 BV). There was a 28% reduction in the number of lower respiratory tract infections in the patients treated with OM-85 BV; this was entirely due to 40% reduction in the number of episodes of acute bronchitis (p < 0.01), with no difference in the number of episodes of pneumonia and bronchopneumonia. A larger number of patients in the OM-85 BV group were free of acute bronchitis throughout the 6 month study period (96 vs 69) and there was a 28% reduction in the number of antibiotic prescriptions in the OM-85 BV treated group. These results suggest that OM-85 BV has a protective effect against acute bronchitis in elderly patients living in institutions. PMID:8013600

  17. Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages

    PubMed Central

    Cocolin, Luca; Manzano, Marisa; Cantoni, Carlo; Comi, Giuseppe

    2001-01-01

    In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family Micrococcaceae and meat contaminants, such as Brochothrix thermosphacta and Enterococcus sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required. PMID:11679334

  18. Application of Locked Nucleic Acid (LNA) Oligonucleotide–PCR Clamping Technique to Selectively PCR Amplify the SSU rRNA Genes of Bacteria in Investigating the Plant-Associated Community Structures

    PubMed Central

    Ikenaga, Makoto; Sakai, Masao

    2014-01-01

    The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide–PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3′ end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide–PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes. PMID:25030190

  19. Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction.

    PubMed

    Vuong, Jeni; Collard, Jean-Marc; Whaley, Melissa J; Bassira, Issaka; Seidou, Issaka; Diarra, Seydou; Oudraogo, Rasmata T; Kambir, Dinanib; Taylor, Thomas H; Sacchi, Claudio; Mayer, Leonard W; Wang, Xin

    2016-01-01

    Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824-135,982 for 5x Omni, and 168-6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0-99.9%, 97.5-99.9%, and 92.9-99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume. PMID:26829233

  20. Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction

    PubMed Central

    Vuong, Jeni; Collard, Jean-Marc; Whaley, Melissa J.; Bassira, Issaka; Seidou, Issaka; Diarra, Seydou; Ouédraogo, Rasmata T.; Kambiré, Dinanibè; Taylor, Thomas H.; Sacchi, Claudio; Mayer, Leonard W.; Wang, Xin

    2016-01-01

    Neisseria meningitidis (Nm), Haemophilus influenzae (Hi), and Streptococcus pneumoniae (Sp) are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR) requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA) and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y) targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824–135,982 for 5x Omni, and 168–6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0–99.9%, 97.5–99.9%, and 92.9–99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume. PMID:26829233

  1. Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly

    PubMed Central

    Sll, Dieter; Steitz, Thomas A.

    2015-01-01

    Here we report the crystal structures of the Thermus thermophilus ribosome at 2.3-2.5-resolution, which have enabled a comprehensive modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields the most complete atomic model of bacterial ribosome. PMID:25775268

  2. Tools for Characterizing Bacterial Protein Synthesis Inhibitors

    PubMed Central

    Orelle, Cédric; Carlson, Skylar; Kaushal, Bindiya; Almutairi, Mashal M.; Liu, Haipeng; Ochabowicz, Anna; Quan, Selwyn; Pham, Van Cuong; Squires, Catherine L.; Murphy, Brian T.

    2013-01-01

    Many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. However, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. Furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. In order to avoid these pitfalls and improve the process of dereplication of chemically complex extracts, we designed a two-pronged approach for the characterization of inhibitors of protein synthesis (ChIPS) that is suitable for the rapid identification of the site and mode of action on the bacterial ribosome. First, we engineered antibiotic-hypersensitive Escherichia coli strains that contain only one rRNA operon. These strains are used for the rapid isolation of resistance mutants in which rRNA mutations identify the site of the antibiotic action. Second, we show that patterns of drug-induced ribosome stalling on mRNA, monitored by primer extension, can be used to elucidate the mode of antibiotic action. These analyses can be performed within a few days and provide a rapid and efficient approach for identifying the site and mode of action of translation inhibitors targeting the bacterial ribosome. Both techniques were validated using a bacterial strain whose culture extract, composed of unknown metabolites, exhibited protein synthesis inhibitory activity; we were able to rapidly detect the presence of the antibiotic chloramphenicol. PMID:24041905

  3. Influence of First-Line Antibiotics on the Antibacterial Activities of Acetone Stem Bark Extract of Acacia mearnsii De Wild. against Drug-Resistant Bacterial Isolates

    PubMed Central

    Olajuyigbe, Olufunmiso O.; Coopoosamy, Roger M.

    2014-01-01

    Background. This study was aimed at evaluating the antibacterial activity of the acetone extract of A. mearnsii and its interactions with antibiotics against some resistant bacterial strains. Methods. The antibacterial susceptibility testing was determined by agar diffusion and macrobroth dilution methods while the checkerboard method was used for the determination of synergy between the antibiotics and the extract. Results. The results showed that the susceptibility of the different bacterial isolates was concentration dependent for the extract and the different antibiotics. With the exception of S. marcescens, the inhibition zones of the extract produced by 20?mg/mL ranged between 18 and 32?mm. While metronidazole did not inhibit any of the bacterial isolates, all the antibiotics and their combinations, except for ciprofloxacin and its combination, did not inhibit Enterococcus faecalis. The antibacterial combinations were more of being antagonistic than of being synergistic in the agar diffusion assay. From the macrobroth dilution, the extract and the antibiotics exerted a varied degree of inhibitory effect on the test organisms. The MIC values of the acetone extract which are in mg/mL are lower than those of the different antibiotics which are in ?g/mL. From the checkerboard assay, the antibacterial combinations showed varied degrees of interactions including synergism, additive, indifference, and antagonism interactions. While antagonistic and additive interactions were 14.44%, indifference interaction was 22.22% and synergistic interaction was 37.78% of the antibacterial combinations against the test isolates. While the additivity/indifference interactions indicated no interactions, the antagonistic interaction may be considered as a negative interaction that could result in toxicity and suboptimal bioactivity. Conclusion. The synergistic effects of the herbal-drug combinations may be harnessed for the discovery and development of more rational evidence-based drug combinations with optimized efficiency in the prevention of multidrug resistance and therapy of multifactorial diseases. PMID:25101132

  4. Extracts of brown seaweeds can attenuate the bacterial lipopolysaccharide-induced pro-inflammatory response in the porcine colon ex vivo.

    PubMed

    Bahar, B; O'Doherty, J V; Hayes, M; Sweeney, T

    2012-12-01

    Bioactive compound-rich brown seaweeds are demonstrated to have numerous health benefits including anti-microbial and immunomodulatory bioactivities in the pig intestine. In this study, the immunomodulating effects of extracts of brown seaweed (Ascophyllum nodosum and Fucus serratus) were evaluated on the porcine colon using a bacterial lipopolysaccharide (LPS) ex vivo model. Approximately 1.5 × 1.5 cm of pig colon (n = 6) was stripped of its overlying muscle layer and incubated in 1 mL Dulbecco's Modified Eagle Medium containing bacterial LPS (10 μg) and seaweed extracts (1 mg). Gene expression of interleukin-8 (IL-8) and interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFA) were measured using quantitative real time PCR. In contrast to the low level of expression of IL-8, IL-6, and TNFA genes in the colonic tissue at 0 h, LPS treatment increased (P < 0.05) the expression of IL-8, IL-6, and TNFA genes to 2.38 ± 0.86, 1.90 ± 0.66, and 1.90 ± 0.57 fold, respectively. This pro-inflammatory response induced by the LPS was suppressed by the extracts of Ascophyllum. Ascophyllum extract reduced (P < 0.05) the expression of IL-8, IL-6, and TNFA genes to 0.99 ± 0.53, 0.75 ± 0.33, and 1.01 ± 0.17 fold, and Fucus extract reduced (P < 0.05) the expression of the corresponding genes to 0.70 ± 0.32, 0.69 ± 0.38, and 1.15 ± 0.25 fold, respectively. It is concluded that the extracts of Ascophyllum and Fucus seaweeds have potential to suppress the pro-inflammatory response induced by the bacterial LPS in the pig colon. PMID:23365280

  5. Selective induction of the glucose-regulated protein grp78 in human monocytes by bacterial extracts (OM-85): a role for calcium as second messenger.

    PubMed

    Jacquier-Sarlin, M R; Dreher, D; Polla, B S

    1996-09-01

    Heat shock/stress proteins (HSP) act as molecular chaperones, protect cells from injury, and are involved in the immune response. We investigated the effects of the immunomodulating bacterial extracts OM-85 on the stress response in normal human peripheral blood monocytes. While OM-85 did not induce the classical HSP, we show here, using 2D gel electrophoresis combined with immunoblotting, the induction of the glucose regulated protein grp78 (the immunoglobulin heavy chain binding protein BiP) along with the described accumulation of pro-interleukin-1 beta. The increased Ca2+ mobilization observed with OM-85 is the likely second messenger for grp78 induction. Recent studies are in favor of a protective role of grp78 against cytokine-mediated cytotoxicity and apoptosis. We suggest that grp78 induction following exposure to OM-85 explains, at least in part, the immunodulatory and protective effects of the bacterial extracts. PMID:8806608

  6. Randomized clinical trial comparing the efficacy of the vaginal use of metronidazole with a Brazilian pepper tree (Schinus) extract for the treatment of bacterial vaginosis.

    PubMed

    Leite, S R R F; Amorim, M M R; Sereno, P F B; Leite, T N F; Ferreira, J A C; Ximenes, R A A

    2011-03-01

    A 7.4% vaginal extract of the Brazilian pepper tree (Schinus terebinthifolius Raddi) was compared with 0.75% vaginal metronidazole, both manufactured by the Hebron Laboratory, for the treatment of bacterial vaginosis, used at bedtime for 7 nights. The condition was diagnosed using the combined criteria of Amsel and Nugent in two groups of 140 and 137 women, aged between 18 and 40 years. Intention-to-treat analysis was performed. Women were excluded from the study if they presented delayed menstruation, were pregnant, were using or had used any topical or systemic medication, presented any other vaginal infections, presented hymen integrity, or if they reported any history suggestive of acute pelvic inflammatory disease. According to Amsel's criteria separately, 29 patients (21.2%) treated with the extract and 87 (62.1%) treated with metronidazole were considered to be cured (P < 0.001). According to Nugent's score separately, 19 women (13.9%) treated with the extract and 79 (56.4%) treated with metronidazole were considered to be cured (P < 0.001). Using the two criteria together, the so-called total cure was observed in 17 women (12.4%) treated with the extract and in 79 women (56.4%) treated with metronidazole (P < 0.001). In conclusion, the cure rate for bacterial vaginosis using a vaginal gel from a pepper tree extract was lower than the rate obtained with metronidazole gel, while side effects were infrequent and non-severe in both groups. PMID:21243318

  7. Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

    PubMed

    Kawase, Jun; Kurosaki, Morito; Kawakami, Yuta; Kashimoto, Takashi; Tsunomori, Yoshie; Sato, Koji; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Etoh, Yoshiki; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2014-01-01

    In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR. PMID:25410559

  8. The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology.

    PubMed

    Sohrabi, Mohsen; Nair, Raj G; Samaranayake, Lakshman P; Zhang, Li; Zulfiker, Abu Hasanat Md; Ahmetagic, Adnan; Good, David; Wei, Ming Q

    2016-03-01

    Recent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling. Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic-mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozyme-achromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others. PMID:26812577

  9. Influence of fecal sample storage on bacterial community diversity.

    PubMed

    Roesch, Luiz F W; Casella, George; Simell, Olli; Krischer, Jeffrey; Wasserfall, Clive H; Schatz, Desmond; Atkinson, Mark A; Neu, Josef; Triplett, Eric W

    2009-01-01

    Previous studies have identified a correlation, either positive or negative, between specific stool bacteria strains and certain autoimmune diseases. These conflicting data may relate to sample collection. The aim of this work was to evaluate the influence of the collection parameters of time and temperature on bacterial community composition. Samples were taken from healthy children and immediately divided in 5 sub-samples. One sample was frozen immediately at -80 C, while the other aliquots were frozen 12, 24, 48, and 72h later DNA extracted from each sample was used to amplify the 16S rRNA with barcoded primers. The amplified products were pooled and partial 16S rRNA sequences were obtained by pyrosequencing. Person-to-person variability in community diversity was high. A list of those taxa that comprise at least 1% of the community was made for each individual. None of these were present in high numbers in all individuals. The Bacteroides were present in the highest abundance in three of four subjects. A total of 23,701 16S rRNA sequences were obtained with an average of 1,185 reads per sample with an average length of 200 bases. Although pyrosequencing of amplified 16S rRNA identified changes in community composition over time (~10%), little diversity change was observed at 12 hours (3.06%) with gradual changes occurring after 24 (8.61%), 48 (9.72%), and 72 h (10.14%), post collection. PMID:19440250

  10. Effect of dietary supplementation with a Saccharomyces cerevisiae mannan oligosaccharide on the bacterial community structure of broiler cecal contents.

    PubMed

    Corrigan, A; Horgan, K; Clipson, N; Murphy, R A

    2011-09-01

    This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla, Firmicutes, Bacteroidetes, and Proteobacteria; of these, Firmicutes were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure. PMID:21803917

  11. Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses

    PubMed Central

    2011-01-01

    Background Black elderberries (Sambucus nigra L.) are well known as supportive agents against common cold and influenza. It is further known that bacterial super-infection during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against three Gram-positive bacteria and one Gram-negative bacteria responsible for infections of the upper respiratory tract, as well as cell culture experiments for two different strains of influenza virus. Methods The antimicrobial activity of the elderberry extract was determined by bacterial growth experiments in liquid cultures using the extract at concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating the bacteria on agar plates. In addition, the inhibitory potential of the extract on the propagation of human pathogenic H5N1-type influenza A virus isolated from a patient and an influenza B virus strain was investigated using MTT and focus assays. Results For the first time, it was shown that a standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of Streptococcus pyogenes and group C and G Streptococci, and the Gram-negative bacterium Branhamella catarrhalis in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses. Conclusion Rubini elderberry liquid extract is active against human pathogenic bacteria as well as influenza viruses. The activities shown suggest that additional and alternative approaches to combat infections might be provided by this natural product. PMID:21352539

  12. Inhibitory effect of pomegranate (Punica granatum L.) polyphenol extracts on the bacterial growth and survival of clinical isolates of pathogenic Staphylococcus aureus and Escherichia coli.

    PubMed

    Pagliarulo, Caterina; De Vito, Valentina; Picariello, Gianluca; Colicchio, Roberta; Pastore, Gabiria; Salvatore, Paola; Volpe, Maria Grazia

    2016-01-01

    In the present study major polyphenols of pomegranate arils and peel by-products were extracted in 50% (v/v) aqueous ethanol, characterized and used in microbiological assays in order to test antimicrobial activity against clinically isolated human pathogenic microorganisms. Total concentration of polyphenols and in vitro antioxidant properties were determined by the Folin-Ciocalteu and DPPH methods, respectively. The most abundant bioactive molecules, including anthocyanins, catechins, tannins, gallic and ellagic acids were identified by RP-HPLC-DAD, also coupled to off-line matrix assisted laser desorption/ionization (MALDI-TOF) mass spectrometry (MS). The inhibitory spectrum of extracts against test microorganisms was assessed by the agar well-diffusion method. Data herein indicated that both pomegranate aril and peel extracts have an effective antimicrobial activity, as evidenced by the inhibitory effect on the bacterial growth of two important human pathogens, including Staphylococcus aureus and Escherichia coli, which are often involved in foodborne illness. PMID:26213044

  13. Multiple DNA Extractions Coupled with Stable-Isotope Probing of Anthracene-Degrading Bacteria in Contaminated Soil▿†

    PubMed Central

    Jones, Maiysha D.; Singleton, David R.; Sun, Wei; Aitken, Michael D.

    2011-01-01

    In many of the DNA-based stable-isotope probing (SIP) studies published to date in which soil communities were investigated, a single DNA extraction was performed on the soil sample, usually using a commercial DNA extraction kit, prior to recovering the 13C-labeled (heavy) DNA by density-gradient ultracentrifugation. Recent evidence suggests, however, that a single extraction of a soil sample may not lead to representative recovery of DNA from all of the organisms in the sample. To determine whether multiple DNA extractions would affect the DNA yield, the eubacterial 16S rRNA gene copy number, or the identification of anthracene-degrading bacteria, we performed seven successive DNA extractions on the same aliquot of contaminated soil either untreated or enriched with [U-13C]anthracene. Multiple extractions were necessary to maximize the DNA yield and 16S rRNA gene copy number from both untreated and anthracene-enriched soil samples. Sequences within the order Sphingomonadales, but unrelated to any previously described genus, dominated the 16S rRNA gene clone libraries derived from 13C-enriched DNA and were designated “anthracene group 1.” Sequences clustering with Variovorax spp., which were also highly represented, and sequences related to the genus Pigmentiphaga were newly associated with anthracene degradation. The bacterial groups collectively identified across all seven extracts were all recovered in the first extract, although quantitative PCR analysis of SIP-identified groups revealed quantitative differences in extraction patterns. These results suggest that performing multiple DNA extractions on soil samples improves the extractable DNA yield and the number of quantifiable eubacterial 16S rRNA gene copies but have little qualitative effect on the identification of the bacterial groups associated with the degradation of a given carbon source by SIP. PMID:21398486

  14. The anti-adhesive mode of action of a purified mushroom (Lentinus edodes) extract with anticaries and antigingivitis properties in two oral bacterial phatogens

    PubMed Central

    2014-01-01

    Background In previous works we have shown that a low-molecular-mass (LMM) fraction from mushroom (Lentinus edodes) homogenate interferes with binding of Streptococcus mutans to hydroxyapatite and Prevotella intermedia to gingival cells. Additionally, inhibition of biofilm formation of both odonto- and periodonto-pathogenic bacteria and detachment from preformed biofilms have been described for this compound. Further purification of mushroom extract has been recently achieved and a sub-fraction (i.e. # 5) has been identified as containing the majority of the mentioned biological activities. The aim of this study was to characterise the bacterial receptors for the purified mushroom sub-fraction #5 in order to better elucidate the mode of action of this compound when interfering with bacterial adhesion to host surfaces or with bacteria-bacteria interactions in the biofilm state. Methods Candidate bacterial molecules to act as target of this compound were bacterial surface molecules involved in cell adhesion and biofilm formation, and, thus, we have considered cell wall associated proteins (CWPs), teichoic acid (TA) and lipoteichoic acid (LTA) of S. mutans, and outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of P. intermedia. Results Fifteen S. mutans CWPs and TA were capable of binding sub-fraction #5, while LTA did not. As far as P. intermedia is concerned, we show that five OMPs interact with sub-fraction # 5. Capacity of binding to P. intermedia LPS was also studied but in this case negative results were obtained. Conclusions Binding sub-fraction # 5 to surface molecules of S. mutans or P. intermedia may result in inactivation of their physiological functions. As a whole, these results indicate, at molecular level, the bacterial surface alterations affecting adhesion and biofim formation. For these antimicrobial properties, the compound may find use in daily oral hygiene. PMID:24564835

  15. Supraglacial bacterial community structures vary across the Greenland ice sheet.

    PubMed

    Cameron, Karen A; Stibal, Marek; Zarsky, Jakub D; Gözdereliler, Erkin; Schostag, Morten; Jacobsen, Carsten S

    2016-02-01

    The composition and spatial variability of microbial communities that reside within the extensive (>200 000 km(2)) biologically active area encompassing the Greenland ice sheet (GrIS) is hypothesized to be variable. We examined bacterial communities from cryoconite debris and surface ice across the GrIS, using sequence analysis and quantitative PCR of 16S rRNA genes from co-extracted DNA and RNA. Communities were found to differ across the ice sheet, with 82.8% of the total calculated variation attributed to spatial distribution on a scale of tens of kilometers separation. Amplicons related to Sphingobacteriaceae, Pseudanabaenaceae and WPS-2 accounted for the greatest portion of calculated dissimilarities. The bacterial communities of ice and cryoconite were moderately similar (global R = 0.360, P = 0.002) and the sampled surface type (ice versus cryoconite) did not contribute heavily towards community dissimilarities (2.3% of total variability calculated). The majority of dissimilarities found between cryoconite 16S rRNA gene amplicons from DNA and RNA was calculated to be the result of changes in three taxa, Pseudanabaenaceae, Sphingobacteriaceae and WPS-2, which together contributed towards 80.8 ± 12.6% of dissimilarities between samples. Bacterial communities across the GrIS are spatially variable active communities that are likely influenced by localized biological inputs and physicochemical conditions. PMID:26691594

  16. [Anti-bacterial activity of extracts from fungi collected from mangrove Rhizophora mangle (Rhizophoraceae) roots in Venezuela].

    PubMed

    Castillo-Machalskis, Isabel; D'Armas, Haydelba; Malaver, Nora; Núñez, Maximiano

    2007-01-01

    The antibacterial activity of marine fungi extracts was evaluated by a test of efficiency. The fungi were previously inoculated in Malt Agar (EMA) extract with 50% of seawater and growth for 60 days. Triplicate antibiograms were carried out with the extracts. An ANOVA I with a posteriori Duncan test were applied to the diameters of inhibition zones. The extracts of Aspergillus ochraceus 3MCMC3 and Penicillium citrinum (14) 4MCMC16 present wide spectral antibacterial properties, inhibiting 100% and 80% of the developing germs, especially Pseudomonas aeruginosa 9027. The extract of Penicillium (1) 3MLLC5 had the highest efficiency on this strain; P. aeruginosa 9027 and Escherichia coli 10536 were the most sensitive germs when treated with these extracts. These marine fungi can be an important source of antibacterial secondary metabolites. PMID:19086381

  17. PCR detection and identification of bacterial contaminants in ocular samples from post-operative endophthalmitis.

    TOXLINE Toxicology Bibliographic Information

    Abrishami M; Hashemi B; Abrishami M; Abnous K; Razavi-Azarkhiavi K; Behravan J

    2015-04-01

    BACKGROUND: Bacterial endophthalmitis is a sight-threatening complication of ocular surgery which requires urgent medical consideration including comprehensive diagnosis. Polymerase chain reaction (PCR) as a sensitive molecular method has been extensively used for detection of microbial species in clinical specimens.AIM: The aim of this study was to identify the causative organisms of endophthalmitis in our patient population using a procedure based on PCR.MATERIALS AND METHODS: Vitreous samples from 32 patients with post-operative endophthalmitis were collected. Total vitreous DNA was extracted and then assessed by agarose gel electrophoresis. Bacterial 16S rRNA gene was amplified from genomic DNA using PCR with a pair of HAD2 universal primers. Library of PCR products from 16S rRNA, cloned into the pTZ57R/T vector. The ligated products were then transformed into E. coli DH5? strain and grown in the LB-ampicillin/X-Gal/IPTG plate.RESULTS: From the total of 32 vitreous samples, 18 specimens were positive, illustrating the presence of bacterial infection (56.4 %). Twelve species including Escherichia coli, Enterobacter cloacae, Bacillus subtilis, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia trachomatis, Staphylococcus aureus, Neisseria meningitides, Staphylococcus epidermidis, Pseudomonas aeruginosa and Bacillus cereus were identified using BLAST for known 16S rRNA sequences.CONCLUSION: Polymerase chain reaction (PCR) accompanied with cloning and sequencing approved to be sensitive and specific. The rapid molecular technique was useful in detection of 12 major microbial species, in infectious endophthalmitis.

