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1

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)

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.

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

2007-07-01

2

Evaluating Bias of Illumina-Based Bacterial 16S rRNA Gene Profiles  

PubMed Central

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

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

2014-01-01

3

Evaluating Bias of Illumina-Based Bacterial 16S rRNA Gene Profiles.  

PubMed

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

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

2014-09-15

4

Bacterial diversity in a finished compost and vermicompost: differences revealed by cultivation-independent analyses of PCR-amplified 16S rRNA genes  

Microsoft Academic Search

Bacterial communities are important catalysts in the production of composts. Here, it was analysed whether the diversity of bacteria in finished composts is stable and specific for the production process. Single-strand conformation polymorphism (SSCP) based on polymerase chain reaction amplified partial 16S rRNA genes was used to profile and analyse bacterial communities found in total DNA extracted from finished composts.

Letizia Fracchia; Anja B. Dohrmann; Maria Giovanna Martinotti; Christoph C. Tebbe

2006-01-01

5

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 ...

6

Interaction of Bartonella henselae with endothelial cells results in rapid bacterial rRNA synthesis and replication.  

PubMed

Bartonella henselae is a slow-growing microorganism and the causative pathogen of bacillary angiomatosis in man. Here, we analysed how interaction of B. henselae with endothelial cells might affect bacterial growth. For this purpose, bacterial rRNA production and ribosome content was determined by fluorescence in situ hybridization (FISH) using rRNA-targeted fluorescence-labelled oligonucleotide probes. B. henselae grown on agar plates showed no detectable rRNA content by means of FISH, whereas B. henselae co-cultured with endothelial cells showed a rapid increase of rRNA production within the first 18 h after inoculation. The increased rRNA synthesis was paralleled by a approximately 1000-fold intracellular bacterial replication, whereas bacteria grown on agar base showed only a approximately 10-fold replication within the first 48 h of culture. Pretreatment of host cells with paraformaldehyde prevented adhesion, invasion, intracellular replication and bacterial rRNA synthesis of B. henselae. In contrast, inhibition of host cell protein synthesis by cycloheximide did not affect bacterial adhesion and invasion, but prevented intracellular replication although bacterial rRNA content was increased. Inhibition of actin polymerization by cytochalasin D did not affect adhesion, invasion, increased rRNA content or intracellular replication of B. henselae. These results demonstrate that rRNA synthesis and replication of B. henselae is promoted by viable host cells with intact de novo protein synthesis. PMID:11207598

Kempf, V A; Schaller, M; Behrendt, S; Volkmann, B; Aepfelbacher, M; Cakman, I; Autenrieth, I B

2000-10-01

7

Direct extraction and purification of rRNA for ecological studies.  

PubMed

Microgram quantities of rRNA were recovered from natural microbial communities in sediment, soil, and water with a lysozyme-hot phenol direct extraction method. Gel filtration with Sephadex G-75 spun columns readily removed humic-like contaminants without any measurable loss of rRNA and rendered RNA extracts of sufficient quality for molecular procedures. PMID:7683186

Moran, M A; Torsvik, V L; Torsvik, T; Hodson, R E

1993-03-01

8

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 ...

9

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 ...

10

16S rRNA survey revealed complex bacterial communities and evidence of bacterial interference on human adenoids.  

PubMed

Adenoid microbiota plays an important role in the development of various infectious and non-infectious diseases of the upper airways, such as otitis media, adenotonsillitis, rhinosinusitis and adenoid hypertrophy. Studies have suggested that adenoids could act as a potential reservoir of opportunistic pathogens. However, previous bacterial surveys of adenoids were mainly culture based and therefore might only provide an incomplete and potentially biased assessment of the microbial diversity. To develop an in-depth and comprehensive understanding of the adenoid microbial communities and test the 'pathogen reservoir hypothesis', we carried out a 16S rRNA based, culture-independent survey of bacterial communities on 67 human adenoids removed by surgery. Our survey revealed highly diverse adenoid bacterial communities distinct from those of other body habitats. Despite large interpersonal variations, adenoid microbiota shared a core set of taxa and can be classified into at least five major types based on its bacterial species composition. Our results support the 'pathogen reservoir hypothesis' as we found common pathogens of otitis media to be both prevalent and abundant. Co-occurrence analyses revealed evidence consistent with the bacterial interference theory in that multiple common pathogens showed 'non-coexistence' relationships with non-pathogenic members of the commensal microflora. PMID:23113966

Ren, Tiantian; Glatt, Dominique Ulrike; Nguyen, Tam Nhu; Allen, Emma Kaitlynn; Early, Stephen V; Sale, Michele; Winther, Birgit; Wu, Martin

2013-02-01

11

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

Microsoft Academic Search

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA\\u000a extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic\\u000a analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly\\u000a 62% of all DNA sequences analyzed.

Randy P. Revetta; Robin S. Matlib

2011-01-01

12

Comparison of bacterial communities in the Solimões and Negro River tributaries of the Amazon River based on small subunit rRNA gene sequences.  

PubMed

The microbiota of the Amazon River basin has been little studied. We compared the structure of bacterial communities of the Solimões and Negro Rivers, the main Amazon River tributaries, based on analysis of 16S rRNA gene sequences. Water was sampled with a 3-L Van Dorn collection bottle; samples were collected at nine different points/depths totaling 27 L of water from each river. Total DNA was extracted from biomass retained by a 0.22-?m filter after sequential filtration of the water through 0.8- and 0.22-?m filters. The 16S rRNA gene was amplified by PCR, cloned and sequenced, and the sequences were analyzed with the PHYLIP and DOTUR programs to obtain the operational taxonomic units (OTUs) and to calculate the diversity and richness indices using the SPADE program. Taxonomic affiliation was determined using the naive Bayesian rRNA Classifier of the RDP II (Ribosomal Database Project). We recovered 158 sequences from the Solimões River grouped into 103 OTUs, and 197 sequences from the Negro River library grouped into 90 OTUs by the DOTUR program. The Solimões River was found to have a greater diversity of bacterial genera, and greater estimated richness of 446 OTUs, compared with 242 OTUs from the Negro River, as calculated by ACE estimator. The Negro River has less bacterial diversity, but more 16S rRNA gene sequences belonging to the bacterial genus Polynucleobacter were detected; 56 sequences from this genus were found (about 30% of the total sequences). We suggest that a more in-depth investigation be made to elucidate the role played by these bacteria in the river environment. These differences in bacterial diversity between Solimões and Negro Rivers could be explained by differences in organic matter content and pH of the rivers. PMID:22183948

Peixoto, J C C; Leomil, L; Souza, J V; Peixoto, F B S; Astolfi-Filho, S

2011-01-01

13

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

Microsoft Academic Search

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

ANDREAS FELSKE; ARTHUR WOLTERINK; ROBERT VAN LIS; ANTOON D. L. AKKERMANS; Hesselink van Suchtelenweg

1998-01-01

14

Levels of Bacterial Community Diversity in Four Arid Soils Compared by Cultivation and 16S rRNA Gene Cloning  

Microsoft Academic Search

Techniques based on amplification of 16S rRNA genes for comparing bacterial communities are now widely used in microbial ecology, but calibration of these techniques with traditional tools, such as cultivation, has been conspicuously absent. In this study, we compared levels of bacterial community diversity in two pinyon rhizosphere soil samples and two between-tree (interspace) soil samples by analyzing 179 cultivated

JOHN DUNBAR; SHANNON TAKALA; SUSAN M. BARNS; JODY A. DAVIS; CHERYL R. KUSKE

1999-01-01

15

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

Microsoft Academic Search

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)

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

2003-01-01

16

Phylogenetic analysis of 16S rRNA gene sequences reveals distal gut bacterial diversity in wild wolves (Canis lupus).  

PubMed

The aim of this study was to describe the microbial communities in the distal gut of wild wolves (Canis lupus). Fecal samples were collected from three healthy unrelated adult wolves captured at the nearby of Dalai Lake Nature Reserve in Inner Mongolia of China. The diversity of fecal bacteria was investigated by constructing PCR-amplified 16S rRNA gene clone libraries using the universal bacterial primers 27 F and 1493 R. A total of 307 non-chimeric near-full-length 16S rRNA gene sequences were analyzed and 65 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified. Seventeen OTUs (26%) showed less than 98% sequence similarity to 16S rRNA gene sequences were reported previously. Five different bacterial phyla were identified, with the majority of OTUs being classified within the phylum Firmicutes (60%), followed by Bacteroidetes (16.9%), Proteobacteria (9.2%), Fusobacteria (9.2%) and Actinobacteria (4.6%). The majority of clones fell within the order Clostridiales (53.8% of OTUs). It was predominantly affiliated with five families: Lachnospiraceae was the most diverse bacterial family in this order, followed by Ruminococcaceae, Clostridiaceae, Peptococcaceae and Peptostreptococcaceae. PMID:20306230

Zhang, Honghai; Chen, Lei

2010-12-01

17

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

PubMed

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

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

2009-08-01

18

Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.  

PubMed

Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit 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. PMID:9097448

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

1997-04-01

19

Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa.  

PubMed Central

Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhibit 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. PMID:9097448

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

1997-01-01

20

Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids.  

PubMed

Given the potential for a variety of bacterial pathogens to occur in variably stabilized sewage sludge (biosolids), an understanding of pathogen diversity and abundance is necessary for accurate assessment of infective risk when these products are land applied. 16S rDNA was PCR amplified from genomic DNA extracted from municipal wastewater residuals (mesophilic- and thermophilic-phased anaerobic digestion (MAD and TPAD), composting (COM)), and agricultural soil (SOIL), and these amplicons were sequenced using massively parallel pyrosequencing technology. Resulting libraries contained an average of 30,893 16S rDNA sequences per sample with an average length of 392 bases. FASTUNIFRAC-based comparisons of population phylogenetic distance demonstrated similarities between the populations of different treatment plants performing the same stabilization method (e.g. different MAD samples), and population differences among samples from different biosolids stabilization methods (COM, MAD, and TPAD). Based on a 0.03 Jukes-Cantor distance to 80 potential bacterial pathogens, all samples contained pathogens and enrichment ranged from 0.02% to 0.1% of sequences. Most (61%) species identified were opportunistic pathogens of the genera Clostridium and Mycobacterium. As risk sciences continue to evolve to address scenarios that include multiple pathogen exposure, the analysis described here can be used to determine the diversity of pathogens in an environmental sample. This work provides guidance for prioritizing subsequent culturable and quantitative analysis, and for the first time, ensuring that potentially significant pathogens are not left out of risk estimations. PMID:20566210

Bibby, Kyle; Viau, Emily; Peccia, Jordan

2010-07-01

21

Bacteria and Bacterial rRNA Genes Associated with the Development of Colitis in IL-10–/– Mice  

PubMed Central

Background Microorganisms appear to play important yet ill-defined roles in the etiology of inflammatory bowel disease (IBD). This study utilized a novel population-based approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL-10–/– mice. Methods Mice were housed in 2 environments: a community mouse facility where the mice were fed nonsterile chow (Room 3) and a limited access facility where the mice were fed sterile chow (Room 4). Every month the disease activity levels were assessed and fecal bacterial compositions were analyzed. At the end of the experiments histological and bacterial analyses were performed on intestinal tissue. Results Although disease activity increased over time in both environments, it progressed at a faster rate in Room 3 than Room 4. Culture and culture-independent bacterial analyses identified several isolates and phylotypes associated with colitis. Two phylotypes (GpC2 and Gp66) were distinguished by their negative associations with disease activity in fecal and tissue samples. Notably, rRNA genes from these phylotypes had high sequence identity (99%) to an rRNA gene from a previously described flagellated Clostridium (Lachnospiraceae bacterium A4). Conclusions The negative associations of these 2 phylotypes (GpC2 and Gp66) suggest that these bacteria were being immunologically targeted, consistent with prior findings that the Lachnospiraceae bacterium A4 bears a prevalent flagellar antigen for disease-associated immunity in murine immune colitis and human Crohn's disease. Identification of these associations suggests that the experimental approach used in this study will have considerable utility in elucidating the host–microbe interactions underlying IBD. PMID:18381614

Presley, Laura L.; Bent, Elizabeth; Wei, Bo; Braun, Jonathan; Schiller, Neal L.; Straus, Daniel S.; Borneman, James

2013-01-01

22

Phylogenetic diversity of bacterial symbionts of Solemya hosts based on comparative sequence analysis of 16S rRNA genes.  

PubMed Central

The bacterial endosymbionts of two species of the bivalve genus Solemya from the Pacific Ocean, Solemya terraeregina and Solemya pusilla, were characterized. Prokaryotic cells resembling gram-negative bacteria were observed in the gills of both host species by transmission electron microscopy. The ultrastructure of the symbiosis in both host species is remarkably similar to that of all previously described Solemya spp. By using sequence data from 16S rRNA, the identity and evolutionary origins of the S. terraeregina and S. pusilla symbionts were also determined. Direct sequencing of PCR-amplified products from host gill DNA with primers specific for Bacteria 16S rRNA genes gave a single, unambiguous sequence for each of the two symbiont species. In situ hybridization with symbiont-specific oligonucleotide probes confirmed that these gene sequences belong to the bacteria residing in the hosts gills. Phylogenetic analyses of the 16S rRNA gene sequences by both distance and parsimony methods identify the S. terraeregina and S. pusilla symbionts as members of the gamma subdivision of the Proteobacteria. In contrast to symbionts of other bivalve families, which appear to be monophyletic, the S. terraeregina and S. pusilla symbionts share a more recent common ancestry with bacteria associating endosymbiotically with bivalves of the superfamily Lucinacea than with other Solemya symbionts (host species S. velum, S. occidentalis, and S. reidi). Overall, the 16S rRNA gene sequence data suggest that the symbionts of Solemya hosts represent at least two distinct bacterial lineages within the gamma-Proteobacteria. While it is increasingly clear that all extant species of Solemya live in symbiosis with specific bacteria, the associations appear to have multiple evolutionary origins. PMID:8979342

Krueger, D M; Cavanaugh, C M

1997-01-01

23

Bacterial Population Dynamics in a Laboratory Activated Sludge Reactor Monitored by Pyrosequencing of 16S rRNA  

PubMed Central

The microbial population in a laboratory activated sludge reactor was monitored for 245 d at 75 time points by pyrosequencing of 16S rRNA. Synthetic wastewater was used as the influent, and the reactor was operated under the same conditions throughout the experiment. The behaviors of different bacterial operational taxonomic units (OTUs) were observed. Multiple OTUs showed periodic propagation and recession. One of the OTUs showed sharp recession, which suggests that cells in the OTU were selectively killed. The behaviors of different phylogenetic lineages of Candidatus ‘Accumulibacter phosphatis’ were also visualized. It was clearly demonstrated that pyrosequencing with barcoded primers is a very effective tool to clarify the dynamics of the bacterial population in activated sludge. PMID:23100021

Satoh, Hiroyasu; Oshima, Kenshiro; Suda, Wataru; Ranasinghe, Purnika; Li, Ning; Gunawardana, Egodaha Gedara Wasana; Hattori, Masahira; Mino, Takashi

2013-01-01

24

Soil Bacterial Diversity Screening Using Single 16S rRNA Gene V Regions Coupled with Multi-Million Read Generating Sequencing Technologies  

PubMed Central

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

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

2012-01-01

25

Bacterial diversity in Adirondack mountain lakes as revealed by 16S rRNA gene sequences.  

PubMed

Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA. Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%). Additionally, a number of the sequences were similar to those of the order Verrucomicrobiales. However, few of the sequence types are closely related to those of characterized species. The relative contributions of the groups of sequences differed among the lakes, suggesting that bacterial population structure varies and that it may be possible to relate aquatic bacterial community structure to water chemistry. PMID:9212443

Hiorns, W D; Methé, B A; Nierzwicki-Bauer, S A; Zehr, J P

1997-07-01

26

Bacterial Diversity in Adirondack Mountain Lakes as Revealed by 16S rRNA Gene Sequences  

Microsoft Academic Search

Bacterial communities of seven lakes in the Adirondack Mountains of New York State were characterized by amplification and sequencing of 16S ribosomal DNA. Analysis of over 100 partial sequences revealed a diverse collection of lineages, largely of the class Proteobacteria (19% alpha subdivision, 31% beta subdivision, and 9% gamma subdivision), the phylum Cytophaga-Flavobacteria-Bacteroides (15%), and the order Actinomycetales (18%). Additionally,

WILLIAM D. HIORNS; BARBARA A. METHE; SANDRA A. NIERZWICKI-BAUER; JONATHAN P. ZEHR; Darrin Fresh

1997-01-01

27

16S rRNA sequencing in routine bacterial identification: A 30-month experiment  

Microsoft Academic Search

Accurate identification of bacterial isolates is an essential task in clinical microbiology. Phenotypic methods are time-consuming and either fail to identify some bacteria such as Gram-positive rods entirely or at least fail to do so in some clinical situations. 16S rDNA sequencing is a recent method of identification which offers a useful alternative. In this study, we investigate the usefulness

S. Mignard; J. P. Flandrois

2006-01-01

28

Macroalgal extracts induce bacterial assemblage shifts and sublethal tissue stress in Caribbean corals.  

PubMed

Benthic macroalgae can be abundant on present-day coral reefs, especially where rates of herbivory are low and/or dissolved nutrients are high. This study investigated the impact of macroalgal extracts on both coral-associated bacterial assemblages and sublethal stress response of corals. Crude extracts and live algal thalli from common Caribbean macroalgae were applied onto the surface of Montastraea faveolata and Porites astreoides corals on reefs in both Florida and Belize. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons was used to examine changes in the surface mucus layer (SML) bacteria in both coral species. Some of the extracts and live algae induced detectable shifts in coral-associated bacterial assemblages. However, one aqueous extract caused the bacterial assemblages to shift to an entirely new state (Lobophora variegata), whereas other organic extracts had little to no impact (e.g. Dictyota sp.). Macroalgal extracts more frequently induced sublethal stress responses in M. faveolata than in P. astreoides corals, suggesting that cellular integrity can be negatively impacted in selected corals when comparing co-occurring species. As modern reefs experience phase-shifts to a higher abundance of macroalgae with potent chemical defenses, these macroalgae are likely impacting the composition of microbial assemblages associated with corals and affecting overall reef health in unpredicted and unprecedented ways. PMID:23028648

Morrow, Kathleen M; Ritson-Williams, Raphael; Ross, Cliff; Liles, Mark R; Paul, Valerie J

2012-01-01

29

Composition and Dynamics of Bacterial Communities of a Drinking Water Supply System as Assessed by RNA- and DNA-Based 16S rRNA Gene Fingerprinting  

PubMed Central

Bacterial community dynamics of a whole drinking water supply system (DWSS) were studied from source to tap. Raw water for this DWSS is provided by two reservoirs with different water characteristics in the Harz mountains of Northern Germany. Samples were taken after different steps of treatment of raw water (i.e., flocculation, sand filtration, and chlorination) and at different points along the supply system to the tap. RNA and DNA were extracted from the sampled water. The 16S rRNA or its genes were partially amplified by reverse transcription-PCR or PCR and analyzed by single-strand conformation polymorphism community fingerprints. The bacterial community structures of the raw water samples from the two reservoirs were very different, but no major changes of these structures occurred after flocculation and sand filtration. Chlorination of the processed raw water strongly affected bacterial community structure, as reflected by the RNA-based fingerprints. This effect was less pronounced for the DNA-based fingerprints. After chlorination, the bacterial community remained rather constant from the storage containers to the tap. Furthermore, the community structure of the tap water did not change substantially for several months. Community composition was assessed by sequencing of abundant bands and phylogenetic analysis of the sequences obtained. The taxonomic compositions of the bacterial communities from both reservoirs were very different at the species level due to their different limnologies. On the other hand, major taxonomic groups, well known to occur in freshwater, such as Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes, were found in both reservoirs. Significant differences in the detection of the major groups were observed between DNA-based and RNA-based fingerprints irrespective of the reservoir. Chlorination of the drinking water seemed to promote growth of nitrifying bacteria. Detailed analysis of the community dynamics of the whole DWSS revealed a significant influence of both source waters on the overall composition of the drinking water microflora and demonstrated the relevance of the raw water microflora for the drinking water microflora provided to the end user. PMID:16517632

Eichler, Stefan; Christen, Richard; Holtje, Claudia; Westphal, Petra; Botel, Julia; Brettar, Ingrid; Mehling, Arndt; Hofle, Manfred G.

2006-01-01

30

Extraction of Bacterial RNA from Soil: Challenges and Solutions  

PubMed Central

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

Wang, Yong; Hayatsu, Masahito; Fujii, Takeshi

2012-01-01

31

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

PubMed Central

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

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

1992-01-01

32

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

PubMed

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

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

2013-09-01

33

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

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.

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

34

Identification of bacterial species associated with the sheep scab mite (Psoroptes ovis) by using amplified genes coding for 16S rRNA.  

PubMed

This was the first molecular study of the bacterial flora of the sheep scab mite (Psoroptes ovis). A sequence analysis of genes coding for 16S rRNA revealed that Serratia marcescens and bacteria closely related to Staphylococcus intermedius or Staphylococcus chromogens and Alloiococcus otitidis were present. These bacteria were associated with skin lesions, dermatitis, and otitis media caused by P. ovis. PMID:10473440

Hogg, J C; Lehane, M J

1999-09-01

35

Identification of Bacterial Species Associated with the Sheep Scab Mite (Psoroptes ovis) by Using Amplified Genes Coding for 16S rRNA  

PubMed Central

This was the first molecular study of the bacterial flora of the sheep scab mite (Psoroptes ovis). A sequence analysis of genes coding for 16S rRNA revealed that Serratia marcescens and bacteria closely related to Staphylococcus intermedius or Staphylococcus chromogens and Alloiococcus otitidis were present. These bacteria were associated with skin lesions, dermatitis, and otitis media caused by P. ovis. PMID:10473440

Hogg, J. C.; Lehane, M. J.

1999-01-01

36

Phylogenetic characterization and in situ localization of the bacterial symbiont of shipworms (Teredinidae: Bivalvia) by using 16S rRNA sequence analysis and oligodeoxynucleotide probe hybridization.  