  18. Diagnosis of fulminant pneumonia caused by Legionella pneumophila serogroup 8 with the sequence analysis of the 16S rRNA gene.

    PubMed

    Kawanami, Toshinori; Yatera, Kazuhiro; Fukuda, Kazumasa; Yamasaki, Kei; Kunimoto, Masamizu; Nagata, Shuya; Nishida, Chinatsu; Ishimoto, Hiroshi; Ogawa, Midori; Taniguchi, Hatsumi; Mukae, Hiroshi

    2011-01-01

    Pneumonia is the fourth leading cause of death in Japan. Accurate and rapid detection of the causative pathogen(s) is necessary and important for appropriate antimicrobial treatment, especially in patients with rapidly progressive pneumonia or immunocompromised patients. Conventional methods, such as cultivations, detection of urinary antigens or PCR amplification of specific genes, inevitably require the precise presumption of potential pathogens in each case, and pneumonia caused by unanticipated microorganisms might lead to inadequate antimicrobial treatments and unfortunate consequences. We herein report an immunocompromised female patient (69 years old) with fulminant pneumonia caused by Legionella (L.) pneumophila serogroup 8. Ordinary cultivation methods and urinary antigen detection failed to identify the causative organisms. Accordingly, DNA was extracted from the bronchoalveolar lavage fluid and used for the PCR-based cloning of the bacterial 16S rRNA gene. Sequencing analysis of the isolated clones revealed the predominance of L. pneumophila. Based on this information, the patient received an appropriate and successful antimicrobial treatment. In addition, L. pneumophila serogroup 8 was identified with culturing the bronchoalveolar lavage fluid and serotyping with L. pneumophila antisera. The 16S rRNA gene sequencing analysis can reveal the potential pathogens without any presumption about the organism, and can evaluate the kinds and ratio of bacterial species in each specimen. In conclusion, this cultivation-independent method is a potential diagnostic modality for pneumonia, especially in patients with rapidly progressive pneumonia or those who are immunocompromised. PMID:21878746

  19. Effect of Dietary Supplementation with a Saccharomyces cerevisiae Mannan Oligosaccharide on the Bacterial Community Structure of Broiler Cecal Contents▿†

    PubMed Central

    Corrigan, A.; Horgan, K.; Clipson, N.; Murphy, R. A.

    2011-01-01

    This study investigated the effects of dietary supplementation with a prebiotic mannan oligosaccharide (MOS) on broiler performance, bacterial community structure, and phylogenetic populations of cecal contents. Bird performance data were collected, and cecal samples were extracted from randomly caught poults from each treatment group every 7 days from hatching to the age of 42 days. Weight gain, feed consumption, and feed efficiency ratios did not differ significantly between groups. Automated ribosomal intergenic spacer analysis (ARISA) of the bacterial communities in birds receiving MOS-supplemented diets indicated that dietary supplementation with MOS at either of 2 levels significantly altered the bacterial community structure from that of the control group on all sample days. The phylogenetic identities of bacteria contained within the cecum were determined by constructing a 16S rRNA gene clone library. A total of 594 partial 16S rRNA gene sequences from the cecal contents were analyzed and compared for the three dietary treatments. The dominant bacteria of the cecum belonged to three phyla, Firmicutes, Bacteroidetes, and Proteobacteria; of these, Firmicutes were the most dominant in all treatment groups. Statistical analysis of the bacterial 16S rRNA gene clone libraries showed that the compositions of the clone libraries from broilers receiving MOS-supplemented diets were, in most cases, significantly different from that of the control group. It can be concluded that in this trial MOS supplementation significantly altered the cecal bacterial community structure. PMID:21803917

  20. Bacterial clearance, heterophil function, and hematological parameters of transport stressed turkey poults supplemented with dietary yeast extract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeast extracts contain biological response modifiers that may be useful as alternatives to antibiotics for controlling pathogens in poultry production and mitigating the deleterious effects of production stressors. A standardized yeast extract feed supplement, Alphamune (YE), was added to turkey po...

  1. Benthic bacterial diversity in submerged sinkhole ecosystems.

    PubMed

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

    2010-01-01

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

  2. Bacterial communities established in bauxite residues with different restoration histories.

    PubMed

    Schmalenberger, Achim; O'Sullivan, Orla; Gahan, Jacinta; Cotter, Paul D; Courtney, Ronan

    2013-07-01

    Bauxite residue is the alkaline byproduct generated when alumina is extracted from bauxite ores and is commonly deposited in impoundments. These sites represent hostile environments with increased salinity and alkalinity and little prospect of revegetation when left untreated. This study reports the establishment of bacterial communities in bauxite residues with and without restoration amendments (compost and gypsum addition, revegetation) in samples taken in 2009 and 2011 from 0 to 10 cm depth. DNA fingerprint analysis of bacterial communities based on 16S rRNA gene fragments revealed a significant separation of the untreated site and the amended sites in both sampling years. 16S amplicon analysis (454 FLX pyrosequencing) revealed significantly lower alpha diversities in the unamended in comparison to the amended sites and hierarchical clustering separated the unamended site from the amended sites. The taxonomic analysis revealed that the restoration resulted in the accumulation of bacterial populations typical for soils including Acidobacteriaceae, Nitrosomonadaceae, and Caulobacteraceae. In contrast, the unamended site was dominated by taxonomic groups including Beijerinckiaceae, Xanthomonadaceae, Acetobacteraceae, and Chitinophagaceae, repeatedly associated with alkaline salt lakes and sediments. While bacterial communities developed in the initially sterile bauxite residue, only the restoration treatments created diverse soil-like bacterial communities alongside diverse vegetation on the surface. PMID:23745718

  3. Identification of the metabolically active members of a bacterial community in a polychlorinated biphenyl-polluted moorland soil.

    PubMed

    Nogales, B; Moore, E R; Abraham, W R; Timmis, K N

    1999-06-01

    The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription-polymerase chain reaction (RT-PCR) amplification products of 16S rRNA generated from total RNA extracts. Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs. Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga-Acidobacterium phylum (14%), the Actinobacteria (6.5%) and the Planctomycetales (2%). Three cloned sequence types were not affiliated to any described phylogenetic group. An unusual feature of this soil was the abundance of sequence types within the beta-subclass of the Proteobacteria, most of which were similar to the 16S rRNA gene sequences of species from only two genera, Burkholderia and Variovorax. Three other numerous 16S rRNA sequence types were similar to the sequences of Sphingomonas species, members of the Rhodopila globiformis group and Acidobacterium capsulatum. Some of the sequence types retrieved were similar to the 16S rRNA sequences of bacterial isolates able to degrade a variety of organic pollutants, including PCBs. As the PCB contamination is the major source of measurable carbon in this soil, some of the 16S rRNA sequence types detected and presumed to represent the metabolically active members of the community indicate the organisms likely to be involved, directly or indirectly, in the utilization of the PCBs as carbon and energy sources. PMID:11207739

  4. Medicinal plant extracts as anti-Escherichia coli O157:H7 agents and their effects on bacterial cell aggregation.

    PubMed

    Voravuthikunchai, Supayang Piyawan; Limsuwan, Surasak

    2006-10-01

    Ethanolic extracts of eight Thai medicinal plants (representing five families) that are used as traditional remedies for treating diarrhea were examined with a salt aggregation test for their ability to modulate cell surface hydrophobicity of enterohemorrhagic Escherichia coli strains, including E. coli O157:H7. Four of these medicinal plants, Acacia catechu, Peltophorum pterocarpum, Punica granatum, and Quercus infectoria, have high bacteriostatic and bactericidal activities. The ethanolic extract of Q. infectoria was the most effective against all strains of E. coli, with MICs of 0.12 to 0.98 mg/ml and MBCs of 0.98 to 3.91 mg/ml. The ethanolic extract of P. granatum had MICs of 0.49 to 1.95 mg/ml and MBCs of 1.95 to 3.91 mg/ml. Ethanolic extracts of Q. infectoria, P. pterocarpum, and P. granatum were among the most effective extracts against the two strains of E. coli O157:H7. The other four plants, Andrographis paniculata, Pluchia indica, Tamarindus indica, and Walsura robusta, did not have high bacteriostatic and bactericidal activities but were able to affect hydrophobicity characteristics on their outermost surface. All plants except Q. infectoria had some ability to increase cell surface hydrophobicity. There appears to be no correlation between antibacterial activity and cell aggregative properties. PMID:17066910

  5. Compilation of 5S rRNA and 5S rRNA gene sequences

    PubMed Central

    Specht, Thomas; Wolters, Jrn; Erdmann, Volker A.

    1991-01-01

    This is an update for the 5S rRNA sequences of the BERLIN RNA DATABANK last published in 1990 (1). The new entry consists of 25 eubacterial and 2 eukaryotic 5S rRNA sequences and 10 plant 5S rRNA pseudogenes (Table 1). Thus the BERLIN RNA DATABANK contains as of Febuary 1, 1991 the 5S rRNA sequences of 44 archaebacteria, 292 eubacteria, 20 plastids, 6 mitochondria, 321 eukaryotes and 21 eukaryotic pseudogenes. The BERLIN RNA DATABANK uses the format of the EMBL Nucleotide Sequence Data Library complemented by a Sequence Alignment (SA) field including secondary structure information. PMID:2041804

  6. Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity.

    PubMed

    Yasir, Muhammad; Aslam, Zubair; Kim, Seon Won; Lee, Seon-Woo; Jeon, Che Ok; Chung, Young Ryun

    2009-10-01

    Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi. PMID:19423335

  7. Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge, Rhopaloeides odorabile.

    PubMed

    Simister, Rachel; Taylor, Michael W; Tsai, Peter; Fan, Lu; Bruxner, Timothy J; Crowe, Mark L; Webster, Nicole

    2012-12-01

    Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so-called sponge-specific clusters (SCs) or sponge- and coral-specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)-based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31C) had no effect on the present and/or active portions of the R.?odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two-thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change. PMID:23106937

  8. Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis

    PubMed Central

    2012-01-01

    Background The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis. Results Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks. Conclusions Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis. PMID:23186268

  9. Polyarthritis in the rat: effects of tolerance and sensitization to the bacterial extract OM-89 with a possible mode of action.

    PubMed

    Willis, D; Moore, A R; Gowland, G; Willoughby, A

    1995-12-01

    The Bacterial Extract OM-89 has been shown to significantly reduce paw swelling in an avridine-induced polyarthritis (AvPA) in female Wistar rats whether given orally for 3 weeks or as a series of i.p. injections. Paw swelling was also markedly reduced in groups of adult rats which had received i.p. injections of OM-89 as neonates. Three separate tolerance-inducing regimes were used and all were equally effective. It is suggested that OM-89 can modulate the undesirable immunological reactions to endogenous antigens and since oral administration has been shown to be clinically effective in the human, that the induction of oral tolerance could be a mechanism of action of OM-89. PMID:8608354

  10. Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.

    PubMed

    Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

    2014-05-01

    In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI. PMID:24615272

  11. An Unspliced Group I Intron in 23S rRNA Links Chlamydiales, Chloroplasts, and Mitochondria

    PubMed Central

    Everett, Karin D. E.; Kahane, Simona; Bush, Robin M.; Friedman, Maureen G.

    1999-01-01

    Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simkania negevensis ZT and Parachlamydia acanthamoebae strains. The present study of Chlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU · 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function. PMID:10438738

  12. Taxonomy of bacterial fish pathogens

    PubMed Central

    2011-01-01

    Bacterial taxonomy has progressed from reliance on highly artificial culture-dependent techniques involving the study of phenotype (including morphological, biochemical and physiological data) to the modern applications of molecular biology, most recently 16S rRNA gene sequencing, which gives an insight into evolutionary pathways (= phylogenetics). The latter is applicable to culture-independent approaches, and has led directly to the recognition of new uncultured bacterial groups, i.e. "Candidatus", which have been associated as the cause of some fish diseases, including rainbow trout summer enteritic syndrome. One immediate benefit is that 16S rRNA gene sequencing has led to increased confidence in the accuracy of names allocated to bacterial pathogens. This is in marked contrast to the previous dominance of phenotyping, and identifications, which have been subsequently challenged in the light of 16S rRNA gene sequencing. To date, there has been some fluidity over the names of bacterial fish pathogens, with some, for example Vibrio anguillarum, being divided into two separate entities (V. anguillarum and V. ordalii). Others have been combined, for example V. carchariae, V. harveyi and V. trachuri as V. harveyi. Confusion may result with some organisms recognized by more than one name; V. anguillarum was reclassified as Beneckea and Listonella, with Vibrio and Listonella persisting in the scientific literature. Notwithstanding, modern methods have permitted real progress in the understanding of the taxonomic relationships of many bacterial fish pathogens. PMID:21314902

  13. Chronic N-amended soils exhibit an altered bacterial community structure in Harvard Forest, MA, USA.

    PubMed

    Turlapati, Swathi A; Minocha, Rakesh; Bhiravarasa, Premsai S; Tisa, Louis S; Thomas, William K; Minocha, Subhash C

    2013-02-01

    At the Harvard Forest, Petersham, MA, the impact of 20 years of annual ammonium nitrate application to the mixed hardwood stand on soil bacterial communities was studied using 16S rRNA genes pyrosequencing. Amplification of 16S rRNA genes was done using DNA extracted from 30 soil samples (three treatments × two horizons × five subplots) collected from untreated (control), low N-amended (50 kg ha(-1) year(-1)) and high N-amended (150 kg ha(-1) year(-1)) plots. A total of 1.3 million sequences were processed using qiime. Although Acidobacteria represented the most abundant phylum based on the number of sequences, Proteobacteria were the most diverse in terms of operational taxonomic units (OTUs). UniFrac analyses revealed that the bacterial communities differed significantly among soil horizons and treatments. Microsite variability among the five subplots was also evident. Nonmetric multidimensional scaling ordination of normalized OTU data followed by permutational manova further confirmed these observations. Richness indicators and indicator species analyses revealed higher bacterial diversity associated with N amendment. Differences in bacterial diversity and community composition associated with the N treatments were also observed at lower phylogenetic levels. Only 28-35% of the 6 936 total OTUs identified were common to three treatments, while the rest were specific to one treatment or common to two. PMID:22974374

  14. Effect of dietary prebiotic (mannan oligosaccharide) supplementation on the caecal bacterial community structure of turkeys.

    PubMed

    Corrigan, A; Horgan, K; Clipson, N; Murphy, R A

    2012-10-01

    The identification of specific bacterial species influenced by mannan oligosaccharide (MOS) supplementation may assist in the formulation of new and improved diets that promote intestinal health and improve bird performance, offering suitable alternatives to antimicrobials in feed for sustainable poultry production. This study has been conducted to evaluate the use of a MOS compound derived from the yeast cell wall of Saccharomyces cerevisiae on turkey performance, bacterial community structure and their phylogenetic associations. A 42-day turkey trial was carried out on birds fed control and MOS-supplemented diets. Bird performance data (weight gains, feed consumption and feed efficiency ratios) were collected, and caecal contents were extracted from randomly caught poults on days 28, 35 and 42 posthatch. Bird performance data showed no improvements as a result of dietary supplementation. Automated ribosomal intergenic spacer analysis (ARISA) revealed the bacterial community structure to be significantly altered on days 28 and 35 posthatch but not day 42 as a result of dietary supplementation. This technique was coupled with 16S rRNA gene sequence analysis to elucidate phylogenetic identities of bacteria. The dominant bacteria of the caecum on all days in both treatment groups were members of phylum Firmicutes, followed by the Bacteroidetes and Proteobacteria phyla, respectively. Statistical analysis of the 16S rRNA gene libraries showed that the composition of the MOS clone library differed significantly to the control on day 35 posthatch. It can be concluded that MOS alters the bacterial community structure in the turkey caecum. PMID:22538976

  15. Anti-Bacterial effect of Aqueous Garlic Extract (AGE) determined by Disc Diffusion Method against Escherichia coli.

    PubMed

    Saha, S; Saha, S K; Hossain, M A; Paul, S K; Gomes, R R; Imtiaz, M; Islam, M M; Nahar, H; Begum, S A; Mirza, T T

    2016-01-01

    The study was performed to determine the antibacterial effect of aqueous extract of garlic (Allium sativum) against standard strain of Escherichia coli ATCC 25922. An interventional study was conducted in Department of Pharmacology and Therapeutics in collaboration with Department of Microbiology, Mymensingh Medical College, Mymensingh. Antibacterial effect of AGE was determined by disc diffusion method. Sensitivity of AGE determined in disc diffusion and the zone of inhibition (ZOI) was 4mm, 10mm and 20mm at 25μg/10μl, 50μg/10μl and 100μg/10μl concentrations respectively. From the findings it is clearly determined the extract has definite antibacterial effect upon Escherichia coli. Further studies are required to detect and isolate the active ingredients present in the Garlic extract as well as detail steps of mechanism responsible for antibacterial effect. Then their effects against the studied organism should be studied in vivo separately and its toxicity profile should also be taken into account. PMID:26931244

  16. Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling

    PubMed Central

    Schmalenberger, Achim; Schwieger, Frank; Tebbe, Christoph C.

    2001-01-01

    Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (?, ?, and ? subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking universal primers can affect a PCR-based microbial community analysis. PMID:11472932

  17. Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

    PubMed Central

    Chen, Yuhuang; Liu, Chang; Xiao, Yuchun; Li, Xu; Su, Mingming; Jing, Huaiqi; Wang, Xin

    2016-01-01

    API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method. PMID:26808495

  18. Changes in cytokine and nitric oxide secretion by rat alveolar macrophages after oral administration of bacterial extracts.

    PubMed Central

    Broug-Holub, E; Persoons, J H; Schornagel, K; Kraal, G

    1995-01-01

    Oral administration of the bacterial immunomodulator Broncho-Vaxom (OM-85), a lysate of eight bacteria strains commonly causing respiratory disease, has been shown to enhance the host defence of the respiratory tract. In this study we examined the effect of orally administered (in vivo) OM-85 on stimulus-induced cytokine and nitric oxide secretion by rat alveolar macrophages in vitro. The results show that alveolar macrophages isolated from OM-85-treated rats secreted significantly more nitric oxide, tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta upon in vitro stimulation with lipopolysaccharide (LPS), whereas, in contrast, LPS-induced IL-6 secretion was significantly lower. The observed effects of in vivo OM-85 treatment on stimulus-induced cytokine secretion in vitro are not due to a direct effect of OM-85 on the cells, because in vitro incubation of alveolar macrophages with OM-85 did not result in altered activity, nor did direct intratracheal instillation of OM-85 in the lungs of rats result in altered alveolar macrophage activity in vitro. It is hypothesized that oral administration of OM-85 leads to priming of alveolar macrophages in such a way that immune responses are non-specifically enhanced upon stimulation. The therapeutic action of OM-85 may therefore result from an enhanced clearance of infectious bacteria from the respiratory tract due to increased alveolar macrophage activity. PMID:7648713

  19. Changes in cytokine and nitric oxide secretion by rat alveolar macrophages after oral administration of bacterial extracts.

    PubMed

    Broug-Holub, E; Persoons, J H; Schornagel, K; Kraal, G

    1995-08-01

    Oral administration of the bacterial immunomodulator Broncho-Vaxom (OM-85), a lysate of eight bacteria strains commonly causing respiratory disease, has been shown to enhance the host defence of the respiratory tract. In this study we examined the effect of orally administered (in vivo) OM-85 on stimulus-induced cytokine and nitric oxide secretion by rat alveolar macrophages in vitro. The results show that alveolar macrophages isolated from OM-85-treated rats secreted significantly more nitric oxide, tumour necrosis factor-alpha (TNF-alpha) and IL-1 beta upon in vitro stimulation with lipopolysaccharide (LPS), whereas, in contrast, LPS-induced IL-6 secretion was significantly lower. The observed effects of in vivo OM-85 treatment on stimulus-induced cytokine secretion in vitro are not due to a direct effect of OM-85 on the cells, because in vitro incubation of alveolar macrophages with OM-85 did not result in altered activity, nor did direct intratracheal instillation of OM-85 in the lungs of rats result in altered alveolar macrophage activity in vitro. It is hypothesized that oral administration of OM-85 leads to priming of alveolar macrophages in such a way that immune responses are non-specifically enhanced upon stimulation. The therapeutic action of OM-85 may therefore result from an enhanced clearance of infectious bacteria from the respiratory tract due to increased alveolar macrophage activity. PMID:7648713

  20. Role of the 5.8S rRNA in ribosome translocation.

    PubMed Central

    Abou Elela, S; Nazar, R N

    1997-01-01

    Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation. PMID:9108162

  1. Bacterial diversity in hyperarid Atacama Desert soils

    NASA Astrophysics Data System (ADS)

    Connon, Stephanie A.; Lester, Elizabeth D.; Shafaat, Hannah S.; Obenhuber, Donald C.; Ponce, Adrian

    2007-12-01

    Surface and subsurface soil samples analyzed for this investigation were collected from the hyperarid Yungay region in the Atacama Desert, Chile. This report details the bacterial diversity derived from DNA and PLFA extracted directly from these extremely desiccated soils. Actinobacteria, Proteobacteria, Firmicutes and TM7 division bacteria were detected. Ninety-four percent of the 16S rRNA genes cloned from these soils belong to the Actinobacteria phylum, and the majority of these were most closely related to the genus Frankia. A 24-hour water activity (aw) time course showed a diurnal cycle that peaked at 0.52 in the early predawn hours, and ranged from 0.01-0.08 during the day. All measured water activity values were below the levels required for microbial growth or enzyme activity. Total organic carbon (TOC) concentrations were above the limit of detection and below the limit of quantification (i.e., 200 ?g/g < TOC < 1000 ?g/g), and phospholipid fatty acid (PLFA) concentrations ranged from 2 105 to 7 106 cell equivalents per gram of soil. Soil extracts analyzed for culturable biomass yielded mostly no growth on R2A media; the highest single extract yielded 47 colony forming units (CFU) per gram of soil.

  2. Mulberry leaf extract mediated synthesis of gold nanoparticles and its anti-bacterial activity against human pathogens

    NASA Astrophysics Data System (ADS)

    Adavallan, K.; Krishnakumar, N.