PubMed Central

It has been proposed that a bacterium isolated from the gills of shipworms (teredinid mollusks) is, by virtue of its ability both to degrade cellulose and to fix dinitrogen, the symbiont that enables these mollusks to utilize wood as their principal food source. The phylogenetic affiliation of four of these bacteria isolated from wood-boring bivalve mollusks was determined by 16S rRNA sequence analysis by using the reverse transcriptase method with six oligodeoxynucleotide primers. The four bacterial strains tested had indistinguishable 16S rRNA sequences, supporting the previous conclusion, based on phenotypic characterization, that these isolates represent a single species. Evolutionary distance matrix analysis of the RNA sequence indicated that the bacterial symbiont falls within the gamma-3 subdivision of the Proteobacteria and is distinct from other known bacterial genera. In situ localization of the bacterial symbiont in tissue sections of the shipworm Lyrodus pedicellatus was determined by using a 16S rRNA-directed oligodeoxynucleotide hybridization probe specific for the bacterium isolated from shipworm gill tissue. Fluorescence microscopy showed that the specific probe bound to L. pedicellatus tissue at sites coincident with the location of symbiont cells and that it did not bind to other host tissues. This technique provided direct visual evidence that the cellulolytic, nitrogen-fixing bacterial isolates were the symbionts observed within the gill of L. pedicellatus. Images PMID:1722662

Distel, D L; DeLong, E F; Waterbury, J B

1991-01-01

37

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment  

PubMed Central

High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions – provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results. PMID:24146837

Guo, Feng; Ju, Feng; Cai, Lin; Zhang, Tong

2013-01-01

38

Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients  

PubMed Central

Chronic bronchopulmonary bacterial infections remain the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Recent community sequencing work has now shown that the bacterial community in the CF lung is polymicrobial. Identifying bacteria in the CF lung through sequencing can be costly and is not practical for many laboratories. Molecular techniques such as terminal restriction fragment length polymorphism or amplicon length heterogeneity-polymerase chain reaction (LH-PCR) can provide many laboratories with the ability to study CF bacterial communities without costly sequencing. The aim of this study was to determine if the use of LH-PCR with multiple hypervariable regions of the 16S rRNA gene could be used to identify organisms found in sputum DNA. This work also determined if LH-PCR could be used to observe the dynamics of lung infections over a period of time. Nineteen samples were analysed with the V1 and the V1_V2 region of the 16S rRNA gene. Based on the amplicon size present in the V1_V2 region, Pseudomonas aeruginosa was confirmed to be in all 19 samples obtained from the patients. The V1 region provided a higher power of discrimination between bacterial profiles of patients. Both regions were able to identify trends in the bacterial population over a period of time. LH profiles showed that the CF lung community is dynamic and that changes in the community may in part be driven by the patient's antibiotic treatment. LH-PCR is a tool that is well suited for studying bacterial communities and their dynamics. PMID:20368138

2010-01-01

39

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

PubMed

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

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

2013-12-01

40

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)

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.

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

1999-08-01

41

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

PubMed Central

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

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

2004-01-01

42

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

PubMed Central

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

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

2001-01-01

43

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

PubMed Central

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

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

2012-01-01

44

From learning taxonomies to phylogenetic learning: Integration of 16S rRNA gene data into FAME-based bacterial classification  

PubMed Central

Background Machine learning techniques have shown to improve bacterial species classification based on fatty acid methyl ester (FAME) data. Nonetheless, FAME analysis has a limited resolution for discrimination of bacteria at the species level. In this paper, we approach the species classification problem from a taxonomic point of view. Such a taxonomy or tree is typically obtained by applying clustering algorithms on FAME data or on 16S rRNA gene data. The knowledge gained from the tree can then be used to evaluate FAME-based classifiers, resulting in a novel framework for bacterial species classification. Results In view of learning in a taxonomic framework, we consider two types of trees. First, a FAME tree is constructed with a supervised divisive clustering algorithm. Subsequently, based on 16S rRNA gene sequence analysis, phylogenetic trees are inferred by the NJ and UPGMA methods. In this second approach, the species classification problem is based on the combination of two different types of data. Herein, 16S rRNA gene sequence data is used for phylogenetic tree inference and the corresponding binary tree splits are learned based on FAME data. We call this learning approach 'phylogenetic learning'. Supervised Random Forest models are developed to train the classification tasks in a stratified cross-validation setting. In this way, better classification results are obtained for species that are typically hard to distinguish by a single or flat multi-class classification model. Conclusions FAME-based bacterial species classification is successfully evaluated in a taxonomic framework. Although the proposed approach does not improve the overall accuracy compared to flat multi-class classification, it has some distinct advantages. First, it has better capabilities for distinguishing species on which flat multi-class classification fails. Secondly, the hierarchical classification structure allows to easily evaluate and visualize the resolution of FAME data for the discrimination of bacterial species. Summarized, by phylogenetic learning we are able to situate and evaluate FAME-based bacterial species classification in a more informative context. PMID:20113515

2010-01-01

45

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

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

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

2004-01-01

46

Bacterial community structure in kimchi, a Korean fermented vegetable food, as revealed by 16S rRNA gene analysis  

Microsoft Academic Search

Kimchi is a traditional Korean food fermented from a variety of vegetables. We elucidated the microbial community structure of five commercially produced kimchis made from Chinese cabbage by examining culture-independent 16S rRNA gene clone libraries. Most of the clones (347 out of 348) belonged to lactic acid bacteria and included several species of the genera Lactobacillus, Leuconostoc and Weissella. Weissella

Myungjin Kim; Jongsik Chun

2005-01-01

47

Bacterial population changes in a membrane bioreactor for graywater treatment monitored by denaturing gradient gel electrophoretic analysis of 16S rRNA gene fragments.  

PubMed

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 (BOD(5)), 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

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

2003-02-01

48

Massive parallel 16S rRNA gene pyrosequencing reveals highly diverse fecal bacterial and fungal communities in healthy dogs and cats.  

PubMed

This study evaluated the fecal microbiota of 12 healthy pet dogs and 12 pet cats using bacterial and fungal tag-encoded FLX-Titanium amplicon pyrosequencing. A total of 120,406 pyrosequencing reads for bacteria (mean 5017) and 5359 sequences (one pool each for dogs and cats) for fungi were analyzed. Additionally, group-specific 16S rRNA gene clone libraries for Bifidobacterium spp. and lactic acid-producing bacteria (LAB) were constructed. The most abundant bacterial phylum was Firmicutes, followed by Bacteroidetes in dogs and Actinobacteria in cats. The most prevalent bacterial class in dogs and cats was Clostridia, dominated by the genera Clostridium (clusters XIVa and XI) and Ruminococcus. At the genus level, 85 operational taxonomic units (OTUs) were identified in dogs and 113 OTUs in cats. Seventeen LAB and eight Bifidobacterium spp. were detected in canine feces. Ascomycota was the only fungal phylum detected in cats, while Ascomycota, Basidiomycota, Glomeromycota, and Zygomycota were identified in dogs. Nacaseomyces was the most abundant fungal genus in dogs; Saccharomyces and Aspergillus were predominant in cats. At the genus level, 33 different fungal OTUs were observed in dogs and 17 OTUs in cats. In conclusion, this study revealed a highly diverse bacterial and fungal microbiota in canine and feline feces. PMID:21261668

Handl, Stefanie; Dowd, Scot E; Garcia-Mazcorro, Jose F; Steiner, Jörg M; Suchodolski, Jan S

2011-05-01

49

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

PubMed Central

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

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

2014-01-01

50

16S rRNA Restriction Fragment Length Polymorphism Analysis of Bacterial Diversity as a Biomarker of Ecological Health in Polluted Sediments from New Bedford Harbor, Massachusetts, USA  

Microsoft Academic Search

A polymerase chain reaction (PCR)-based method was developed to compare bacterial diversity among environmental sites with varying degrees of anthropogenic impact. New Bedford Harbor, MA, a US Environmental Protection Agency-designated Superfund hazardous waste site, was studied to assess changes in bacterial diversity resulting from long-term inputs of organic and inorganic pollutants. Total DNA was extracted from surficial sediments sampled from

Jonathan J. Sorci; Joseph D. Paulauskis; Timothy E. Ford

1999-01-01

51

Sampling of Intestinal Microbiota and Targeted Amplification of Bacterial 16S rRNA Genes for Microbial Ecologic Analysis.  

PubMed

Dysbiosis of host-associated commensal microbiota is emerging as an important factor in risk and phenotype of immunologic, metabolic, and behavioral diseases. Accurate analysis of microbial composition and functional state in humans or mice requires appropriate collection and pre-processing of biospecimens. Methods to sample luminal and mucosal microbiota from human or mouse intestines and to profile microbial phylogenetic composition using 16S rRNA sequencing are presented here. Data generated using the methods in this unit can be used for downstream quantitative analysis of microbial ecology. © 2014 by John Wiley & Sons, Inc. PMID:25367129

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

2014-01-01

52

Taxonomic composition of the particle-attached and free-living bacterial assemblages in the Northwest Mediterranean Sea analyzed by pyrosequencing of the 16S rRNA  

PubMed Central

Abstract Free-living (FL) and particle-attached (PA) bacterial assemblages in the Northwest Mediterranean Sea were studied using pyrosequencing data of the 16S rRNA. We have described and compared the richness, the distribution of Operational Taxonomic Units (OTUs) within the two fractions, the spatial distribution, and the taxonomic composition of FL and PA bacterial assemblages. The number of OTUs in the present work was two orders of magnitude higher than in previous studies. Only 25% of the total OTUs were common to both fractions, whereas 49% OTUs were exclusive to the PA fraction and 26% to the FL fraction. The OTUs exclusively present in PA or FL assemblages were very low in abundance (6% of total abundance). Detection of the rare OTUs revealed the larger richness of PA bacteria that was hidden in previous studies. Alpha-Proteobacteria dominated the FL bacterial assemblage and gamma-Proteobacteria dominated the PA fraction. Bacteroidetes were important in the PA fraction mainly at the coast. The high number of sequences in this study detected additional phyla from the PA fraction, such as Actinobacteria, Firmicutes, and Verrucomicrobia. PMID:23723056

Crespo, Bibiana G; Pommier, Thomas; Fernández-Gómez, Beatriz; Pedrós-Alió, Carlos

2013-01-01

53

Detection of Verrucomicrobia in a Pasture Soil by PCR-Mediated Amplification of 16S rRNA Genes  

PubMed Central

Oligonucleotide primers were designed and used to amplify, by PCR, partial 16S rRNA genes of members of the bacterial division Verrucomicrobia in DNA extracted from a pasture soil. By applying most-probable-number theory to the assay, verrucomicrobia appeared to contribute some 0.2% of the soil DNA. Amplified ribosomal DNA restriction analysis of 53 cloned PCR-amplified partial 16S rRNA gene fragments and comparative sequence analysis of 21 nonchimeric partial 16S rRNA genes showed that these primers amplified only 16S rRNA genes of members of the Verrucomicrobia in DNA extracted from the soil. PMID:10473454

O'Farrell, Katrina A.; Janssen, Peter H.

1999-01-01

54

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

PubMed Central

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 45°C, 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

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

2012-01-01

55

Study of anaerobic ammonium oxidation bacterial community in the aged refuse bioreactor with 16S rRNA gene library technique.  

PubMed

In order to investigate the anaerobic ammonium-oxidation (Anammox) nitrogen removal pathway of the aged refuse bioreactor treating landfill leachate, a lab-scale bioreactor was established and run for 35 weeks, the performance of the bioreactor and its bacterial community structure of Planctomycetes were analyzed. The results showed that the average TN removal rate of landfill leachate could be reached to 89%. 16S rRNA gene library of Planctomycetes revealed that Anammox sequences accounted for 28.3% of the total Planctomycetes sequences in the bioreactor, and previously recognized Anammox bacterium Candidatus Kuenenia stuttgartiensis was the only detected Anammox species in the reactor. It was also found that Anammox bacteria distributed at different sites of the bioreactor while mostly concentrated in the middle and low-middle part. Results above confirmed that Anammox process could happen in aged refuse bioreactor treating landfill leachate and provided an alternative nitrogen removal pathway in practical landfills. PMID:23489564

Wang, Chao; Xie, Bing; Han, Lu; Xu, Xiaofan

2013-10-01

56

Bacterial communities of traditional salted and fermented seafoods from Jeju Island of Korea using 16S rRNA gene clone library analysis.  

PubMed

Jeotgal, which is widely consumed as a nutritional supplement in Korea, is traditional type of preserved seafood that is prepared by salting and fermenting. Here, we report on the bacterial community structure and diversity of jeotgal obtained from the Korean island of Jeju, which has a subtropical climate. Two samples of Jeotgal were collected from Jeju, made from either damselfish (Chromis notata; jari-dom-jeot, J1 and J2) or silver-stripe round herring (Spratelloides gracilis; ggot-myulchi-jeot, K1 and K2). The physical characteristics (pH and salinity) were assessed and the bacterial communities characterized using 16S rRNA gene-clone library analysis and cultural isolation. No difference was found in the community composition between the J and K fermented seafoods. Both fermented seafoods had relatively high salinity (26% to 33%) and high pH values (pH 6.08 to 6.72). Based on the 16S rRNA gene sequences, the halophilic lactic-acid bacteria Tetragenococcus halophilus and T. muriaticus were observed to be dominant in the J and K fermented seafoods, accompanied by halophilic bacteria including Halanaerobium spp., Halomonas spp., and Chromohalobacter spp. When compared with 7 other types of fermented seafood from a previous study, the communities of the J and K fermented seafoods were separated by the most influential group, the genus Tetragenococcus. The results suggest that these 2 types of traditional salted fermented seafood from Jeju have distinct communities dominated by Tetragenococcus spp., which are derived from the raw ingredients and are dependent on the physical conditions. This may explain how the seafoods that are made in Jeju may differ from other jeotgals. PMID:24689962

Kim, Min-Soo; Park, Eun-Jin

2014-05-01

57

Lactic acid bacterial extract as a biogenic mineral growth modifier  

NASA Astrophysics Data System (ADS)

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.

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

2009-04-01

58

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

PubMed Central

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

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

2002-01-01

59

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

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.

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

2014-01-01

60

Diversity, Dynamics, and Activity of Bacterial Communities during Production of an Artisanal Sicilian Cheese as Evaluated by 16S rRNA Analysis†  

PubMed Central

The diversity and dynamics of the microbial communities during the manufacturing of Ragusano cheese, an artisanal cheese produced in Sicily (Italy), were investigated by a combination of classical and culture-independent approaches. The latter included PCR, reverse transcriptase-PCR (RT-PCR), and denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes (rDNA). Bacterial and Lactobacillus group-specific primers were used to amplify the V6 to V8 and V1 to V3 regions of the 16S rRNA gene, respectively. DGGE profiles from samples taken during cheese production indicated dramatic shifts in the microbial community structure. Cloning and sequencing of rDNA amplicons revealed that mesophilic lactic acid bacteria (LAB), including species of Leuconostoc, Lactococcus lactis, and Macrococcus caseolyticus were dominant in the raw milk, while Streptococcus thermophilus prevailed during lactic fermentation. Other thermophilic LAB, especially Lactobacillus delbrueckii and Lactobacillus fermentum, also flourished during ripening. Comparison of the rRNA-derived patterns obtained by RT-PCR to the rDNA DGGE patterns indicated a substantially different degree of metabolic activity for the microbial groups detected. Identification of cultivated LAB isolates by phenotypic characterization and 16S rDNA analysis indicated a variety of species, reflecting to a large extent the results obtained from the 16S rDNA clone libraries, with the significant exception of the Lactobacillus delbrueckii species, which dominated in the ripening cheese but was not detected by cultivation. The present molecular approaches combined with culture can effectively describe the complex ecosystem of natural fermented dairy products, giving useful information for starter culture design and preservation of artisanal fermented food technology. PMID:11916708

Randazzo, Cinzia L.; Torriani, Sandra; Akkermans, Antoon D. L.; de Vos, Willem M.; Vaughan, Elaine E.

2002-01-01

61

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

PubMed Central

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

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

2014-01-01

62

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

PubMed Central

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

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

2004-01-01

63

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

PubMed

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

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

64

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

PubMed Central

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

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

65

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

PubMed Central

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

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

2014-01-01

66

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

PubMed Central

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

Bolhuis, Henk; Stal, Lucas J

2011-01-01

67

Effects of season and experimental warming on the bacterial community in a temperate mountain forest soil assessed by 16S rRNA gene pyrosequencing  

PubMed Central

Climate warming may induce shifts in soil microbial communities possibly altering the long-term carbon mineralization potential of soils. We assessed the response of the bacterial community in a forest soil to experimental soil warming (+4 °C) in the context of seasonal fluctuations. Three experimental plots were sampled in the fourth year of warming in summer and winter and compared to control plots by 16S rRNA gene pyrosequencing. We sequenced 17 308 amplicons per sample and analysed operational taxonomic units at genetic distances of 0.03, 0.10 and 0.25, with respective Good's coverages of 0.900, 0.977 and 0.998. Diversity indices did not differ between summer, winter, control or warmed samples. Summer and winter samples differed in community structure at a genetic distance of 0.25, corresponding approximately to phylum level. This was mainly because of an increase of Actinobacteria in winter. Abundance patterns of dominant taxa (> 0.06% of all reads) were analysed individually and revealed, that seasonal shifts were coherent among related phylogenetic groups. Seasonal community dynamics were subtle compared to the dynamics of soil respiration. Despite a pronounced respiration response to soil warming, we did not detect warming effects on community structure or composition. Fine-scale shifts may have been concealed by the considerable spatial variation. PMID:22670891

Kuffner, Melanie; Hai, Brigitte; Rattei, Thomas; Melodelima, Christelle; Schloter, Michael; Zechmeister-Boltenstern, Sophie; Jandl, Robert; Schindlbacher, Andreas; Sessitsch, Angela

2012-01-01

68

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

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

Malygin, Alexey A.; Karpova, Galina G.

2010-01-01

69

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...

70

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

SciTech Connect

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.

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

2009-09-01

71

RNA end-labeling and RNA ligase activities can produce a circular rRNA in whole cell extracts from trypanosomes.  

PubMed Central

We have found two enzymatic activities in whole cell extracts from Trypanosoma brucei; an end-labeling reaction involving a single uridine at the 3' end of ribosomal RNAs (rRNAs) and an RNA ligase activity joining 5' monophosphates to 3' hydroxyl groups. The RNA ligase acts upon one of the small rRNAs (180 nucleotides) from the trypanosome ribosomal repeat unit, forming a circular RNA. The specific circularization of this small rRNA is probably dependent on the secondary structure of the molecule and is not detectable in vivo. Images PMID:2437529

White, T C; Borst, P

1987-01-01

72

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

PubMed

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

Canto-Canché, B; Tzec-Simá, M; Vázquez-Loría, J I; Espadas-Álvarez, H; Chí-Manzanero, B H; Rojas-Herrera, R; Valdez-Ojeda, R; Alzate-Gaviria, L

2013-01-01

73

Application of a strain-specific rRNA oligonucleotide probe targeting Pseudomonas fluorescens Ag1 in a mesocosm study of bacterial release into the environment.  

PubMed Central

Sequence analysis of domains 3 and 4 of 23S rRNA from Pseudomonas fluorescens Ag1 was carried out to allow the design of a strain-specific rRNA oligonucleotide probe targeting this strain. The specificity of the probe, Ps-Ag1, was assessed by dot blot analysis and whole-cell hybridization, and it was found to be specific for P. fluorescens Ag1. The correlation between the ribosomal content of P. fluorescens Ag1 and growth rate was determined during balanced growth conditions with generation times ranging from 1.2 to 31.8 h. Hybridization of the rRNA-targeting probes combined with charged coupled device-enhanced microscopy was used to determine the rRNA content. The total RNA content per cell was determined by staining with acridine orange and charged coupled device-enhanced microscopy. After 2 h under carbon starvation conditions, the rRNA content per cell decreased to 45% of the content of an exponentially growing cell. After 1 day of carbon starvation, the rRNA content had decreased to 20%. When cells were grown at different temperatures, it was found that the rRNA content per cell was only dependent on the substrate in the temperature range from 5 to 30 degrees C. P. fluorescens Ag1 was used in a mesocosm release experiment. The strain could be detected by use of the oligonucleotide probe targeting rRNA for 8 days in the water column and for 10 days on solid surfaces. The standard curve correlating growth rate with rRNA content was used to estimate the physiological activity of P. fluorescens Ag1 in the mesocosm experiment. PMID:7538276

Boye, M; Ahl, T; Molin, S

1995-01-01

74

Soybean extracts facilitate bacterial agglutination and prevent biofilm formation on orthodontic wire.  

PubMed

Soybean is an essential food ingredient that contains a class of organic compounds known as isoflavones. It is also well known that several plant agglutinins interfere with bacterial adherence to smooth surfaces. However, little is known about the effects of soybean extracts or genistein (a purified isoflavone from soybean) on bacterial biofilm formation. We evaluated the effects of soybean (Glycine max) extracts, including fermented soybean and genistein, on streptococcal agglutination and attachment onto stainless steel orthodontic wire. After cultivating streptococci in biofilm medium containing soybean extracts and orthodontic wire, the viable bacteria attached to the wire were counted. Phase-contrast microscopy and scanning electron microscopy (SEM) analyses were conducted to evaluate bacterial agglutination and attachment. Our study showed that soybean extracts induce agglutination between streptococci, which results in bacterial precipitation. Conversely, viable bacterial counting and SEM image analysis of Streptococcus mutans attached to the orthodontic wire show that bacterial attachment decreases significantly when soybean extracts were added. However, there was no significant change in pre-attached S. mutans biofilm in response to soybean. A possible explanation for these results is that increased agglutination of planktonic streptococci by soybean extracts results in inhibition of bacterial attachment onto the orthodontic wire. PMID:24456364

Lee, Heon-Jin; Kwon, Tae-Yub; Kim, Kyo-Han; Hong, Su-Hyung

2014-01-01

75

Comparative Analysis of Eukaryotic Marine Microbial Assemblages from 18S rRNA Gene and Gene Transcript Clone Libraries by Using Different Methods of Extraction  

PubMed Central

Eukaryotic marine microbes play pivotal roles in biogeochemical nutrient cycling and ecosystem function, but studies that focus on the protistan biogeography and genetic diversity lag-behind studies of other microbes. 18S rRNA PCR amplification and clone library sequencing are commonly used to assess diversity that is culture independent. However, molecular methods are not without potential biases and artifacts. In this study, we compare the community composition of clone libraries generated from the same water sample collected at the San Pedro Ocean Time Series (SPOTs) station in the northwest Pacific Ocean. Community composition was assessed using different cell lysis methods (chemical and mechanical) and the extraction of different nucleic acids (DNA and RNA reverse transcribed to cDNA) to build Sanger ABI clone libraries. We describe specific biases for ecologically important phylogenetic groups resulting from differences in nucleic acid extraction methods that will inform future designs of eukaryotic diversity studies, regardless of the target sequencing platform planned. PMID:22447590

Koid, Amy; Nelson, William C.; Mraz, Amy

2012-01-01

76

Bacterial diversity of extremely alkaline bauxite residue site of alumina industrial plant using culturable bacteria and residue 16S rRNA gene clones.  

PubMed

Bauxite residue (red mud), generated during the extraction of alumina from bauxite ore is characterized by high pH, high concentrations of soluble ions with low or virtually no organic matter. These extreme conditions along with numerous nutrient deficiencies, limit the microbial growth and vegetation establishment. In the present study, diversity of both cultivable and non-cultivable bacteria present in the red mud was investigated by 16S rDNA sequence analyses. The cultivable bacteria were identified as Agromyces indicus, Bacillus litoralis, B. anthracis, Chungangia koreensis, Kokuria flava, K. polaris, Microbacterium hominis, Planococcus plakortidis, Pseudomonas alcaliphila and Salinococcus roseus based on their 16S rDNA sequence analysis. These isolates were alkali tolerant, positive for one or more of the enzyme activities tested, able to produce organic acids and oxidize wide range of carbon substrates. For non-cultivable diversity of bacteria, DNA was extracted from the bauxite residue samples and 16S rDNA clone library was constructed. The 16S rDNA clones of this study showed affiliation to three major phyla predominant being betaproteobacteria (41.1%) followed by gammaproteobacteria (37.5%) and bacteroidetes (21.4%). We are reporting for the first time about the bacterial diversity of this unique and extreme environment. PMID:24817611

Krishna, Pankaj; Babu, A Giridhar; Reddy, M Sudhakara

2014-07-01

77

Expansion of the aminoglycoside-resistance 16S rRNA (m(1)A1408) methyltransferase family: expression and functional characterization of four hypothetical enzymes of diverse bacterial origin.  