    2014-06-01

    Gold nanoparticles (Au-NPs) were synthesized at room temperature using Morus alba (mulberry) leaf extract as reducing and stabilizing agent. The development of plant mediated synthesis of nanoparticles is gaining importance due to its simplicity, low cost, non-toxicity, eco-friendliness, long term stability and reproducible aqueous synthesis method to obtain a self-assembly of nearly monodispersed Au-NPs. The formation and morphology of biosynthesized nanoparticles are investigated with the help of UV-Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) techniques. Au-NPs formation was screened by UV-Vis spectroscopy through color conversion due to surface plasmon resonance band at 538 nm for Au-NPs. DLS studies revealed that the average size of Au-NPs was 50 nm. TEM studies showed the particles to be nearly spherical with few irregular shapes and particle size ranges 15-53 nm. The AFM image clearly shows the surface morphology of the well-dispersed Au-NPs with less than 50 nm. The high crystallinity of nanoparticles is evident from bright circular spots in the selected area electron diffraction (SAED) pattern. X-ray diffraction pattern showed high purity and face-centered cubic structure of Au-NPs. The FT-IR results indicate the presence of different functional groups present in the biomolecule capping the nanoparticles. Further, biosynthesized Au-NPs show strong zone of inhibition against Vibrio cholera (gram-negative) and Staphylococcus aureus (gram-positive) whereas, chemically synthesized Au-NPs and mulberry leaf extract exhibit a fair zone of inhibition.

  3. Determining fungi rRNA copy number by PCR.

    PubMed

    Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

    2013-04-01

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment. PMID:23543828

  4. Determining Fungi rRNA Copy Number by PCR

    PubMed Central

    Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

    2013-01-01

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment. PMID:23543828

  5. Microbial rRNA: rDNA gene ratios may be unexpectedly low due to extracellular DNA preservation in soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We tested a method of estimating the activity of detectable individual bacterial and archaeal OTUs within a community by calculating ratios of absolute 16S rRNA to rDNA copy numbers. We investigated phylogenetically coherent patterns of activity among soil prokaryotes in non-growing soil communitie...

  6. Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

  7. Pilot-scale chitin extraction from shrimp shell waste by deproteination and decalcification with bacterial enrichment cultures.

    PubMed

    Bajaj, Mini; Freiberg, Andrea; Winter, Josef; Xu, Youmei; Gallert, Claudia

    2015-11-01

    Extraction of chitin from mechanically pre-purified shrimp shells can be achieved by successive NaOH/HCl treatment, protease/HCl treatment or by environmentally friendly fermentation with proteolytic/lactic acid bacteria (LAB). For the last mentioned alternative, scale-up of shrimp shell chitin purification was investigated in 0.25 L (F1), 10 L (F2), and 300 L (F3) fermenters using an anaerobic, chitinase-deficient, proteolytic enrichment culture from ground meat for deproteination and a mixed culture of LAB from bio-yoghurt for decalcification. Protein removal in F1, F2, and F3 proceeded in parallel within 40 h at an efficiency of 89-91 %. Between 85 and 90 % of the calcit was removed from the shells by LAB in another 40 h in F1, F2, and F3. After deproteination of shrimp shells in F3, spent fermentation liquor was re-used for a next batch of 30-kg shrimp shells in F4 (300 L) which eliminated 85.5 % protein. The purity of the resulting chitin was comparable in F1, F2, F3, and F4. Viscosities of chitosan, obtained after chitin deacetylation and of chitin, prepared biologically or chemically in the laboratory, were much higher than those of commercially available chitin and chitosan. PMID:26227412

  8. The Majority of In Vitro Macrophage Activation Exhibited by Extracts of Some Immune Enhancing Botanicals is Due to Bacterial Lipoproteins and Lipopolysaccharides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacteriall...

  9. Metatranscriptomics reveals overall active bacterial composition in caries lesions

    PubMed Central

    Simn-Soro, Aurea; Guillen-Navarro, Miriam; Mira, Alex

    2014-01-01

    Background Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms. Objective To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay. Design Non-cavitated enamel caries lesions (n=15) and dentin caries lesions samples (n=12) were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition. Results A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.020.73% of the total bacterial community. Conclusions The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci detected confirms that they are a minority and questions its importance as the main etiological agent of tooth decay. Future experimental work should be performed to confirm the cariogenicity of the identified bacteria. PMID:25626770

  10. Rapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat Agents

    PubMed Central

    Yang, Samuel; Rothman, Richard E.; Hardick, Justin; Kuroki, Marcos; Hardick, Andrew; Doshi, Vishal; Ramachandran, Padmini; Gaydos, Charlotte A.

    2013-01-01

    Objectives To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. Methods The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. Results The UniProbe detected the presence of all tested Eubacteria (31 / 31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. Conclusions A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents. PMID:18370996

  11. The intestinal bacterial community in the food waste-reducing larvae of Hermetia illucens.

    PubMed

    Jeon, Hyunbum; Park, Soyoung; Choi, Jiyoung; Jeong, Gilsang; Lee, Sang-Beom; Choi, Youngcheol; Lee, Sung-Jae

    2011-05-01

    As it is known that food waste can be reduced by the larvae of Hermetia illucens (Black soldier fly, BSF), the scientific and commercial value of BSF larvae has increased recently. We hypothesised that the ability of catabolic degradation by BSF larvae might be due to intestinal microorganisms. Herein, we analysed the bacterial communities in the gut of BSF larvae by pyrosequencing of extracting intestinal metagenomic DNA from larvae that had been fed three different diets. The 16S rRNA sequencing results produced 9737, 9723 and 5985 PCR products from larval samples fed food waste, cooked rice and calf forage, respectively. A BLAST search using the EzTaxon program showed that the bacterial community in the gut of larvae fed three different diets was mainly composed of the four phyla with dissimilar proportions. Although the composition of the bacterial communities depended on the different nutrient sources, the identified bacterial strains in the gut of BSF larvae represented unique bacterial species that were unlike the intestinal microflora of other insects. Thus, our study analysed the structure of the bacterial communities in the gut of BSF larvae after three different feedings and assessed the application of particular bacteria for the efficient degradation of organic compounds. PMID:21267722

  12. Effects of a hydrosoluble bacterial extract from Escherichia coli (OM-89) on cytokine production by peripheral blood mononuclear cells from healthy subjects and patients with rheumatoid arthritis.

    PubMed

    Franchimont, N; Vrindts-Gevaert, Y; Collette, J; Franchimont, P

    1991-01-01

    OM-89 is a bacterial extract from escherichia coli, proposed as an immunomodulating drug for the treatment of rheumatoid arthritis (RA). Since immunological mechanisms may play a role in its action, the immunological effects of OM-89 were evaluated in vitro on peripheral blood mononuclear cells (PBMC) derived from healthy subjects and RA patients. Results indicated that in the absence of OM-89, production of the monokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) is increased, while that of the lymphokines interleukin-2 (IL-2) and interferon-gamma (IFN-gamma is decreased by phytohemagglutinin (PHA)-stimulated PBMC from RA patients as compared with PBMC from healthy subjects. In the presence of PHA, OM-89 enhanced the production of IL-1 beta, TNF-alpha, IL-2, and IFN-gamma. IL-1 beta and IL-2 curves obtained using increasing amounts of OM-89 did not differ depending on the source of PBMC. By contrast, in the presence of increasing amounts of OM-89, TNF-alpha secretion significantly higher and IFN-gamma secretion significantly lower with PBMC from RA patients compared to PBMC from healthy subjects. These data indicate that OM-89 acts on monocytes and T cells directly and/or indirectly and suggest a possible clinical activity by OM-89 in RA relative to its immunological properties. PMID:2068542

  13. Transforming growth factor-beta and natural killer T-cells are involved in the protective effect of a bacterial extract on type 1 diabetes.

    PubMed

    Alyanakian, Marie-Alexandra; Grela, Franoise; Aumeunier, Aude; Chiavaroli, Carlo; Gouarin, Christine; Bardel, Emilie; Normier, Grard; Chatenoud, Lucienne; Thieblemont, Nathalie; Bach, Jean-Franois

    2006-01-01

    The onset of type 1 diabetes in NOD mice is delayed by oral administration of a bacterial extract (OM-85) and can be completely prevented by its intraperitoneal administration. Optimal prevention is observed when starting treatment at 3 or 6 weeks of age, and some effect is still observed with treatment at 10 weeks of age. Using genetically deficient mice and cytokine-neutralizing monoclonal antibodies, we demonstrate here that the therapeutic effect does not involve T-helper type 2 cytokines (interleukin [IL]-4 and -10) but is tightly dependent on transforming growth factor (TGF)-beta. Natural killer T-cells also participate in the therapeutic effect because CD1d(-/-) NOD mice are partially resistant to the protective effect of OM-85. The question remains of the specificity of the protective effect of OM-85, which may include proinflammatory components. It will thus be important to further characterize the molecular components that afford protection from type 1 diabetes. Lipopolysaccharide is excluded, but other Toll-like receptor (TLR) agonists could be involved because OM-85 stimulated dendritic cells and induced TGF-beta production by splenocytes in a TLR-2-, TLR-4-, and MyD88-dependent fashion. PMID:16380491

  14. Technical challenges in metatranscriptomic studies applied to the bacterial communities of freshwater ecosystems.

    PubMed

    Pascault, Nomie; Loux, Valentin; Derozier, Sandra; Martin, Vronique; Debroas, Didier; Maloufi, Selma; Humbert, Jean-Franois; Leloup, Julie

    2015-04-01

    Metatranscriptome analysis relates to the transcriptome of microbial communities directly sampled in the environment. Accessing the mRNA pool in natural bacterial communities presents some technical challenges such as the RNA extraction, rRNA depletion, and the choice of the high-throughput sequencing technique. The lack of technical details in scientific articles is a major problem to correctly obtained mRNA from a microbial community and thus the corresponding sequencing data. In our study, we present the methodological procedure that was developed in order to access to the metatranscriptome of the microbial communities during two cyanobacterial blooms successively occurring in a freshwater eutrophic lake. Each procedure step was detailed and discussed with regard to the choices and difficulties encountered and to the recent literature. Finally, the two major limits for metatranscriptomic approaches targeting bacterial communities from natural environments were (i) the removal of rRNA in order to increase the putative mRNA reads number after sequencing, and (ii) for most of the bacterial communities living in natural environments, the lack of reference genomes in databases that leads to the non-assignation of numerous reads. Once these challenges overcome, we managed to access putative mRNA of dominant species, i.e. cyanobacteria (from 6 to 72% of mRNA assigned), and of the surrounding bacteria (from 1 to 5% of mRNA assigned). PMID:25216965

  15. Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis.

    PubMed

    Stets, Maria Isabel; Pinto, Artur Soares; Huergo, Luciano Fernandes; de Souza, Emanuel Maltempi; Guimares, Vandeir Francisco; Alves, Alexessander Couto; Steffens, Maria Berenice Reynaud; Monteiro, Rose Adele; Pedrosa, Fbio de Oliveira; Cruz, Leonardo Magalhes

    2013-06-10

    Whole-cell mass spectrometry analysis is a powerful tool to rapidly identify microorganisms. Several studies reported the successful application of this technique to identify a variety of bacterial species with a discriminatory power at the strain level, mainly for bacteria of clinical importance. In this study we used matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to assess the diversity of wheat-associated bacterial isolates. Wheat plants cultivated in non-sterile vermiculite, under greenhouse conditions were used for bacterial isolation. Total cellular extracts of 138 isolates were analyzed by MALDI-TOF MS and the mass spectra were used to cluster the isolates. Taxonomic identification and phylogenetic reconstruction based on 16S rRNA gene sequences showed the presence of Pseudomonas, Pantoea, Acinetobacter, Enterobacter and Curtobacterium. The 16S rRNA gene sequence analyses were congruent with the clusterization from mass spectra profile. Moreover, MALDI-TOF whole cell mass profiling allowed a finer discrimination of the isolates, suggesting that this technique has the potential of differentiating bacterial isolates at the strain level. PMID:23591594

  16. Bacterial profiles of root caries in elderly patients.

    PubMed

    Preza, Dorita; Olsen, Ingar; Aas, Jrn A; Willumsen, Tiril; Grinde, Bjrn; Paster, Bruce J

    2008-06-01

    Culture-based studies have shown that Streptococcus mutans and lactobacilli are associated with root caries (RC). The purpose of the present study was to assess the bacterial diversity of RC in elderly patients by use of culture-independent molecular techniques and to determine the associations of specific bacterial species or bacterial communities with healthy and carious roots. Plaque was collected from root surfaces of 10 control subjects with no RC and from 11 subjects with RC. The bacterial 16S rRNA genes from extracted DNA were PCR amplified, cloned, and sequenced to determine species identity. From a total of 3,544 clones, 245 predominant species or phylotypes were observed, representing eight bacterial phyla. The majority (54%) of the species detected have not yet been cultivated. Species of Selenomonas and Veillonella were common in all samples. The healthy microbiota included Fusobacterium nucleatum subsp. polymorphum, Leptotrichia spp., Selenomonas noxia, Streptococcus cristatus, and Kingella oralis. Lactobacilli were absent, S. mutans was present in one, and Actinomyces spp. were present in 50% of the controls. In contrast, the microbiota of the RC subjects was dominated by Actinomyces spp., lactobacilli, S. mutans, Enterococcus faecalis, Selenomonas sp. clone CS002, Atopobium and Olsenella spp., Prevotella multisaccharivorax, Pseudoramibacter alactolyticus, and Propionibacterium sp. strain FMA5. The bacterial profiles of RC showed considerable subject-to-subject variation, indicating that the microbial communities are more complex than previously presumed. The data suggest that putative etiological agents of RC include not only S. mutans, lactobacilli, and Actinomyces but also species of Atopobium, Olsenella, Pseudoramibacter, Propionibacterium, and Selenomonas. PMID:18385433

  17. Bacterial Profiles of Root Caries in Elderly Patients?

    PubMed Central

    Preza, Dorita; Olsen, Ingar; Aas, Jrn A.; Willumsen, Tiril; Grinde, Bjrn; Paster, Bruce J.

    2008-01-01

    Culture-based studies have shown that Streptococcus mutans and lactobacilli are associated with root caries (RC). The purpose of the present study was to assess the bacterial diversity of RC in elderly patients by use of culture-independent molecular techniques and to determine the associations of specific bacterial species or bacterial communities with healthy and carious roots. Plaque was collected from root surfaces of 10 control subjects with no RC and from 11 subjects with RC. The bacterial 16S rRNA genes from extracted DNA were PCR amplified, cloned, and sequenced to determine species identity. From a total of 3,544 clones, 245 predominant species or phylotypes were observed, representing eight bacterial phyla. The majority (54%) of the species detected have not yet been cultivated. Species of Selenomonas and Veillonella were common in all samples. The healthy microbiota included Fusobacterium nucleatum subsp. polymorphum, Leptotrichia spp., Selenomonas noxia, Streptococcus cristatus, and Kingella oralis. Lactobacilli were absent, S. mutans was present in one, and Actinomyces spp. were present in 50% of the controls. In contrast, the microbiota of the RC subjects was dominated by Actinomyces spp., lactobacilli, S. mutans, Enterococcus faecalis, Selenomonas sp. clone CS002, Atopobium and Olsenella spp., Prevotella multisaccharivorax, Pseudoramibacter alactolyticus, and Propionibacterium sp. strain FMA5. The bacterial profiles of RC showed considerable subject-to-subject variation, indicating that the microbial communities are more complex than previously presumed. The data suggest that putative etiological agents of RC include not only S. mutans, lactobacilli, and Actinomyces but also species of Atopobium, Olsenella, Pseudoramibacter, Propionibacterium, and Selenomonas. PMID:18385433

  18. Variable rRNA gene copies in extreme halobacteria

    SciTech Connect

    Sanz, J.L.; Marin, I.; Ramirez, L.; Amils, R. ); Abad, J.P.; Smith, C.L. )

    1988-08-25

    Using PFG electrophoresis techniques, the authors have examined the organization of rRNA gene in halobacterium species. The results show that the organization of rRNA genes among closely related halobacteria is quite heterogeneous. This contrasts with the high degree of conservation of rRNA sequence. The possible mechanism of such rRNA gene amplification and its evolutionary implications are discussed.

  19. Direct 5S rRNA assay for monitoring mixed-culture bioprocesses

    SciTech Connect

    Stoner, D.L.; Bulmer, D.K.; Ward, T.E.

    1996-06-01

    This study demonstrates the efficacy of a direct 5S rRNA assay for the characterization of mixed microbial populations by using as an example the bacteria associated with acidic mining environments. The direct 5S rRNA assay described herein represents a nonselective, direct molecular method for monitoring and characterizing the predominant, metabolically active members of a microbial population. The foundation of the assay is high-resolution denaturing gradient gel electrophoresis in denaturing gradient gel electrophoresis (DGGE), which is used to separate 5S rRNA species during electrophoresis in denaturing gradient gels. With mixtures of RNA extracted from laboratory cultures, the upper practical limit for detection in the current experimental system has been estimated to be greater than 15 different species. With this method, the resolution was demonstrated to be effective at least to the species level. The strength of this approach was demonstrated by the ability to discriminate between Thiobacillus ferrooxidans ATCC 19859 and Thiobacillus thiooxidans ATCC 8085, two very closely related species. Migration patterns for the 5S rRNA from members of the genus Thiobacillus were readily distinguishable from those of the general Acidiphilium and Leptospirillum. In conclusion, the 5S rRNA assay represents a powerful method by which the structure of a microbial population within acidic environments can be assessed. 40 refs., 12 figs., 1 tab.

  20. The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction

    PubMed Central

    Fritz, Brian R.; Jewett, Michael C.

    2014-01-01

    In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3′ modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants. PMID:24792158

  1. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    SciTech Connect

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Baelum, Jacob; Tas, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Phillip; Prieme, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

  2. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    PubMed Central

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Taş, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-01-01

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below −10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface. PMID:25983731

  3. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    SciTech Connect

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Taş, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy number of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.

  4. Distinct summer and winter bacterial communities in the active layer of Svalbard permafrost revealed by DNA- and RNA-based analyses

    DOE PAGESBeta

    Schostag, Morten; Stibal, Marek; Jacobsen, Carsten S.; Bælum, Jacob; Taş, Neslihan; Elberling, Bo; Jansson, Janet K.; Semenchuk, Philipp; Priemé, Anders

    2015-04-30

    The active layer of soil overlaying permafrost in the Arctic is subjected to dramatic annual changes in temperature and soil chemistry, which likely affect bacterial activity and community structure. We studied seasonal variations in the bacterial community of active layer soil from Svalbard (78°N) by co-extracting DNA and RNA from 12 soil cores collected monthly over a year. PCR amplicons of 16S rRNA genes (DNA) and reverse transcribed transcripts (cDNA) were quantified and sequenced to test for the effect of low winter temperature and seasonal variation in concentration of easily degradable organic matter on the bacterial communities. The copy numbermore » of 16S rRNA genes and transcripts revealed no distinct seasonal changes indicating potential bacterial activity during winter despite soil temperatures well below -10°C. Multivariate statistical analysis of the bacterial diversity data (DNA and cDNA libraries) revealed a season-based clustering of the samples, and, e.g., the relative abundance of potentially active Cyanobacteria peaked in June and Alphaproteobacteria increased over the summer and then declined from October to November. The structure of the bulk (DNA-based) community was significantly correlated with pH and dissolved organic carbon, while the potentially active (RNA-based) community structure was not significantly correlated with any of the measured soil parameters. A large fraction of the 16S rRNA transcripts was assigned to nitrogen-fixing bacteria (up to 24% in June) and phototrophic organisms (up to 48% in June) illustrating the potential importance of nitrogen fixation in otherwise nitrogen poor Arctic ecosystems and of phototrophic bacterial activity on the soil surface.« less

  5. Survival of free-living Acholeplasma in aerated pig manure slurry revealed by 13C-labeled bacterial biomass probing

    PubMed Central

    Hanajima, Dai; Aoyagi, Tomo; Hori, Tomoyuki

    2015-01-01

    Many studies have been performed on microbial community succession and/or predominant taxa during the composting process; however, the ecophysiological roles of microorganisms are not well understood because microbial community structures are highly diverse and dynamic. Bacteria are the most important contributors to the organic-waste decomposition process, while decayed bacterial cells can serve as readily digested substrates for other microbial populations. In this study, we investigated the active bacterial species responsible for the assimilation of dead bacterial cells and their components in aerated pig manure slurry by using 13C-labeled bacterial biomass probing. After 3 days of forced aeration, 13C-labeled and unlabeled dead Escherichia coli cell suspensions were added to the slurry. The suspensions contained 13C-labeled and unlabeled bacterial cell components, possibly including the cell wall and membrane, as well as intracellular materials. RNA extracted from each slurry sample 2 h after addition of E. coli suspension was density-resolved by isopycnic centrifugation and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of bacterial 16S rRNA genes. In the heavy isotopically labeled RNA fraction, the predominant 13C-assimilating population was identified as belonging to the genus Acholeplasma, which was not detected in control heavy RNA. Acholeplasma spp. have limited biosynthetic capabilities and possess a wide variety of transporters, resulting in their metabolic dependence on external carbon and energy sources. The prevalence of Acholeplasma spp. was further confirmed in aerated pig manure slurry from four different pig farms by pyrosequencing of 16S rRNA genes; their relative abundance was ∼4.4%. Free-living Acholeplasma spp. had a competitive advantage for utilizing dead bacterial cells and their components more rapidly relative to other microbial populations, thus allowing the survival and prevalence of Acholeplasma spp. in pig manure slurry. PMID:26583009

  6. Deoxygenation alters bacterial diversity and community composition in the oceans largest oxygen minimum zone

    NASA Astrophysics Data System (ADS)

    Beman, J. Michael; Carolan, Molly T.

    2013-10-01

    Oceanic oxygen minimum zones (OMZs) have a central role in biogeochemical cycles and are expanding as a consequence of climate change, yet how deoxygenation will affect the microbial communities that control these cycles is unclear. Here we sample across dissolved oxygen gradients in the oceans largest OMZ and show that bacterial richness displays a unimodal pattern with decreasing dissolved oxygen, reaching maximum values on the edge of the OMZ and decreasing within it. Rare groups on the OMZ margin are abundant at lower dissolved oxygen concentrations, including sulphur-cycling Chromatiales, for which 16S rRNA was amplified from extracted RNA. Microbial species distribution models accurately replicate community patterns based on multivariate environmental data, demonstrate likely changes in distributions and diversity in the eastern tropical North Pacific Ocean, and highlight the sensitivity of key bacterial groups to deoxygenation. Through these mechanisms, OMZ expansion may alter microbial composition, competition, diversity and function, all of which have implications for biogeochemical cycling in OMZs.

  7. Direct PCR amplification of the 16S rRNA gene from single microbial cells isolated from an Antarctic iceberg using laser microdissection microscopy

    NASA Astrophysics Data System (ADS)

    Yanagihara, Katsuhiko; Niki, Hironori; Baba, Tomoya

    2011-09-01

    Here, we describe a technique that allows the genetic linage analysis of 16S rRNA genes in bacteria observed under a microscope. The technique includes the isolation of microbial cells using a laser microdissection microscope, lysis of the cells, and amplification of the 16S rRNA genes in the isolated cells without interference by bacterial DNA contamination from the experimental environment or reagents. Using this technique, we successfully determined 15 16S rRNA gene sequences in cells isolated from an Antarctic iceberg. These sequences showed 94%-100% identity to their closest strains, which included bacteria that occur in aqueous, marine, and soil environments.

  8. Effects on enteric methane production and bacterial and archaeal communities by the addition of cashew nut shell extract or glycerol-an in vitro evaluation.

    PubMed

    Danielsson, Rebecca; Werner-Omazic, Anna; Ramin, Mohammad; Schnrer, Anna; Griinari, Mikko; Dicksved, Johan; Bertilsson, Jan

    2014-09-01

    The objective of the study was to evaluate the effect of cashew nut shell extract (CNSE) and glycerol (purity >99%) on enteric methane (CH4) production and microbial communities in an automated gas in vitro system. Microbial communities from the in vitro system were compared with samples from the donor cows, in vivo. Inoculated rumen fluid was mixed with a diet with a 60:40 forage:concentrate ratio and, in total, 5 different treatments were set up: 5mg of CNSE (CNSE-L), 10mg of CNSE (CNSE-H), 15mmol of glycerol/L (glycerol-L), and 30mmol of glycerol/L (glycerol-H), and a control without feed additive. Gas samples were taken at 2, 4, 8, 24, 32, and 48h of incubation, and the CH4 concentration was measured. Samples of rumen fluid were taken for volatile fatty acid analysis and for microbial sequence analyses after 8, 24, and 48h of incubation. In vivo rumen samples from the cows were taken 2h after the morning feeding at 3 consecutive days to compare the in vitro system with in vivo conditions. The gas data and data from microbial sequence analysis (454 sequencing) were analyzed using a mixed model and principal components analysis. These analyses illustrated that CH4 production was reduced with the CNSE treatment, by 8 and 18%, respectively, for the L and H concentration. Glycerol instead increased CH4 production by 8 and 12%, respectively, for the L and H concentration. The inhibition with CNSE could be due to the observed shift in bacterial population, possibly resulting in decreased production of hydrogen or formate, the methanogenic substrates. Alternatively the response could be explained by a shift in the methanogenic community. In the glycerol treatments, no main differences in bacterial or archaeal population were detected compared with the in vivo control. Thus, the increase in CH4 production may be explained by the increase in substrate in the in vitro system. The reduced CH4 production in vitro with CNSE suggests that CNSE can be a promising inhibitor of CH4 formation in the rumen of dairy cows. PMID:24996274

  9. Profiling of ornithine lipids in bacterial extracts of Rhodobacter sphaeroides by reversed-phase liquid chromatography with electrospray ionization and multistage mass spectrometry (RPLC-ESI-MS(n)).

    PubMed

    Granafei, Sara; Losito, Ilario; Trotta, Massimo; Italiano, Francesca; de Leo, Vincenzo; Agostiano, Angela; Palmisano, Francesco; Cataldi, Tommaso R I

    2016-01-15

    Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the ?-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R.sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation (CID) multistage mass spectrometry (MS(n), n=2-4). OLs signatures found in two R.sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinations of fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra of monoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing 18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([M-H](-) at m/z 717.5) in R.sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amide-linked to ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [M-H](-) ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R.sphaeroides. PMID:26709304

  10. Comparison of bacterial community in aqueous and oil phases of water-flooded petroleum reservoirs using pyrosequencing and clone library approaches.

    PubMed

    Wang, Li-Ying; Ke, Wen-Ji; Sun, Xiao-Bo; Liu, Jin-Feng; Gu, Ji-Dong; Mu, Bo-Zhong

    2014-05-01

    Bacterial communities in both aqueous and oil phases of water-flooded petroleum reservoirs were characterized by molecular analysis of bacterial 16S rRNA genes obtained from Shengli Oil Field using DNA pyrosequencing and gene clone library approaches. Metagenomic DNA was extracted from the aqueous and oil phases and subjected to polymerase chain reaction amplification with primers targeting the bacterial 16S rRNA genes. The analysis by these two methods showed that there was a large difference in bacterial diversity between the aqueous and oil phases of the reservoir fluids, especially in the reservoirs with lower water cut. At a high phylogenetic level, the predominant bacteria detected by these two approaches were identical. However, pyrosequencing allowed the detection of more rare bacterial species than the clone library method. Statistical analysis showed that the diversity of the bacterial community of the aqueous phase was lower than that of the oil phase. Phylogenetic analysis indicated that the vast majority of sequences detected in the water phase were from members of the genus Arcobacter within the Epsilonproteobacteria, which is capable of degrading the intermediates of hydrocarbon degradation such as acetate. The oil phase of reservoir fluid samples was dominated by members of the genus Pseudomonas within the Gammaproteobacteria and the genus Sphingomonas within the Alphaproteobacteria, which have the ability to degrade crude oil through adherence to hydrocarbons under aerobic conditions. In addition, many anaerobes that could degrade the component of crude oil were also found in the oil phase of reservoir fluids, mainly in the reservoir with lower water cut. These were represented by Desulfovibrio spp., Thermodesulfovibrio spp., Thermodesulforhabdus spp., Thermotoga spp., and Thermoanaerobacterium spp. This research suggested that simultaneous analysis of DNA extracted from both aqueous and oil phases can facilitate a better understanding of the bacterial communities in water-flooded petroleum reservoirs. PMID:24413919

  11. Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.

    PubMed Central

    Taylor, D E; Ge, Z; Purych, D; Lo, T; Hiratsuka, K

    1997-01-01

    In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer. PMID:9420030

  12. Bacterial gastroenteritis

    MedlinePLUS

    Infectious diarrhea - bacterial gastroenteritis; Acute gastroenteritis; Gastroenteritis - bacterial ... the sickness. All types of food poisoning cause diarrhea . Other symptoms include: Abdominal cramps Abdominal pain Bloody ...

  13. Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.

    PubMed

    Kahlisch, Leila; Henne, Karsten; Grbe, Lothar; Brettar, Ingrid; Hfle, Manfred G

    2012-02-01

    The question which bacterial species are present in water and if they are viable is essential for drinking water safety but also of general relevance in aquatic ecology. To approach this question we combined propidium iodide/SYTO9 staining ("live/dead staining" indicating membrane integrity), fluorescence-activated cell sorting (FACS) and community fingerprinting for the analysis of a set of tap water samples. Live/dead staining revealed that about half of the bacteria in the tap water had intact membranes. Molecular analysis using 16S rRNA and 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of drinking water bacteria before and after FACS sorting revealed: (1) the DNA- and RNA-based overall community structure differed substantially, (2) the community retrieved from RNA and DNA reflected different bacterial species, classified as 53 phylotypes (with only two common phylotypes), (3) the percentage of phylotypes with intact membranes or damaged cells were comparable for RNA- and DNA-based analyses, and (4) the retrieved species were primarily of aquatic origin. The pronounced difference between phylotypes obtained from DNA extracts (dominated by Betaproteobacteria, Bacteroidetes, and Actinobacteria) and from RNA extracts (dominated by Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes, and Cyanobacteria) demonstrate the relevance of concomitant RNA and DNA analyses for drinking water studies. Unexpected was that a comparable fraction (about 21%) of phylotypes with membrane-injured cells was observed for DNA- and RNA-based analyses, contradicting the current understanding that RNA-based analyses represent the actively growing fraction of the bacterial community. Overall, we think that this combined approach provides an interesting tool for a concomitant phylogenetic and viability analysis of bacterial species of drinking water. PMID:21845446

  14. The murine lung microbiome in relation to the intestinal and vaginal bacterial communities

    PubMed Central

    2013-01-01

    Background This work provides the first description of the bacterial population of the lung microbiota in mice. The aim of this study was to examine the lung microbiome in mice, the most used animal model for inflammatory lung diseases such as COPD, cystic fibrosis and asthma. Bacterial communities from broncho-alveolar lavage fluids and lung tissue were compared to samples taken from fecal matter (caecum) and vaginal lavage fluid from female BALB/cJ mice. Results Using a customized 16S rRNA sequencing protocol amplifying the V3-V4 region our study shows that the mice have a lung microbiome that cluster separately from mouse intestinal microbiome (caecum). The mouse lung microbiome is dominated by Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Cyanobacteria overlapping the vaginal microbiome. We also show that removal of host tissue or cells from lung fluid during the DNA extraction step has an impact on the resulting bacterial community profile. Sample preparation needs to be considered when choosing an extraction method and interpreting data. Conclusions We have consistently amplified bacterial DNA from mouse lungs that is distinct from the intestinal microbiome in these mice. The gut microbiome has been extensively studied for its links to development of disease. Here we suggest that also the lung microbiome could be important in relation to inflammatory lung diseases. Further research is needed to understand the contribution of the lung microbiome and the gut-lung axis to the development of lung diseases such as COPD and asthma. PMID:24373613

  15. Bacterial communities in the phylloplane of Prunus species.

    PubMed

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

    2015-04-01

    Bacterial populations in the phylloplane of four different Prunus species were investigated by 16 S rRNA pyrosequencing. Bioinformatic analysis identified an average of 510 operational taxonomic units belonging to 159 genera in 76 families. The two genera, Sphingomonas and Methylobacterium, were dominant in the phylloplane of four Prunus species. Twenty three genera were commonly identified in the four Prunus species, indicating a high level of bacterial diversity dependent on the plant species. Our study based on 16 S rRNA sequencing reveals the complexity of bacterial diversity in the phylloplane of Prunus species in detail. PMID:25515303

  16. Genotoxicity evaluation of Guibi-Tang extract using an in vitro bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus test

    PubMed Central

    2014-01-01

    Background Guibi-Tang is a traditional herbal prescription made from 12 different herbs that is used in the treatment of amnesia and poor memory. Methods In the present study, we evaluated the acute oral toxicity and genotoxic potential of Guibi-Tang water extract (GBT) at doses up to 2000?g/plate an using a bacterial reverse mutation test (Ames test) with Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537, and Escherichia coli strain WP2uvrA. Acute toxicity and genotoxic potential were measured in the presence and absence of an exogenous source of metabolic activation, in an in vitro chromosome aberration assay with Chinese hamster lung (CHL) cells, and in an in vivo micronucleus test using ICR mice bone marrow as recommended by the Korean Food and Drug Administration. An acute oral toxicity test of GBT was performed in Sprague Dawley rats. The Ames test showed that GBT did not induce gene mutations in S. typhimurium or in E. coli in the presence or absence of S9 activation. Results GBT did not significantly increase the number of structural aberrations in CHL cells with or without S9 activation. The oral administration of GBT at a dose of up to 2000mg/kg caused no significant increase in the number of micronucleated polychromatic erythrocytes or in the mean ratio of polychromatic to total erythrocytes. Conclusions However, as we did not identify the components of GBT responsible for these effects, other assays are needed to confirm its genotoxicity. PMID:24985139

  17. Preventive treatment of chronic bronchitis: a meta-analysis of clinical trials with a bacterial extract (OM-85 BV) and a cost-effectiveness analysis.

    PubMed

    Bergemann, R; Brandt, A; Zoellner, U; Donner, C F

    1994-09-01

    Chronic bronchitis is a lifelong disease with significant effects on the patient and on the costs to health insurance institutions. Acute exacerbations in chronic bronchitic patients may have a negative impact on patients' quality of life and on the progression of the disease, particularly in more severe patients. The clinical efficacy of the immunoactive bacterial extract OM-85 BV has been shown in several clinical trials, a cost-effectiveness evaluation (CEA) of its use in chronic bronchitic patients has not been fulfilled so far. In this study a meta-analysis on the preventive treatment of acute exacerbations with OM-85 BV and a CEA, focusing on direct costs only, was performed. The meta-analysis showed a mean value of 0.6 prevented acute exacerbations per 6 months per patient, and a reduction of 9 days in antibiotic treatment per 6 months per patient. CEA evaluated the different cost elements. The mean direct cost (consultations, antibiotics, etc.) for the treatment of 1 acute exacerbation in chronic bronchitic patients was calculated as 143, 459 Lira. Thus for 0.6 prevented acute exacerbations per 6 months per patient a reduction in cost of 86,075 Lira (0.6-143,459) could be expected. The additional costs for the preventive treatment with OM-85 BV, based on prices for Italy, are 34,980 Lira per patient per 6 months. In conclusion, the effective cost savings per patient per 6 months are 51, 095 Lira. The sensitivity analysis revealed only one critical parameter, i.e. the clinical effectiveness of OM-85 BV. Even assuming 0.3 prevented exacerbations per 6 months per patient, the preventive treatment still proved to be cost effective. PMID:8000415

  18. Bacterial diversity in a deep-subsurface clay environment.

    PubMed Central

    Boivin-Jahns, V; Ruimy, R; Bianchi, A; Daumas, S; Christen, R

    1996-01-01

    The presence of bacteria in a deep clay sediment was analyzed in a 20-m-long core horizontally drilled from a mine gallery at a depth of 224 m in the Boom clay formation (Mol, Belgium). This clay deposit is the result of a marine sedimentary process that occurred 35 million years ago. Bacterial activities were estimated by measuring respiration on [14C]glucose. Using the same samples, universal primers for the genes coding for eubacterial 16S rRNA were used to amplify extracted DNA. PCR products were then cloned, sequenced, and analyzed by molecular phylogeny. Our data showed a decrease in bacterial densities as a function of distance from the gallery, with few bacteria detectable by culture at more than 80 cm from the gallery wall. PCR experiments showed the presence of bacteria in all samples, and phylogenetic analyses were then used to tentatively identify these organisms. Because of low bacterial densities in deep clay samples, direct counts and enumeration of viable bacteria on diverse culture media remained negative. All experiments, both cultures and PCR, demonstrated the difficulty of analyzing samples that contain only a few poorly active bacteria as it is difficult to avoid a small contamination by active bacteria during sampling. Since the porosity of the Boom clay formation is less than the expected size of bacteria, it is possible that some of the bacteria present in this 35-million-year-old deep clay deposit derive from cells initially trapped during the sedimentation process. PMID:8795233

  19. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis.

    PubMed

    Bahl, Martin Iain; Bergstrm, Anders; Licht, Tine Rask

    2012-04-01

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology. PMID:22325006

  20. Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota.

    PubMed

    Bartosch, Sabine; Fite, Alemu; Macfarlane, George T; McMurdo, Marion E T

    2004-06-01

    Fecal bacteria were studied in healthy elderly volunteers (age, 63 to 90 years; n = 35) living in the local community, elderly hospitalized patients (age, 66 to 103; n = 38), and elderly hospitalized patients receiving antibiotic treatment (age, 65 to 100; n = 21). Group- and species-specific primer sets targeting 16S rRNA genes were used to quantitate intestinal bacteria by using DNA extracted from feces and real-time PCR. The principal difference between healthy elderly volunteers and both patient cohorts was a marked reduction in the Bacteroides-Prevotella group following hospitalization. Reductions in bifidobacteria, Desulfovibrio spp., Clostridium clostridiiforme, and Faecalibacterium prausnitzii were also found in the hospitalized patients. However, total 16S rRNA gene copy numbers (per gram of wet weight of feces) were generally lower in the stool samples of the two groups of hospitalized patients compared to the number in the stool samples of elderly volunteers living in the community, so the relative abundance (percentage of the group- and species-specific rRNA gene copies in relation to total bacterial rRNA gene copies) of bifidobacteria, Desulfovibrio spp., C. clostridiiforme, and F. prausnitzii did not change. Antibiotic treatment resulted in further reductions in the numbers of bacteria and their prevalence and, in some patients, complete elimination of certain bacterial communities. Conversely, the numbers of enterobacteria increased in the hospitalized patients who did not receive antibiotics, and due to profound changes in fecal microbiotas during antibiotic treatment, the opportunistic species Enterococcus faecalis proliferated. PMID:15184159

  1. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis.

    PubMed

    Cole, J R; Wang, Q; Cardenas, E; Fish, J; Chai, B; Farris, R J; Kulam-Syed-Mohideen, A S; McGarrell, D M; Marsh, T; Garrity, G M; Tiedje, J M

    2009-01-01

    The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/. PMID:19004872

  2. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis

    PubMed Central

    Cole, J. R.; Wang, Q.; Cardenas, E.; Fish, J.; Chai, B.; Farris, R. J.; Kulam-Syed-Mohideen, A. S.; McGarrell, D. M.; Marsh, T.; Garrity, G. M.; Tiedje, J. M.

    2009-01-01

    The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/. PMID:19004872

  3. Bacteriocin from Bacillus subtilis as a novel drug against diabetic foot ulcer bacterial pathogens

    PubMed Central

    Joseph, Baby; Dhas, Berlina; Hena, Vimalin; Raj, Justin

    2013-01-01

    Objective To isolate and identify Bacillus subtilis (B. subtilis) from soil and to characterize and partially purify the bacteriocin. To evaluate the antimicrobial activity against four diabetic foot ulcer bacterial pathogens. Methods Genotypic identification was done based on Bergey's manual of systemic bacteriology. Antimicrobial susceptibility test was done by Kirby-Bauer disc diffusion method. Colonies were identified by colony morphology and biochemical characterization and also compared with MTCC 121 strain. Further identification was done by 16S rRNA sequencing. Inhibitory activities of partially purified bacteriocin on all the DFU isolates were done by agar well diffusion method. The strain was identified to produce bacteriocin by stab overlay assay. Bacteriocin was extracted by organic solvent extraction using chloroform, further purified by HPLC and physical, and chemical characterization was performed. Results The four isolates showed high level of resistance to amoxyclav and sensitivity to ciprofloxacin. HPLC purification revealed that the extracts are bacteriocin. The phylogenetic tree analysis results showed that the isolate was 99% related to B. subtilis BSF01. The results reveled activity to all the four isolates and high level of activity was seen in case of Klebsiella sp. Conclusions Partially purified bacteriocin was found to have antimicrobial activity against the four diabetic foot ulcer bacterial pathogens, which can thus be applied as a better drug molecule on further studies. The strain B. subtilis are found to be safe for use and these antimicrobial peptides can be used as an antimicrobial in humans to treat DFU bacterial pathogens. PMID:24093784

  4. Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer

    PubMed Central

    Ruegger, Paul M.; Clark, Robin T.; Weger, John R.; Braun, Jonathan; Borneman, James

    2014-01-01

    Current high throughput sequencing (HTS) methods are limited in their ability to resolve bacteria at or below the genus level. While the impact of this limitation may be relatively minor in whole-community analyses, it constrains the use of HTS as a tool for identifying and examining individual bacteria of interest. The limited resolution is a consequence of both short read lengths and insufficient sequence variation within the commonly targeted variable regions of the small-subunit rRNA (SSU) gene. The goal of this work was to improve the resolving power of bacterial HTS. We developed an assay targeting the hypervariable rRNA internal transcribed spacer (ITS) region residing between the SSU and large-subunit (LSU) rRNA genes. Comparisons of the ITS region and two SSU regions using annotated bacterial genomes in GenBank showed much greater resolving power is possible with the ITS region. This report presents a new HTS method for analyzing bacterial composition with improved capabilities. The greater resolving power enabled by the ITS region arises from its high sequence variation across a wide range of bacterial taxa and an associated decrease in taxonomic heterogeneity within its OTUs. Although the method should be adaptable to any HTS platform, this report presents PCR primers, amplification parameters, and protocols for Illumina-based analyses. PMID:25034229

  5. Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome

    PubMed Central

    Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jrn A; Paster, Bruce J; DeSantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

    2010-01-01

    AIM: To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. METHODS: A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. RESULTS: Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species. Assuming that a typical polymerase chain reaction can tolerate 2 mismatches between a primer and a template, the modified 347F and 803R primers should be able to anneal 98% and 99.6% of all 16S rRNA genes in the RDP database. CONCLUSION: 347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes. PMID:20806429

  6. Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6

    SciTech Connect

    Allison E. Ray; Stephanie A. Connon; Peter P. Sheridan; Jeremy Gilbreath; Malcolm S. Shields; Deborah T. Newby; Yoshiko Fujita; Timothy S. Magnuson

    2010-06-01

    The determination of variation in 16S rRNA gene sequences is perhaps the most common method for assessing microbial community diversity. However, the occurrence of multiple copies of 16S rRNA genes within some organisms can bias estimates of microbial diversity. During phylogenetic characterization of a metal-transforming, fermentative bacterium (strain UFO1) isolated from the Field Research Center (FRC) in Oak Ridge, TN, we detected an apparent 16S rRNA pseudogene. The putative 16S rRNA pseudogene was first detected in clone libraries constructed with 16S rRNA genes amplified from UFO1 genomic DNA. Sequencing revealed two distinct 16S rRNA genes, with one differing from the other by a 100 bp insert near the 5 end. Ribosomal RNA was extracted from strain UFO1 and analyzed by RT-qPCR with insert and non-insert specific primers; however, only the non-insert 16S rRNA sequence was expressed. Reverse-transcribed rRNA from strain UFO1 was also used to construct a cDNA library. Of 190 clones screened by PCR, none contained the 16S rRNA gene with the 100 bp insert. Examination of GenBank 16S rRNA gene sequences revealed that the same insert sequence was present in other clones, including those from an environmental library constructed from FRC enrichments. These findings demonstrate the existence of widely disparate copies of the 16S rRNA gene in the same species and a putative 16S rRNA pseudogene, which may confound 16S rRNA-based methods for assessments of microbial diversity in environmental samples.

  7. Bacterial populations colonizing and degrading rice straw in anoxic paddy soil.

    PubMed

    Weber, S; Stubner, S; Conrad, R

    2001-03-01

    Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the alpha, beta, and gamma Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa. PMID:11229927

  8. Bacterial Populations Colonizing and Degrading Rice Straw in Anoxic Paddy Soil

    PubMed Central

    Weber, Sabine; Stubner, Stephan; Conrad, Ralf

    2001-01-01

    Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the ?, ?, and ? Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa. PMID:11229927

  9. Nested PCR Biases in Interpreting Microbial Community Structure in 16S rRNA Gene Sequence Datasets

    PubMed Central

    Yu, Guoqin; Fadrosh, Doug; Goedert, James J.; Ravel, Jacques; Goldstein, Alisa M.

    2015-01-01

    Background Sequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention. Results In this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon’s index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool). Conclusions Nested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies. PMID:26196512

  10. Impact of Phanerochaete chrysosporium on the Functional Diversity of Bacterial Communities Associated with Decaying Wood.