PubMed

The global dissemination, potential activity in diverse species and broad resistance spectrum conferred by the aminoglycoside-resistance ribosomal RNA methyltransferases make them a significant potential new threat to the efficacy of aminoglycoside antibiotics in the treatment of serious bacterial infections. The N1 methylation of adenosine 1408 (m(1)A1408) confers resistance to structurally diverse aminoglycosides, including kanamycin, neomycin and apramycin. The limited analyses to date of the enzymes responsible have identified common features but also potential differences in their molecular details of action. Therefore, with the goal of expanding the known 16S rRNA (m(1)A1408) methyltransferase family as a platform for developing a more complete mechanistic understanding, we report here the cloning, expression and functional analyses of four hypothetical aminoglycoside-resistance rRNA methyltransferases from recent genome sequences of diverse bacterial species. Each of the genes produced a soluble, folded protein with a secondary structure, as determined from circular dichroism (CD) spectra, consistent with enzymes for which high-resolution structures are available. For each enzyme, antibiotic minimum inhibitory concentration (MIC) assays revealed a resistance spectrum characteristic of the known 16S rRNA (m(1)A1408) methyltransferases and the modified nucleotide was confirmed by reverse transcription as A1408. In common with other family members, higher binding affinity for the methylation reaction by-product S-adenosylhomocysteine (SAH) than the cosubstrate S-adenosyl-L-methionine (SAM) was observed for three methyltransferases, while one unexpectedly showed no measurable affinity for SAH. Collectively, these results confirm that each hypothetical enzyme is a functional 16S rRNA (m(1)A1408) methyltransferase but also point to further potential mechanistic variation within this enzyme family. PMID:24963996

Witek, Marta A; Conn, Graeme L

2014-09-01

78

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

Microsoft Academic Search

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

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

2009-01-01

79

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

PubMed

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

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

2013-01-01

80

Diagnostic Utility of Broad Range Bacterial 16S rRNA Gene PCR with Degradation of Human and Free Bacterial DNA in Bloodstream Infection Is More Sensitive Than an In-House Developed PCR without Degradation of Human and Free Bacterial DNA  

PubMed Central

We compared a commercial broad range 16S rRNA gene PCR assay (SepsiTest) to an in-house developed assay (IHP). We assessed whether CD64 index, a biomarker of bacterial infection, can be used to exclude patients with a low probability of systemic bacterial infection. From January to March 2010, 23 patients with suspected sepsis were enrolled. CD64 index, procalcitonin, and C-reactive protein were measured on admission. Broad range 16S rRNA gene PCR was performed from whole blood (SepsiTest) or blood plasma (IHP) and compared to blood culture results. Blood samples spiked with Staphylococcus aureus were used to assess sensitivity of the molecular assays in vitro. CD64 index was lower in patients where possible sepsis was excluded than in patients with microbiologically confirmed sepsis (P = 0.004). SepsiTest identified more relevant pathogens than blood cultures (P = 0.008); in three patients (13%) results from blood culture and SepsiTest were congruent, whereas in four cases (17.4%) relevant pathogens were detected by SepsiTest only. In vitro spiking experiments suggested equal sensitivity of SepsiTest and IHP. A diagnostic algorithm using CD64 index as a decision maker to perform SepsiTest shows improved detection of pathogens in patients with suspected blood stream infection and may enable earlier targeted antibiotic therapy. PMID:25120284

Rogina, Petra; Kofol, Romina; Kaasch, Achim

2014-01-01

81

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

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

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

82

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

Microsoft Academic Search

Three 16S rRNA gene clone libraries (P1L, P4L and P8L) were constructed using three soil samples (P1S, P4S and P8S) collected\\u000a 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.

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

2011-01-01

83

Wild Mushroom Extracts as Inhibitors of Bacterial Biofilm Formation  

PubMed Central

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.

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

2014-01-01

84

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

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

Dang, Hongyue; Lovell, Charles R.

2000-01-01

85

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

PubMed

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, cachaça 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 cachaça 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

Lacerda, Inayara C A; Gomes, Fátima C O; Borelli, Beatriz M; Faria, César L L; Franco, Gloria R; Mourão, Marina M; Morais, Paula B; Rosa, Carlos A

2011-04-01

86

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

PubMed Central

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, cachaça 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 cachaça 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

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

2011-01-01

87

Supercritical Fluid Extraction of Bacterial and Archaeal Lipid Biomarkers from Anaerobically Digested Sludge  

PubMed Central

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile. PMID:22489140

Hanif, Muhammad; Atsuta, Yoichi; Fujie, Koichi; Daimon, Hiroyuki

2012-01-01

88

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

PubMed

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

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

2010-05-01

89

Microfluidic device for bacterial genome extraction and analysis  

NASA Astrophysics Data System (ADS)

Although single molecule DNA manipulation and analysis techniques are emerging, methods for whole genome extraction from single cells, genomic length DNA handling and analytics is still to be developed. Here we present a microfabricated device to address some of these needs. This microfluidic chip is suitable for culturing bacteria and subsequently retrieve their genetic content. As a next step, the extracted DNA can be introduced in a nanostructured segment of the chip for precise handling, stretching and analysis. We hope that similar microdevices can be useful in studying genetic aspects of the cell lifecycle in a variety of organisms.

Galajda, Peter; Riehn, Robert; Wang, Yan-Mei; Keymer, Juan; Golding, Ido; Cox, Edward C.; Austin, Robert H.

2006-03-01

90

Evidence for indigenous Streptomyces populations in a marine environment determined with a 16S rRNA probe.  

PubMed Central

A 16S rRNA genus-specific probe was used to determine whether Streptomyces populations are an indigenous component of marine sediment bacterial communities. Previous debates have suggested that marine Streptomyces isolates are derived not from resident populations but from spores of terrestrial species which have been physically transported to marine ecosystems but remain dormant until isolation. Rigorously controlled hybridization of rRNA extracted from coastal marsh sediments with the genus-specific probe indicated that Streptomyces rRNA accounted for 2 to 5% of the sediment community rRNA and that spores are not the source of the hybridization signal. Streptomyces populations must therefore be at least the 26th most abundant genus-level source of bacterial rRNA. the relative amounts of rRNAs from Streptomyces spp. and members of the Bacteria (69 to 79%) and Archaea (4 to 7%) domains were highly consistent in these marine sediments throughout an annual cycle, indicating that the species composition of sediment bacterial communities may be more stable than recent studies suggest for marine planktonic bacterial communities. Laboratory studies designed to investigate the possible functional roles of Streptomyces populations in coastal sediments demonstrated that population levels of this genus changed relatively rapidly (within a time frame of 6 weeks) in response to manipulation of substrate availability. Amendments of intact sediment cores with two compounds (vanillic acid and succinic acid) consistently resulted in Streptomyces populations contributing an increased percentage of rRNA (6 to 15%) to the total bacterial rRNA pool. PMID:7487005

Moran, M A; Rutherford, L T; Hodson, R E

1995-01-01

91

Automated Purification and Suspension Array Detection of 16S rRNA from Soil and Sediment Extracts Using Tunable Surface Microparticles  

SciTech Connect

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA (~104 cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA (~105 cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system.

Chandler, Darrell P.; Jarrell, Ann E.

2004-05-01

92

Automated Purification and Suspension Array Detection of 16S rRNA from Soil and Sediment Extracts by Using Tunable Surface Microparticles  

PubMed Central

Autonomous, field-deployable molecular detection systems require seamless integration of complex biochemical solutions and physical or mechanical processing steps. In an attempt to simplify the fluidic requirements for integrated biodetection systems, we used tunable surface microparticles both as an rRNA affinity purification resin in a renewable microcolumn sample preparation system and as the sensor surface in a flow cytometer detector. The tunable surface detection limits in both low- and high-salt buffers were 1 ng of total RNA (?104 cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA (?105 cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system. PMID:15128511

Chandler, Darrell P.; Jarrell, Ann E.

2004-01-01

93

Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis  

E-print Network

constituent of bacterial ribosomes present at high copy numbers (103 -104 per actively growing cell).3,4 Targeting these biomarkers can potentially increase assay sensitivity compared with the assays targeting centrifugation steps, buffer exchange(s), lysing, and associated multistep processes of nucleic acid extraction

Santiago, Juan G.

94

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

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

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

2014-08-01

95

Extraction, purification and identification of bacterial signal molecules based on N-acyl homoserine lactones  

PubMed Central

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

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

2011-01-01

96

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

PubMed Central

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

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

2013-01-01

97

Use of bioluminescent bacterial sensors as an alternative method for measuring heavy metals in soil extracts  

Microsoft Academic Search

The luminescence based bacterial sensor strains Pseudomonas fluorescens OS8 (pTPT11) for mercury detection and Pseudomonas fluorescens OS8 (pTPT31) for arsenite detection were used in testing their application in detecting heavy metals in soil extracts. Three different soil types (humus, mineral and clay) were spiked with 1, 100 or 500?gg?1 Hg2+ or As3+. Samples were taken 1, 14 and 30 days

Tiina Petänen; Martin Romantschuk

2002-01-01

98

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)

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.

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

99

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

PubMed

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

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

2014-08-01

100

Phylogenetic clustering of soil microbial communities by 16S rRNA but not 16S rRNA genes.  

PubMed

We evaluated phylogenetic clustering of bacterial and archaeal communities from redox-dynamic subtropical forest soils that were defined by 16S rRNA and rRNA gene sequences. We observed significant clustering for the RNA-based communities but not the DNA-based communities, as well as increasing clustering over time of the highly active taxa detected by only rRNA. PMID:22286992

DeAngelis, Kristen M; Firestone, Mary K

2012-04-01

101

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

NASA Astrophysics Data System (ADS)

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.

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

2010-12-01

102

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification.  

PubMed

Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial identification. Five bacterial strains (two Escherichia coli, two Bacillus spp. and one Brevibacillus laterosporus) were studied in this investigation. Axenically grown bacterial cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream that was sprayed into the ionization chamber of the ESI-MS. The spectra produced contained reproducible information, which was useful for discriminating between the bacteria. Tandem mass spectrometry was used to characterize further the peaks, and at least three classes of macromolecules, namely phospholipids, glycolipids, and proteins, were found to contribute most to the spectral information. Bacterial extracts stored under different conditions gave very similar mass spectra for each of the five bacterial strains, indicating that the extracts were stable even at room temperature for up to 24 h, with no loss of information content, which has obvious implications for automated high-throughput analysis. An analysis of the components of the extracting solvent mixture and their effects on the spectral information showed that acetonitrile contributes most significantly to the extraction process and hence to the information content of the spectra. PMID:11838015

Vaidyanathan, Seetharaman; Kell, Douglas B; Goodacre, Royston

2002-02-01

103

Improved detection of bacterial antigens by latex agglutination after rapid extraction from body fluids.  

PubMed Central

Nonspecific agglutination of antibody-coated latex particles, unrelated to the presence of specific bacterial antigens, is a major difficulty with commercial latex particle agglutination tests. Rheumatoid and other factors are known to interfere with latex tests. We studied the use of six chelating, reducing, and anticoagulatory reagents in a rapid extraction of antigen procedure to free heat-stable antigens of Haemophilus influenzae type b and group B streptococcus which had been added to human sera. We also screened sera for the incidence of nonspecific agglutination from the three following groups: 123 patients with positive serology tests, 112 hospitalized patients, and 87 blood donors. The rapid extraction of antigen procedure involved a 1:4 dilution of the sera with each of the six reagents, incubation at 100 degrees C for 3 min, and centrifugation at 13,000 X g for 5 min. Two commercial latex kits were tested (Bactigen and Wellcogen). Nonspecific agglutination was entirely eliminated by each of the six extraction reagents. Sera from 52% of the patients with positive serology tests, 29% of the hospitalized patients, and 28% of the blood donors showed nonspecific agglutination with Bactigen before extraction. Nonspecific agglutination was eliminated in all but one sample after the rapid extraction of antigen procedure. This simple, rapid extraction procedure eliminated nonspecific reactions in cerebrospinal fluids and amniotic fluids and reduced this problem in urines and sera with each commercial kit used on clinical specimens. PMID:6439735

Smith, L P; Hunter, K W; Hemming, V G; Fischer, G W

1984-01-01

104

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

SciTech Connect

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 communitie

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

2009-03-01

105

Phylogenetic Analysis of Rumen Bacteria by Comparative Sequence Analysis of Cloned 16S rRNA Genes ß  

Microsoft Academic Search

Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage\\/corn silage\\/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In

Marc F Whitford; Robert J Forster; Cheryl E Beard; Jianhua Gong; Ronald M Teather

1998-01-01

106

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.  

PubMed

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods. PMID:16621089

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-09-18

107

Inhibition of bacterial quorum sensing and biofilm formation by extracts of neotropical rainforest plants.  

PubMed

Bacterial biofilms are responsible for many persistent infections by many clinically relevant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilms are much more resistant to conventional antibiotics than their planktonic counterparts. Quorum sensing, an intercellular communication system, controls pathogenesis and biofilm formation in most bacterial species. Quorum sensing provides an important pharmacological target since its inhibition does not provide a selective pressure for resistance. In this study, we investigated the quorum sensing and biofilm inhibitory activities of 126 plant extracts from 71 species collected from neotropical rainforests in Costa Rica. Quorum sensing and biofilm interference were assessed using a modified disc diffusion bioassay with Chromobacterium violaceum ATCC 12,472 and a spectrophotometric bioassay with Pseudomonas aeruginosa PA14, respectively. Species with significant anti-quorum sensing and/or anti-biofilm activities belonged to the Meliaceae, Melastomataceae, Lepidobotryaceae, Sapindaceae, and Simaroubaceae families. IC50 values ranged from 45 to 266 µg/mL. Extracts of these active species could lead to future development of botanical treatments for biofilm-associated infections. PMID:24488718

Ta, Chieu Anh; Freundorfer, Marie; Mah, Thien-Fah; Otárola-Rojas, Marco; Garcia, Mario; Sanchez-Vindas, Pablo; Poveda, Luis; Maschek, J Alan; Baker, Bill J; Adonizio, Allison L; Downum, Kelsey; Durst, Tony; Arnason, John T

2014-03-01

108

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

PubMed Central

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 1500–2500 m), which constitute valuable candidates for further isolation and characterization of bioactive molecules. PMID:24305113

2013-01-01

109

Enzymatic degradation of trichloroethylene using enzyme extracts isolated from a bacterial consortium.  

PubMed

Degradation of trichloroethylene (TCE) using crude enzyme extracts from a bacterial consortium was examined for wastewater treatment. The effects of pH, chemical induction, and cofactor were investigated. Enzyme extracts showed an optimal activity (3.03 +/- 0.03 mg of TCE/[mg of protein x d]) at neutral pH (6.5-7.5). In an attempt to increase the production of effective enzymes for TCE degradation, chemical induction using both toluene and TCE in the growth of the bacterium consortium was conducted. Although the induction increased the overall production of protein by about fourfold, the activity of the extracts was only slightly improved (up to 3.40 mg of TCE/[mg of protein x d]), indicating that the induction did not specifically enhance the production of TCE-degrading enzymes. Interestingly, the addition of a cofactor (up to 0.02 mg/mL), NADH, led to an initial reaction rate of 5.30 +/- 0.05 mg of TCE/(mg of protein x d). This observation demonstrated that the availability of the cofactor played an important role in determining the overall degradation reaction rates. The observations with NADH were in agreement with the assumption that toluene monooxygenases (which are NADH dependent) are the key enzymes for the degradation reactions. PMID:15304768

El-Zahab, Bilal; Meza, Liliana; Cutright, Teresa; Wang, Ping

2004-06-01

110

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

PubMed

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

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

111

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

ERIC Educational Resources Information Center

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)

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

1986-01-01

112

Aminoglycoside resistance genes sgm and kgmB protect bacterial but not yeast small ribosomal subunits in vitro despite high conservation of the rRNA A-site  

PubMed Central

The aminoglycoside resistance genes sgm from Micromonospora zionensis and kgmB from Streptomyces tenebrarius were cloned into a yeast expression vector to test whether the encoded prokaryotic methylases can modify the 18S rRNA A-site and thus confer resistance to G-418. Despite the detectable presence of mRNAs in yeast cells, neither G-418-resistant yeast transformants nor positive western blot signals were obtained. Neither methylase was capable of methylating 40S subunits despite very high conservation of the antibiotic rRNA binding sites. However, the results provide novel insight into the action of Sgm by showing that it methylates the same site as KgmB, i.e. G1405 in 16S rRNA. PMID:18930134

Tomic, Tatjana Ilic; Moric, Ivana; Conn, Graeme L.; Vasiljevic, Branka

2008-01-01

113

Bacterial diversity and soil enzyme activity in diseased and disease free apple rhizosphere soils  

Microsoft Academic Search

A culture-independent survey of the bacterial diversity in rhizosphere soils of diseased (scab) and disease-free apple trees\\u000a was conducted to assess the role of bacteria in disease suppression. Community DNA was extracted from soil samples and amplified\\u000a by PCR using primers specific for bacterial 16S rRNA gene sequences. Clone libraries were constructed with the PCR products\\u000a and analysed based on

Veerubommu Shanmugam; Rajni Verma; Subramani Rajkumar; Digvijay Singh Naruka

114

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

PubMed Central

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 17 mm, 17 mm and 15 mm 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 16–10 mm 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 11–12.5 mm, The extracts of C. intybus and C. sativus were found to be effective with zone of inhibition 11–6 mm 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 (25 mcg). 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

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

2013-01-01

115

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

PubMed

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

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

2014-01-01

116

Using Bacterial Extract along with Differential Gene Expression in Acropora millepora Larvae to Decouple the Processes of Attachment and Metamorphosis  

PubMed Central

Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP), isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA) Neogoniolithon fosliei, induced coral larval metamorphosis (100%) with little or no attachment (0–2%). To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR) and employed 47 genes of interest (GOI), selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p<0.05) in gene expression profiles of 24 GOI, 12 hours post exposure. Fourteen of those GOI also presented differences in expression (p<0.05) following exposure to the threshold concentration of bacterial TBP-containing extract. The specificity of the bacterial TBP-containing extract to induce the metamorphic stage in A. millepora larvae without attachment, using a robust, low cost, accurate, ecologically relevant and highly reproducible RT-qPCR assay, allowed partially decoupling of the transcriptomic processes of attachment and metamorphosis. The bacterial TBP-containing extract provided a unique opportunity to monitor the regulation of genes exclusively involved in the process of metamorphosis, contrasting previous gene expression studies that utilized cues, such as crustose coralline algae, biofilms or with GLW-amide neuropeptides that stimulate the entire onset of larval metamorphosis and attachment. PMID:22655067

Siboni, Nachshon; Abrego, David; Seneca, Francois; Motti, Cherie A.; Andreakis, Nikos; Tebben, Jan; Blackall, Linda L.; Harder, Tilmann

2012-01-01

117

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

PubMed Central

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

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

2001-01-01

118

Identification of multiple physicochemical and structural properties associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts  

PubMed Central

Bacterial extracts are widely used to synthesize recombinant proteins. Vast data volumes have been accumulated in cell-free expression databases, covering a whole range of existing proteins. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with protein solubility and aggregation. In the present paper, an approach to identify the multiple physicochemical and structural properties of amino acid sequences associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts is presented. The method includes: (1) categorical assessment of expression data; (2) calculation and prediction of multiple properties of expressed sequences; (3) correlation of the individual properties with the expression scores; and (4) evaluation of statistical significance of the observed correlations. Using this method, a number of significant correlations between calculated and predicted properties of amino acid sequences and their propensity for soluble cell-free expression have been revealed. PMID:24999341

Tokmakov, Alexander A.

2014-01-01

119

Absence of mutagenic effects of a particular Symphytum officinale L. liquid extract in the bacterial reverse mutation assay.  

PubMed

Comfrey (Symphytum officinale L.) root is traditionally used for the topical treatment of contusions, strains and sprains. Besides allantoin and rosmarinic acid, which are discussed as pharmacologically active principles, the drug contains pyrrolizidine alkaloids (PAs) known for their hepatotoxic, carcinogenic and mutagenic properties. The topical herbal medicinal products Kytta-Salbe f and Kytta-Plasma f contain a PA-free liquid extract from comfrey root as active substance. The aim of this study was to demonstrate the absence of genotoxic effects of this special extract in the bacterial reverse mutation assay (Ames test). Briefly, comfrey root liquid extract was investigated for its ability to induce gene mutations in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation using the mammalian microsomal fraction S9 mix. Reference mutagens were used to check the validity of the experiments. Comfrey root fluid extract showed no biologically relevant increases in revertant colony numbers of any of the five tester strains, neither in the presence nor in the absence of metabolic activation. In conclusion, the comfrey root fluid extract contained in Kytta-Salbe f and Kytta-Plasma f was not mutagenic in the bacterial reverse mutation assay. PMID:19827020

Benedek, Birgit; Ziegler, Andreas; Ottersbach, Peter

2010-03-01

120

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

PubMed Central

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 4–76 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

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

2014-01-01

121

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA.  

PubMed

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms. PMID:11295460

Dees, P M.; Ghiorse, W C.

2001-04-01

122

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

Microsoft Academic Search

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

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

2007-01-01

123

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

PubMed Central

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 µg/mL with respect to the positive control (vincristine sulphate) which was 0.91 µg/mL. Among all studied extracts, Xanthium indicum displayed the highest cytotoxic effect with LC50 value of 8.447 µg/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

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

2013-01-01

124

Evaluation of the Effect of Green Tea Extract on Mouth Bacterial Activity in the Presence of Propylene Glycol  

PubMed Central

Background Compounds present in green tea have proved to inhibit the growth and activity of bacteria associated with infections. Objectives To assess the effects of green tea leaves extract in presence of propylene glycol on the aerobic mouth bacteria load. Materials and Methods Saliva of 25 volunteer girl students aging 20-25 years were selected and evaluated by a mouthwash sample containing 1% tannin, as the most effective antibacterial complex in green tea. Comparative studies were also conducted between green tea mouthwashes containing 1% tannin and a similar sample with 10% propylene glycol added during extraction. This comparison was applied for a chlorhexidine 0.2% sample as a chemical mouthwash brand, too. Results There was a meaningful difference between the green tea mouthwashes containing 10% propylene glycol and the simple green tea extract (P < 0.05). Significant difference was also seen between the herbal and chemical mouthwashes (P < 0.05). The extract 1% tannin containing 10% propylene glycol reduced the aerobic mouth bacterial load of the student salvia about 64 percent. The pH monotonousness in different days and temperatures approved the stability of tannin in liquid water medium. Conclusions Using green tea extract as a herbal mouthwash is safe and harmless specially for children and pregnant women. This result led us to suppose that green tea may prevent plaque formation on teeth, coming over halitosis due to mouth infection, too. These effects need to be approved in an in vivo trial as a second study. PMID:24624155

Moghbel, Abdolhossein; Farjzadeh, Ahmad; Aghel, Nasrin; Agheli, Homaun; Raisi, Nafiseh

2012-01-01

125

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

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

Mohit, Vani; Archambault, Philippe; Toupoint, Nicolas

2014-01-01

126

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

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

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

2014-01-01

127

Elucidating the Key Member of a Linuron-Mineralizing Bacterial Community by PCR and Reverse Transcription-PCR Denaturing Gradient Gel Electrophoresis 16S rRNA Gene Fingerprinting and Cultivation  

PubMed Central

A bacterial community from Danish agricultural soil was enriched with linuron [N-(3,4-dichlorophenyl)-N?-methoxy-N?-methylurea] as the sole carbon and nitrogen source. The community mineralized [ring-U-14C]linuron completely to 14CO2 and 14C-biomass. Denaturing gradient gel electrophoresis analysis and cultivation revealed that a Variovorax sp. was responsible for the mineralization activity. PMID:16000836

S?rensen, Sebastian R.; Rasmussen, Jim; Jacobsen, Carsten S.; Jacobsen, Ole S.; Juhler, Rene K.; Aamand, Jens

2005-01-01

128

Anti-viral and anti-bacterial activities of an extract of blackcurrants (Ribes nigrum L.).  