    PubMed

    Hervé, Vincent; Ketter, Elodie; Pierrat, Jean-Claude; Gelhaye, Eric; Frey-Klett, Pascale

    2016-01-01

    Bacteria and fungi naturally coexist in various environments including forest ecosystems. While the role of saprotrophic basidiomycetes in wood decomposition is well established, the influence of these fungi on the functional diversity of the wood-associated bacterial communities has received much less attention. Based on a microcosm experiment, we tested the hypothesis that both the presence of the white-rot fungus Phanerochaete chrysosporium and the wood, as a growth substrate, impacted the functional diversity of these bacterial communities. Microcosms containing sterile sawdust were inoculated with a microbial inoculum extracted from a forest soil, in presence or in absence of P. chrysosporium and subsequently, three enrichment steps were performed. First, bacterial strains were isolated from different microcosms previously analyzed by 16S rRNA gene-based pyrosequencing. Strains isolated from P. chrysosporium mycosphere showed less antagonism against this fungus compared to the strains isolated from the initial forest soil inoculum, suggesting a selection by the fungus of less inhibitory bacterial communities. Moreover, the presence of the fungus in wood resulted in a selection of cellulolytic and xylanolytic bacterial strains, highlighting the role of mycospheric bacteria in wood decomposition. Additionally, the proportion of siderophore-producing bacteria increased along the enrichment steps, suggesting an important role of bacteria in iron mobilization in decaying-wood. Finally, taxonomic identification of 311 bacterial isolates revealed, at the family level, strong similarities with the high-throughput sequencing data as well as with other studies in terms of taxonomic composition of the wood-associated bacterial community, highlighting that the isolated strains are representative of the wood-associated bacterial communities. PMID:26824755

  11. Impact of Phanerochaete chrysosporium on the Functional Diversity of Bacterial Communities Associated with Decaying Wood

    PubMed Central

    Hervé, Vincent; Ketter, Elodie; Pierrat, Jean-Claude; Gelhaye, Eric; Frey-Klett, Pascale

    2016-01-01

    Bacteria and fungi naturally coexist in various environments including forest ecosystems. While the role of saprotrophic basidiomycetes in wood decomposition is well established, the influence of these fungi on the functional diversity of the wood-associated bacterial communities has received much less attention. Based on a microcosm experiment, we tested the hypothesis that both the presence of the white-rot fungus Phanerochaete chrysosporium and the wood, as a growth substrate, impacted the functional diversity of these bacterial communities. Microcosms containing sterile sawdust were inoculated with a microbial inoculum extracted from a forest soil, in presence or in absence of P. chrysosporium and subsequently, three enrichment steps were performed. First, bacterial strains were isolated from different microcosms previously analyzed by 16S rRNA gene-based pyrosequencing. Strains isolated from P. chrysosporium mycosphere showed less antagonism against this fungus compared to the strains isolated from the initial forest soil inoculum, suggesting a selection by the fungus of less inhibitory bacterial communities. Moreover, the presence of the fungus in wood resulted in a selection of cellulolytic and xylanolytic bacterial strains, highlighting the role of mycospheric bacteria in wood decomposition. Additionally, the proportion of siderophore-producing bacteria increased along the enrichment steps, suggesting an important role of bacteria in iron mobilization in decaying-wood. Finally, taxonomic identification of 311 bacterial isolates revealed, at the family level, strong similarities with the high-throughput sequencing data as well as with other studies in terms of taxonomic composition of the wood-associated bacterial community, highlighting that the isolated strains are representative of the wood-associated bacterial communities. PMID:26824755

  12. Patterns of bacterial diversity across a range of Antarctic terrestrial habitats.

    PubMed

    Yergeau, Etienne; Newsham, Kevin K; Pearce, David A; Kowalchuk, George A

    2007-11-01

    Although soil-borne bacteria represent the world's greatest source of biological diversity, it is not well understood whether extreme environmental conditions, such as those found in Antarctic habitats, result in reduced soil-borne microbial diversity. To address this issue, patterns of bacterial diversity were studied in soils sampled along a > 3200 km southern polar transect spanning a gradient of increased climate severity over 27 degrees of latitude. Vegetated and fell-field plots were sampled at the Falkland (51 degrees S), South Georgia (54 degrees S), Signy (60 degrees S) and Anchorage Islands (67 degrees S), while bare frost-sorted soil polygons were examined at Fossil Bluff (71 degrees S), Mars Oasis (72 degrees S), Coal Nunatak (72 degrees S) and the Ellsworth Mountains (78 degrees S). Bacterial 16S rRNA gene sequences were recovered subsequent to direct DNA extraction from soil, polymerase chain reaction amplification and cloning. Although bacterial diversity was observed to decline with increased latitude, habitat-specific patterns appeared to also be important. Namely, a negative relationship was found between bacterial diversity and latitude for fell-field soils, but no such pattern was observed for vegetated sites. The Mars Oasis site, previously identified as a biodiversity hotspot within this region, proved exceptional within the study transect, with unusually high bacterial diversity. In independent analyses, geographical distance and vegetation cover were found to significantly influence bacterial community composition. These results provide insight into the factors shaping the composition of bacterial communities in Antarctic terrestrial habitats and support the notion that bacterial diversity declines with increased climatic severity. PMID:17922752

  13. Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB▿

    PubMed Central

    Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

    2011-01-01

    Despite the increasing use of 16S rRNA gene sequencing, interpretation of 16S rRNA gene sequence results is one of the most difficult problems faced by clinical microbiologists and technicians. To overcome the problems we encountered in the existing databases during 16S rRNA gene sequence interpretation, we built a comprehensive database, 16SpathDB (http://147.8.74.24/16SpathDB) based on the 16S rRNA gene sequences of all medically important bacteria listed in the Manual of Clinical Microbiology and evaluated its use for automated identification of these bacteria. Among 91 nonduplicated bacterial isolates collected in our clinical microbiology laboratory, 71 (78%) were reported by 16SpathDB as a single bacterial species having >98.0% nucleotide identity with the query sequence, 19 (20.9%) were reported as more than one bacterial species having >98.0% nucleotide identity with the query sequence, and 1 (1.1%) was reported as no match. For the 71 bacterial isolates reported as a single bacterial species, all results were identical to their true identities as determined by a polyphasic approach. For the 19 bacterial isolates reported as more than one bacterial species, all results contained their true identities as determined by a polyphasic approach and all of them had their true identities as the “best match in 16SpathDB.” For the isolate (Gordonibacter pamelaeae) reported as no match, the bacterium has never been reported to be associated with human disease and was not included in the Manual of Clinical Microbiology. 16SpathDB is an automated, user-friendly, efficient, accurate, and regularly updated database for 16S rRNA gene sequence interpretation in clinical microbiology laboratories. PMID:21389154

  14. Bacterial and archaeal populations associated with freshwater ferromanganous micronodules and sediments.

    PubMed

    Stein, L Y; La Duc, M T; Grundl, T J; Nealson, K H

    2001-01-01

    Biology is believed to play a large role in the cycling of iron and manganese in many freshwater environments, but specific microbial groups indigenous to these systems have not been well characterized. To investigate the populations of Bacteria and Archaea associated with metal-rich sediments from Green Bay, WI, we extracted nucleic acids and analysed the phylogenetic relationships of cloned 16S rRNA genes. Because nucleic acids have not been routinely extracted from metal-rich samples, we investigated the bias inherent in DNA extraction and gene amplification from pure MnO2 using defined populations of whole cells or naked DNA. From the sediments, we screened for manganese-oxidizing bacteria using indicator media and found three isolates that were capable of manganese oxidation. In the phylogenetic analysis of bacterial 16S rRNA gene clones, we found two groups related to known metal-oxidizing genera, Leptothrix of the beta-Proteobacteria and Hyphomicrobium of the alpha-Proteobacteria, and a Fe(III)-reducing group related to the Magnetospirillum genus of the alpha-Proteobacteria. Groups related to the metal-reducing delta-Proteobacteria constituted 22% of the gene clones. In addition, gene sequences from one group of methanogens and a group of Crenarchaeota, identified in the archaeal gene clone library, were related to those found previously in Lake Michigan sediments. PMID:11225719

  15. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences.

    PubMed

    Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

    2015-02-25

    The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

  16. Common 5S rRNA variants are likely to be accepted in many sequence contexts

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

    2003-01-01

    Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.

  17. Capnocytophaga canimorsus meningitis diagnosed by 16S rRNA PCR.

    PubMed

    Beernink, T M J; Wever, P C; Hermans, M H A; Bartholomeus, M G T

    2016-04-01

    Capnocytophaga canimorsus is a common Gram-negative anaerobic bacterium from the oral flora of dogs, typically transmitted to humans by dog bites. We report a case of C. canimorsus meningitis where there was (on presentation) no apparent predisposing risk factor and in whom we used 16S rRNA PCR gene sequencing to identify the pathogen quickly and to switch to appropriate antibiotic therapy. Physicians should be aware of potential C. canimorsus meningitis if conventional cerebrospinal fluid bacterial culture is negative but Gram staining identifies bacteria, especially in patients with a recent dog bite or known immunodeficiency. PMID:26608220

  18. 5 S rRNA: structure and interactions.

    PubMed Central

    Szyma?ski, Maciej; Barciszewska, Miros?awa Z; Erdmann, Volker A; Barciszewski, Jan

    2003-01-01

    5 S rRNA is an integral component of the large ribosomal subunit in all known organisms. Despite many years of intensive study, the function of 5 S rRNA in the ribosome remains unknown. Advances in the analysis of ribosome structure that have revealed the crystal structures of large ribosomal subunits and of the complete ribosome from various organisms put the results of studies on 5 S rRNA in a new perspective. This paper summarizes recently published data on the structure and function of 5 S rRNA and its interactions in complexes with proteins, within and outside the ribosome. PMID:12564956

  19. Bacterial Vaginosis

    MedlinePLUS

    ... vaginosis can increase your chance of getting an STD. What is bacterial vaginosis? Bacterial vaginosis (BV) is ... contributes to BV. BV is not considered an STD, but having BV can increase your chances of ...

  20. Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences.

    PubMed

    Lu, Jingrang; Domingo, Jorge Santo

    2008-10-01

    The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the microbial composition of avian fecal microbial communities. PMID:18974945

  1. Microbial community of salt crystals processed from Mediterranean seawater based on 16S rRNA analysis.

    PubMed

    Baati, Houda; Guermazi, Sonda; Gharsallah, Neji; Sghir, Abdelghani; Ammar, Emna

    2010-01-01

    Phylogenetic analysis of 16S rRNA was used to investigate for the first time the structure of the microbial community that inhabits salt crystals retrieved from the bottom of a solar saltern, located in the coastal area of the Mediterranean Sea (Sfax, Tunisia). This community lives in an extremely salty environment of 250-310 g/L total dissolved salt. A total of 78 bacterial 16S rRNA clone sequences making up to 21 operational taxonomic units (OTUs), determined by the DOTUR program to 97% sequence similarity, was analyzed. These OTUs were affiliated to Bacteroidetes (71.4% of OTUs), and gamma-Proteobacteria and alpha-Proteobacteria (equally represented by 14.2% of the OTUs observed). The archaeal community composition appeared more diverse with 68 clones, resulting in 44 OTUs, all affiliated with the Euryarchaeota phylum. Of the bacterial and archaeal clones showing <97% 16S rRNA sequence identity with sequences in public databases, 47.6% and 84.1% respectively were novel clones. Both rarefaction curves and diversity measurements (Simpson, Shannon-Weaver, Chao) showed a more diverse archaeal than bacterial community at the Tunisian solar saltern pond. The analysis of an increasing clone's number may reveal additional local diversity. PMID:20130693

  2. Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria.

    PubMed

    Schabereiter-Gurtner, Claudia; Lubitz, Werner; Rlleke, Sabine

    2003-02-01

    Ticks play an important role in the transmission of arthropod-borne diseases of viral, protozoal and bacterial origin. The present article describes a molecular-biological based method, which facilitated the broad-range analyses of bacterial communities in ixodid ticks (Ixodes ricinus). DNA was extracted both from single ticks and pooled adult ticks. Eubacterial 16S rRNA gene fragments (16S rDNA) were amplified by polymerase chain reaction (PCR) with broad-range ribosomal primers. Sequences spanning the hypervariable V3 region of the 16S rDNA and representing individual bacterial taxons were separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were exised, cloned and sequenced. In addition, we set up a 16S rDNA clone library which was screened by DGGE. Sequences were compared with sequences of known bacteria listed in the GenBank database. A number of bacteria were affiliated with the genera Rickettsia, Bartonella, and Borrelia, which are known to be pathogenic and transmitted by ticks. Two sequences were related to the yet to be cultivated Haemobartonella. To our knowledge, Haemobartonella has never been directly detected in I. ricinus. In addition, members of the genera Staphylococcus, Rhodococcus, Pseudomonas, and Moraxella were detected, which have not been identified in ticks so far. Two bacteria were most closely related to a rickettsial endosymbiont of an Acanthamoeba sp., and to an endosymbiont (Legionellaceae, Coxiella group) of the microarthropod Folsomia candida. The results prove that 16S rDNA genotyping in combination with DGGE analysis is a promising approach for the detection and identification of bacteria infecting ticks, regardless of whether these bacteria are fastidious, obligate intracellular or noncultivable. PMID:12459246

  3. Assessment of microbial dynamics in the Pearl River Estuary by 16S rRNA terminal restriction fragment analysis

    NASA Astrophysics Data System (ADS)

    Wu, Madeline; Song, Liansheng; Ren, Jianping; Kan, Jianjun; Qian, Pei-Yuan

    2004-10-01

    We have evaluated the feasibility of using the terminal restriction fragment length polymorphism (T-RFLP) pattern of polymerase chain reaction (PCR) amplified 16S rRNA sequences to track the changes of the free-living bacterial community for the Pearl River Estuary surface waters. The suitability of specific PCR primers, PCR bias induced by thermal cycles, and field-sampling volumes were critically evaluated in laboratory tests. We established a workable protocol and obtained TRF patterns that reflected the changes in the bacterial population. The temporal dynamics over a 24 h period were examined at one anchored station, as well as the spatial distribution pattern of the bacterial community at several stations, covering the transects along the river discharge direction and across the river plume. The TRF pattern revealed 9 dominant bacterial groups. Changes in their relative abundance reflecting the changes in the bacterial community composition were documented. Many culturable species were isolated from each field sample and a portion of the 16S rRNA gene for each species was sequenced. The species was identified based on sequence data comparison. In this region, the dominant species belong to the γ-subdivision of proteobacteria and the Bacillus/Clostridium group of Firmicutes. We also detected the wide spread distribution of Acinetobacter spp.; many of these species are known nosocomial pathogen for humans.

  4. Dynamics of bacterial and fungal communities associated with eggshells during incubation

    PubMed Central

    Grizard, Stéphanie; Dini-Andreote, Francisco; Tieleman, B Irene; Salles, Joana F

    2014-01-01

    Microorganisms are closely associated with eggs and may play a determinant role in embryo survival. Yet, the majority of studies focusing on this association relied on culture-based methodology, eventually leading to a skewed assessment of microbial communities. By targeting the 16S rRNA gene and internal transcribed spacer (ITS) region, we, respectively, described bacterial and fungal communities on eggshells of the homing pigeon Columba livia. We explored their structure, abundance, and composition. Firstly, we showed that sampling technique affected the outcome of the results. While broadly used, the egg swabbing procedure led to a lower DNA extraction efficiency and provided different profiles of bacterial communities than those based on crushed eggshell pieces. Secondly, we observed shifts in bacterial and fungal communities during incubation. At late incubation, bacterial communities showed a reduction in diversity, while their abundance increased, possibly due to the competitive advantage of some species. When compared to their bacterial counterparts, fungal communities also decreased in diversity at late incubation. In that case, however, the decline was associated with a diminution of their overall abundance. Conclusively, our results showed that although incubation might inhibit microbial growth when compared to unincubated eggs, we observed the selective growth of specific bacterial species during incubation. Moreover, we showed that fungi are a substantial component of the microbial communities associated with eggshells and require further investigations in avian ecology. Identifying the functional roles of these microorganisms is likely to provide news insights into the evolutionary strategies that control embryo survival. We aimed to describe the dynamics of bacterial and fungal communities on homing pigeon eggshell surfaces. We investigated these communities at early and late incubation stages. PMID:24772289

  5. Bacteria evade immune recognition via TLR13 and binding of their 23S rRNA by MLS antibiotics by the same mechanisms

    PubMed Central

    Hochrein, Hubertus; Kirschning, Carsten J.

    2013-01-01

    The immune system recognizes pathogens and other danger by means of pattern recognition receptors. Recently, we have demonstrated that the orphan Toll-like receptor 13 (TLR13) senses a defined sequence of the bacterial rRNA and that bacteria use specific mechanisms to evade macrolide lincosamide streptogramin (MLS) antibiotics detection via TLR13. PMID:23802068

  6. Bacterial Sialidase

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Data shows that elevated sialidase in bacterial vaginosis patients correlates to premature births in women. Bacterial sialidase also plays a significant role in the unusual colonization of Pseudomonas aeruginosa in cystic fibrosis patients. Crystals of Salmonella sialidase have been reproduced and are used for studying the inhibitor-enzyme complexes. These inhibitors may also be used to inhibit a trans-sialidase of Trypanosome cruzi, a very similar enzyme to bacterial sialidase, therefore preventing T. cruzi infection, the causitive agent of Chagas' disease. The Center for Macromolecular Crystallography suggests that inhibitors of bacterial sialidases can be used as prophylactic drugs to prevent bacterial infections in these critical cases.

  7. A mitochondrial rRNA dimethyladenosine methyltransferase in Arabidopsis

    PubMed Central

    Richter, Uwe; Kühn, Kristina; Okada, Sachiko; Brennicke, Axel; Weihe, Andreas; Börner, Thomas

    2010-01-01

    S-adenosyl-l-methionine-dependent rRNA dimethylases mediate the methylation of two conserved adenosines near the 3′ end of the rRNA in the small ribosomal subunits of bacteria, archaea and eukaryotes. Proteins related to this family of dimethylases play an essential role as transcription factors (mtTFBs) in fungal and animal mitochondria. Human mitochondrial rRNA is methylated and human mitochondria contain two related mtTFBs, one proposed to act as rRNA dimethylase, the other as transcription factor. The nuclear genome of Arabidopsis thaliana encodes three dimethylase/mtTFB-like proteins, one of which, Dim1B, is shown here to be imported into mitochondria. Transcription initiation by mitochondrial RNA polymerases appears not to be stimulated by Dim1B in vitro. In line with this finding, phylogenetic analyses revealed Dim1B to be more closely related to a group of eukaryotic non-mitochondrial rRNA dimethylases (Dim1s) than to fungal and animal mtTFBs. We found that Dim1B was capable of substituting the E. coli rRNA dimethylase activity of KsgA. Moreover, we observed methylation of the conserved adenines in the 18S rRNA of Arabidopsis mitochondria; this modification was not detectable in a mutant lacking Dim1B. These data provide evidence: (i) for rRNA methylation in Arabidopsis mitochondria; and (ii) that Dim1B is the enzyme catalyzing this process. PMID:19929881

  8. Optimized microbial DNA extraction from diarrheic stools

    PubMed Central

    2012-01-01

    Background The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction. Findings A total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4%) non-lyophilised (Ct value 28.78??9.1) versus 39/41 (95.1%) lyophilised aliquots (Ct value 22.04??5.5); bacterial 16S rRNA was detected in 33/41 (80.5%) non-lyophilised (Ct value 28.11??5.9) versus 40/41 (97.6%) lyophilised aliquots (Ct value 24.94??6.6); and S. enterica was detected in 6/6 (100%) non-lyophilized and lyophilized aliquots (Ct value 26.98??4.55 and 26.16??4.97, respectively). S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p?=?0.00043) and Bacteria (p?=?0.015). Conclusion Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea. PMID:23273000

  9. Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.

    PubMed

    Neria-Gonzlez, Isabel; Wang, En Tao; Ramrez, Florina; Romero, Juan M; Hernndez-Rodrguez, Csar

    2006-06-01

    Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions. PMID:16765858

  10. High-throughput 16S rRNA gene sequencing reveals alterations of intestinal microbiota in myalgic encephalomyelitis/chronic fatigue syndrome patients.

    PubMed

    Frémont, Marc; Coomans, Danny; Massart, Sebastien; De Meirleir, Kenny

    2013-08-01

    Human intestinal microbiota plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. Intestinal dysfunction is a frequent complaint in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients, and previous reports suggest that dysbiosis, i.e. the overgrowth of abnormal populations of bacteria in the gut, is linked to the pathogenesis of the disease. We used high-throughput 16S rRNA gene sequencing to investigate the presence of specific alterations in the gut microbiota of ME/CFS patients from Belgium and Norway. 43 ME/CFS patients and 36 healthy controls were included in the study. Bacterial DNA was extracted from stool samples, PCR amplification was performed on 16S rRNA gene regions, and PCR amplicons were sequenced using Roche FLX 454 sequencer. The composition of the gut microbiota was found to differ between Belgian controls and Norwegian controls: Norwegians showed higher percentages of specific Firmicutes populations (Roseburia, Holdemania) and lower proportions of most Bacteroidetes genera. A highly significant separation could be achieved between Norwegian controls and Norwegian patients: patients presented increased proportions of Lactonifactor and Alistipes, as well as a decrease in several Firmicutes populations. In Belgian subjects the patient/control separation was less pronounced, however some abnormalities observed in Norwegian patients were also found in Belgian patients. These results show that intestinal microbiota is altered in ME/CFS. High-throughput sequencing is a useful tool to diagnose dysbiosis in patients and could help designing treatments based on gut microbiota modulation (antibiotics, pre and probiotics supplementation). PMID:23791918

  11. Antimicrobial activity of various extracts of Ocimum basilicum L. and observation of the inhibition effect on bacterial cells by use of scanning electron microscopy.

    PubMed

    Kaya, Ilhan; Yigit, Nazife; Benli, Mehlika

    2008-01-01

    The antimicrobial activities of chloroform, acetone and two different concentrations of methanol extracts of Ocimum basilicum L. were studied. These extracts were tested in vitro against 10 bacteria and 4 yeasts strains by the disc diffusion method. The results indicated that the methanol extracts of O. basilucum exhibited the antimicrobial activity against tested microorganisms. While the chloroform and acetone extracts had no effect, the methanol extracts showed inhibition zones against strains of Pseudomonas aeruginosa, Shigella sp., Listeria monocytogenes, Staphylococcus aureus and two different strains of Escherichia coli. The cells of microorganisms, which were treated and untreated with plant extracts, were observed by using the scanning electron microscope. It was observed that the treated cells were damaged. PMID:20161958

  12. Comparison of the Rhizosphere Bacterial Communities of Zigongdongdou Soybean and a High-Methionine Transgenic Line of This Cultivar

    PubMed Central

    Ji, Jun; Wu, Haiying; Meng, Fang; Zhang, Mingrong; Zheng, Xiaobo; Wu, Cunxiang; Zhang, Zhengguang

    2014-01-01

    Previous studies have shown that methionine from root exudates affects the rhizosphere bacterial population involved in soil nitrogen fixation. A transgenic line of Zigongdongdou soybean cultivar (ZD91) that expresses Arabidopsis cystathionine γ-synthase resulting in an increased methionine production was examined for its influence to the rhizosphere bacterial population. Using 16S rRNA gene-based pyrosequencing analysis of the V4 region and DNA extracted from bacterial consortia collected from the rhizosphere of soybean plants grown in an agricultural field at the pod-setting stage, we characterized the populational structure of the bacterial community involved. In total, 87,267 sequences (approximately 10,908 per sample) were analyzed. We found that Acidobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Planctomycetes, Gemmatimonadetes, Firmicutes, and Verrucomicrobia constitute the dominant taxonomic groups in either the ZD91 transgenic line or parental cultivar ZD, and that there was no statistically significant difference in the rhizosphere bacterial community structure between the two cultivars. PMID:25079947

  13. Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome

    PubMed Central

    Yuan, Sanqing; Cohen, Dora B.; Ravel, Jacques; Abdo, Zaid; Forney, Larry J.

    2012-01-01

    Background DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled. Methodology/Principal Findings In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used. Conclusions/Significance Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells. PMID:22457796

  14. Bead Array Direct rRNA Capture Assay (rCapA) for Amplification Free Speciation of Mycobacterium Cultures

    PubMed Central

    de Ronde, Hans; Gonzlez Alonso, Paula; van Soolingen, Dick; Klatser, Paul R.; Anthony, Richard M.

    2012-01-01

    Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay. PMID:22396779

  15. Comparing the identification of Clostridium spp. by two Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry platforms to 16S rRNA PCR sequencing as a reference standard: a detailed analysis of age of culture and sample preparation.

    PubMed

    Chean, Roy; Kotsanas, Despina; Francis, Michelle J; Palombo, Enzo A; Jadhav, Snehal R; Awad, Milena M; Lyras, Dena; Korman, Tony M; Jenkin, Grant A

    2014-12-01

    We compared the identification of Clostridium species using mass spectrometry by two different Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) platforms (Bruker MS and Vitek MS) against 16S rRNA sequencing as the reference standard. We then examined the impact of different sample preparations and (on one of those platforms) age of bacterial colonial growth on the performance of the MALDI-TOF MS systems. We identified 10 different species amongst the 52 isolates by 16S rRNA sequencing, with Clostridium perfringens the most prevalent (n=30). Spectrometric analysis using Vitek MS correctly speciated 47/52 (90.4%) isolates and was not affected by the sample preparation used. Performance of the Bruker MS was dependent on sample preparation with correct speciation obtained for 36 of 52 (69.2%) isolates tested using the Direct Transfer [DT] protocol, but all 52 (100%) isolates were correctly speciated using either an Extended Direct Transfer [EDT] or a Full Formic Extraction [EX] protocol. We then examined the effect of bacterial colonial growth age on the performance of Bruker MS and found substantial agreement in speciation using DT (Kappa=0.62, 95% CI: 0.46-0.75), almost perfect agreement for EDT (Kappa=0.94, 95% CI: 0.86-1.00) and exact agreement for EX (Kappa=1.00) between different days. PMID:25230331

  16. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  17. Bacterial Endocarditis

    MedlinePLUS

    ... skin, mouth, intestines or urinary tract enter the bloodstream (usually during a dental or medical procedure) and infect the heart. Causes & Risk Factors Who gets bacterial endocarditis? Although ...

  18. The Potential of Metatranscriptomics for Identifying Screening Targets for Bacterial Vaginosis

    PubMed Central

    Twin, Jimmy; Bradshaw, Catriona S.; Garland, Suzanne M.; Fairley, Christopher K.; Fethers, Katherine; Tabrizi, Sepehr N.

    2013-01-01

    Background The ribosomal RNA content of a sample collected from a woman with bacterial vaginosis (BV) was analysed to determine the active microbial community, and to identify potential targets for further screening. Methodology/Principal Findings The sample from the BV patient underwent total RNA extraction, followed by physical subtraction of human rRNA and whole transcriptome amplification. The metatranscriptome was sequenced using Roche 454 titanium chemistry. The bioinformatics pipeline MG-RAST and desktop DNA analysis platforms were utilised to analyse results. Bacteria of the genus Prevotella (predominately P. amnii) constituted 36% of the 16S rRNA reads, followed by Megasphaera (19%), Leptotrichia/Sneathia (8%) and Fusobacterium (8%). Comparison of the abundances of several bacteria to quantitative PCR (qPCR) screening of extracted DNA revealed comparable relative abundances. This suggests a correlation between what was present and transcriptionally active in this sample: however distinct differences were seen when compared to the microbiome determined by 16S rRNA gene amplicon sequencing. To assess the presence of P. amnii in a larger pool of samples, 90 sexually active women were screened using qPCR. This bacterium was found to be strongly associated with BV (P<0.001, OR 23.3 (95%CI:2.9190.7)) among the 90 women. Conclusions/Significance This study highlighted the potential of metatranscriptomics as a tool for characterising metabolically active microbiota and identifying targets for further screening. Prevotella amnii was chosen as an example target, being the most metabolically active species present in the single patient with BV, and was found to be detected at a high concentration by qPCR in 31% of cohort with BV, with an association with both oral and penile-vaginal sex. PMID:24086764

  19. Chronic bacterial prostatitis: efficacy of short-lasting antibiotic therapy with prulifloxacin (Unidrox) in association with saw palmetto extract, lactobacillus sporogens and arbutin (Lactorepens)

    PubMed Central

    2014-01-01

    Background Bacterial prostatitis (BP) is a common condition accounting responsible for about 5-10% of all prostatitis cases; chronic bacterial prostatitis (CBP) classified as type II, are less common but is a condition that significantly hampers the quality of life, (QoL) because not only is it a physical condition but also a psychological distress. Commonly patients are treated with antibiotics alone, and in particular fluoroquinolones are suggested by the European Urology guidelines. This approach, although recommended, may not be enough. Thus, a multimodal approach to the prolonged antibiotic therapy may be helpful. Methods 210 patients affected by chronic bacterial prostatitis were enrolled in the study. All patients were positive to Meares-Stamey test and symptoms duration was?>?3months. The purpose of the study was to evaluate the efficacy of a long lasting therapy with a fluoroquinolone in association with a nutraceutical supplement (prulifloxacin 600mg for 21days and an association of Serenoa repens 320mg, Lactobacillus Sporogens 200mg, Arbutin 100mg for 30days). Patients were randomized in two groups (A and B) receiving respectively antibiotic alone and an association of antibiotic plus supplement. Results Biological recurrence at 2months in Group A was observed in 21 patients (27.6%) and in Group B in 6 patients (7.8%). Uropathogens found at the first follow-up were for the majority Gram (E. coli and Enterobacter spp.). A statistically significant difference was found at the time of the follow-up between Group A and B in the NIH-CPSI questionnaire score, symptoms evidence and serum PSA. Conclusions Broad band, short-lasting antibiotic therapy in association with a nutritional supplement (serenoa repens, lactobacillus sporogens and arbutin) show better control and recurrence rate on patients affected by chronic bacterial prostatitits in comparison with antibiotic treatment alone. Trial registration NCT02130713 Date of trial Registration: 30/04/2014 PMID:25038794

  20. Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)

    NASA Astrophysics Data System (ADS)

    MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

    2012-12-01

    Small-subunit ribosomal RNA (SSU rRNA) is a phylogenetically informative molecule found in all species. Because it is poorly preserved in most environments, it is a useful marker for active microbial populations. We are using the natural-abundance stable carbon isotopic composition of specific microbial groups to help identify the carbon substrates contributing to microbial biomass in a variety of marine environments. At Guaymas Basin, hydrothermal fluids interact with abundant sedimentary organic carbon to produce natural gas and petroleum. Where this reaches the sediment surface, it can support dense patches of seafloor life, including Beggiatoa mats. We report here on the stable carbon isotopic composition of SSU rRNA from a Beggiatoa mat transect, a cold background site, a warm site with high oil concentration, and a second Beggiatoa mat. The central part of the transect mat overlay the steepest temperature gradient, and was visually dominated by orange Beggiatoa. This was fringed by white Beggiatoa mat and bare, but still warm, sediment. Methane concentrations were saturating beneath the orange and white mats and at the oily site, lower beneath bare sediment, and below detection at the background site. Our initial hypotheses were that rRNA isotopic composition would be strongly influenced by methane supply, and that archaeal rRNA might be lighter than bacterial due to contributions from methanogens and anaerobic methane oxidizers. We used biotin-labeled oligonucleotides to capture Bacterial and Archaeal SSU rRNA for isotopic determination. Background-site rRNA was isotopically heaviest, and bacterial RNA from below 2 cm at the oily site was lightest, consistent with control by methane. Within the transect mat, however, the pattern was more complicated; at some sediment depths, rRNA from the mat periphery was isotopically lightest. Part of this may be due to the spatially and temporally variable paths followed by hydrothermal fluid, which can include horizontal flow. There was no consistent isotopic difference between rRNAs captured by the two probes, although RNA recoveries were too low for isotopic determinations at depths where methanogens and methane oxidizers are expected. Our prediction that rRNA stable carbon isotopic composition would correlate with methane supply was borne out by the comparison between background and mat sediments, but may be an oversimplification for sites within hydrothermal features. Future work will include the isotopic characterization of other potential carbon substrates, such as acetate. We are also investigating cold-seep sediments and brine pools in the Gulf of Mexico, where methane is significantly more 13C-depleted than at Guaymas Basin and may therefore leave a stronger imprint on microbial biomass.table carbon isotopes of rRNA captured with Bacterial and Archaeal probes at mat transect and background sites.

  1. 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. PMID:23316189

  2. Standardized ginger (Zingiber officinale) extract reduces bacterial load and suppresses acute and chronic inflammation in Mongolian gerbils infected with cagA+Helicobacter pylori

    PubMed Central

    Gaus, Kristen; Huang, Yue; Israel, Dawn A.; Pendland, Susan L.; Adeniyi, Bolanle A.; Mahady, Gail B.

    2010-01-01

    Previous investigations demonstrated that a standardized extract of ginger rhizome inhibited the growth of Helicobacter pylori in vitro with a minimum inhibitory concentration in the range 0.78 to 12.5 ?g/mL. In the present work, the extract was tested in a rodent model of H. pylori-induced disease, the Mongolian gerbil, to examine the effects of the extract on both prevention and eradication of infection. The extract was administered to Mongolian gerbils at a daily dose of 100 mg/kg body weight in rations either 3 weeks prior to infection or 6 weeks post-infection. Treatment with the standardized ginger extract reduced H. pylori load as compared with controls and significantly (P<0.05) reduced both acute and chronic muscosal and submucosal inflammation, cryptitis, as well as epithelial cell degeneration and erosion induced by H. pylori. Importantly, the extract did not increase morbidity or mortality. Further investigations of the mechanism demonstrated that the ginger extract inhibited the activity of cyclooxygenase-2, with 50% inhibitory concentration (IC50) of 8.5 ?g/mL in vitro, inhibited the nuclear factor-?B transcriptional response in kBZ Jurkat cells (human T lymphocytes) with an IC50 of 24.6 ?g/mL, and significantly inhibited the release of interleukin (IL)-1?, IL-6, IL-8, and tumor necrosis factor-? from lipopolysaccharide-stimulated human peripheral blood mononuclear cells with IC50 values of 3.89, 7.7, 8.5, and 8.37 ?g/mL, respectively. These results suggest ginger extracts may be useful for development as agents to reduce H. pylori-induced inflammation and as for gastric cancer chemoprevention. PMID:20376296

  3. Evaluation of Microbial Bacterial and Fungal Diversity in Cerebrospinal Fluid Shunt Infection

    PubMed Central

    Simon, Tamara D.; Pope, Christopher E.; Browd, Samuel R.; Ojemann, Jeffrey G.; Riva-Cambrin, Jay; Mayer-Hamblett, Nicole; Rosenfeld, Margaret; Zerr, Danielle M.; Hoffman, Lucas

    2014-01-01

    Background Cerebrospinal fluid shunt infection can be recalcitrant. Recurrence is common despite appropriate therapy for the pathogens identified by culture. Improved diagnostic and therapeutic approaches are required, and culture-independent molecular approaches to cerebrospinal fluid shunt infections have not been described. Objectives To identify the bacteria and fungi present in cerebrospinal fluid from children with cerebrospinal fluid shunt infection using a high-throughput sequencing approach, and to compare those results to those from negative controls and conventional culture. Methods This descriptive study included eight children ?18 years old undergoing treatment for culture-identified cerebrospinal fluid shunt infection. After routine aerobic culture of each cerebrospinal fluid sample, deoxyribonucleic acid (DNA) extraction was followed by amplification of the bacterial 16S rRNA gene and the fungal ITS DNA region tag-encoded FLX-Titanium amplicon pyrosequencing and microbial phylogenetic analysis. Results The microbiota analyses for the initial cerebrospinal fluid samples from all eight infections identified a variety of bacteria and fungi, many of which did not grow in conventional culture. Detection by conventional culture did not predict the relative abundance of an organism by pyrosequencing, but in all cases, at least one bacterial taxon was detected by both conventional culture and pyrosequencing. Individual bacterial species fluctuated in relative abundance but remained above the limits of detection during infection treatment. Conclusions Numerous bacterial and fungal organisms were detected in these cerebrospinal fluid shunt infections, even during and after treatment, indicating diverse and recalcitrant shunt microbiota. In evaluating cerebrospinal fluid shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches offer a promising alternative diagnostic approach. More effective treatment of cerebrospinal fluid shunt infections is needed to reduce unacceptably high rates of reinfection, and this work suggests that one effective strategy may be reduction of the diverse microbiota present in infection. PMID:24421877

  4. 16S rRNA partial gene sequencing for the differentiation and molecular subtyping of Listeria species.

    PubMed

    Hellberg, Rosalee S; Martin, Keely G; Keys, Ashley L; Haney, Christopher J; Shen, Yuelian; Smiley, R Derike

    2013-12-01

    Use of 16S rRNA partial gene sequencing within the regulatory workflow could greatly reduce the time and labor needed for confirmation and subtyping of Listeria monocytogenes. The goal of this study was to build a 16S rRNA partial gene reference library for Listeria spp. and investigate the potential for 16S rRNA molecular subtyping. A total of 86 isolates of Listeria representing L. innocua, L. seeligeri, L. welshimeri, and L. monocytogenes were obtained for use in building the custom library. Seven non-Listeria species and three additional strains of Listeria were obtained for use in exclusivity and food spiking tests. Isolates were sequenced for the partial 16S rRNA gene using the MicroSeq ID 500 Bacterial Identification Kit (Applied Biosystems). High-quality sequences were obtained for 84 of the custom library isolates and 23 unique 16S sequence types were discovered for use in molecular subtyping. All of the exclusivity strains were negative for Listeria and the three Listeria strains used in food spiking were consistently recovered and correctly identified at the species level. The spiking results also allowed for differentiation beyond the species level, as 87% of replicates for one strain and 100% of replicates for the other two strains consistently matched the same 16S type. PMID:24010602

  5. Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences

    PubMed Central

    Lay, Christophe; Ho, Eliza Xin Pei; Low, Louie; Hibberd, Martin Lloyd; Nagarajan, Niranjan

    2013-01-01

    The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization. PMID:23579286

  6. Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification

    PubMed Central

    Lyra, Anna; Rinttilä, Teemu; Nikkilä, Janne; Krogius-Kurikka, Lotta; Kajander, Kajsa; Malinen, Erja; Mättö, Jaana; Mäkelä, Laura; Palva, Airi

    2009-01-01

    AIM: To study whether selected bacterial 16S ribosomal RNA (rRNA) gene phylotypes are capable of distinguishing irritable bowel syndrome (IBS). METHODS: The faecal microbiota of twenty volunteers with IBS, subdivided into eight diarrhoea-predominant (IBS-D), eight constipation-predominant (IBS-C) and four mixed symptom-subtype (IBS-M) IBS patients, and fifteen control subjects, were analysed at three time-points with a set of fourteen quantitative real-time polymerase chain reaction assays. All assays targeted 16S rRNA gene phylotypes putatively associated with IBS, based on 16S rRNA gene library sequence analysis. The target phylotypes were affiliated with Actinobacteria, Bacteroidetes and Firmicutes. Eight of the target phylotypes had less than 95% similarity to cultured bacterial species according to their 16S rRNA gene sequence. The data analyses were made with repeated-measures ANCOVA-type modelling of the data and principle component analysis (PCA) with linear mixed-effects models applied to the principal component scores. RESULTS: Bacterial phylotypes Clostridium cocleatum 88%, Clostridium thermosuccinogenes 85%, Coprobacillus catenaformis 91%, Ruminococcus bromii-like, Ruminococcus torques 91%, and R. torques 93% were detected from all samples analysed. A multivariate analysis of the relative quantities of all 14 bacterial 16S rRNA gene phylotypes suggested that the intestinal microbiota of the IBS-D patients differed from other sample groups. The PCA on the first principal component (PC1), explaining 30.36% of the observed variation in the IBS-D patient group, was significantly altered from all other sample groups (IBS-D vs control, P = 0.01; IBS-D vs IBS-M, P = 0.00; IBS-D vs IBS-C, P = 0.05). Significant differences were also observed in the levels of distinct phylotypes using relative values in proportion to the total amount of bacteria. A phylotype with 85% similarity to C. thermosuccinogenes was quantified in significantly different quantities among the IBS-D and control subjects (-4.08 ± 0.90 vs -3.33 ± 1.16, P = 0.04) and IBS-D and IBS-M subjects (-4.08 ± 0.90 vs -3.08 ± 1.38, P = 0.05). Furthermore, a phylotype with 94% similarity to R. torques was more prevalent in IBS-D patients’ intestinal microbiota than in that of control subjects (-2.43 ± 1.49 vs -4.02 ± 1.63, P = 0.01). A phylotype with 93% similarity to R. torques was associated with control samples when compared with IBS-M (-2.41 ± 0.53 vs -2.92 ± 0.56, P = 0.00). Additionally, a R. bromii-like phylotype was associated with IBS-C patients in comparison to control subjects (-1.61 ± 1.83 vs -3.69 ± 2.42, P = 0.01). All of the above mentioned phylotype specific alterations were independent of the effect of time. CONCLUSION: Significant phylotype level alterations in the intestinal microbiotas of IBS patients were observed, further emphasizing the possible contribution of the gastrointestinal microbiota in IBS. PMID:20014457

  7. A randomized, double-blind, placebo-controlled trial to assess the bacterial anti-adhesion effects of cranberry extract beverages.

    PubMed

    Kaspar, Kerrie L; Howell, Amy B; Khoo, Christina

    2015-04-01

    In this study, we examined the ex vivo urinary anti-adhesion activity of low-calorie cranberry extract beverages in both a pilot study (n = 10) and a randomized, double-blind, placebo controlled clinical trial (n = 59). In the pilot study, subjects consumed a cranberry extract beverage (CEB) or a cranberry extract and juice beverage (CEJB), compared to placebo. Both cranberry beverages utilized a standardized cranberry extract powder at a level equivalent to low-calorie cranberry juice cocktail (LCJC) on a PAC content basis. Clean-catch urine samples collected at baseline and post intervention were tested for anti-adhesion activity utilizing a mannose-resistant human red blood cell hemagglutination assay specific for P-fimbriated E. coli. Results from the pilot study indicated that ex vivo anti-adhesion activity for both cranberry treatments were higher (p < 0.05) than placebo. In the clinical trial, we compared CEJB to LCJC and a placebo beverage. Post-consumption urine from both cranberry treatment groups showed significantly higher (p < 0.05) anti-adhesion activity compared to placebo. There were no differences observed in anti-adhesion activity between CJEB and LCJC, indicating similar bioactivity. Therefore, acute beverage consumption of cranberry extract and/or juice provides ex vivo anti-adhesion activity, which may help to improve urinary tract health. PMID:25723356

  8. Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans

    NASA Technical Reports Server (NTRS)

    Jurtshuk, R. J.; Blick, M.; Bresser, J.; Fox, G. E.; Jurtshuk, P. Jr

    1992-01-01

    A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

  9. Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

    PubMed Central

    Van Puyvelde, Sandra; De Block, Tessa; Maltha, Jessica; Palpouguini, Lompo; Tahita, Marc; Tinto, Halidou; Jacobs, Jan; Deborggraeve, Stijn

    2016-01-01

    Background Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. Methodology/Principal Findings The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Conclusions/Significance Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination. PMID:26927306

  10. Bacterial population structure and dynamics during the development of almond drupes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: To describe the bacterial populations and their dynamics during the development of almond drupes. Methods and Results: We examined 16S rRNA gene libraries derived from the bacterial populations on almond drupes at three stages of development: 1) when the drupes were full sized, but before embr...

  11. Dominant obligate anaerobes revealed in lower respiratory tract infection in horses by 16S rRNA gene sequencing.

    PubMed

    Kinoshita, Yuta; Niwa, Hidekazu; Katayama, Yoshinari; Hariu, Kazuhisa

    2014-04-01

    Obligate anaerobes are important etiological agents in pneumonia or pleuropneumonia in horses, because they are isolated more commonly from ill horses that have died or been euthanized than from those that survive. We performed bacterial identification and antimicrobial susceptibility testing for obligate anaerobes to establish effective antimicrobial therapy. We used 16S rRNA gene sequencing to identify 58 obligate anaerobes and compared the results with those from a phenotypic identification kit. The identification results of 16S rRNA gene sequencing were more reliable than those of the commercial kit. We concluded that genera Bacteroides and Prevotella-especially B. fragilis and P. heparinolytica-are dominant anaerobes in lower respiratory tract infection in horses; these organisms were susceptible to metronidazole, imipenem and clindamycin. PMID:24366152

  12. Dominant Obligate Anaerobes Revealed in Lower Respiratory Tract Infection in Horses by 16S rRNA Gene Sequencing

    PubMed Central

    KINOSHITA, Yuta; NIWA, Hidekazu; KATAYAMA, Yoshinari; HARIU, Kazuhisa

    2013-01-01

    ABSTRACT Obligate anaerobes are important etiological agents in pneumonia or pleuropneumonia in horses, because they are isolated more commonly from ill horses that have died or been euthanized than from those that survive. We performed bacterial identification and antimicrobial susceptibility testing for obligate anaerobes to establish effective antimicrobial therapy. We used 16S rRNA gene sequencing to identify 58 obligate anaerobes and compared the results with those from a phenotypic identification kit. The identification results of 16S rRNA gene sequencing were more reliable than those of the commercial kit. We concluded that genera Bacteroides and Prevotellaespecially B. fragilis and P. heparinolyticaare dominant anaerobes in lower respiratory tract infection in horses; these organisms were susceptible to metronidazole, imipenem and clindamycin. PMID:24366152

  13. Distribution of Intervening Sequences in the Genes for 23S rRNA and rRNA Fragmentation among Strains of the Salmonella Reference Collection B (SARB) and SARC Sets

    PubMed Central

    Pabbaraju, Kanti; Miller, Wayne L.; Sanderson, Kenneth E.