PubMed

The inhibitory effects of an extract of the blackcurrant (Ribes nigrum L.) against pathogens associated with oral, nasopharyngeal and upper respiratory infectious diseases; namely respiratory syncytial virus (RSV), influenza virus A and B (IFV-A and IFV-B), adenovirus (AdV), herpes simplex virus type 1, Haemophilus influenzae type B, Streptococcus pneumoniae and Streptococcus mutans, were investigated. Less than 1% concentration of extract of blackcurrant inhibited replication of RSV, IFV-A and -B and HSV-1 by over 50% and a 10% extract inhibited adsorption of these viruses onto the cell surface by over 95%. The effects on AdV were much less pronounced; the half minimal inhibitory concentration of AdV replication was 2.54 ± 0.26, and a 10% concentration of the extract inhibited AdV adsorption on the cell surface by 72.9 ± 3.4%. The antibacterial activities of the blackcurrant were evaluated based on its efficacy as a disinfectant. A 10% extract disinfected 99.8% of H. Influenzae type B and 78.9% of S. pneumoniae in 10 min, but had no demonstrable effect against S. mutans. The blackcurrant extract still showed antiviral and antibacterial activities after the pH had been made neutral with sodium hydroxide, suggesting that these activities are not the result of acidic reactions or of components precipitated at a neutral pH. These findings demonstrate the potential of blackcurrant extract as a functional food for oral care. PMID:22985050

Ikuta, Kazufumi; Hashimoto, Koichi; Kaneko, Hisatoshi; Mori, Shuichi; Ohashi, Kazutaka; Suzutani, Tatsuo

2012-12-01

129

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

PubMed Central

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 ?g 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

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

130

Inhibition of bacterial quorum sensing by extracts from aquatic fungi: first report from marine endophytes.  

PubMed

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 ?g 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

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

2014-01-01

131

Analysis of bacterial community diversity in anaerobic fluidized bed bioreactors treating 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) using 16S rRNA gene clone libraries.  

PubMed

Clone libraries were used to evaluate the effects of 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) on bacterial populations within three anaerobic bioreactors. Prior to the addition of DNAN and MNA greater than 69% of the clones in each reactor were identified as a single Desulfuromonales species. However, after 60 days of treatment the Desulfuromonales distribution decreased to less than 13% of the distribution and a clone identified as a Levilinea sp. became the dominant organism at greater than 27% of the clone distribution in each reactor suggesting the species may play an important roll in the reduction of DNAN and MNA. PMID:21566358

Arnett, Clint M; Rodriguez, Giselle; Maloney, Stephen W

2009-01-01

132

Estimation of the Abundance of an Uncultured Soil Bacterial Strain by a Competitive Quantitative PCR Method  

Microsoft Academic Search

Strain EA25 was identified in a clone library of bacterial 16S rRNA gene sequences that had been amplified from DNA extracted from soil collected in eastern Washington State. EA25 was subsequently shown to be related to members of the genera Planctomyces and Chlamydia and most closely related (93% similarity) to strain MC18, a strain identified in an Australian soil sample

SOO-YOUN LEE; JOHN BOLLINGER; DAVID BEZDICEK; ANDANDREW OGRAM

1996-01-01

133

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

SciTech Connect

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.

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

1987-01-01

134

Retrospective observational study to investigate Sinerga, a multifactorial nutritional product, and bacterial extracts in the prevention of recurrent respiratory infections in children.  

PubMed

In this retrospective observational clinical study, 167 children, aged 3 to 7 years, of both sexes, with a clinical history of recurrent respiratory infections, administered with bacterial extracts of first and second generation or Sinerga a nutritional product containing palmitoylethanolamide, bovine colostrum, phenylethylamine and the new generation of probiotic kluyveromyces FM B0399, were observed. The goal of the study was to compare the supplementation with Sinerga with the supplementation with bacterial extracts, for the effect on the frequency of episodes of respiratory infection that had resulted in a prescription for antibiotics. The study focused retrospectively on the months from March 2013 to November 2012. The results showed a greater reduction in the frequency of respiratory infections with antibiotic therapy in the group of children supplemented with Sinerga than in the group treated with bacterial extracts. In particular, it was observed that 49.3% of the children supplemented with Sinerga, against 5% of those supplemented with extracts, had no infectious episodes requiring the administration of an antibiotic. 100% of subjects supplemented with Sinerga have had no more than two episodes of respiratory infection, while this condition, in the cohort treated with bacterial extracts, was observed in only 51% of cases. PMID:25280039

Nigro, A; Nicastro, A; Trodella, R

2014-01-01

135

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  

Microsoft Academic Search

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

LUCA COCOLIN; MARISA MANZANO; CARLO CANTONI; GIUSEPPE COMI

2001-01-01

136

The anti-biofilm potential of pomegranate (Punica granatum L.) extract against human bacterial and fungal pathogens.  

PubMed

Infectious diseases caused by bacteria and fungi are the major cause of morbidity and mortality across the globe. Multi-drug resistance in these pathogens augments the complexity and severity of the diseases. Various studies have shown the role of biofilms in multi-drug resistance, where the pathogen resides inside a protective coat made of extracellular polymeric substances. Since biofilms directly influence the virulence and pathogenicity of a pathogen, it is optimal to employ a strategy that effectively inhibits the formation of biofilm. Pomegranate is a common food and is also used traditionally to treat various ailments. This study assessed the anti-biofilm activity of a methanolic extract of pomegranate against bacterial and fungal pathogens. Methanolic extract of pomegranate was shown to inhibit the formation of biofilms by Staphylococcus aureus, methicillin resistant S. aureus, Escherichia coli, and Candida albicans. Apart from inhibiting the formation of biofilm, pomegranate extract disrupted pre-formed biofilms and inhibited germ tube formation, a virulence trait, in C. albicans. Characterization of the methanolic extract of pomegranate revealed the presence of ellagic acid (2,3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione) as the major component. Ellagic acid is a bioactive tannin known for its antioxidant, anticancer, and anti-inflammatory properties. Further studies revealed the ability of ellagic acid to inhibit the growth of all species in suspension at higher concentrations (>75??g?ml(-1)) and biofilm formation at lower concentrations (<40??g?ml(-1)) which warrants further investigation of the potential of ellagic acid or peel powders of pomegranate for the treatment of human ailments. PMID:23906229

Bakkiyaraj, Dhamodharan; Nandhini, Janarthanam Rathna; Malathy, Balakumar; Pandian, Shunmugiah Karutha

2013-09-01

137

Inhibition of Bacterial Quorum Sensing-Regulated Behaviors by Tremella fuciformis Extract  

Microsoft Academic Search

Quorum sensing (QS), or the control of gene expression in response to cell density, is used by both gram-negative and gram-positive\\u000a bacteria to regulate a variety of physiological functions. Increasing evidence implies that certain eukaryotes produce QS-inhibitory\\u000a compounds. In this work, we tested Tremella fuciformis for their ability to inhibit QS-regulated behaviors. T. fuciformis fruiting bodies were dried and extracted

H. Zhu; S. J. Sun

2008-01-01

138

Bacterial chromosome extraction and isolation Christelle Prinz,a Jonas O. Tegenfeldt,a Robert H. Austin,a Edward C. Coxb and James  

E-print Network

Bacterial chromosome extraction and isolation Christelle Prinz,a Jonas O. Tegenfeldt,a Robert H in a microfabricated environment and trap the E. coli chromosome. We characterize the conditions needed for efficient cell chromosomes using the techniques of diffusive mixing and dielectrophoretic trapping. While

139

Evaluation of novel carbon nano-tube particles in the bacterial and viral DNA and RNA extraction from the clinical samples  

Microsoft Academic Search

Molecular techniques have become the most im- portant methods of detecting bacterial and viral pathogens. However, current genomic extraction methods are currently limited in term of automation. In this study, carbon nano-tube was used as the vector to trap DNA and RNA molecules. The capability of carbon nano-tube to trap DNA and RNA was evaluated using samples (TB and HBV

Nguyen KC; Vo DXA; Hoang HN; Ho LTT; Pham HV

2010-01-01

140

Office Space Bacterial Abundance and Diversity in Three Metropolitan Areas  

PubMed Central

People in developed countries spend approximately 90% of their lives indoors, yet we know little about the source and diversity of microbes in built environments. In this study, we combined culture-based cell counting and multiplexed pyrosequencing of environmental ribosomal RNA (rRNA) gene sequences to investigate office space bacterial diversity in three metropolitan areas. Five surfaces common to all offices were sampled using sterile double-tipped swabs, one tip for culturing and one for DNA extraction, in 30 different offices per city (90 offices, 450 total samples). 16S rRNA gene sequences were PCR amplified using bar-coded “universal” bacterial primers from 54 of the surfaces (18 per city) and pooled for pyrosequencing. A three-factorial Analysis of Variance (ANOVA) found significant differences in viable bacterial abundance between offices inhabited by men or women, among the various surface types, and among cities. Multiplex pyrosequencing identified more than 500 bacterial genera from 20 different bacterial divisions. The most abundant of these genera tended to be common inhabitants of human skin, nasal, oral or intestinal cavities. Other commonly occurring genera appeared to have environmental origins (e.g., soils). There were no significant differences in the bacterial diversity between offices inhabited by men or women or among surfaces, but the bacterial community diversity of the Tucson samples was clearly distinguishable from that of New York and San Francisco, which were indistinguishable. Overall, our comprehensive molecular analysis of office building microbial diversity shows the potential of these methods for studying patterns and origins of indoor bacterial contamination. “[H]umans move through a sea of microbial life that is seldom perceived except in the context of potential disease and decay.” – Feazel et al. (2009). PMID:22666400

Hewitt, Krissi M.; Gerba, Charles P.; Maxwell, Sheri L.; Kelley, Scott T.

2012-01-01

141

Development of an oligonucleotide probe targeting 16S rRNA and its application for detection and quantitation of the ruminal bacterium Synergistes jonesii in a mixed-population chemostat.  

PubMed Central

Radiolabelled and fluorescent-dye-conjugated oligonucleotide probes which targeted rRNA sequences were developed for the enumeration of the ruminal bacterium Synergistes jonesii 78-1 in mixed culture. Two probes were tested, and both were highly specific for the respective complementary sequences of the target organism. Individual cells of S. jonesii in pure and mixed cultures were clearly visualized in situ by hybridization with the fluorescent-dye-conjugated probe but could not be detected in natural samples. Therefore the radiolabelled probe was used to monitor the population of S. jonesii introduced into a chemostat which simulated the rumen ecosystem. The S. jonesii probe did not hybridize to RNA extracted from the culture prior to inoculation with the target organism. After inoculation, S. jonesii rRNA represented 4.5% of the total bacterial rRNA and then rapidly declined to < 0.2% before increasing to about 1% of the total bacterial rRNA during the following 3 weeks. This study demonstrates that rRNA-targeted probes could be used for tracking organisms introduced into the rumen ecosystem. Images PMID:7686002

McSweeney, C S; Mackie, R I; Odenyo, A A; Stahl, D A

1993-01-01

142

Tools for Characterizing Bacterial Protein Synthesis Inhibitors  

PubMed Central

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

Orelle, Cedric; 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

143

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

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

Ikenaga, Makoto; Sakai, Masao

2014-09-17

144

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

PubMed Central

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 80°C, 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

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

2013-01-01

145

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

PubMed Central

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

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

2014-01-01

146

Direct electrochemical interaction between a modified gold electrode and a bacterial membrane extract  

PubMed Central

A novel electrochemical approach is described for redox-active membrane proteins. A total membrane extract (in the form of vesicles) of Bacillus subtilis is tethered onto gold surfaces modified with cholesterol based thiols. The membrane vesicles remain intact on the surface and do not rupture or fuse to form a planar bilayer. Oxidation/reduction signals are obtained of the natural co-enzyme, menaquinone-7, located in the membrane. The membrane protein, succinate menaquinone oxidoreductase (SQR), remains in the vesicles and is able to reduce fumarate using menaquinone as mediator. The catalysis of the reverse reaction (oxidation of succinate), which is the natural catalytic function of SQR, is almost absent with menaquinone. However, adding the co-enzyme ubiquinone, which has a reduction potential that is about 0.2 V higher, restores the succinate oxidation activity. PMID:15697298

Jeuken, Lars J.C.; Connell, Simon D.; Nurnabi, Mohammed; O'Reilly, John; Henderson, Peter J.F.; Evans, Stephen D.; Bushby, Richard J.

2013-01-01

147

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

PubMed Central

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

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

2008-01-01

148

Detection of rifampin- and ciprofloxacin-resistant Mycobacterium tuberculosis by using species-specific assays for precursor rRNA.  

PubMed Central

rRNA precursor (pre-rRNA) molecules carry terminal stems which are removed during rRNA synthesis to form the mature rRNA subunits. Their abundance in bacterial cells can be markedly affected by antibiotics which directly or indirectly inhibit RNA synthesis. We evaluated the feasibility of rapidly detecting antibiotic-resistant Mycobacterium tuberculosis strains by measuring the effects of brief in vitro antibiotic exposure on mycobacterial pre-rRNA. By hybridizing extracted M. tuberculosis nucleic acid with radiolabeled nucleic acid probes specific for pre-16S rRNA stem sequences, we detected clear responses to rifampin and ciprofloxacin within 24 and 48 h, respectively, of exposure of cultured cells to these drugs. Detectable pre-rRNA was depleted in susceptible cells but remained abundant in resistant cells. In contrast, no measurable responses to isoniazid or ethambutol were observed. Probes for pre-rRNA were specific for the M. tuberculosis complex when tested against a panel of eight Mycobacterium species and 48 other bacteria. After 24 h of incubation with rifampin, resistant M. tuberculosis strains were detectable in a reverse transcriptase PCR assay for pre-rRNA with a calculated lower limit of sensitivity of approximately 10(2) cells. Susceptible cells were negative in this assay at over 500 times the calculated lower limit of sensitivity. This general approach may prove useful for rapidly testing the susceptibility of slowly growing Mycobacterium species to the rifamycin and fluoroquinolone drugs and, with possible modifications, to other drugs as well. PMID:8843282

Cangelosi, G A; Brabant, W H; Britschgi, T B; Wallis, C K

1996-01-01

149

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

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

Olajuyigbe, Olufunmiso O.; Coopoosamy, Roger M.

2014-01-01

150

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

Microsoft Academic Search

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\\u000a during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract\\u000a (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against\\u000a three Gram-positive bacteria

Christian Krawitz; Mobarak Abu Mraheil; Michael Stein; Can Imirzalioglu; Eugen Domann; Stephan Pleschka; Torsten Hain

2011-01-01

151

Barcoding marine nematodes: an improved set of nematode 18S rRNA primers to overcome eukaryotic co-interference  

Microsoft Academic Search

Nematodes form an important component of many benthic marine ecosystems and DNA barcoding approaches could provide an insight\\u000a into nematode community composition from different environments globally. We have amplified nematode 18S rRNA sequences using\\u000a standard nematode18S rRNA primers from environmental DNA extracted from intertidal sediment collected from New Jersey coast,\\u000a USA to test whether the published marine nematode 18S rRNA

Punyasloke BhaduryMelanie; Melanie C. Austen

2010-01-01

152

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

PubMed Central

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

2011-01-01

153

Multiple DNA extractions coupled with stable-isotope probing of anthracene-degrading bacteria in contaminated soil.  

PubMed

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 (13)C-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-(13)C]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 (13)C-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

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

2011-05-01

154

Inhibitory effects on bacterial growth and beta-ketoacyl-ACP reductase by different species of maple leaf extracts and tannic acid.  

PubMed

It is important to develop new antibiotics aimed at novel targets. The investigation found that the leaf extracts from five maples (Acer platanoides, Acer campestre, Acer rubrum, Acer saccharum and Acer truncatum Bunge collected in Denmark, Canada and China) and their component tannic acid displayed antibacterial ability against 24 standard bacteria strains with the minimum inhibitory concentration of 0.3-8.0 mg/mL. Unlike the standard antibiotic levofloxacin (LFX), these samples inhibited Gram-positive bacteria more effectively than they inhibited Gram-negative bacteria. These samples effectively inhibited two antidrug bacterial strains. The results show that these samples inhibit bacteria by a different mechanism from LFX. These samples potently inhibited b-ketoacyl-ACP reductase (FabG), which is an important enzyme in bacterial fatty acid synthesis. Tannic acid showed the strongest inhibition on FabG with a half inhibition concentration of 0.78 microM (0.81 microg/mL). Furthermore, tannic acid and two maple leaf extracts showed time-dependent irreversible inhibition of FabG. These three samples also exhibited better inhibition on bacteria. It is suggested that FabG is the antibacteria target of maple leaf extracts and tannic acid, and both reversible and irreversible inhibitions of FabG are important for the antibacterial effect. PMID:19444866

Wu, Dan; Wu, Xiao-Dong; You, Xue-Fu; Ma, Xiao-Feng; Tian, Wei-Xi

2010-01-01

155

Bioefficacy of larvicdial and pupicidal properties of Carica papaya (Caricaceae) leaf extract and bacterial insecticide, spinosad, against chikungunya vector, Aedes aegypti (Diptera: Culicidae).  

PubMed

The present study was carried out to establish the properties of Carica papaya leaf extract and bacterial insecticide, spinosad on larvicidal and pupicidal activity against the chikungunya vector, Aedes aegypti. The medicinal plants were collected from the area around Bharathiar University, Coimbatore, India. C. papaya leaf was washed with tap water and shade-dried at room temperature. An electrical blender powdered the dried plant materials (leaves). The powder (500 g) of the leaf was extracted with 1.5 l of organic solvents of methanol for 8 h using a Soxhlet apparatus and then filtered. The crude leaf extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal and pupicidal effects after 24 h of exposure; however, the highest larval and pupal mortality was found in the leaf extract of methanol C. papaya against the first- to fourth-instar larvae and pupae of values LC(50)?=?I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, and pupae was 440.65 ppm, respectively, and bacterial insecticide, spinosad against the first to fourth instar larvae and pupae of values LC(50)?=?I instar was 51.76 ppm, II instar was 61.87 ppm, III instar was 74.07 ppm, and IV instar was 82.18 ppm, and pupae was 93.44 ppm, respectively. Moreover, combined treatment of values of LC(50)?=?I instar was 55.77 ppm, II instar was 65.77 ppm, III instar was 76.36 ppm, and IV instar was 92.78 ppm, and pupae was 107.62 ppm, respectively. No mortality was observed in the control. The results that the leaves extract of C. papaya and bacterial insecticide, Spinosad is promising as good larvicidal and pupicidal properties of against chikungunya vector, A. aegypti. This is an ideal eco-friendly approach for the control of chikungunya vector, A. aegypti as target species of vector control programs. PMID:21750871

Kovendan, Kalimuthu; Murugan, Kadarkarai; Naresh Kumar, Arjunan; Vincent, Savariar; Hwang, Jiang-Shiou

2012-02-01

156

Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability  

PubMed Central

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi. PMID:11571146

Ranjard, L.; Poly, F.; Lata, J.-C.; Mougel, C.; Thioulouse, J.; Nazaret, S.

2001-01-01

157

Bacterial communities established in bauxite residues with different restoration histories.  

PubMed

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

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

2013-07-01

158

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

PubMed

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

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

1999-06-01

159

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

PubMed

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 (31°C) 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 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change. PMID:23106937

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

2012-12-01

160

Statistical optimization of medium composition for bacterial cellulose production by Gluconacetobacter hansenii UAC09 using coffee cherry husk extract--an agro-industry waste.  

PubMed

During the production of grape wine, the formation of thick leathery pellicle/bacterial cellulose (BC) at the airliquid interface was due to the bacterium, which was isolated and identified as Gluconacetobacter hansenii UAC09. Cultural conditions for bacterial cellulose production from G. hansenii UAC09 were optimized by central composite rotatable experimental design. To economize the BC production, coffee cherry husk (CCH) extract and corn steep liquor (CSL) were used as less expensive sources of carbon and nitrogen, respectively. CCH and CSL are byproducts from the coffee processing and starch processing industry, respectively. The interactions between pH (4.5- 8.5), CSL (2-10%), alcohol (0.5-2%), acetic acid (0.5- 2%), and water dilution rate to CCH ratio (1:1 to 1:5) were studied using response surface methodology. The optimum conditions for maximum BC production were pH (6.64), CSL (10%), alcohol (0.5%), acetic acid (1.13%), and water to CCH ratio (1:1). After 2 weeks of fermentation, the amount of BC produced was 6.24 g/l. This yield was comparable to the predicted value of 6.09 g/l. This is the first report on the optimization of the fermentation medium by RSM using CCH extract as the carbon source for BC production by G. hansenii UAC09. PMID:21791961

Rani, Mahadevaswamy Usha; Rastogi, Navin K; Appaiah, K A Anu

2011-07-01

161

Taxonomy of bacterial fish pathogens  

PubMed Central

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

2011-01-01

162

Direct 5S rRNA Assay for Monitoring Mixed-Culture Bioprocesses  

PubMed Central

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 (DGGE), which is used to separate 5S rRNA species extracted from collected biomass. Separation is based on the unique migration behavior of each 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 genera 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. PMID:16535333

Stoner, D. L.; Browning, C. K.; Bulmer, D. K.; Ward, T. E.; MacDonell, M. T.

1996-01-01

163

Chronic N-amended soils exhibit an altered bacterial community structure in Harvard Forest, MA, USA.  

PubMed

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

Turlapati, Swathi A; Minocha, Rakesh; Bhiravarasa, Premsai S; Tisa, Louis S; Thomas, William K; Minocha, Subhash C

2013-02-01

164

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

PubMed Central

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

2012-01-01

165

Evaluation and optimization of nucleic acid extraction methods for the molecular analysis of bacterial communities associated with corroded carbon steel  

Microsoft Academic Search

Different DNA and RNA extraction approaches were evaluated and protocols optimized on in situ corrosion products from carbon steel in marine environments. Protocols adapted from the PowerSoil DNA\\/RNA Isolation methods resulted in the best nucleic acid (NA) extraction performances (ie combining high NA yield, quality, purity, representativeness of microbial community and processing time efficiency). The PowerSoil RNA Isolation Kit was

Florence Marty; Jean-François Ghiglione; Sandrine Païssé; Hervé Gueuné; Laurent Quillet; Mark C. M. van Loosdrecht; Gerard Muyzer

2012-01-01

166

Characterization of Microbial Diversity by Determining Terminal Restriction Fragment Length Polymorphisms of Genes Encoding 16S rRNA  

Microsoft Academic Search

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5* end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction

WEN-TSO LIU; TERENCE L. MARSH; HANS CHENG; LARRY J. FORNEY

1997-01-01

167

Evaluation of 16S rRNA Gene-Based PCR Assays for Genus-Level Identification of Helicobacter Species?  