    2000-01-01

    Intervening sequences (IVSs) occur sporadically in several bacterial genera in the genes for 23S rRNA at relatively conserved locations. They are cleaved after transcription and lead to the presence of fragmented rRNA, which is incorporated into the ribosomes without religation but is nevertheless functional. The fragmentation of rRNA and the number of IVSs in all 72 strains of the Salmonella Reference Collection B set and 16 strains of the Salmonella Reference Collection C set, which have been established on the basis of multilocus enzyme electrophoresis (MLEE), were analyzed in the present study. Fragmentation of 23S rRNA was restricted to conserved cleavage sites located at bp 550 (helix 25) and bp 1170 (helix 45), locations where IVSs have been reported. Random cleavage at sites where IVSs could not be detected was not seen. Uncleaved IVSs were not detected in any case; thus, the IVSs invariably led to rRNA fragmentation, indicating a strong selection for maintenance of RNase III cleavage sites. The distribution of the number of IVSs carried by the different strains in the seven rrl genes is diverse, and the pattern of IVS possession could not be related to the MLEE pattern among the various Salmonella strains tested; this indicates that the IVSs are frequently exchanged between strains by lateral transfer. All eight subspecies of the genus Salmonella, including subspecies V represented by Salmonella bongori, have IVSs in both helix 25 and helix 45; this indicates that IVSs entered the genus after its divergence from Escherichia coli (more than 100 million years ago) but before separation of the genus Salmonella into many forms or that they were in the ancestor but have been lost from Escherichia. PMID:10714998

  14. Effects of a dietary yeast extract on hematological parameters, heterophil function, and bacterial clearance in turkey poults challenged with Escherichia coli and subjected to transport stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop nutritional methods for controlling pathogens in poultry production. A standardized yeast extract supplement, Alphamune (YE), was added to turkey poult diets. Male poults were challenged by air sac injection with 60 cfu of E. coli at 1 week of age. At 3 weeks of age chal...

  15. Effects of a dietary yeast extract on hematological parameters, heterophil function, and bacterial clearance in turkey poults challenged with Escherichia coli and subjected to transport stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop nutritional methods for controlling pathogens in poultry production. A standardized yeast extract supplement, Alphamune™ (YE), was added to turkey poult diets. Male poults were challenged by air sac injection with 60 cfu of E. coli at 1 week of age. At 3 weeks of age chal...

  16. Bacterial Diversity at an Acid Mine Drainage Site in Maine

    NASA Astrophysics Data System (ADS)

    Gaynor, J.; Sawyer, T.; Riley, F. E.; Moulton, K. D.; Rothschild, L. J.; Duboise, S. M.

    2010-04-01

    Bacterial diversity in acidic mine drainage at a historic Maine iron mining site was investigated by isolation of environmental DNA, PCR amplification of the V3 region of the 16S rRNA gene, denaturing gradient gel electrophoresis, and DNA sequencing.

  17. Bacterial Keratitis

    MedlinePLUS

    ... Kierstan Boyd Reviewed by: Devin A Harrison MD Mar. 01, 2015 Bacterial keratitis is an infection of ... Medications Aug 18, 2014 Pink Eye and School Mar 11, 2014 Sun Exposure Mar 10, 2014 Leer ...

  18. Bacterial Vaginosis

    MedlinePLUS

    ... incubation period for bacterial vaginosis. How Is the Diagnosis Made? Your childs pediatrician can make the diagnosis ... Editorial Policy This site complies with the HONcode standard for trustworthy health information: verify here. Copyright 2016 ...

  19. Molecular Phylogenetic Diversity and Spatial Distribution of Bacterial Communities in Cooling Stage during Swine Manure Composting

    PubMed Central

    Guo, Yan; Zhang, Jinliang; Yan, Yongfeng; Wu, Jian; Zhu, Nengwu; Deng, Changyan

    2015-01-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and subsequent sub-cloning and sequencing were used in this study to analyze the molecular phylogenetic diversity and spatial distribution of bacterial communities in different spatial locations during the cooling stage of composted swine manure. Total microbial DNA was extracted, and bacterial near full-length 16S rRNA genes were subsequently amplified, cloned, RFLP-screened, and sequenced. A total of 420 positive clones were classified by RFLP and near-full-length 16S rDNA sequences. Approximately 48 operational taxonomic units (OTUs) were found among 139 positive clones from the superstratum sample; 26 among 149 were from the middle-level sample and 35 among 132 were from the substrate sample. Thermobifida fusca was common in the superstratum layer of the pile. Some Bacillus spp. were remarkable in the middle-level layer, and Clostridium sp. was dominant in the substrate layer. Among 109 OTUs, 99 displayed homology with those in the GenBank database. Ten OTUs were not closely related to any known species. The superstratum sample had the highest microbial diversity, and different and distinct bacterial communities were detected in the three different layers. This study demonstrated the spatial characteristics of the microbial community distribution in the cooling stage of swine manure compost. PMID:25925066

  20. Effects of functionalized and raw multi-walled carbon nanotubes on soil bacterial community composition.

    PubMed

    Kerfahi, Dorsaf; Tripathi, Binu M; Singh, Dharmesh; Kim, Hyoki; Lee, Sujin; Lee, Junghoon; Adams, Jonathan M

    2015-01-01

    Carbon nanotubes (CNTs) are widely used in industry, but their environmental impacts on soil microbial communities are poorly known. In this paper, we compare the effect of both raw and acid treated or functionalized (fCNTs) multi-walled carbon nanotubes (MWCNTs) on soil bacterial communities, applying different concentrations of MWCNTs (0 g/g, 50 g/g, 500 g/g and 5000 g/g) to a soil microcosm system. Soil DNA was extracted at 0, 2 and 8 weeks and the V3 region of the 16S rRNA gene was PCR-amplified and sequenced using paired-end Illumina bar-coded sequencing. The results show that bacterial diversity was not affected by either type of MWCNT. However, overall soil bacterial community composition, as illustrated by NMDS, was affected only by fMWCNT at high concentrations. This effect, detectable at 2 weeks, remained equally strong by 8 weeks. In the case of fMWCNTs, overall changes in relative abundance of the dominant phyla were also found. The stronger effect of fMWCNTs could be explained by their intrinsically acidic nature, as the soil pH was lower at higher concentrations of fMWCNTs. Overall, this study suggests that fMWCNTs may at least temporarily alter microbial community composition on the timescale of at least weeks to months. It appears, by contrast, that raw MWCNTs do not affect soil microbial community composition. PMID:25825905

  1. Effects of Functionalized and Raw Multi-Walled Carbon Nanotubes on Soil Bacterial Community Composition

    PubMed Central

    Kerfahi, Dorsaf; Tripathi, Binu M.; Singh, Dharmesh; Kim, Hyoki; Lee, Sujin; Lee, Junghoon; Adams, Jonathan M.

    2015-01-01

    Carbon nanotubes (CNTs) are widely used in industry, but their environmental impacts on soil microbial communities are poorly known. In this paper, we compare the effect of both raw and acid treated or functionalized (fCNTs) multi-walled carbon nanotubes (MWCNTs) on soil bacterial communities, applying different concentrations of MWCNTs (0 µg/g, 50 µg/g, 500 µg/g and 5000 µg/g) to a soil microcosm system. Soil DNA was extracted at 0, 2 and 8 weeks and the V3 region of the 16S rRNA gene was PCR-amplified and sequenced using paired-end Illumina bar-coded sequencing. The results show that bacterial diversity was not affected by either type of MWCNT. However, overall soil bacterial community composition, as illustrated by NMDS, was affected only by fMWCNT at high concentrations. This effect, detectable at 2 weeks, remained equally strong by 8 weeks. In the case of fMWCNTs, overall changes in relative abundance of the dominant phyla were also found. The stronger effect of fMWCNTs could be explained by their intrinsically acidic nature, as the soil pH was lower at higher concentrations of fMWCNTs. Overall, this study suggests that fMWCNTs may at least temporarily alter microbial community composition on the timescale of at least weeks to months. It appears, by contrast, that raw MWCNTs do not affect soil microbial community composition. PMID:25825905

  2. Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes

    PubMed Central

    Javadi Nobandegani, Mohammad Bagher; Saud, Halimi Mohd; Yun, Wong Mui

    2015-01-01

    Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field. PMID:25632387

  3. Phylogenetic relationship of phosphate solubilizing bacteria according to 16S rRNA genes.

    PubMed

    Javadi Nobandegani, Mohammad Bagher; Saud, Halimi Mohd; Yun, Wong Mui

    2015-01-01

    Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field. PMID:25632387

  4. Diversity and biogeography of bacterial assemblages in surface sediments across the San Pedro Basin, Southern California Borderlands.

    PubMed

    Hewson, Ian; Jacobson Meyers, Myrna E; Fuhrman, Jed A

    2007-04-01

    Sediment bacteria play important roles in the biogeochemistry of ocean sediments; however, factors influencing assemblage composition have not been extensively studied. We examined extractable sediment bacterial abundance, the composition of bacterial assemblages using a high-throughput molecular fingerprinting approach, and several sediment biogeochemical parameters (organic matter content and alkaline phosphatase activity), along a 35 km transect from Point Fermin, Southern California, to Santa Catalina Island, across the approximately 900-m-deep San Pedro Basin. Automated rRNA intergenic spacer analysis (ARISA) demonstrated that in two spatially isolated shallow (approximately < 60 m, on opposite sides of the channel) sediment environments, assemblages were more similar to each other than to deeper communities. Distinct communities existed in deeper and shallower sediments, and stations within the deep basin over 2 km apart contained remarkably similar assemblage fingerprints. The relative contribution to total amplified DNA fluorescence of operational taxonomic units (OTUs) was significantly correlated to that of other OTUs in few comparisons (2.7% of total), i.e. few bacterial types were found together or apart consistently. The relative proportions within assemblages of only a few OTU were significantly correlated to measured physicochemical parameters (organic matter content and wet/dry weight ratio of sediments) or enzyme (alkaline phosphatase) activities. A low percentage of shared OTU between shallow and deep sediments, and the presence of similar, but spatially isolated assemblages suggests that bacterial OTU may be widely dispersed over scales of a few kilometres, but that environmental conditions select for particular assemblages. PMID:17359264

  5. Assessing the effects of salmon farming seabed enrichment using bacterial community diversity and high-throughput sequencing.

    PubMed

    Dowle, Eddy; Pochon, Xavier; Keeley, Nigel; Wood, Susanna A

    2015-08-01

    Aquaculture is an extremely valuable and rapidly expanding sector of the seafood industry. The sediment below active aquaculture farms receives inputs of organic matter from uneaten food and faecal material and this has led to concerns related to environmental sustainability. The impacts of organic enrichment on macrobenthic infauna are well characterized; however, much less is known about effect on bacterial communities. In this study, sediment, macrobenthic infauna samples and environmental data were collected along an enrichment gradient radiating out from a Chinook salmon (Oncorhynchus tshawytscha) farm (Marlborough Sounds; New Zealand). DNA and RNA were extracted and 16S rRNA metabarcodes from bacterial communities characterized using high-throughput sequencing. Desulfobacterales dominated at the cage (DNA and RNA), and at sites 50 m (DNA and RNA) and 150 m (RNA) from the farm. In contrast, unclassified bacteria from the class Gammaproteobacteria were the most abundant taxa at control sites (625 and 4000 m). Pronounced differences among DNA and RNA samples occurred at the cage site where Desulfobacterales abundance was markedly higher in RNA samples. There were strong correlations between shifts in bacterial communities and total organic matter and redox. This suggests that bacterial composition is strongly influenced by organic enrichment, a trait that may make them useful for assessing impacts associated with aquaculture farms. PMID:26207046

  6. Development of a novel long-range 16S rRNA universal primer set for metagenomic analysis of gastrointestinal microbiota in newborn infants.

    PubMed

    Ku, Hye-Jin; Lee, Ju-Hoon

    2014-06-28

    Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias. PMID:24722376

  7. Changes in rRNA Levels during Stress Invalidates Results from mRNA Blotting: Fluorescence In Situ rRNA Hybridization Permits Renormalization for Estimation of Cellular mRNA Levels

    PubMed Central

    Hansen, Martin C.; Nielsen, Allan K.; Molin, Sren; Hammer, Karin; Kilstrup, Mogens

    2001-01-01

    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value. PMID:11466277

  8. The impact of date palm fruits and their component polyphenols, on gut microbial ecology, bacterial metabolites and colon cancer cell proliferation.

    PubMed

    Eid, Noura; Enani, Sumia; Walton, Gemma; Corona, Giulia; Costabile, Adele; Gibson, Glenn; Rowland, Ian; Spencer, Jeremy P E

    2014-01-01

    The fruit of the date palm (Phoenix dactylifera L.) is a rich source of dietary fibre and polyphenols. We have investigated gut bacterial changes induced by the whole date fruit extract (digested date extract; DDE) and its polyphenol-rich extract (date polyphenol extract; DPE) using faecal, pH-controlled, mixed batch cultures mimicking the distal part of the human large intestine, and utilising an array of microbial group-specific 16S rRNA oligonucleotide probes. Fluorescence microscopic enumeration indicated that there was a significant increase in the growth of bifidobacteria in response to both treatments, whilst whole dates also increased bacteroides at 24h and the total bacterial counts at later fermentation time points when compared with DPE alone. Bacterial metabolism of whole date fruit led to the production of SCFA, with acetate significantly increasing following bacterial incubation with DDE. In addition, the production of flavonoid aglycones (myricetin, luteolin, quercetin and apigenin) and the anthocyanidin petunidin in less than 1h was also observed. Lastly, the potential of DDE, DPE and metabolites to inhibit Caco-2 cell growth was investigated, indicating that both were capable of potentially acting as antiproliferative agents in vitro, following a 48h exposure. This potential to inhibit growth was reduced following fermentation. Together these data suggest that consumption of date fruits may enhance colon health by increasing beneficial bacterial growth and inhibiting the proliferation of colon cancer cells. This is an early suggestion that date intake by humans may aid in the maintenance of bowel health and even the reduction of colorectal cancer development. PMID:26101614

  9. In-Vitro, Anti-Bacterial Activities of Aqueous Extracts of Acacia catechu (L.F.)Willd, Castanea sativa, Ephedra sinica stapf and shilajita mumiyo Against Gram Positive and Gram Negative Bacteria

    PubMed Central

    Dashtdar, Mehrab; Dashtdar, Mohammad Reza; Dashtdar, Babak; shirazi, Mohammad khabaz; Khan, Saeed Ahmad

    2013-01-01

    Objective: Evaluations of the in-vitro anti-bacterial activities of aqueous extracts of Acacia catechu (L.F.)Willd, Castanea sativa, Ephedra sinica stapf and Shilajita mumiyo against gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumonia) and gram-negative bacteria (Escherichia coli, klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa) are reasonable since these ethnomedicinal plants have been used in Persian folk medicine for treating skin diseases, venereal diseases, respiratory problems and nervous disorders for ages. Methods: The well diffusion method (KB testing) with a concentration of 250 ?g/disc was used for evaluating the minimal inhibitory concentrations (MIC). Maximum synergistic effects of different combinations of components were also observed. Results: A particular combination of Acacia catechu (L.F.) Willd, Castanea sativa, Ephedra sinica stapf and shilajita mumiyo extracts possesses an outstanding anti-bacterial activity. It's inhibiting effect on microorganisms is significant when compared to the control group (P< 0.05). Staphylococcus aureus was the most sensitive microorganism. The highest antibacterial activity against gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumonia) or gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and Pseudomonas aeruginosa) was exerted by formula number 2 (Table1). Conclusion: The results reveal the presence of antibacterial activities of Acacia catechu, Castanea sativa husk, Ephedra sp. and Mumiyo against gram-positive and gram-negative bacteria. Synergistic effects in a combined formula, especially in formula number 2 (ASLAN?) can lead to potential sources of new antiseptic agents for treatment of acute or chronic skin ulcers. These results considering the significant antibacterial effect of the present formulation, support ethno-pharmacological uses against diarrheal and venereal diseases and demonstrate use of these plants to treat infectious diseases. PMID:25780663

  10. The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase.

    PubMed Central

    Vester, B; Hansen, L H; Douthwaite, S

    1995-01-01

    The ErmE methyltransferase confers resistance to MLS antibiotics by specifically dimethylating adenine 2058 (A2058, Escherichia coli numbering) in bacterial 23S rRNA. To define nucleotides in the rRNA that are part of the motif recognized by ErmE, we investigated both in vivo and in vitro the effects of mutations around position A2058 on methylation. Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE. No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058. Breaking the neighboring G2057-C2611 Watson-Crick base pair by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents and it was shown that the A2057 and U2611 mutations alone and in combination alter the reactivity of A2058 and adjacent bases. However, mutagenizing position G-->A2032 in an adjacent loop, which has been implicated to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents. The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE. Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the ErmE methyltransferase. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6 PMID:7489511

  11. Potential applications of next generation DNA sequencing of 16S rRNA gene amplicons in microbial water quality monitoring.

    PubMed

    Vierheilig, J; Savio, D; Ley, R E; Mach, R L; Farnleitner, A H; Reischer, G H

    2015-01-01

    The applicability of next generation DNA sequencing (NGS) methods for water quality assessment has so far not been broadly investigated. This study set out to evaluate the potential of an NGS-based approach in a complex catchment with importance for drinking water abstraction. In this multi-compartment investigation, total bacterial communities in water, faeces, soil, and sediment samples were investigated by 454 pyrosequencing of bacterial 16S rRNA gene amplicons to assess the capabilities of this NGS method for (i) the development and evaluation of environmental molecular diagnostics, (ii) direct screening of the bulk bacterial communities, and (iii) the detection of faecal pollution in water. Results indicate that NGS methods can highlight potential target populations for diagnostics and will prove useful for the evaluation of existing and the development of novel DNA-based detection methods in the field of water microbiology. The used approach allowed unveiling of dominant bacterial populations but failed to detect populations with low abundances such as faecal indicators in surface waters. In combination with metadata, NGS data will also allow the identification of drivers of bacterial community composition during water treatment and distribution, highlighting the power of this approach for monitoring of bacterial regrowth and contamination in technical systems. PMID:26606090

  12. Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing.

    PubMed

    Pajarillo, Edward Alain B; Chae, Jong Pyo; Balolong, Marilen P; Kim, Hyeun Bum; Seo, Kang-Seok; Kang, Dae-Kyung

    2015-04-01

    This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs. PMID:25656184

  13. Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing

    PubMed Central

    Pajarillo, Edward Alain B.; Chae, Jong Pyo; Balolong, Marilen P.; Kim, Hyeun Bum; Seo, Kang-Seok; Kang, Dae-Kyung

    2015-01-01

    This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1–V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs. PMID:25656184

  14. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.

    PubMed

    Corless, C E; Guiver, M; Borrow, R; Edwards-Jones, V; Kaczmarski, E B; Fox, A J

    2000-05-01

    A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical. PMID:10790092

  15. Contamination and Sensitivity Issues with a Real-Time Universal 16S rRNA PCR

    PubMed Central

    Corless, C. E.; Guiver, M.; Borrow, R.; Edwards-Jones, V.; Kaczmarski, E. B.; Fox, A. J.

    2000-01-01

    A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical. PMID:10790092

  16. 16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

    NASA Astrophysics Data System (ADS)

    Ma, Haiyan; Jiang, Guoliang; Wu, Zhiqiang; Wang, Xin

    2009-06-01

    Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.

  17. Different bacterial populations associated with the roots and rhizosphere of rice incorporate plant-derived carbon.

    PubMed

    Hernndez, Marcela; Dumont, Marc G; Yuan, Quan; Conrad, Ralf

    2015-03-01

    Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH4, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with (13)CO2 for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with (13)C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the "Spartobacteria" and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (?20%) than did those in the rhizosphere (?4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment. PMID:25616793

  18. Different Bacterial Populations Associated with the Roots and Rhizosphere of Rice Incorporate Plant-Derived Carbon

    PubMed Central

    Hernández, Marcela; Yuan, Quan; Conrad, Ralf

    2015-01-01

    Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH4, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with 13CO2 for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with 13C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the “Spartobacteria” and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (∼20%) than did those in the rhizosphere (∼4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment. PMID:25616793

  19. Community Analysis of Chronic Wound Bacteria Using 16S rRNA Gene-Based Pyrosequencing: Impact of Diabetes and Antibiotics on Chronic Wound Microbiota

    PubMed Central

    Price, Lance B.; Liu, Cindy M.; Melendez, Johan H.; Frankel, Yelena M.; Engelthaler, David; Aziz, Maliha; Bowers, Jolene; Rattray, Rogan; Ravel, Jacques; Kingsley, Chris; Keim, Paul S.; Lazarus, Gerald S.; Zenilman, Jonathan M.