PubMed Central

The inclusivity, exclusivity, and detection limit of six 16S rRNA gene-based Helicobacter genus-specific PCR assays were examined. Five out of six assays were 100% inclusive, but the tests varied considerably in their exclusivity (9.1 to 95.5%). The clinical detection limit varied between 103 and 1 viable bacterial cell per reaction mixture. PMID:18337392

Moyaert, H.; Pasmans, F.; Ducatelle, R.; Haesebrouck, F.; Baele, M.

2008-01-01

168

Identification of Oral Peptostreptococcus Isolates by PCR-Restriction Fragment Length Polymorphism Analysis of 16S rRNA Genes  

PubMed Central

Oral Peptostreptococcus isolates tentatively identified by conventional microbiological culture methods were identified to the species level by a combination of PCR amplification of 16S rRNA genes and restriction enzyme analysis of the amplified products. This method is a reliable and rapid alternative to conventional methods for identification of these bacterial species. PMID:12958299

Riggio, Marcello P.; Lennon, Alan

2003-01-01

169

Target-Specific Capture Enhances Sensitivity of Electrochemical Detection of Bacterial Pathogens ? †  

PubMed Central

We report the concentration and purification of bacterial 16S rRNA by the use of a biotinylated DNA target-specific capture (TSC) probe. For both cultivated bacterial and urine specimens from urinary tract infection patients, TSC resulted in a 5- to 8-fold improvement in the sensitivity of bacterial detection in a 16S rRNA electrochemical sensor assay. PMID:21940468

Patel, Mayank; Gonzalez, Rodrigo; Halford, Colin; Lewinski, Michael A.; Landaw, Elliot M.; Churchill, Bernard M.; Haake, David A.

2011-01-01

170

Bacterial protein signals are associated with Crohn's disease  

PubMed Central

Objective No Crohn’s disease (CD) molecular maker has advanced to clinical use, and independent lines of evidence support a central role of the gut microbial community in CD. Here we explore the feasibility of extracting bacterial protein signals relevant to CD, by interrogating myriads of intestinal bacterial proteomes from a small number of patients and healthy controls. Design We first developed and validated a workflow—including extraction of microbial communities, two-dimensional difference gel electrophoresis (2D-DIGE), and LC-MS/MS—to discover protein signals from CD-associated gut microbial communities. Then we used selected reaction monitoring (SRM) to confirm a set of candidates. In parallel, we used 16S rRNA gene sequencing for an integrated analysis of gut ecosystem structure and functions. Results Our 2D-DIGE-based discovery approach revealed an imbalance of intestinal bacterial functions in CD. Many proteins, largely derived from Bacteroides species, were over-represented, while under-represented proteins were mostly from Firmicutes and some Prevotella members. Most overabundant proteins could be confirmed using SRM. They correspond to functions allowing opportunistic pathogens to colonise the mucus layers, breach the host barriers and invade the mucosae, which could still be aggravated by decreased host-derived pancreatic zymogen granule membrane protein GP2 in CD patients. Moreover, although the abundance of most protein groups reflected that of related bacterial populations, we found a specific independent regulation of bacteria-derived cell envelope proteins. Conclusions This study provides the first evidence that quantifiable bacterial protein signals are associated with CD, which can have a profound impact on future molecular diagnosis. PMID:24436141

Juste, Catherine; Kreil, David P; Beauvallet, Christian; Guillot, Alain; Vaca, Sebastian; Carapito, Christine; Mondot, Stanislas; Sykacek, Peter; Sokol, Harry; Blon, Florence; Lepercq, Pascale; Levenez, Florence; Valot, Benoit; Carre, Wilfrid; Loux, Valentin; Pons, Nicolas; David, Olivier; Schaeffer, Brigitte; Lepage, Patricia; Martin, Patrice; Monnet, Veronique; Seksik, Philippe; Beaugerie, Laurent; Ehrlich, S Dusko; Gibrat, Jean-Francois; Van Dorsselaer, Alain; Dore, Joel

2014-01-01

171

Analysis of a change in bacterial community in different environments with addition of chitin or chitosan.  

PubMed

The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands of the 31 species indicated that 20 species belonged to the division Proteobacteria, and that the genus Cellvibrio was apparently predominant among them (7/20). The 16S rRNA sequences of the 16 deduced species (16/31) showed less than 98% similarities to those of previously identified bacteria, indicating that the species were derived from unidentified bacteria. The total community DNAs extracted from bacterial cells adsorbed on the surface of flakes of chitin and chitosan placed in a river, a moat, or soil were subjected to PCR-DGGE to examine the extent of diversity of chitinolytic bacteria among different environments. The predominant species significantly differed between the chitin and chitosan placed in the river and moat, but not so much between those placed in the soil. The large difference between the diversities of the three bacterial communities indicated that a wide variety of bacteria including unidentified ones are involved in the degradation of chitin and chitosan in the above-mentioned natural environments. PMID:20347770

Sato, Kazuaki; Azama, Yasuhito; Nogawa, Masahiro; Taguchi, Goro; Shimosaka, Makoto

2010-05-01

172

Analysis of structure and composition of bacterial core communities in mature drinking water biofilms and bulk water of a citywide network in Germany.  

PubMed

The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity. PMID:22389373

Henne, Karsten; Kahlisch, Leila; Brettar, Ingrid; Höfle, Manfred G

2012-05-01

173

Analysis of Structure and Composition of Bacterial Core Communities in Mature Drinking Water Biofilms and Bulk Water of a Citywide Network in Germany  

PubMed Central

The bacterial core communities of bulk water and corresponding biofilms of a more than 20-year-old drinking water network were compared using 16S rRNA single-strand confirmation polymorphism (SSCP) fingerprints based on extracted DNA and RNA. The structure and composition of the bacterial core community in the bulk water was highly similar (>70%) across the city of Braunschweig, Germany, whereas all biofilm samples contained a unique community with no overlapping phylotypes from bulk water. Biofilm samples consisted mainly of Alphaproteobacteria (26% of all phylotypes), Gammaproteobacteria (11%), candidate division TM6 (11%), Chlamydiales (9%), and Betaproteobacteria (9%). The bulk water community consisted primarily of Bacteroidetes (25%), Betaproteobacteria (20%), Actinobacteria (16%), and Alphaproteobacteria (11%). All biofilm communities showed higher relative abundances of single phylotypes and a reduced richness compared to bulk water. Only biofilm communities sampled at nearby sampling points showed similar communities irrespective of support materials. In all of our bulk water studies, the community composition determined from 16S rRNA was completely different from the 16S rRNA gene-based community composition, whereas in biofilms both molecular fractions resulted in community compositions that were similar to each other. We hypothesize that a higher fraction of active bacterial phylotypes and a better protection from oxidative stress in drinking water biofilms are responsible for this higher similarity. PMID:22389373

Henne, Karsten; Kahlisch, Leila; Brettar, Ingrid

2012-01-01

174

Determining Fungi rRNA Copy Number by PCR  

PubMed Central

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 ?-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

Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

2013-01-01

175

Anti-bacterial activity of ethanolic extract of Indoneesiella echioides (L) nees. evaluated by the filter paper disc method.  

PubMed

The study was carried out to investigate the antibacterial activity of the ethanolic extracts of Indoneesiella echioides (L) Nees. was evaluated by the filter paper disc method. This method is based on the diffusion of an antibiotic from a filter paper disc through the solidified culture media of a Petri dish used for study. The growth of inoculated is inhibited entirely in a circular area "Zone around the filter" paper disc containing a solution of antibiotic and the plant extract. The microorganisms used were: 1. Staphylococcus aureus (Gram positive 2. Escherichia coli (Gram negative). The organisms were maintained on nutrient agar slants. These were tested using nutrient broth. One loop full of the respective cultures was taken in slants which were maintained below 40 degrees C were taken and inoculated in the broth and incubated at 37 degrees C for 24 hrs and were observed for the growth of the organism with naked eye for their turbid nature. It was compared with that of sterile broth. The presence of turbidity indicated growth and suitability of the culture for further work. PMID:19339219

Qadrie, Zulfkar Latief; Jacob, Beena; Anandan, R; Rajkapoor, B; Rahamathulla, Mohammed

2009-04-01

176

Deoxygenation alters bacterial diversity and community composition in the ocean’s largest oxygen minimum zone  

NASA Astrophysics Data System (ADS)

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.

Beman, J. Michael; Carolan, Molly T.

2013-10-01

177

Multispectral bacterial identification  

NASA Astrophysics Data System (ADS)

A multi spectral optical technique was developed to simultaneously classify individual bacterial cells within mixed populations. Multi spectral Bacterial Identification (mBID) combines innovations in microscopy with a software analysis program to measure and characterize the fluorescence signals from multiplexed 16S ribosomal RNA probes hybridized to populations of different bacteria. Software was developed to identify individual bacteria at the level of species within these mixed populations. TO test the feasibility of mBID, we examined the fluorescence emissions from a mixture of probes specific for individual species of known bacteria from the American Type Culture Collection. Currently, up to seven species can be detected simultaneously by fluorescence microscopy. An eighth signal was reserved for a universal probe to control for fluorescence intensity. mBID can also be used to identify uncultured microorganisms. We plan to couple this new multi spectral technology to existing identification technologies that utilize 16S rRNA sequence alignment. Using this integrated identification protocol, bacteria that may be associated with chronic conditions will be identified first by analyzing their 16S rDNA sequences and then by visualizing them with flourescent probes hybridized to their 16S rRNA in situ.

Tanner, Michael A.; Coleman, William J.; Everett, Christine L.; Robles, Steven J.; Dilworth, Michael R.; Yang, Mary M.; Youvan, Douglas C.

2000-04-01

178

Coamplification of Eukaryotic DNA with 16S rRNA Gene-Based PCR Primers: Possible Consequences for Population Fingerprinting of Complex Microbial Communities  

Microsoft Academic Search

The main aim of this study was to evaluate the specificity of three commonly used 16S rRNA gene-based polymerase chain reaction\\u000a (PCR) primer sets for bacterial community analysis of samples contaminated with eukaryotic DNA. The specificity of primer\\u000a sets targeting the V3, V3-V5, and V6-V8 hypervariable regions of the 16S rRNA gene was investigated in silico and by community fingerprinting

Geert Huys; Tom Vanhoutte; Marie Joossens; Amal S. Mahious; Evie De Brandt; Severine Vermeire; Jean Swings

2008-01-01

179

Recognition of Potentially Novel Human Disease-Associated Pathogens by Implementation of Systematic 16S rRNA Gene Sequencing in the Diagnostic Laboratory? †  

PubMed Central

Clinical isolates that are difficult to identify by conventional means form a valuable source of novel human pathogens. We report on a 5-year study based on systematic 16S rRNA gene sequence analysis. We found 60 previously unknown 16S rRNA sequences corresponding to potentially novel bacterial taxa. For 30 of 60 isolates, clinical relevance was evaluated; 18 of the 30 isolates analyzed were considered to be associated with human disease. PMID:20631113

Keller, Peter M.; Rampini, Silvana K.; Buchler, Andrea C.; Eich, Gerhard; Wanner, Roger M.; Speck, Roberto F.; Bottger, Erik C.; Bloemberg, Guido V.

2010-01-01

180

Application of SmartGene IDNS Software to Partial 16S rRNA Gene Sequences for a Diverse Group of Bacteria in a Clinical Laboratory  

Microsoft Academic Search

Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bac- terial clinical isolates was

Keith E. Simmon; Ann C. Croft; Cathy A. Petti

2006-01-01

181

Assessing the viability of bacterial species in drinking water by combined cellular and molecular analyses.  

PubMed

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

Kahlisch, Leila; Henne, Karsten; Gröbe, Lothar; Brettar, Ingrid; Höfle, Manfred G

2012-02-01

182

Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography  

PubMed Central

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification. PMID:16332863

Ginige, Maneesha P.; Keller, Jurg; Blackall, Linda L.

2005-01-01

183

The murine lung microbiome in relation to the intestinal and vaginal bacterial communities  

PubMed Central

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

2013-01-01

184

Molecular analysis of the bacterial drinking water community with respect to live/dead status.  

PubMed

The assessment of the physiological state of the bacteria in drinking water is a critical issue, especially with respect to the presence of pathogenic bacteria. Though molecular methods can provide insight into the taxonomic composition of the drinking water microflora, the question if a bacterial species is alive or dead still needs to be addressed. To distinguish live and dead bacteria at the taxonomic level, we combined three methods: i) a staining procedure indicating membrane-injured cells (using SYTO9 and Propidium Iodide) that is considered to distinguish between live and dead cells, ii) Fluorescence Activated Cell Sorting (FACS) of the membrane injured and intact bacteria, and iii) molecular analyses of the RNA extracted from the bacteria before and after sorting to analyse the bacterial community at the species level. By staining and FACS analysis the drinking water bacteria could be separated according to their different membrane integrities, and RNA could be extracted from the live and dead sorted bacterial fractions. 16S rRNA based fingerprints revealed a diverse bacterial community in the drinking water samples with the majority being represented by 31 identified phylotypes. Most of the phylotypes referenced belonged to the phyla Proteobacteria (Alpha-, Beta-, Gamma-), Cyanobacteria and Bacteroidetes, and were mostly related to freshwater bacteria. 90% of the total phylotypes could be recovered after FAC-Sorting; 32% of the phylotypes occurred only in the "live" sorted fraction, 21% only in the "dead" sorted fraction, and 46% occurred in both fractions. PMID:20057086

Kahlisch, L; Henne, K; Groebe, L; Draheim, J; Höfle, M G; Brettar, I

2010-01-01

185

CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence  

PubMed Central

Summary Mycobacterium tuberculosis is arguably the world’s most successful infectious agent due to its ability to control its own cell growth within the host. Bacterial growth rate is closely coupled to rRNA transcription, which in E. coli is regulated through DksA and (p)ppGpp. The mechanisms of rRNA transcriptional control in mycobacteria, which lack DksA, are undefined. Here we identify CarD as an essential mycobacterial protein that controls rRNA transcription. Loss of CarD is lethal for mycobacteria in culture and during infection of mice. CarD depletion leads to sensitivity to killing by oxidative stress, starvation, and DNA damage, accompanied by failure to reduce rRNA transcription. CarD can functionally replace DksA for stringent control of rRNA transcription, even though CarD associates with a distinct site on RNA polymerase. These findings highlight a new molecular mechanism for regulating rRNA transcription in mycobacteria that is critical for M. tuberculosis pathogenesis. PMID:19596241

Stallings, Christina L.; Stephanou, Nicolas C.; Chu, Linda; Hochschild, Ann; Nickels, Bryce E.; Glickman, Michael S.

2009-01-01

186

Ribosomal Database Project: data and tools for high throughput rRNA analysis  

PubMed Central

Ribosomal Database Project (RDP; http://rdp.cme.msu.edu/) provides the research community with aligned and annotated rRNA gene sequence data, along with tools to allow researchers to analyze their own rRNA gene sequences in the RDP framework. RDP data and tools are utilized in fields as diverse as human health, microbial ecology, environmental microbiology, nucleic acid chemistry, taxonomy and phylogenetics. In addition to aligned and annotated collections of bacterial and archaeal small subunit rRNA genes, RDP now includes a collection of fungal large subunit rRNA genes. RDP tools, including Classifier and Aligner, have been updated to work with this new fungal collection. The use of high-throughput sequencing to characterize environmental microbial populations has exploded in the past several years, and as sequence technologies have improved, the sizes of environmental datasets have increased. With release 11, RDP is providing an expanded set of tools to facilitate analysis of high-throughput data, including both single-stranded and paired-end reads. In addition, most tools are now available as open source packages for download and local use by researchers with high-volume needs or who would like to develop custom analysis pipelines. PMID:24288368

Cole, James R.; Wang, Qiong; Fish, Jordan A.; Chai, Benli; McGarrell, Donna M.; Sun, Yanni; Brown, C. Titus; Porras-Alfaro, Andrea; Kuske, Cheryl R.; Tiedje, James M.

2014-01-01

187

Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria  

PubMed Central

The gene encoding the small subunit rRNA serves as a prominent tool for the phylogenetic analysis and classification of Bacteria and Archaea owing to its high degree of conservation and its fundamental function in living organisms. Here we show that the 16S rRNA genes of not-yet-cultivated large sulfur bacteria, among them the largest known bacterium Thiomargarita namibiensis, regularly contain numerous self-splicing introns of variable length. The 16S rRNA genes can thus be enlarged to up to 3.5 kb. Remarkably, introns have never been identified in bacterial 16S rRNA genes before, although they are the most frequently sequenced genes today. This may be caused in part by a bias during the PCR amplification step that discriminates against longer homologs, as we show experimentally. Such length heterogeneity of 16S rRNA genes has so far never been considered when constructing 16S rRNA-based clone libraries, even though an elongation of rRNA genes due to intervening sequences has been reported previously. The detection of elongated 16S rRNA genes has profound implications for common methods in molecular ecology and may cause systematic biases in several techniques. In this study, catalyzed reporter deposition–fluorescence in situ hybridization on both ribosomes and rRNA precursor molecules as well as in vitro splicing experiments were performed and confirmed self-splicing of the introns. Accordingly, the introns do not inhibit the formation of functional ribosomes. PMID:22371583

Salman, Verena; Amann, Rudolf; Shub, David A.; Schulz-Vogt, Heide N.

2012-01-01

188

Bacteriocin from Bacillus subtilis as a novel drug against diabetic foot ulcer bacterial pathogens  

PubMed Central

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

Joseph, Baby; Dhas, Berlina; Hena, Vimalin; Raj, Justin

2013-01-01

189

Identification of oral bacterial DNA in synovial fluid of arthritis patients with native and failed prosthetic joints  

PubMed Central

Objective We examined the presence of bacterial DNA in synovial fluids of native or aseptically failed prosthetic joints from patients having periodontal disease and arthritis to determine if there is bacterial spread from the oral cavity to the joints. Methods A total of 36 subjects were enrolled in the study. Among these, 11 were diagnosed with rheumatoid arthritis (RA), and 25 with osteoarthritis (OA). Eight patients with OA and are with RA had failed prostheses. Synovial fluid was aspirated from the affected hip or knee joint. Pooled subgingival plaque samples were collected followed by clinical periodontal examination. Bacterial DNA was extracted from the collected synovial fluid and dental plaque samples followed by polymerase chain reactions (PCR) and DNA sequence analysis of the 16S-23S rRNA genes. Results Of the 36 subjects, bacterial DNA was detected in the synovial fluid samples from five patients (13.9%), two with rheumatoid arthritis (one native and one failed prosthetic joints) and three with osteoarthritis (one native and two failed prosthetic joints). Of these five patients, two were diagnosed with periodontitis and had identical bacterial clones (Fusobacterium nucleatum and Serratia proteamaculans, respectively) detected in both the synovial fluid and dental plaque samples. Conclusions The present findings of this bacterial DNA in synovial fluid suggest the possibility of infection translocating from the periodontal tissue to the synovium. We suggest that patients with arthritis or failed prosthetic joints be examined for the presence of periodontal diseases and that be treated accordingly. PMID:22426587

Temoin, Stephanie; Chakaki, Alia; Askari, Ali; El-Halaby, Ahmed; Fitzgerald, Steven; Marcus, Randall E.; Han, Yiping W.; Bissada, Nabil F.

2013-01-01

190

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

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

Danielsson, Rebecca; Werner-Omazic, Anna; Ramin, Mohammad; Schnürer, Anna; Griinari, Mikko; Dicksved, Johan; Bertilsson, Jan

2014-09-01

191

Improved resolution of bacteria by high throughput sequence analysis of the rRNA internal transcribed spacer.  

PubMed

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

Ruegger, Paul M; Clark, Robin T; Weger, John R; Braun, Jonathan; Borneman, James

2014-10-01

192

Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability  

Microsoft Academic Search

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fun- gal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal tran- scribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified

L. Ranjard; F. Poly; J.-C. Lata; C. Mougel; J. Thioulouse; S. Nazaret

2001-01-01

193

Automated Identification of Medically Important Bacteria by 16S rRNA Gene Sequencing Using a Novel Comprehensive Database, 16SpathDB?  

PubMed Central

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

Woo, Patrick C. Y.; Teng, Jade L. L.; Yeung, Juilian M. Y.; Tse, Herman; Lau, Susanna K. P.; Yuen, Kwok-Yung

2011-01-01

194

The phenolic acids from bacterial degradation of the mangiferin aglycone are quantified in the feces of pigs after oral ingestion of an extract of Cyclopia genistoides (honeybush tea)  

Microsoft Academic Search

Polyphenols are cleaved by bacterial enzymes to form phenolic acid metabolites in the colon, where they may exert physiologic effects. For norathyriol, the aglycone of mangiferin, one of the major phenolic compounds present in Cyclopia genistoides (honeybush), a further bacterial degradation is likely; but knowledge of the importance of this metabolic process is very limited. Based on a hypothesized cleavage

Constance Bock; Waldemar Ternes

2010-01-01

195

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 ...

196

16S rRNA Terminal Restriction Fragment Length Polymorphism for the Characterization of the Nasopharyngeal Microbiota  

PubMed Central

A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs. PMID:23284951

Brugger, Silvio D.; Frei, Laurence; Frey, Pascal M.; Aebi, Suzanne; Muhlemann, Kathrin; Hilty, Markus

2012-01-01

197

16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.  

PubMed

A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs. PMID:23284951

Brugger, Silvio D; Frei, Laurence; Frey, Pascal M; Aebi, Suzanne; Mühlemann, Kathrin; Hilty, Markus

2012-01-01

198

Bacterial conjunctivitis  

PubMed Central

Clinical question What is the best treatment for bacterial conjunctivitis? Results Topical antibiotics expedite recovery from bacterial conjunctivitis. The choice of antibiotic usually does not affect outcome. Implementation Recognition of key distinguishing features of bacterial conjunctivitis Pitfalls that can be recognized in the history and physical examinationChoice of antibioticWhen to refer for specialist treatment. PMID:21188158

Hutnik, Cindy; Mohammad-Shahi, Mohammad H

2010-01-01

199

Dynamics of bacterial and fungal communities associated with eggshells during incubation.  

PubMed

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

Grizard, Stéphanie; Dini-Andreote, Francisco; Tieleman, B Irene; Salles, Joana F

2014-04-01

200

Dynamics of bacterial and fungal communities associated with eggshells during incubation  

PubMed Central

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

Grizard, Stephanie; Dini-Andreote, Francisco; Tieleman, B Irene; Salles, Joana F

2014-01-01

201

Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species  

Microsoft Academic Search

To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify

Lisa R. Fogarty; Mary A. Voytek

2005-01-01

202

Determinants of Acidobacteria activity inferred from the relative abundances of 16S rRNA transcripts in German grassland and forest soils.  