    2009-01-01

    Background Bacterial colonization is hypothesized to play a pathogenic role in the non-healing state of chronic wounds. We characterized wound bacteria from a cohort of chronic wound patients using a 16S rRNA gene-based pyrosequencing approach and assessed the impact of diabetes and antibiotics on chronic wound microbiota. Methodology/Principal Findings We prospectively enrolled 24 patients at a referral wound center in Baltimore, MD; sampled patients' wounds by curette; cultured samples under aerobic and anaerobic conditions; and pyrosequenced the 16S rRNA V3 hypervariable region. The 16S rRNA gene-based analyses revealed an average of 10 different bacterial families in wounds—approximately 4 times more than estimated by culture-based analyses. Fastidious anaerobic bacteria belonging to the Clostridiales family XI were among the most prevalent bacteria identified exclusively by 16S rRNA gene-based analyses. Community-scale analyses showed that wound microbiota from antibiotic treated patients were significantly different from untreated patients (p = 0.007) and were characterized by increased Pseudomonadaceae abundance. These analyses also revealed that antibiotic use was associated with decreased Streptococcaceae among diabetics and that Streptococcaceae was more abundant among diabetics as compared to non-diabetics. Conclusions/Significance The 16S rRNA gene-based analyses revealed complex bacterial communities including anaerobic bacteria that may play causative roles in the non-healing state of some chronic wounds. Our data suggest that antimicrobial therapy alters community structure—reducing some bacteria while selecting for others. PMID:19649281

  20. Bacterial rheotaxis.

    PubMed

    Marcos; Fu, Henry C; Powers, Thomas R; Stocker, Roman

    2012-03-27

    The motility of organisms is often directed in response to environmental stimuli. Rheotaxis is the directed movement resulting from fluid velocity gradients, long studied in fish, aquatic invertebrates, and spermatozoa. Using carefully controlled microfluidic flows, we show that rheotaxis also occurs in bacteria. Excellent quantitative agreement between experiments with Bacillus subtilis and a mathematical model reveals that bacterial rheotaxis is a purely physical phenomenon, in contrast to fish rheotaxis but in the same way as sperm rheotaxis. This previously unrecognized bacterial taxis results from a subtle interplay between velocity gradients and the helical shape of flagella, which together generate a torque that alters a bacterium's swimming direction. Because this torque is independent of the presence of a nearby surface, bacterial rheotaxis is not limited to the immediate neighborhood of liquid-solid interfaces, but also takes place in the bulk fluid. We predict that rheotaxis occurs in a wide range of bacterial habitats, from the natural environment to the human body, and can interfere with chemotaxis, suggesting that the fitness benefit conferred by bacterial motility may be sharply reduced in some hydrodynamic conditions. PMID:22411815

  1. Control of rRNA transcription in Escherichia coli.

    PubMed Central

    Condon, C; Squires, C; Squires, C L

    1995-01-01

    The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium. This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis. In this review, we summarize four general areas of E. coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination. We also cite similar mechanisms in other bacteria and eukaryotes. The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy. One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control. Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions. Lastly, clues as to the role of antitermination in rRNA operons have begun to appear. Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response. PMID:8531889

  2. Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples

    PubMed Central

    Francioli, Davide; Reitz, Thomas; Buscot, Franois; Schloter, Michael; Krger, Dirk

    2015-01-01

    Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected. PMID:25749323

  3. Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale.

    PubMed

    Webster, Gordon; Newberry, Carole J; Fry, John C; Weightman, Andrew J

    2003-10-01

    Investigations into the deep marine environment have demonstrated the presence of a significant microbial biomass buried deep within sediments on a global scale. It is now believed that this deep biosphere plays a major role in the global cycling of elements and contains a large reservoir of organic carbon. This paper reports the development of a DNA extraction protocol that addresses the particular problems faced in applying molecular ecological techniques to samples containing very low biomass. Sediment samples were collected from different geographical locations within the Pacific Ocean and include the Ocean Drilling Program (ODP) Leg 190, Nankai Trough Accretionary Prism. Seven DNA extraction protocols were tested and a commercially available DNA extraction kit with modifications was shown to produce higher yields of polymerase chain reaction (PCR)-amplifiable DNA than standard laboratory methods. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene diversity revealed that template DNA from these extremely low biomass sediment samples was susceptible to PCR bias and random amplification. We propose that it is essential to screen 16S rRNA gene products for bacterial diversity by DGGE or other rapid fingerprinting methods, prior to their use in establishing a representative clone library of deep sub-seafloor bacteria. This represents a cautionary approach to analysis of microbial diversity in such sub-seafloor ecosystems. PMID:14500007

  4. Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

    PubMed Central

    Suzuki, Marcelino T.; Taylor, Lance T.; DeLong, Edward F.

    2000-01-01

    Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5′-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. PMID:11055900

  5. Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns*

    PubMed Central

    Atherly, Todd; Ziemer, Cherie J

    2014-01-01

    One-hundred-and-three isolates of Bacteroides ovatus,B. thetaiotaomicron, and B. xylanisolvens were recovered from cow, goat, human, and pig fecal enrichments with cellulose or xylan/pectin. Isolates were compared using 16S rRNA gene sequencing, repetitive sequence-based polymerase chain reaction (rep-PCR), and phenotypic microarrays. Analysis of 16S rRNA gene sequences revealed high sequence identity in these Bacteroides; with distinct phylogenetic groupings by bacterial species but not host origin. Phenotypic microarray analysis demonstrated these Bacteroides shared the ability to utilize many of the same carbon substrates, without differences due to species or host origin, indicative of their broad carbohydrate fermentation abilities. Limited nitrogen substrates were utilized; in addition to ammonia, guanine, and xanthine, purine derivatives were utilized by most isolates followed by a few amino sugars. Only rep-PCR analysis demonstrated host-specific patterns, indicating that genomic changes due to coevolution with host did not occur by mutation in the 16S rRNA gene or by a gain or loss of carbohydrate utilization genes within these Bacteroides. This is the first report to indicate that host-associated genomic differences are outside of 16S rRNA gene and carbohydrate utilization genes and suggest conservation of specific bacterial species with the same functionality across mammalian hosts for this Bacteroidetes clade. PMID:24532571

  6. Molecular diversity of drinking water bacterial communities using 16S rRNA gene sequence analyses

    EPA Science Inventory

    Our understanding of the microbial community structure of drinking water distribution system has relied on culture-based methods. However, recent studies have suggested that the majority of bacteria inhabiting distribution systems are unable to grow on artificial media. The goal ...

  7. A Comparison between Transcriptome Sequencing and 16S Metagenomics for Detection of Bacterial Pathogens in Wildlife

    PubMed Central

    Razzauti, Maria; Galan, Maxime; Bernard, Maria; Maman, Sarah; Klopp, Christophe; Charbonnel, Nathalie; Vayssier-Taussat, Muriel; Eloit, Marc; Cosson, Jean-François

    2015-01-01

    Background Rodents are major reservoirs of pathogens responsible for numerous zoonotic diseases in humans and livestock. Assessing their microbial diversity at both the individual and population level is crucial for monitoring endemic infections and revealing microbial association patterns within reservoirs. Recently, NGS approaches have been employed to characterize microbial communities of different ecosystems. Yet, their relative efficacy has not been assessed. Here, we compared two NGS approaches, RNA-Sequencing (RNA-Seq) and 16S-metagenomics, assessing their ability to survey neglected zoonotic bacteria in rodent populations. Methodology/Principal Findings We first extracted nucleic acids from the spleens of 190 voles collected in France. RNA extracts were pooled, randomly retro-transcribed, then RNA-Seq was performed using HiSeq. Assembled bacterial sequences were assigned to the closest taxon registered in GenBank. DNA extracts were analyzed via a 16S-metagenomics approach using two sequencers: the 454 GS-FLX and the MiSeq. The V4 region of the gene coding for 16S rRNA was amplified for each sample using barcoded universal primers. Amplicons were multiplexed and processed on the distinct sequencers. The resulting datasets were de-multiplexed, and each read was processed through a pipeline to be taxonomically classified using the Ribosomal Database Project. Altogether, 45 pathogenic bacterial genera were detected. The bacteria identified by RNA-Seq were comparable to those detected by 16S-metagenomics approach processed with MiSeq (16S-MiSeq). In contrast, 21 of these pathogens went unnoticed when the 16S-metagenomics approach was processed via 454-pyrosequencing (16S-454). In addition, the 16S-metagenomics approaches revealed a high level of coinfection in bank voles. Conclusions/Significance We concluded that RNA-Seq and 16S-MiSeq are equally sensitive in detecting bacteria. Although only the 16S-MiSeq method enabled identification of bacteria in each individual reservoir, with subsequent derivation of bacterial prevalence in host populations, and generation of intra-reservoir patterns of bacterial interactions. Lastly, the number of bacterial reads obtained with the 16S-MiSeq could be a good proxy for bacterial prevalence. PMID:26284930

  8. Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp.

    PubMed Central

    Trust, T J; Logan, S M; Gustafson, C E; Romaniuk, P J; Kim, N W; Chan, V L; Ragan, M A; Guerry, P; Gutell, R R

    1994-01-01

    The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria. Images PMID:8045890

  9. A bacterial enrichment study and overview of the extractable lipids from paleosols in the Dry Valleys, Antarctica: implications for future Mars reconnaissance.

    PubMed

    Hart, Kris M; Szpak, Michal T; Mahaney, William C; Dohm, James M; Jordan, Sean F; Frazer, Andrew R; Allen, Christopher C R; Kelleher, Brian P

    2011-05-01

    The Dry Valleys of Antarctica are one of the coldest and driest environments on Earth with paleosols in selected areas that date to the emplacement of tills by warm-based ice during the Early Miocene. Cited as an analogue to the martian surface, the ability of the Antarctic environment to support microbial life-forms is a matter of special interest, particularly with the upcoming NASA/ESA 2018 ExoMars mission. Lipid biomarkers were extracted and analyzed by gas chromatography--mass spectrometry to assess sources of organic carbon and evaluate the contribution of microbial species to the organic matter of the paleosols. Paleosol samples from the ice-free Dry Valleys were also subsampled and cultivated in a growth medium from which DNA was extracted with the explicit purpose of the positive identification of bacteria. Several species of bacteria were grown in solution and the genus identified. A similar match of the data to sequenced DNA showed that Alphaproteobacteria, Gammaproteobacteria, Bacteriodetes, and Actinobacteridae species were cultivated. The results confirm the presence of bacteria within some paleosols, but no assumptions have been made with regard to in situ activity at present. These results underscore the need not only to further investigate Dry Valley cryosols but also to develop reconnaissance strategies to determine whether such likely Earth-like environments on the Red Planet also contain life. PMID:21545270

  10. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

    PubMed Central

    Dedduwa-Mudalige, Gayani N. P.; Chow, Christine S.

    2015-01-01

    Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA) intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA) including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human. PMID:26370969

  11. Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins.

    PubMed

    Dedduwa-Mudalige, Gayani N P; Chow, Christine S

    2015-01-01

    Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA) intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA) including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human. PMID:26370969

  12. Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments.

    PubMed

    Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

    2006-01-01

    Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained. PMID:16391020

  13. Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective

    NASA Technical Reports Server (NTRS)

    Gutell, R. R.; Larsen, N.; Woese, C. R.

    1994-01-01

    The 16S and 23S rRNA higher-order structures inferred from comparative analysis are now quite refined. The models presented here differ from their immediate predecessors only in minor detail. Thus, it is safe to assert that all of the standard secondary-structure elements in (prokaryotic) rRNAs have been identified, with approximately 90% of the individual base pairs in each molecule having independent comparative support, and that at least some of the tertiary interactions have been revealed. It is interesting to compare the rRNAs in this respect with tRNA, whose higher-order structure is known in detail from its crystal structure (36) (Table 2). It can be seen that rRNAs have as great a fraction of their sequence in established secondary-structure elements as does tRNA. However, the fact that the former show a much lower fraction of identified tertiary interactions and a greater fraction of unpaired nucleotides than the latter implies that many of the rRNA tertiary interactions remain to be located. (Alternatively, the ribosome might involve protein-rRNA rather than intramolecular rRNA interactions to stabilize three-dimensional structure.) Experimental studies on rRNA are consistent to a first approximation with the structures proposed here, confirming the basic assumption of comparative analysis, i.e., that bases whose compositions strictly covary are physically interacting. In the exhaustive study of Moazed et al. (45) on protection of the bases in the small-subunit rRNA against chemical modification, the vast majority of bases inferred to pair by covariation are found to be protected from chemical modification, both in isolated small-subunit rRNA and in the 30S subunit. The majority of the tertiary interactions are reflected in the chemical protection data as well (45). On the other hand, many of the bases not shown as paired in Fig. 1 are accessible to chemical attack (45). However, in this case a sizeable fraction of them are also protected against chemical modification (in the isolated rRNA), which suggests that considerable higher-order structure remains to be found (although all of it may not involve base-base interactions and so may not be detectable by comparative analysis). The agreement between the higher-order structure of the small-subunit rRNA and protection against chemical modification is not perfect, however; some bases shown to covary canonically are accessible to chemical modification (45).(ABSTRACT TRUNCATED AT 400 WORDS).

  14. Phylogeny of parasitiform mites (Acari) based on rRNA.

    PubMed

    Klompen, Hans; Lekveishvili, Mariam; Black, William C

    2007-06-01

    Acari (mites and ticks) form one the most diverse lineages of arthropods, but basal relationships in the group are still poorly understood. The current study addresses this issue for one of its two main lineages, the order Parasitiformes. Relationships are examined at the subordinal and infraordinal level using complete 18S and partial 28S nuclear rRNA sequence data. Most currently recognized lineages are recovered with good support, suggesting that nuclear rRNA, and specifically 18S rRNA, is very well suited for analyzing relationships at this level in this lineage. These results were found despite quite variable rates of sequence evolution, with rates "ratcheting up" from relatively low in most non-mite arachnid lineages, to intermediate in Pseudoscorpiones, the mite order Acariformes, and the parasitiform suborders Opilioacarida, Holothyrida, and Ixodida, to high in the parasitiform suborder Mesostigmata. The most species rich mesostigmatid infraorder, Dermanyssina, shows huge distances to the outgroups, but remarkably low within-group divergence in nuclear rRNA. This suggests the possibility of a relatively recent origin of this lineage. PMID:17197202

  15. 23S RRNA OPERON DIFFERENCES IN ISOLATES OF CAMPYLOBACTER SPP.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lateral transfer of genes appears to occur between Campylobacter species, but the extent of that transfer is not fully appreciated. The presence or absence of an intervening sequence (IVS) in the 23S rRNA has been noted in Campylobacter coli and Campylobacter jejuni. As part of a survey, we used P...

  16. New Screening Software Shows that Most Recent Large 16S rRNA Gene Clone Libraries Contain Chimeras

    PubMed Central

    Ashelford, Kevin E.; Chuzhanova, Nadia A.; Fry, John C.; Jones, Antonia J.; Weightman, Andrew J.

    2006-01-01

    A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/. PMID:16957188

  17. Molecular identification of Capnocytophaga spp. via 16S rRNA PCR-restriction fragment length polymorphism analysis.

    PubMed

    Ciantar, Marilou; Newman, Hubert N; Wilson, Michael; Spratt, David A

    2005-04-01

    Capnocytophaga spp. have been implicated as putative periodontal pathogens associated with various periodontal diseases. Although the genus is known to contain five human oral isolates, accurate identification to species level of these organisms recovered from subgingival plaque has been hampered by the lack of a reliable method. Hence, most studies to date have reported these isolates as Capnocytophaga spp. Previous attempts at identification were based on biochemical tests; however, the results were inconclusive. Considering the differing virulence features of the respective isolates, it is crucial to identify these isolates to species level. The universal and conservative nature of the 16S rRNA gene has provided an accurate method for bacterial identification. The aim of this study was to identify Capnocytophaga spp. via restriction enzyme analysis of this gene (16S rRNA PCR-restriction fragment length polymorphism). The results (backed up by 16S rRNA gene sequencing) showed that this method reliably identifies all named Capnocytophaga spp. to species level. PMID:15815015

  18. Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

    SciTech Connect

    Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua

    2009-10-09

    ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

  19. Phylogenetic mapping of bacterial morphology

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  20. Curcuma and Scutellaria plant extracts protect chickens against inflammation and Salmonella Enteritidis infection.

    PubMed

    Varmuzova, Karolina; Matulova, Marta Elsheimer; Gerzova, Lenka; Cejkova, Darina; Gardan-Salmon, Delphine; Panhéleux, Marina; Robert, Fabrice; Sisak, Frantisek; Havlickova, Hana; Rychlik, Ivan

    2015-09-01

    After a ban on the use of antibiotics as growth promoters in farm animals in the European Union in 2006, an interest in alternative products with antibacterial or anti-inflammatory properties has increased. In this study, we therefore tested the effects of extracts from Curcuma longa and Scutellaria baicalensis used as feed additives against cecal inflammation induced by heat stress or Salmonella Enteritidis (S. Enteritidis) infection in chickens. Curcuma extract alone was not enough to decrease gut inflammation induced by heat stress. However, a mixture of Curcuma and Scutellaria extracts used as feed additives decreased gut inflammation induced by heat or S. Enteritidis, decreased S. Enteritidis counts in the cecum but was of no negative effect on BW or humoral immune response. Using next-generation sequencing of 16S rRNA we found out that supplementation of feed with the 2 plant extracts had no effect on microbiota diversity. However, if the plant extract supplementation was provided to the chickens infected with S. Enteritidis, Faecalibacterium, and Lactobacillus, both bacterial genera with known positive effects on gut health were positively selected. The supplementation of chicken feed with extracts from Curcuma and Scutelleria thus may be used in poultry production to effectively decrease gut inflammation and increase chicken performance. PMID:26188032

  1. Novel Major Bacterial Candidate Division within a Municipal Anaerobic Sludge Digester

    PubMed Central

    Chouari, Rakia; Le Paslier, Denis; Dauga, Catherine; Daegelen, Patrick; Weissenbach, Jean; Sghir, Abdelghani

    2005-01-01

    In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages. PMID:15812049

  2. Effect in vitro of a bacterial extract (OM-89) on interleukin 1 and interleukin 2 production by peripheral blood mononuclear cells from healthy subjects and rheumatoid arthritis patients.

    PubMed

    Clot, J; Andary, M

    1990-01-01

    The effect of a lyophilized extract from Escherichia coli strains (OM-89) on interleukin 1 and interleukin 2 production was studied by using peripheral blood mononuclear cells (PBMC) from healthy volunteers and from patients suffering from rheumatoid arthritis (RA) since, in this autoimmune disease, an abnormal cytokine network has been already described. The secretion of interleukin 1 (IL-1) was investigated in supernatants of monocytes purified by adherence, and measured by the C3H/HeJ thymocyte co-mitogenic assay. OM-89 was able to induce the secretion of IL-1 by normal and RA monocytes to about half of the level reached when the same cells were stimulated by lipopolysaccharide. The production of interleukin 2 (IL-2) was investigated in supernatants of PBMC, stimulated or not by phytohaemagglutinin (PHA) and mixed or not with various concentrations of OM-89. The level of IL-2 in supernatants, as measured by the stimulation of the CTLL2 murine cell line, was lower in RA supernatants than in control ones. In the presence of PHA and OM-89, the IL-2 production was enhanced and normalized in supernatants from RA patients. Such data may help to explain the clinical improvement previously reported in RA patients orally treated with OM-89. PMID:2292468

  3. Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

    PubMed Central

    Nübel, Ulrich; Garcia-Pichel, Ferran; Kühl, Michael; Muyzer, Gerard

    1999-01-01

    We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied. PMID:9925563

  4. Development of an Analysis Pipeline Characterizing Multiple Hypervariable Regions of 16S rRNA Using Mock Samples

    PubMed Central

    Barb, Jennifer J.; Oler, Andrew J.; Kim, Hyung-Suk; Chalmers, Natalia; Wallen, Gwenyth R.; Cashion, Ann; Munson, Peter J.; Ames, Nancy J.

    2016-01-01

    Objectives There is much speculation on which hypervariable region provides the highest bacterial specificity in 16S rRNA sequencing. The optimum solution to prevent bias and to obtain a comprehensive view of complex bacterial communities would be to sequence the entire 16S rRNA gene; however, this is not possible with second generation standard library design and short-read next-generation sequencing technology. Methods This paper examines a new process using seven hypervariable or V regions of the 16S rRNA (six amplicons: V2, V3, V4, V6-7, V8, and V9) processed simultaneously on the Ion Torrent Personal Genome Machine (Life Technologies, Grand Island, NY). Four mock samples were amplified using the 16S Ion Metagenomics Kit™ (Life Technologies) and their sequencing data is subjected to a novel analytical pipeline. Results Results are presented at family and genus level. The Kullback-Leibler divergence (DKL), a measure of the departure of the computed from the nominal bacterial distribution in the mock samples, was used to infer which region performed best at the family and genus levels. Three different hypervariable regions, V2, V4, and V6-7, produced the lowest divergence compared to the known mock sample. The V9 region gave the highest (worst) average DKL while the V4 gave the lowest (best) average DKL. In addition to having a high DKL, the V9 region in both the forward and reverse directions performed the worst finding only 17% and 53% of the known family level and 12% and 47% of the genus level bacteria, while results from the forward and reverse V4 region identified all 17 family level bacteria. Conclusions The results of our analysis have shown that our sequencing methods using 6 hypervariable regions of the 16S rRNA and subsequent analysis is valid. This method also allowed for the assessment of how well each of the variable regions might perform simultaneously. Our findings will provide the basis for future work intended to assess microbial abundance at different time points throughout a clinical protocol. PMID:26829716

  5. Global Patterns in Bacterial Diversity

    NASA Astrophysics Data System (ADS)

    Lozupone, C.; Knight, R.

    2007-12-01

    Microbes are difficult to culture. Consequently, the primary source of information about a fundamental evolutionary topic, life's diversity, is the environmental distribution of gene sequences. We report the most comprehensive analysis of the environmental distribution of bacteria to date, based on 21,752 16S rRNA sequences compiled from 111 studies of diverse physical environments. We clustered the samples based on similarities in the phylogenetic lineages that they contain and found that, surprisingly, the major environmental determinant of microbial community composition is salinity rather than extremes of temperature, pH, or other physical and chemical factors represented in our samples. We find that sediments are more phylogenetically diverse than any other environment type. Surprisingly, soil, which has high species-level diversity, has below-average phylogenetic diversity. This work provides a framework for understanding the impact of environmental factors on bacterial evolution and for the direction of future sequencing efforts to discover new lineages.

  6. Potential of Kalopanax septemlobus leaf extract in synthesis of silver nanoparticles for selective inhibition of specific bacterial strain in mixed culture.

    PubMed

    Salunke, Bipinchandra K; Sawant, Shailesh S; Kim, Beom Soo

    2014-09-01

    Silver nanoparticles (AgNPs) were synthesised using Kalopanax septemlobus plant leaf extracts. UV-visible spectrophotometric, Fourier-transform infrared, electron dispersive X-ray spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses confirmed synthesis of AgNPs. TEM micrographs revealed presence of well-dispersed AgNPs predominantly of small size and different shapes with an average particle size of 30.8 nm. Antimicrobial susceptibility tests of AgNP treatments revealed variability in sensitivity of bacteria Bacillus cereus and Saccharophagus degradans under study. Minimum inhibitory concentration (MIC) values of the AgNPs for B. cereus and S. degradans were found to be 30 and 10 ?g/mL, respectively. The mixed culture of B. cereus and S. degradans treated with AgNPs at 10 ?g/mL showed increase in growth with time, suggesting survival of bacteria in liquid media. The plating of mixed culture before AgNP treatment showed presence of both bacteria, but 24-h-old mixed culture treated with AgNPs at the concentration of 10 ?g/mL showed presence of B. cereus colonies. SEM micrographs revealed damage to S. degradans cells but no effect on B. cereus cells after AgNP treatment. Confocal microscopic observations of AgNP-treated mixed cultures by Nile blue A staining indicated intact polyhydroxyalkanoates producing flourescent cells of B. cereus but damage and deformities in S. degradans cells. This study suggests that AgNPs can selectively inhibit growth of S. d