PubMed

16S rRNA genes and transcripts of Acidobacteria were investigated in 57 grassland and forest soils of three different geographic regions. Acidobacteria contributed 9-31% of bacterial 16S rRNA genes whereas the relative abundances of the respective transcripts were 4-16%. The specific cellular 16S rRNA content (determined as molar ratio of rRNA : rRNA genes) ranged between 3 and 80, indicating a low in situ growth rate. Correlations with flagellate numbers, vascular plant diversity and soil respiration suggest that biotic interactions are important determinants of Acidobacteria 16S rRNA transcript abundances in soils. While the phylogenetic composition of Acidobacteria differed significantly between grassland and forest soils, high throughput denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism fingerprinting detected 16S rRNA transcripts of most phylotypes in situ. Partial least squares regression suggested that chemical soil conditions such as pH, total nitrogen, C : N ratio, ammonia concentrations and total phosphorus affect the composition of this active fraction of Acidobacteria. Transcript abundance for individual Acidobacteria phylotypes was found to correlate with particular physicochemical (pH, temperature, nitrogen or phosphorus) and, most notably, biological parameters (respiration rates, abundances of ciliates or amoebae, vascular plant diversity), providing culture-independent evidence for a distinct niche specialization of different Acidobacteria even from the same subdivision. PMID:23802854

Foesel, Bärbel U; Nägele, Verena; Naether, Astrid; Wüst, Pia K; Weinert, Jan; Bonkowski, Michael; Lohaus, Gertrud; Polle, Andrea; Alt, Fabian; Oelmann, Yvonne; Fischer, Markus; Friedrich, Michael W; Overmann, Jörg

2014-03-01

203

5 S rRNA: structure and interactions.  

PubMed Central

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

Szymanski, Maciej; Barciszewska, Miroslawa Z; Erdmann, Volker A; Barciszewski, Jan

2003-01-01

204

Bacterial communities in PAH contaminated soils at an electronic-waste processing center in China  

Microsoft Academic Search

Surface soils from Guiyu, China (an intense e-waste processing center) were analyzed for persistent organic pollutants (POPs)\\u000a and variations in composition of the resident bacterial communities. Denaturing Gradient Gel Electrophoresis analysis of bacterial\\u000a 16S rRNA gene showed that e-waste pollution altered the bacterial community structure by promoting changes in species composition\\u000a and species richness. Bacterial diversity was not decreased at

Wen Zhang; Hui Wang; Rui Zhang; Xie-Zhi Yu; Pei-Yuan Qian; M. H. Wong

2010-01-01

205

Bacterial Overgrowth  

Microsoft Academic Search

\\u000a The human gastrointestinal tract typically contains 300–500 bacterial species. Most bacterial species are acquired during\\u000a the birth process and although some changes to the flora may occur during later stages of life, the composition of the intestinal\\u000a microflora remains relatively constant. Small bowel bacterial overgrowth (SBBO) is defined as an excessive increase in the\\u000a number of bacteria in the upper

Rosemary J. Young; Jon A. Vanderhoof

206

Investigation of an Acetate-Fed Denitrifying Microbial Community by Stable Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescent In Situ Hybridization-Microautoradiography  

Microsoft Academic Search

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). ( 13 C)acetate was used in SIP to label the DNA of the denitrifiers. The ( 13 C)DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13 C library

Maneesha P. Ginige; Jurg Keller; Linda L. Blackall

2005-01-01

207

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds  

PubMed Central

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

Adewumi, Gbenga A.; Oguntoyinbo, Folarin A.; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2013-01-01

208

Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.  

PubMed

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

Adewumi, Gbenga A; Oguntoyinbo, Folarin A; Keisam, Santosh; Romi, Wahengbam; Jeyaram, Kumaraswamy

2012-01-01

209

Characterisation of the bacterial community in expressed prostatic secretions from patients with chronic prostatitis/chronic pelvic pain syndrome and infertile men: a preliminary investigation  

PubMed Central

The expressed prostatic secretions (EPSs) of men with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), infertile men and normal men were subjected to microbiological study. EPSs were collected from the subjects, which included 26 normal men, 11 infertile patients and 51 CP/CPPS patients. DNA was extracted from each specimen, and the V3 regions of the 16S rRNA genes were amplified using universal bacterial primers. The results showed that the EPS 16S rRNA gene-positive rate in the CP/CPPS and infertile patients was much higher than in the normal men, but without any difference among the three patient groups. The denaturing gradient gel electrophoresis (DGGE) method was used to characterize the EPS bacterial community structure of the prostate fluid from patients with CP/CPPS or infertility issues. Principal component analysis (PCA) and partial least squares (PLS) analyses of PCR-DGGE profiles revealed that the EPS bacterial community structure differed among the three groups. Three bands were identified as the key factors responsible for the discrepancy between CP/CPPS patients and infertile patients (P<0.05). Two bands were identified as priority factors in the discrepancy of category IIIA and category IIIB prostatitis patients (P<0.05). According to this research, the ecological balance of the prostate and low urethra tract, when considered as a microenvironment, might play an important role in the maintenance of a healthy male reproductive tract. PMID:22635162

Hou, Dong-Sheng; Long, Wen-Min; Shen, Jian; Zhao, Li-Ping; Pang, Xiao-Yan; Xu, Chen

2012-01-01

210

The Potential of Metatranscriptomics for Identifying Screening Targets for Bacterial Vaginosis  

PubMed Central

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:2.9–190.7)) 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

Twin, Jimmy; Bradshaw, Catriona S.; Garland, Suzanne M.; Fairley, Christopher K.; Fethers, Katherine; Tabrizi, Sepehr N.

2013-01-01

211

Bead array direct rRNA capture assay (rCapA) for amplification free speciation of Mycobacterium cultures.  

PubMed

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

de Ronde, Hans; González Alonso, Paula; van Soolingen, Dick; Klatser, Paul R; Anthony, Richard M

2012-01-01

212

Specific identification of Gallibacterium by a PCR using primers targeting the 16S rRNA and 23S rRNA genes.  

PubMed

Gallibacterium was recently established as a new genus including organisms previously reported as Pasteurella anatis, [Actinobacillus] salpingitidis and avian Pasteurella haemolytica-like organisms. The aim of the present study was to develop a PCR method allowing unambiguous identification of Gallibacterium. PCR primers positioned in the 16S rRNA (1133fgal) and 23S rRNA (114r) genes were defined and their specificity was subsequently tested on 122 strains. Twenty-five of the strains represented all of the presently available 15 phenotypic variants of Gallibacterium from different geographical locations, 22 other strains represented other poultry associated bacterial species or bacteria which could pose a differential diagnostic problem including members of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae, and finally 75 Gallibacterium field strains isolated from Mexican chicken egg-layers. Specific amplicons were generated in all 100 Gallibacterium strains tested, whereas none of the non-Gallibacterium strains tested positive. Correct identification was confirmed by hybridization with the Gallibacterium specific probe GAN850. Two internal amplification control strategies were successfully incorporated into the PCR assay, one based on amplification of the house-keeping gene rpoB (sharing target DNA) and another based on addition of trout DNA (foreign target DNA) and amplification with beta-actin specific primers. In conclusion, the described PCR assay enables specific identification of Gallibacterium and will thus stand as a strong alternative to the present diagnostic methods. PMID:17350770

Bojesen, Anders Miki; Vazquez, Maria Elena; Robles, Fransisco; Gonzalez, Carlos; Soriano, Edgardo V; Olsen, John Elmerdahl; Christensen, Henrik

2007-07-20

213

Bacterial amyloids.  

PubMed

Many bacteria can assemble functional amyloid fibers on their cell surface. The majority of bacterial amyloids contribute to biofilm or other community behaviors where cells interact with a surface or with another cell. Bacterial amyloids, like all functional amyloids, share structural and biochemical properties with disease-associated eukaryotic amyloids. The general ability of amyloids to bind amyloid-specific dyes, such as Congo red, and their resistance to denaturation have provided useful tools for scoring and quantifying bacterial amyloid formation. Here, we present basic approaches to study bacterial amyloids by focusing on the well-studied curli amyloid fibers expressed by Enterobacteriaceae. These methods exploit the specific tinctorial and biophysical properties of amyloids. The methods described here are straightforward and can be easily applied by any modern molecular biology lab for the study of other bacterial amyloids. PMID:22528099

Zhou, Yizhou; Blanco, Luz P; Smith, Daniel R; Chapman, Matthew R

2012-01-01

214

16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles  

PubMed Central

Background Sample storage conditions, extraction methods, PCR primers, and parameters are major factors that affect metagenomics analysis based on microbial 16S rRNA gene sequencing. Most published studies were limited to the comparison of only one or two types of these factors. Systematic multi-factor explorations are needed to evaluate the conditions that may impact validity of a microbiome analysis. This study was aimed to improve methodological options to facilitate the best technical approaches in the design of a microbiome study. Three readily available mock bacterial community materials and two commercial extraction techniques, Qiagen DNeasy and MO BIO PowerSoil DNA purification methods, were used to assess procedures for 16S ribosomal DNA amplification and pyrosequencing-based analysis. Primers were chosen for 16S rDNA quantitative PCR and amplification of region V3 to V1. Swabs spiked with mock bacterial community cells and clinical oropharyngeal swabs were incubated at respective temperatures of -80°C, -20°C, 4°C, and 37°C for 4 weeks, then extracted with the two methods, and subjected to pyrosequencing and taxonomic and statistical analyses to investigate microbiome profile stability. Results The bacterial compositions for the mock community DNA samples determined in this study were consistent with the projected levels and agreed with the literature. The quantitation accuracy of abundances for several genera was improved with changes made to the standard Human Microbiome Project (HMP) procedure. The data for the samples purified with DNeasy and PowerSoil methods were statistically distinct; however, both results were reproducible and in good agreement with each other. The temperature effect on storage stability was investigated by using mock community cells and showed that the microbial community profiles were altered with the increase in incubation temperature. However, this phenomenon was not detected when clinical oropharyngeal swabs were used in the experiment. Conclusions Mock community materials originated from the HMP study are valuable controls in developing 16S metagenomics analysis procedures. Long-term exposure to a high temperature may introduce variation into analysis for oropharyngeal swabs, suggestive of storage at 4°C or lower. The observed variations due to sample storage temperature are in a similar range as the intrapersonal variability among different clinical oropharyngeal swab samples. PMID:25228989

2014-01-01

215

Natural-abundance stable carbon isotopes of small-subunit ribosomal RNA (SSU rRNA) from Guaymas Basin (Mexico)  

NASA Astrophysics Data System (ADS)

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.

MacGregor, B. J.; Mendlovitz, H.; Albert, D.; Teske, A. P.

2012-12-01

216

The rhizosphere and PAH amendment mediate impacts on functional and structural bacterial diversity in sandy peat soil  

Microsoft Academic Search

To reveal the degradation capacity of bacteria in PAH polluted soil and rhizosphere we combined bacterial extradiol ring-cleavage dioxygenase and 16S rRNA analysis in Betula pubescens rhizoremediation. Characterisation of the functional bacterial community by RFLP revealed novel environmental dioxygenases, and their putative hosts were studied by 16S rRNA amplification. Plant rhizosphere and PAH amendment effects were detected by the RFLP\\/T-RFLP

Kim Yrjälä; Anna-Kaisa Keskinen; Marja-Leena Åkerman; Carola Fortelius; Timo P. Sipilä

2010-01-01

217

Species Identification and Profiling of Complex Microbial Communities Using Shotgun Illumina Sequencing of 16S rRNA Amplicon Sequences  

PubMed Central

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

Lay, Christophe; Ho, Eliza Xin Pei; Low, Louie; Hibberd, Martin Lloyd; Nagarajan, Niranjan

2013-01-01

218

In vivo evidence that TATA-binding protein/SL1 colocalizes with UBF and RNA polymerase I when rRNA synthesis is either active or inactive  

PubMed Central

Here we show that the TATA-binding protein (TBP) is localized in the nucleoplasm and in the nucleolus of mammalian cells, consistent with its known involvement in transcription by RNA polymerase I, II, and III. In the nucleolus of actively growing cells, TBP colocalizes with upstream binding factor (UBF) and RNA polymerase I at the sites of rRNA transcription. During mitosis, when rRNA synthesis is down-regulated, TBP colocalizes with TBP-associated factors for RNA polymerase I (TAF(I)s), UBF, and RNA polymerase I on the chromosomal regions containing the rRNA genes. Treatment of cells with a low concentration of actinomycin D inhibits rRNA synthesis and causes a redistribution of the rRNA genes that become concentrated in clusters at the periphery of the nucleolus. A similar redistribution was observed for the major components of the rRNA transcription machinery (i.e., TBP, TAF(I)s, UBF, and RNA polymerase I), which still colocalized with each other. Furthermore, anti-TBP antibodies are shown to coimmunoprecipitate TBP and TAF(I)63 in extracts prepared from untreated and actinomycin D- treated cells. Collectively, the data indicate that in vivo TBP/promoter selectivity factor, UBF, and RNA polymerase I remain associated with both active and inactive rRNA genes. PMID:8609157

1996-01-01

219

Evaluation of MolYsis™ Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing.  

PubMed

Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized Staphylococcus aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P<0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood. PMID:24503182

McCann, Chase D; Jordan, Jeanne A

2014-04-01

220

Bacterial Vaginosis  

MedlinePLUS

... Field Search Button Advanced Search NIAID Home Health & Research Topics Labs & Scientific Resources Funding About NIAID News & Events NIAID > Health & Research Topics > Bacterial Vaginosis Skip Website Tools Website Tools Print ...

221

Differentiation of Paenibacillus larvae subsp. larvae, the Cause of American Foulbrood of Honeybees, by Using PCR and Restriction Fragment Analysis of Genes Encoding 16S rRNA  

PubMed Central

A rapid procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American foulbrood (AFB) disease of honeybees (Apis mellifera L.), based on PCR and restriction fragment analysis of the 16S rRNA genes (rDNA) is described. Eighty-six bacterial strains belonging to 39 species of the genera Paenibacillus, Bacillus, Brevibacillus, and Virgibacillus were characterized. Amplified rDNA was digested with seven restriction endonucleases. The combined data from restriction analysis enabled us to distinguish 35 profiles. Cluster analysis revealed that P. larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens formed a group with about 90% similarity; however, the P. larvae subsp. larvae restriction fragment length polymorphism pattern produced by endonuclease HaeIII was found to be unique and distinguishable among other closely related bacteria. This pattern was associated with DNA extracted directly from honeybee brood samples showing positive AFB clinical signs that yielded the restriction profile characteristic of P. larvae subsp. larvae, while no amplification product was obtained from healthy larvae. The method described here is particularly useful because of the short time required to carry it out and because it allows the differentiation of P. larvae subsp. larvae-infected larvae from all other species found in apiarian sources. PMID:12089057

Alippi, Adriana M.; López, Ana Claudia; Aguilar, O. Mario

2002-01-01

222

Effects of pH on Chemical Stability and Deesterification of Fenoxaprop-ethyl by Purified Enzymes, Bacterial Extracts, and Soils  

Microsoft Academic Search

De-esterification is an initial step in the metabolism of certain herbicides, for example, fenoxaprop- ethyl ((()-ethyl 2-(4-((6-chloro-2-benzoxaolyl)oxy)phenoxy)propanoate) (FE). The ethyl-ester bond cleavage of FE to fenoxaprop acid (FA) by purified enzymes, crude bacterial enzyme preparations, and soils was investigated. In similar experiments fluorescein diacetate (FDA) was used as an alternative substrate. FE stability was pH sensitive in acidic buffered solutions;

Robert M. Zablotowicz; Robert E. Hoagland; William J. Staddon; Martin A. Locke

2000-01-01

223

Oligodeoxynucleotide probes for Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequences.  

PubMed Central

Deoxyoligonucleotide probes were constructed for the identification of Campylobacter fetus and Campylobacter hyointestinalis based on 16S rRNA sequence data. Probes were targeted to hypervariable regions of 16S rRNA. Specificity of oligonucleotide probes was tested in a colony blot assay with type strains of 15 Campylobacter and Arcobacter species as well as in a slot blot format using genomic DNA extracted from field strains of C. fetus and C. hyointestinalis. Two oligonucleotides were constructed for C. fetus that hybridized with equal specificity with each of 57 biochemically confirmed isolates of C. fetus but not with any other Campylobacter species. The C. hyointestinalis probe reacted with 47 of 48 biochemically confirmed field isolates of C. hyointestinalis. In Southern blot hybridization of BglII digests of genomic DNA, the respective probes reacted within three restriction fragments of either C. hyointestinalis (7.2, 8.2, and 10.1 kb) or C. fetus (7.0, 7.7, and 9.0 kb). This suggests multiple copies of genes encoding 16S rRNA. Images PMID:1723076

Wesley, I V; Wesley, R D; Cardella, M; Dewhirst, F E; Paster, B J

1991-01-01

224

Variations of bacterial 16S rDNA phylotypes prior to and after chlorination for drinking water production from two surface water treatment plants.  

PubMed

We examined the variations of bacterial populations in treated drinking water prior to and after the final chlorine disinfection step at two different surface water treatment plants. For this purpose, the bacterial communities present in treated water were sampled after granular activated carbon (GAC) filtration and chlorine disinfection from two drinking water treatment plants supplying the city of Paris (France). Samples were analyzed after genomic DNA extraction, polymerase chain reaction (PCR) amplification, cloning, and sequencing of a number of 16S ribosomal RNA (rRNA) genes. The 16S rDNA sequences were clustered into operational taxonomic units (OTUs) and the OTU abundance patterns were obtained for each sample. The observed differences suggest that the chlorine disinfection step markedly affects the bacterial community structure and composition present in GAC water. Members of the Alphaproteobacteria and Betaproteobacteria were found to be predominant in the GAC water samples after phylogenetic analyses of the OTUs. Following the chlorine disinfection step, numerous changes were observed, including decreased representation of Proteobacteria phylotypes. Our results indicate that the use of molecular methods to investigate changes in the abundance of certain bacterial groups following chlorine-based disinfection will aid in further understanding the bacterial ecology of drinking water treatment plants (DWTPs), particularly the disinfection step, as it constitutes the final barrier before drinking water distribution to the consumer's tap. PMID:19908076

Poitelon, Jean-Baptiste; Joyeux, Michel; Welté, Bénédicte; Duguet, Jean-Pierre; Prestel, Eric; DuBow, Michael S

2010-02-01

225

Assessment of bacterial diversity in selected Philippine fermented food products through PCR-DGGE.  

PubMed

The bacterial population in several Philippine fermented food preparations was assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of the 16S rRNA gene (16S rDNA). Genomic DNA was isolated directly from alamang (fermented shrimp paste), burong isda (fermented fish and rice), burong hipon (fermented shrimp and rice), burong mustasa (fermented mustard leaves), tuba (sugar cane wine), suka (vinegar) and sinamak (spiced vinegar) using one of two protocols, namely - MoBio DNA Extraction Kit procedure and a cetyltrimethylammonium bromide-based method. Samples recalcitrant to both methods underwent enrichment in three culture broths prior to DNA isolation. Isolated DNA was amplified using nested primer pairs targeting the bacterial 16S rDNA. PCR products were subjected to DGGE to elucidate the bacterial diversity in each fermented food. 16S rDNA sequence analyses revealed that lactic acid bacteria (LAB) and acetic acid bacteria (AAB) were dominant in the food samples. The LAB identified were Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus panis, Lactobacillus pontis and Weissella cibaria. Identified AAB were Acetobacter pomorum, Acetobacter ghanensis, Acetobacter orientalis, and Acetobacter pasteurianus. Among these, L. fermentum, L. plantarum and W. cibaria are established probiotic bacteria, while L. panis and L. pontis are potential probiotic bacteria. This finding would increase the appeal and significance of local fermented foods to consumers. Furthermore, the majority of the identified bacteria in the study have not been reported before in culture-dependent studies of similar food preparations. As such, some of the bacterial 16S rDNA obtained were cloned to have an initial partial bacterial 16S rDNA library for Philippine fermented foods. PMID:22146687

Dalmacio, L M M; Angeles, A K J; Larcia, L L H; Balolong, M P; Estacio, R C

2011-12-01

226

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis  

PubMed Central

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans. PMID:23719154

Wong, Adam C-N; Chaston, John M; Douglas, Angela E

2013-01-01

227

Longitudinal Changes in the Bacterial Community Composition of the Danube River: a Whole-River Approach  

Microsoft Academic Search

The Danube River is the second longest river in Europe, and its bacterial community composition has never been studied before over its entire length. In this study, bacterial community composition was determined by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified portions of the bacterial 16S rRNA gene from a total of 98 stations on the Danube River (73 stations)

Christian Winter; Thomas Hein; Gerhard Kavka; Robert L. Mach; Andreas H. Farnleitner

2007-01-01

228

[A unique marker of Pseudomonas from the first group of rRNA homology--selective sensitivity to the bacteriostatic action of barium ions].  

PubMed

On the basis of the study of the bacteriostatic action of chlorides and nitrates of barium and 24 other metals on 18 Pseudomonas species of all groups of rRNA homology and on 49 bacterial species belonging to 25 other genera, unique selective sensitivity to the bacteriostatic action of Ba2+ ions has been established in Pseudomonas of the first group of rRNA homology. The marker of barium sensitivity is in line with changes in the classification of Pseudomonas and is of interest for their further more precise systematization. The test for barium sensitivity of bacteria helps simplify the identification of Pseudomonas of the first group of rRNA homology and makes the identification more accurate. PMID:1282762

Sivolodski?, E P

1992-01-01

229

The Unique 16S rRNA Genes of Piezophiles Reflect both Phylogeny and Adaptation? †  

PubMed Central

In the ocean's most extreme depths, pressures of 70 to 110 megapascals prevent the growth of all but the most hyperpiezophilic (pressure-loving) organisms. The physiological adaptations required for growth under these conditions are considered to be substantial. Efforts to determine specific adaptations permitting growth at extreme pressures have thus far focused on relatively few ?-proteobacteria, in part due to the technical difficulties of obtaining piezophilic bacteria in pure culture. Here, we present the molecular phylogenies of several new piezophiles of widely differing geographic origins. Included are results from an analysis of the first deep-trench bacterial isolates recovered from the southern hemisphere (9.9-km depth) and of the first gram-positive piezophilic strains. These new data allowed both phylogenetic and structural 16S rRNA comparisons among deep-ocean trench piezophiles and closely related strains not adapted to high pressure. Our results suggest that (i) the Circumpolar Deep Water acts as repository for hyperpiezophiles and drives their dissemination to deep trenches in the Pacific Ocean and (ii) the occurrence of elongated helices in the 16S rRNA genes increases with the extent of adaptation to growth at elevated pressure. These helix changes are believed to improve ribosome function under deep-sea conditions. PMID:17158629

Lauro, Federico M.; Chastain, Roger A.; Blankenship, Lesley E.; Yayanos, A. Aristides; Bartlett, Douglas H.

2007-01-01

230

Bacterial glycoproteomics.  

PubMed

Glycosylated proteins are ubiquitous components of eukaryote cellular surfaces, where the glycan moieties are implicated in a wide range of cell-cell recognition events. Once thought to be restricted to eukaryotes, glycosylation is now being increasingly reported in prokaryotes. Many of these discoveries have grown from advances in analytical technologies and genome sequencing. This review highlights the capabilities of high-sensitivity mass spectrometry for carbohydrate structure determination of bacterial glycoproteins and the emergence of glycoproteomic strategies that have evolved from proteomics and genomics for the functional analysis of bacterial glycosylation. PMID:16735721

Hitchen, Paul G; Dell, Anne

2006-06-01

231

Origin of the eukaryotic nucleus determined by rate-invariant analysis of rRNA sequences.  

PubMed

The origin of the eukaryotic nucleus is difficult to reconstruct. Eukaryotic organelles (chloroplast, mitochondrion) are eubacterial endosymbionts, but the source of nuclear genes has been obscured by multiple nucleotide substitutions. Using evolutionary parsimony, a newly developed rate-invariant treeing algorithm, the eukaryotic ribosomal rRNA genes are shown to have evolved from the eocytes, a group of extremely thermophilic, sulphur-metabolizing, anucleate cells. The deepest bifurcation yet found separates the reconstructed tree into two taxonomic divisions. These are a proto-eukaryotic group (karyotes) and an essentially bacterial one (parkaryotes). Within the precision of the rooting procedure, the tree is not consistent with either the prokaryotic-eukaryotic or the archaebacterial-eubacterial-eukaryotic groupings. It implies that the last common ancestor of extant life, and the early ancestors of eukaryotes, probably lacked nuclei, metabolized sulphur and lived at near-boiling temperatures. PMID:3340165

Lake, J A

1988-01-14

232

Use of Pyrosequencing of 16S rRNA Fragments to Differentiate between Bacteria Responsible for Neonatal Sepsis  

PubMed Central

Infants admitted to neonatal intensive care units for suspicion of bacterial sepsis receive at least two broad-spectrum antibiotics for a minimum of 48 to 72 hours to cover both gram-positive and gram-negative organisms while awaiting blood culture results. On average, bacterial growth becomes detectable within 12 to 24 hours, with an additional 24 to 48 hours required for identification. We have previously described using a 16S rRNA PCR assay for screening neonatal blood for bacterial DNA. Combining PCR with DNA sequencing could prove a faster means of detecting bacteria than culture-based identification. If successful, antibiotic therapy could be appropriately tailored sooner, thus sparing infants the administration of unnecessary antibiotics. Our goal was to assess the potential of pyrosequencing to differentiate between bacteria commonly associated with neonatal sepsis. To begin, full-length sequencing of the 380-bp 16S rRNA amplicons from representative bacteria was conducted (ABI 3100) and several databases queried. These included Staphylococcus sp., Streptococcus sp., Listeria sp., and numerous gram-negative rods. The sequences from clinical isolates were identical to those present in the published databases for the same bacteria. As a result, an informative 15 bases within the 380-bp amplicon was targeted for pyrosequencing following enrichment culture and PCR amplification. A total of 643 bacterial isolates commonly associated with neonatal sepsis, and 15 PCR-positive, culture-positive neonatal whole blood samples were analyzed by pyrosequencing. Results of DNA sequencing and culture identification were compared. In summary, we were successful at using PCR and pyrosequencing together to accurately differentiate between highly diverse bacterial groups. PMID:15681481

Jordan, Jeanne A.; Butchko, Allyson R.; Beth Durso, Mary

2005-01-01

233

Identification of Thermophilic Bacterial Strains Producing Thermotolerant Hydrolytic Enzymes from Manure Compost  

Microsoft Academic Search

Ten thermophilic bacterial strains were isolated from manure compost. Phylogenetic analysis based on 16S rRNA genes and biochemical\\u000a characterization allowed identification of four different species belonging to four genera: Geobacillus thermodenitrificans, Bacillus smithii, Ureibacillus suwonensis and Aneurinibacillus thermoaerophilus. PCR-RFLP profiles of the 16S-ITS-23S rRNA region allowed us to distinguish two subgroups among the G. thermodenitrificans isolates. Isolates were screened for

David M. Charbonneau; Fatma Meddeb-Mouelhi; Maurice Boissinot; Marc Sirois; Marc Beauregard

234

Bacterial communities of surface and deep hydrocarbon-contaminated waters of the Deepwater Horizon oil spill  

Microsoft Academic Search

We performed a 16S rRNA gene sequencing survey of bacterial communities within oil-contaminated surface water, deep hydrocarbon plume water, and deep water samples above and below the plume to determine spatial and temporal patterns of oil-degrading bacteria growing in response to the Deepwater Horizon oil leak. In addition, we are reporting 16S rRNA sequencing results from time series incubation, enrichment

T. Yang; L. M. Nigro; L. McKay; K. Ziervogel; T. Gutierrez; A. Teske

2010-01-01

235

Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages  

Microsoft Academic Search

The phylogenetic diversity of macroaggregate-attached vs. free-living marine bacteria, co-occurring in the same water mass, was compared. Bacterial diversity and phylogcnetic identity were inferred by analyzing polymerase chain reaction (PCR) amplified, cloned ribosomal RNA (rRNA) genes. Ribosomal RNA genes from macroaggregatc-associated bacteria were fundamentally different from those of free-living bacterio- plankton. Most rRNA types recovered from the free-living bacterioplankton were

EDWARD F. DELONG; DIANA G. FRANKS; ALICE L. ALLDREDGE

1993-01-01

236

A scientific note on the lactic acid bacterial flora within the honeybee subspecies Apis mellifera (Buckfast), A. m. scutellata, A. m. mellifera,  

E-print Network

A scientific note on the lactic acid bacterial flora within the honeybee subspecies Apis mellifera-evolution / 16S rRNA genes It was discovered by Olofsson and Vásquez (2008) that a novel lactic acid bacteria

237

Optimization of a high-throughput CTAB-based protocol for the extraction of qPCR-grade DNA from rumen fluid, plant and bacterial pure cultures.  

PubMed

The quality and yield of extracted DNA are critical for the majority of downstream applications in molecular biology. Moreover, molecular techniques such as quantitative real-time PCR (qPCR) are becoming increasingly widespread; thus, validation and cross-laboratory comparison of data require standardization of upstream experimental procedures. DNA extraction methods depend on the type and size of starting material(s) used. As such, the extraction of template DNA is arguably the most significant variable when cross-comparing data from different laboratories. Here, we describe a reliable, inexpensive and rapid method of DNA purification that is equally applicable to small or large scale or high-throughput purification of DNA. The protocol relies on a CTAB-based buffer for cell lysis and further purification of DNA with phenol : chloroform : isoamyl alcohol. The protocol has been used successfully for DNA purification from rumen fluid and plant cells. Moreover, after slight alterations, the same protocol was used for large-scale extraction of DNA from pure cultures of Gram-positive and Gram-negative bacteria. The yield of the DNA obtained with this method exceeded that from the same samples using commercial kits, and the quality was confirmed by successful qPCR applications. PMID:22029887

Minas, Konstantinos; McEwan, Neil R; Newbold, Charles Jamie; Scott, Karen P

2011-12-01

238

Bacteroides isolated from four mammalian hosts lack host-specific 16S rRNA gene phylogeny and carbon and nitrogen utilization patterns.  

PubMed

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

Atherly, Todd; Ziemer, Cherie J

2014-04-01

239

Bacterial Mining  

Microsoft Academic Search

Bacterial mining (biomining) represents the use of microorganisms to leach out metals from ores or mine tailings (wastes), followed by the subsequent recovery of metals of interest from the leaching solution. This leaching of metals from ores is a natural process, which can be considerably accelerated by inducing and\\/or supporting the microbial activity of certain species with the ability to

I. G. Petrisor; I. Lazar; T. F. Yen

2007-01-01

240

Effect of Elevated Tropospheric Ozone on the Structure of Bacterial Communities Inhabiting the Rhizosphere of Herbaceous Plants Native to Germany†  

PubMed Central

Current elevated concentrations of ozone in the atmosphere, as they are observed during summer seasons, can cause severe effects on plant vegetation. This study was initiated to analyze whether ozone-stressed plants also transfer signals below ground and thereby alter the bacterial community composition in their rhizospheres. Herbaceous plants, native to Germany, with tolerance (Anthoxanthum odoratum, Achillea millefolium, Poa pratensis, Rumex acetosa, and Veronica chamaedrys) and sensitivity (Matricaria chamomilla, Sonchus asper, and Tanacetum vulgare) to ozone, raised in the greenhouse, were exposed in open-top chambers to two different ozone regimes, i.e., “summer stress” and a normal ozone background. DNA of bacterial cells from the rhizospheres was directly extracted, and partial sequences of the 16S rRNA genes were PCR amplified with primers targeting the following phylogenetic groups: Bacteria, ?-Proteobacteria, Actinobacteria, and Pseudomonas, respectively. The diversity of the amplified products was analyzed by genetic profiling based on single-strand conformation polymorphism (SSCP). Neither the tolerant nor the sensitive plants, the latter with visible above-ground damage, showed ozone-induced differences in any of the SSCP profiles, with the single exception of Actinobacteria-targeted profiles from S. asper. To increase the stress, S. asper was germinated and raised in the continuous presence of an elevated level of ozone. SSCP profiles with Bacteria-specific primers combined with gene probe hybridizations indicated an ozone-related increase in a Xanthomonas-related 16S rRNA gene and a decrease in the respective gene from the plant plastids. The fact that only this latter unrealistic scenario caused a detectable effect demonstrated that ozone stress has a surprisingly small effect on the structural diversity of the bacterial community in rhizospheres. PMID:16332747

Dohrmann, Anja B.; Tebbe, Christoph C.

2005-01-01

241

Effect of elevated tropospheric ozone on the structure of bacterial communities inhabiting the rhizosphere of herbaceous plants native to Germany.  

PubMed

Current elevated concentrations of ozone in the atmosphere, as they are observed during summer seasons, can cause severe effects on plant vegetation. This study was initiated to analyze whether ozone-stressed plants also transfer signals below ground and thereby alter the bacterial community composition in their rhizospheres. Herbaceous plants, native to Germany, with tolerance (Anthoxanthum odoratum, Achillea millefolium, Poa pratensis, Rumex acetosa, and Veronica chamaedrys) and sensitivity (Matricaria chamomilla, Sonchus asper, and Tanacetum vulgare) to ozone, raised in the greenhouse, were exposed in open-top chambers to two different ozone regimes, i.e., "summer stress" and a normal ozone background. DNA of bacterial cells from the rhizospheres was directly extracted, and partial sequences of the 16S rRNA genes were PCR amplified with primers targeting the following phylogenetic groups: Bacteria, alpha-Proteobacteria, Actinobacteria, and Pseudomonas, respectively. The diversity of the amplified products was analyzed by genetic profiling based on single-strand conformation polymorphism (SSCP). Neither the tolerant nor the sensitive plants, the latter with visible above-ground damage, showed ozone-induced differences in any of the SSCP profiles, with the single exception of Actinobacteria-targeted profiles from S. asper. To increase the stress, S. asper was germinated and raised in the continuous presence of an elevated level of ozone. SSCP profiles with Bacteria-specific primers combined with gene probe hybridizations indicated an ozone-related increase in a Xanthomonas-related 16S rRNA gene and a decrease in the respective gene from the plant plastids. The fact that only this latter unrealistic scenario caused a detectable effect demonstrated that ozone stress has a surprisingly small effect on the structural diversity of the bacterial community in rhizospheres. PMID:16332747

Dohrmann, Anja B; Tebbe, Christoph C

2005-12-01

242

Phylogenetic mapping of bacterial morphology  

NASA Technical Reports Server (NTRS)

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.

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

1998-01-01

243

Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes  

Microsoft Academic Search

To define and monitor the structure of microbial communities found in the human vagina, a cultivation- independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for

Marco J. L. Coolen; Eduard Post; Catherine C. Davis; Larry J. Forney

2005-01-01

244

Phylogenetic diversity of marine coastal picoplankton 16s rRNA genes cloned from the continental shelf off Cape Hatteras, North Carolina  

Microsoft Academic Search

The phylogenetic diversity of a continental-shelf picoplankton community was examined by analyzing 16s ribosomal RNA (rRNA) genes amplified from environmental DNA with bacterial-specific primers and the polymerase chain reaction (PCR). Picoplankton populations collected from the pycnocline (10 m) over the eastern continental shelf of the United States near Cape Hatteras, North Carolina, served as the source of bulk nucleic acids

Michael S. Rap; Paul F. Kemp; Stephen J. Giovannoni

245

Fast and Accurate Identification ofXenorhabdusandPhotorhabdus Species by Restriction Analysis of PCR-Amplified 16S rRNA Genes  

Microsoft Academic Search

Thirteen bacterial strains of Xenorhabdus and 14 strains of Photorhabdus originating from a wide range of geographical and nematode host sources were typed by analyzing 16S rRNA gene (rDNA) restriction patterns obtained after digestion of PCR-amplified 16S rDNAs. Eight tetrameric restriction endonucleases were exam- ined. A total of 17 genotypes were identified, forming two heterogeneous main clusters after analysis by

B. BRUNEL; A. GIVAUDAN; A. LANOIS; R. J. AKHURST

1997-01-01

246

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 ...

247

Control of rRNA transcription in Escherichia coli.  

PubMed Central

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

Condon, C; Squires, C; Squires, C L

1995-01-01

248

Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments.  

PubMed

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

Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

2006-01-01

249

Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments  

PubMed Central

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°C) and river water (14°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°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°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°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

Kimura, Hiroyuki; Sugihara, Maki; Kato, Kenji; Hanada, Satoshi

2006-01-01

250

In silico Approach to Study Adaptive Divergence in Nucleotide Composition of the 16S rRNA Gene Among Bacteria Thriving Under Different Temperature Regimes.  

PubMed

Abstract Bacteria exist in a wide range of habitats ranging from psychrophilic through mesophilic to thermophilic. These different habitats have distinct environmental restriction for their existence. These microorganisms evolve themselves to survive in a specific habitat through the phenotypic and genotypic changes. In the bacterial domain, in silico analysis of 16S rRNA gene sequences using Mega 5.2 software by computing nucleotide composition, and evaluating their significance by statistical analysis using analysis of variance through Statistical Package for the Social Sciences (SPSS) version 16.0, revealed the habitat-specific bias in the occurrence of four types of nucleosides (A, T, C, and G) in the 16S rRNA gene. This hypothesis is also supported by Duncan's multiple range significance test at p=0.05 and also by the clustering of bacterial species of the same habitat group in the neighbor-joining tree of 150 different bacterial species of different psychrophilic, mesophilic, and thermophilic habitats (50 from each). The results on the probability of substitution (transition and transversion) in 16S rRNA gene sequences suggest that there is a habitat-specific selection pressure that possibly happens at the level of replication and repair process that results in a decreasing frequency of occurrence of adenine and thymine in the order psychrophilic>mesophilic>thermophilic species, and in an increasing frequency of occurrence of cytosine and guanine in the order psychrophilic

Ram, Hari; Kumar, Alok; Thomas, Lebin; Singh, Ved Pal

2014-10-01

251

Phylogenetic analysis of the bacterial communities in marine sediments.  

PubMed

For the phylogenetic analysis of microbial communities present in environmental samples microbial DNA can be extracted from the sample, 16S rDNA can be amplified with suitable primers and the PCR, and clonal libraries can be constructed. We report a protocol that can be used for efficient cell lysis and recovery of DNA from marine sediments. Key steps in this procedure include the use of a bead mill homogenizer for matrix disruption and uniform cell lysis and then purification of the released DNA by agarose gel electrophoresis. For sediments collected from two sites in Puget Sound, over 96% of the cells present were lysed. Our method yields high-molecular-weight DNA that is suitable for molecular studies, including amplification of 16S rRNA genes. The DNA yield was 47 micrograms per g (dry weight) for sediments collected from creosote-contaminated Eagle Harbor, Wash. Primers were selected for the PCR amplification of (eu)bacterial 16S rDNA that contained linkers with unique 8-base restriction sites for directional cloning. Examination of 22 16S rDNA clones showed that the surficial sediments in Eagle Harbor contained a phylogenetically diverse population of organisms from the Bacteria domain (G. J. Olsen, C. R. Woese, and R. Overbeek, J. Bacteriol. 176:1-6, 1994) with members of six major lineages represented: alpha, delta, and gamma Proteobacteria; the gram-positive high G+C content subdivision; clostridia and related organisms; and planctomyces and related organisms. None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA database. The analysis of clonal representives in the first report using molecular techniques to determine the phylogenetic composition of the (eu)bacterial community present in coastal marine sediments. PMID:8899989

Gray, J P; Herwig, R P

1996-11-01

252

Atmospheric cloud water contains a diverse bacterial community  

SciTech Connect

Atmospheric cloud water contains an active microbial community which can impact climate, human health and ecosystem processes in terrestrial and aquatic systems. Most studies on the composition of microbial communities in clouds have been performed with orographic clouds that are typically in direct contact with the ground. We collected water samples from cumulus clouds above the upper U.S. Midwest. The cloud water was analyzed for the diversity of bacterial phylotypes by denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene amplicons. DGGE analyses of bacterial communities detected 17e21 bands per sample. Sequencing confirmed the presence of a diverse bacterial community; sequences from seven bacterial phyla were retrieved. Cloud water bacterial communities appeared to be dominated by members of the cyanobacteria, proteobacteria, actinobacteria and firmicutes.

Kourtev, P. S.; Hill, Kimberly A.; Shepson, Paul B.; Konopka, Allan

2011-06-15

253

Molecular phylogenetic analysis of bacterial community and characterization of Cr(VI) reducers from the sediments of Tantloi hot spring, India  

PubMed Central

Background A geothermal ecosystem located at Tantloi, India has been found to be an interesting habitat for microbes of diverse nature. However, the microbial diversity of this habitat is poorly explored. In this study, a detailed phylogenetic study has been carried out to understand the bacterial diversity of this habitat and to identify prospective metal reducers using culture independent approach. The bacterial diversity of the sediments, which contain undetectable levels of Cr(VI), was analysed with respect to chromium reduction and the strains highly resistant to and efficiently reducing chromium under aerobic conditions were isolated and characterized. Results 16S rRNA gene sequence analysis of Tantloi hot spring microbial community revealed a significant bacterial diversity represented by at least ten taxonomic divisions of Bacteria with clear predominance of Thermus. Similar sequence analysis of rRNA gene library clones derived from bacterial consortia enriched from sediments in presence of Cr(VI) revealed the abundance of the family Bacillaceae. Under aerobic conditions at 65°C, the consortia reduced 1 mM of Cr(VI) completely within 24 h and 5 mM in 6 days. A complete reduction of 1 mM Cr(VI) has been shown by five of our isolates within 36 h. 16S rRNA gene sequences of all the isolates showed high degree of similarity (97-99%) to Bacillaceae with ten of them being affiliated to Anoxybacillus. Crude extract as well as the soluble fraction from isolates TSB-1 and TSB-9 readily reduced Cr(VI); TSB-1 showed higher chromium reductase activity. Conclusion Most of the Tantloi Spring Bacterial (TSB) sequences analyzed in different taxonomic divisions could be related to representatives with known metabolic traits which indicated presence of organisms involved in redox processes of a variety of elements including iron, sulphur and chromium. Approximately 80% of the sequences obtained in this study represented novel phylotypes indicating the possibility of discovery of bacteria with biotechnologically important new biomolecules. Again, highly chromium-resistant and remarkably active Cr(VI)-reducing Anoxybacillus strains isolated in this study could serve as potential candidates for designing chromium bioremediation strategies at high temperatures and also at high chromium concentrations. PMID:25243065

2014-01-01

254

Characterization of rumen bacterial strains isolated from enrichments of rumen content in the presence of propolis.  

PubMed

Propolis presents many biological properties, including antibacterial activities, and has been proposed as an additive in ruminant nutrition. Twenty bacterial strains, previously isolated from enrichments of Brazilian cow rumen contents in the presence of different propolis extracts (LLOS), were characterized using phenotyping and 16S rRNA identification. Seven strains were assigned to Streptococcus sp., most likely S. bovis, and were all degrading starch. One amylolytic lactate-utilizing strain of Selenomonas ruminantium was also found. Two strains of Clostridium bifermentans were identified and showed proteolytic activity. Two strains were assigned to Mitsuokella jalaludinii and were saccharolytic. One strain belonged to a Bacillus species and seven strains were affiliated with Escherichia coli. All of the 20 strains were able to use many sugars, but none of them were able to degrade the polysaccharides carboxymethylcellulose and xylans. The effect of three propolis extracts (LLOS B1, C1 and C3) was tested on the in vitro growth of four representative isolates of S. bovis, E. coli, M. jalaludinii and C. bifermentans. The growth of S. bovis, E. coli and M. jalaludinii was not affected by the three propolis extracts at 1 mg ml(-1). C. bifermentans growth was completely inhibited at this LLOS concentration, but this bacterium was partially resistant at lower concentrations. LLOS C3, with the lower concentration of phenolic compounds, was a little less inhibitory than B1 and C1 on this strain. PMID:25172217

de Aguiar, Sílvia Cristina; Zeoula, Lucia Maria; do Prado, Odimari Pricila Pires; Arcuri, Pedro Braga; Forano, Evelyne

2014-11-01

255

Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions  

PubMed Central

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere. PMID:11010885

Normander, Bo; Prosser, Jim I.

2000-01-01

256

The bacterial community associated with the leech Myzobdella lugubris Leidy 1851 (Hirudinea: Piscicolidae) from Lake Erie, Michigan, USA.  

PubMed

Leeches are widespread in the Great Lakes Basin, yet their potential to harbor disease-causing agents has not been investigated. The purpose of this study was to identify the bacterial community of the commonly occurring leech, Myzobdella lugubris, within the Lake Erie Watershed. Leech samples were collected from the pectoral fins of channel catfish, Ictalurus punctatus, and freshwater drum, Aplodinotus grunniens, from Lake Erie in commercial trap nets and pooled into two samples based on host attachment. Bacteria from within the viscera of M. lugubris were identified by sequencing their 16S rRNA (rDNA) gene of amplified community bacterial DNA extracted from pooled leech homogenate samples and were checked for similarity in two public databases: the Ribosomal Database Project and BLAST. Bacteria belonging to the phylum Bacteroidetes, beta-proteobacteria, Verrucomicrobia, and unclassified Bacteria were present in the leech samples. A large number of bacteria found within leeches attached to channel catfish consisted of sequences that could not be classified beyond the Domain Bacteria. However, many of these sequences were homologous (< 45%) to the phylum Bacteroidetes. One of the five genera detected in the leech homogenates was Flavobacterium psychrophilum, a serious fish pathogen that causes Bacterial Cold Water Disease. While the occurrence of genera varies, bacteria associated with the two fish species were similar. PMID:20597437

Schulz, C; Faisal, M

2010-06-01

257

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

PubMed Central

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

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

2012-01-01

258

Field-Scale Transplantation Experiment To Investigate Structures of Soil Bacterial Communities at Pioneering Sites?†  

PubMed Central

Studies on the effect of environmental conditions on plants and microorganisms are a central issue in ecology, and they require an adequate experimental setup. A strategy often applied in geobotanical studies is based on the reciprocal transplantation of plant species at different sites. We adopted a similar approach as a field-based tool to investigate the relationships of soil bacterial communities with the environment. Soil samples from two different (calcareous and siliceous) unvegetated glacier forefields were reciprocally transplanted and incubated for 15 months between 2009 and 2010. Controls containing local soils were included. The sites were characterized over time in terms of geographical (bedrock, exposition, sunlight, temperature, and precipitation) and physicochemical (texture, water content, soluble and nutrients) features. The incubating local (“home”) and transplanted (“away”) soils were monitored for changes in extractable nutrients and in the bacterial community structure, defined through terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA gene. Concentrations of soluble ions in most samples were more significantly affected by seasons than by the transplantation. For example, NO3? showed a seasonal pattern, increasing from 1 to 3 ?g NO3? (g soil dry weight)?1 after the melting of snow but decreasing to <1 ?g NO3? (g soil dry weight)?1 in autumn. Seasons, and in particular strong precipitation events occurring in the summer of 2010 (200 to 300 mm of rain monthly), were also related to changes of bacterial community structures. Our results show the suitability of this approach to compare responses of bacterial communities to different environmental conditions directly in the field. PMID:21965395

Lazzaro, Anna; Gauer, Andreas; Zeyer, Josef

2011-01-01

259

Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars.  

PubMed

Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space. PMID:22729532

La Duc, Myron T; Vaishampayan, Parag; Nilsson, Henrik R; Torok, Tamas; Venkateswaran, Kasthuri

2012-08-01

260

Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective  

NASA Technical Reports Server (NTRS)

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).

Gutell, R. R.; Larsen, N.; Woese, C. R.

1994-01-01

261

Interactive Effects of Viral and Bacterial Production on Marine Bacterial Diversity  

PubMed Central

A general model of species diversity predicts that the latter is maximized when productivity and disturbance are balanced. Based on this model, we hypothesized that the response of bacterial diversity to the ratio of viral to bacterial production (VP/BP) would be dome-shaped. In order to test this hypothesis, we obtained data on changes in bacterial communities (determined by terminal restriction fragment length polymorphism of 16S rRNA gene) along a wide VP/BP gradient (more than two orders of magnitude), using seawater incubations from NW Mediterranean surface waters, i.e., control and treatments with additions of phosphate, viruses, or both. In December, one dominant Operational Taxonomic Unit accounted for the major fraction of total amplified DNA in the phosphate addition treatment (75±20%, ± S.D.), but its contribution was low in the phosphate and virus addition treatment (23±19%), indicating that viruses prevented the prevalence of taxa that were competitively superior in phosphate-replete conditions. In contrast, in February, the single taxon predominance in the community was held in the phosphate addition treatment even with addition of viruses. We observed statistically robust dome-shaped response patterns of bacterial diversity to VP/BP, with significantly high bacterial diversity at intermediate VP/BP. This was consistent with our model-based hypothesis, indicating that bacterial production and viral-induced mortality interactively affect bacterial diversity in seawater. PMID:24244268

Motegi, Chiaki; Nagata, Toshi; Miki, Takeshi; Weinbauer, Markus G.; Legendre, Louis; Rassoulzadegan, Fereidoun

2013-01-01

262

A bacterial enrichment study and overview of the extractable lipids from paleosols in the Dry Valleys, Antarctica: implications for future Mars reconnaissance.  

PubMed

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

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

263

Phylogeny of parasitiform mites (Acari) based on rRNA  

Microsoft Academic Search

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

Hans Klompen; Mariam Lekveishvili; William C. Black IV

2007-01-01

264

Immunologic properties of bacterial lipopolysaccharide (LPS). II. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS  

PubMed Central

The C3H/HeJ mouse strain, previously shown to be a nonresponder to bacterial lipopolysaccharide (LPS)-induced mitogenesis in vitro, was demonstrated by the present studies to be competent to respond mitogenically to LPS, but only to LPS preparations obtained by selected extraction methods. These preparations appear to be confined to LPS isolated by mild extraction techniques, such as TCA or butanol. In contrast, those obtained by techniques utilizing phenol were only weakly stimulatory or completely nonstimulatory for spleen cells from the C3H/HeJ. All LPS preparations tested, on the other hand, were highly stimulatory for cells from another mouse strain, namely the C3H/St. The critical importance of the method of extraction of LPS on its mitogenic activity for C3H/HeJ cells was stressed by experiments in which LPS was prepared from Escherichia coli K235 using either of two procedures. In these experiments, phenol-extracted LPS, although mitogenic in the C3H/St, was completely nonstimulatory in the C3H/HeJ; whereas, butanol-extracted LPS was highly stimulatory in both strains of mice. This striking difference was attributed to a destructive effect of phenol on LPS, as demonstrated by the fact that treatment of butanol LPS with phenol resulted in a total loss of its mitogenic activity in the C3H/HeJ, but in only a partial loss in the C3H/St. In general, the mitogenic response observed with selected LPS preparations in the C3H/HeJ was quantitatively lower and more transient than that seen with the C3H/St, although qualitatively these responses appeared to be similar. This was evidenced by the observation that in both mouse strains LPS was a specific mitogen for B cells, a property which was also attributed in both strains to the same distinct structural region of the LPS molecule, that is lipid A. A preparation of LPS that failed to stimulate B cells from the C3H/HeJ nonetheless had the capacity to block activation of these B cells by a stimulatory preparation of LPS. These results strongly suggest that mitogenic stimulation of B cells by LPS is a function of the structural integrity of both the LPS molecule and putative B-cell receptors for LPS. PMID:1104747

1975-01-01

265

Temporal dynamics of fibrolytic and methanogenic rumen microorganisms during in situ incubation of switchgrass determined by 16S rRNA gene profiling.  

PubMed

The rumen microbial ecosystem is known for its biomass-degrading and methane-producing phenotype. Fermentation of recalcitrant plant material, comprised of a multitude of interwoven fibers, necessitates the synergistic activity of diverse microbial taxonomic groups that inhabit the anaerobic rumen ecosystem. Although interspecies hydrogen (H2) transfer, a process during which bacterially generated H2 is transferred to methanogenic Archaea, has obtained significant attention over the last decades, the temporal variation of the different taxa involved in in situ biomass-degradation, H2 transfer and the methanogenesis process remains to be established. Here we investigated the temporal succession of microbial taxa and its effect on fiber composition during rumen incubation using 16S rRNA amplicon sequencing. Switchgrass filled nylon bags were placed in the rumen of a cannulated cow and collected at nine time points for DNA extraction and 16S pyrotag profiling. The microbial community colonizing the air-dried and non-incubated (0 h) switchgrass was dominated by members of the Bacilli (recruiting 63% of the pyrotag reads). During in situ incubation of the switchgrass, two major shifts in the community composition were observed: Bacilli were replaced within 30 min by members belonging to the Bacteroidia and Clostridia, which recruited 34 and 25% of the 16S rRNA reads generated, respectively. A second significant shift was observed after 16 h of rumen incubation, when members of the Spirochaetes and Fibrobacteria classes became more abundant in the fiber-adherent community. During the first 30 min of rumen incubation ~13% of the switchgrass dry matter was degraded, whereas little biomass degradation appeared to have occurred between 30 min and 4 h after the switchgrass was placed in the rumen. Interestingly, methanogenic members of the Euryarchaeota (i.e., Methanobacteria) increased up to 3-fold during this period of reduced biomass-degradation, with peak abundance just before rates of dry matter degradation increased again. We hypothesize that during this period microbial-mediated fibrolysis was temporarily inhibited until H2 was metabolized into CH4 by methanogens. Collectively, our results demonstrate the importance of inter-species interactions for the biomass-degrading and methane-producing phenotype of the rumen microbiome-both microbially facilitated processes with global significance. PMID:25101058

Piao, Hailan; Lachman, Medora; Malfatti, Stephanie; Sczyrba, Alexander; Knierim, Bernhard; Auer, Manfred; Tringe, Susannah G; Mackie, Roderick I; Yeoman, Carl J; Hess, Matthias

2014-01-01

266

Temporal dynamics of fibrolytic and methanogenic rumen microorganisms during in situ incubation of switchgrass determined by 16S rRNA gene profiling  

PubMed Central

The rumen microbial ecosystem is known for its biomass-degrading and methane-producing phenotype. Fermentation of recalcitrant plant material, comprised of a multitude of interwoven fibers, necessitates the synergistic activity of diverse microbial taxonomic groups that inhabit the anaerobic rumen ecosystem. Although interspecies hydrogen (H2) transfer, a process during which bacterially generated H2 is transferred to methanogenic Archaea, has obtained significant attention over the last decades, the temporal variation of the different taxa involved in in situ biomass-degradation, H2 transfer and the methanogenesis process remains to be established. Here we investigated the temporal succession of microbial taxa and its effect on fiber composition during rumen incubation using 16S rRNA amplicon sequencing. Switchgrass filled nylon bags were placed in the rumen of a cannulated cow and collected at nine time points for DNA extraction and 16S pyrotag profiling. The microbial community colonizing the air-dried and non-incubated (0 h) switchgrass was dominated by members of the Bacilli (recruiting 63% of the pyrotag reads). During in situ incubation of the switchgrass, two major shifts in the community composition were observed: Bacilli were replaced within 30 min by members belonging to the Bacteroidia and Clostridia, which recruited 34 and 25% of the 16S rRNA reads generated, respectively. A second significant shift was observed after 16 h of rumen incubation, when members of the Spirochaetes and Fibrobacteria classes became more abundant in the fiber-adherent community. During the first 30 min of rumen incubation ~13% of the switchgrass dry matter was degraded, whereas little biomass degradation appeared to have occurred between 30 min and 4 h after the switchgrass was placed in the rumen. Interestingly, methanogenic members of the Euryarchaeota (i.e., Methanobacteria) increased up to 3-fold during this period of reduced biomass-degradation, with peak abundance just before rates of dry matter degradation increased again. We hypothesize that during this period microbial-mediated fibrolysis was temporarily inhibited until H2 was metabolized into CH4 by methanogens. Collectively, our results demonstrate the importance of inter-species interactions for the biomass-degrading and methane-producing phenotype of the rumen microbiome—both microbially facilitated processes with global significance. PMID:25101058

Piao, Hailan; Lachman, Medora; Malfatti, Stephanie; Sczyrba, Alexander; Knierim, Bernhard; Auer, Manfred; Tringe, Susannah G.; Mackie, Roderick I.; Yeoman, Carl J.; Hess, Matthias

2014-01-01

267

RNA-Stable-Isotope Probing Shows Utilization of Carbon from Inulin by Specific Bacterial Populations in the Rat Large Bowel  

PubMed Central

Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect 13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the 13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified. B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism. PMID:24487527

Lawley, Blair; Munro, Karen; Sims, Ian M.; Lee, Julian; Butts, Christine A.; Roy, Nicole

2014-01-01

268

Deoxygenation alters bacterial diversity and community composition in the ocean's largest oxygen minimum zone.  

PubMed

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. PMID:24162368

Beman, J Michael; Carolan, Molly T

2013-01-01

269

Bacterial composition of soils of the Lake Wellman area, Darwin Mountains, Antarctica.  

PubMed

The aim of this study was to examine the bacterial composition of high latitude soils from the Darwin-Hatherton glacier region of Antarctica. Four soil pits on each of four glacial drift sheets were sampled for chemical and microbial analyses. The four drifts-Hatherton, Britannia, Danum, and Isca-ranged, respectively, from early Holocene (10 ky) to mid-Quaternary (ca 900 ky). Numbers of culturable bacteria were low, with highest levels detected in soils from the younger Hatherton drift. DNA was extracted and 16S rRNA gene clone libraries prepared from samples below the desert pavement for each of the four drift sheets. Between 31 and 262 clones were analysed from each of the Hatherton, Britannia, and Danum drifts. Bacterial sequences were dominated by members of the phyla Deinococcus-Thermus, Actinobacteria, and Bacteroidetes. Culturable bacteria, including some that clustered with soil clones (e.g., members of the genera Arthrobacter, Adhaeribacter, and Pontibacter), belonged to Actinobacteria and Bacteroidetes. The isolated bacteria are ideal model organisms for genomic and phenotypic investigations of those attributes that allow bacteria to survive and/or grow in Antarctic soils because they have close relatives that are not tolerant of these conditions. PMID:23820800

Aislabie, Jackie M; Lau, Anna; Dsouza, Melissa; Shepherd, Charis; Rhodes, Phillippa; Turner, Susan J

2013-09-01

270

Identification of bacteria associated with underground parts of Crocus sativus by 16S rRNA gene targeted metagenomic approach.  

PubMed

Saffron (Crocus sativus L), an autumn-flowering perennial sterile plant, reproduces vegetatively by underground corms. Saffron has biannual corm-root cycle that makes it an interesting candidate to study microbial dynamics in its rhizosphere and cormosphere (area under influence of corm). Culture independent 16S rRNA gene metagenomic study of rhizosphere and cormosphere of Saffron during flowering stage revealed presence of 22 genera but none of the genus was common in all the three samples. Bulk soil bacterial community was represented by 13 genera with Acidobacteria being dominant. In rhizosphere, out of eight different genera identified, Pseudomonas was the most dominant genus. Cormosphere bacteria comprised of six different genera, dominated by the genus Pantoea. This study revealed that the bacterial composition of all the three samples is significantly different (P < 0.05) from each other. This is the first report on the identification of bacteria associated with rhizosphere, cormosphere and bulk soil of Saffron, using cultivation independent 16S rRNA gene targeted metagenomic approach. PMID:24989343

Ambardar, Sheetal; Sangwan, Naseer; Manjula, A; Rajendhran, J; Gunasekaran, P; Lal, Rup; Vakhlu, Jyoti

2014-10-01

271

Bacterial ?-glucosidase function and metabolic activity depend on soil management in semiarid rainfed agriculture  

PubMed Central

Genomic and transcriptomic approaches were used to gain insights into the relationship between soil management and bacterial-mediated functions in an olive orchard agroecosystem. Four management practices were assessed in a 30-year trial in a semiarid Mediterranean region. Transcriptional activity of bacterial 16S rRNA genes increased in noncovered soils, indicating higher microbial maintenance requirements to thrive in less favorable environmental conditions. The 16S rRNA transcript:gene copy ratio confirmed this assumption and pointed toward a much higher constitutive expression from rRNA operons in noncovered soils and to even higher expression levels when spontaneous vegetation was removed chemically. As described for 16S rRNA, potential transcription did not reveal the real transcription of bacterial ?-glucosidase genes, and higher gene expression in noncovered soils plus herbicides was evidenced. Since no relationship between total or soluble organic carbon and bacterial ?-glucosidase transcription was found, the above hypothesis could indicate either that soluble organic carbon is not the main pool of enzyme-inducing substrates or that constitutive production of bacterial ?-glucosidase enzymes increases as soil conditions worsen. PMID:22837821

Canizares, Rosa; Moreno, Beatriz; Benitez, Emilio

2012-01-01

272

Potential of Kalopanax septemlobus leaf extract in synthesis of silver nanoparticles for selective inhibition of specific bacterial strain in mixed culture.  

PubMed

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. degradans and retain B. cereus at MIC of S. degradans. This report is a case study for selective inhibition of one bacteria and growth of the other in a culture using plant-synthesized silver nanoparticles. PMID:25085530

Salunke, Bipinchandra K; Sawant, Shailesh S; Kim, Beom Soo

2014-09-01

273

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities  

PubMed Central

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ?1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods. PMID:24441163

Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

2014-01-01

274

Bacterial Population Structure of the Jute-Retting Environment  

Microsoft Academic Search

Jute is one of the most versatile bast fibers obtained through the process of retting, which is a result of decomposition\\u000a of stalks by the indigenous microflora. However, bacterial communities associated with the retting of jute are not well characterized.\\u000a To investigate the presence of microorganisms during the process of jute retting, full-cycle rRNA approach was followed, and\\u000a two 16S

Tulika K. Munshi; Bharat B. Chattoo

2008-01-01

275

Bacterial biodiversity from Roopkund Glacier, Himalayan mountain ranges, India  

Microsoft Academic Search

The bacterial diversity of two soil samples collected from the periphery of the Roopkund glacial lake and one soil sample\\u000a from the surface of the Roopkund Glacier in the Himalayan ranges was determined by constructing three 16S rRNA gene clone\\u000a libraries. The three clone libraries yielded a total of 798 clones belonging to 25 classes.\\u000a Actinobacteria was the most predominant

Suman Pradhan; T. N. R. Srinivas; Pavan Kumar Pindi; K. Hara Kishore; Z. Begum; Pawan Kumar Singh; Ashish Kumar Singh; M. S. Pratibha; Arun K. Yasala; G. S. N. Reddy; S. Shivaji

2010-01-01

276

A microfluidic system for rapid bacterial pathogen detection  

Microsoft Academic Search

We previously demonstrated application of a MEMS electrochemical biosensor to detect pathogenic bacteria in urine. Using custom-designed DNA oligonucleotide probes specific for bacterial 16S rRNA, species-specific detection was achieved within 45 minutes. However, the urine sample preparation protocol is still a labor-intensive 7 to 10 step process. This paper outlines our initial efforts to develop PDMS-based pump and mixer designs

J. D. H. Mai; R. S. Gaster; A. Wu; Wei Gu; K. E. Mach; J. C. Liao

2007-01-01

277

The Effect of Primer Choice and Short Read Sequences on the Outcome of 16S rRNA Gene Based Diversity Studies  

PubMed Central

Different regions of the bacterial 16S rRNA gene evolve at different evolutionary rates. The scientific outcome of short read sequencing studies therefore alters with the gene region sequenced. We wanted to gain insight in the impact of primer choice on the outcome of short read sequencing efforts. All the unknowns associated with sequencing data, i.e. primer coverage rate, phylogeny, OTU-richness and taxonomic assignment, were therefore implemented in one study for ten well established universal primers (338f/r, 518f/r, 799f/r, 926f/r and 1062f/r) targeting dispersed regions of the bacterial 16S rRNA gene. All analyses were performed on nearly full length and in silico generated short read sequence libraries containing 1175 sequences that were carefully chosen as to present a representative substitute of the SILVA SSU database. The 518f and 799r primers, targeting the V4 region of the 16S rRNA gene, were found to be particularly suited for short read sequencing studies, while the primer 1062r, targeting V6, seemed to be least reliable. Our results will assist scientists in considering whether the best option for their study is to select the most informative primer, or the primer that excludes interferences by host-organelle DNA. The methodology followed can be extrapolated to other primers, allowing their evaluation prior to the experiment. PMID:23977026

Heylen, Kim; Sessitsch, Angela; De Vos, Paul

2013-01-01

278

Changes in Bacterial Communities Accompanied by Aggregation in a Fed-Batch Composting Reactor  

Microsoft Academic Search

The contents of fed-batch composting (FBC) reactors often aggregate after prolonged operation. This process leads to irreversible\\u000a breakdown of the decomposition reaction and possible alteration of the bacterial communities. We compared the structures of\\u000a bacterial communities in reactors under aggregate and optimal conditions. The results of 16S rRNA gene clone analysis showed\\u000a that populations of the family Bacillaceae (such as

Keiko Watanabe; Norio Nagao; Tatsuki Toda; Norio Kurosawa

2008-01-01

279

Bacterial phylogeny based on comparative sequence analysis.  

PubMed

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information. PMID:9588802

Ludwig, W; Strunk, O; Klugbauer, S; Klugbauer, N; Weizenegger, M; Neumaier, J; Bachleitner, M; Schleifer, K H

1998-04-01

280

Changes in bacterial communities of the marine sponge Mycale laxissima on transfer into aquaculture.  

PubMed

The changes in bacterial communities associated with the marine sponge Mycale laxissima on transfer to aquaculture were studied using culture-based and molecular techniques. M. laxissima was maintained alive in flowthrough and closed recirculating aquaculture systems for 2 years and 1 year, respectively. The bacterial communities associated with wild and aquacultured sponges, as well as the surrounding water, were assessed using 16S rRNA gene clone library analysis and denaturing gradient gel electrophoresis (DGGE). Bacterial richness and diversity were measured using DOTUR computer software, and clone libraries were compared using S-LIBSHUFF. DGGE analysis revealed that the diversity of the bacterial community of M. laxissima increased when sponges were maintained in aquaculture and that bacterial communities associated with wild and aquacultured M. laxissima were markedly different than those of the corresponding surrounding water. Clone libraries of bacterial 16S rRNA from sponges confirmed that the bacterial communities changed during aquaculture. These communities were significantly different than those of seawater and aquarium water. The diversity of bacterial communities associated with M. laxissima increased significantly in aquaculture. Our work shows that it is important to monitor changes in bacterial communities when examining the feasibility of growing sponges in aquaculture systems because these communities may change. This could have implications for the health of sponges or for the production of bioactive compounds by sponges in cases where these compounds are produced by symbiotic bacteria rather than by the sponges themselves. PMID:18156319

Mohamed, Naglaa M; Enticknap, Julie J; Lohr, Jayme E; McIntosh, Scott M; Hill, Russell T

2008-02-01

281

Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria.  

PubMed

Using the loop-mediated isothermal amplification (LAMP) method, we developed a rapid assay for detection of 16S rRNA methylase genes (rmtA, rmtB, and armA), and investigated 16S rRNA methylase-producing strains among clinical isolates. Primer Explorer V3 software was used to design the LAMP primers. LAMP primers were prepared for each gene, including two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop primers (LF and LB). Detection was performed with the Loopamp DNA amplification kit. For all three genes (rmtA, rmtB, and armA), 10(2) copies/tube could be detected with a reaction time of 60 min. When nine bacterial species (65 strains saved in National Institute of Infectious Diseases) were tested, which had been confirmed to possess rmtA, rmtB, or armA by PCR and DNA sequencing, the genes were detected correctly in these bacteria with no false negative or false positive results. Among 8447 clinical isolates isolated at 36 medical institutions, the LAMP method was conducted for 191 strains that were resistant to aminoglycosides based on the results of antimicrobial susceptibility tests. Eight strains were found to produce 16S rRNA methylase (0.09%), with rmtB being identified in three strains (0.06%) of 4929 isolates of Enterobacteriaceae, rmtA in three strains (0.10%) of 3284 isolates of Pseudomonas aeruginosa, and armA in two strains (0.85%) of 234 isolates of Acinetobacter spp. At present, the incidence of strains possessing 16S rRNA methylase genes is very low in Japan. However, when Gram-negative bacteria showing high resistance to aminoglycosides are isolated by clinical laboratories, it seems very important to investigate the status of 16S rRNA methylase gene-harboring bacilli and monitor their trends among Japanese clinical settings. PMID:25179393

Nagasawa, Mitsuaki; Kaku, Mitsuo; Kamachi, Kazunari; Shibayama, Keigo; Arakawa, Yoshichika; Yamaguchi, Keizo; Ishii, Yoshikazu

2014-10-01

282

Bacterial vaginosis.  

PubMed Central

Bacterial vaginosis (BV) is the most common of the vaginitides affecting women of reproductive age. It appears to be due to an alteration in the vaginal ecology by which Lactobacillus spp., the predominant organisms in the healthy vagina, are replaced by a mixed flora including Prevotella bivia, Prevotella disiens, Porphyromonas spp., Mobiluncus spp., and Peptostreptococcus spp. All of these organisms except Mobiluncus spp. are also members of the endogenous vaginal flora. While evidence from treatment trials does not support the notion that BV is sexually transmitted, recent studies have shown an increased risk associated with multiple sexual partners. It has also been suggested that the pathogenesis of BV may be similar to that of urinary tract infections, with the rectum serving as a reservoir for some BV-associated flora. The organisms associated with BV have also been recognized as agents of female upper genital tract infection, including pelvic inflammatory disease, and the syndrome BV has been associated with adverse outcome of pregnancy, including premature rupture of membranes, chorioamnionitis, and fetal loss; postpartum endometritis; cuff cellulitis; and urinary tract infections. The mechanisms by which the BV-associated flora causes the signs of BV are not well understood, but a role for H2O2-producing Lactobacillus spp. in protecting against colonization by catalase-negative anaerobic bacteria has been recognized. These and other aspects of BV are reviewed. PMID:1747864

Spiegel, C A

1991-01-01

283

Isolation of a Substantial Proportion of Forest Soil Bacterial Communities Detected via Pyrotag Sequencing  

PubMed Central

We isolated 1,264 bacterial strains from forest soils previously surveyed via pyrosequencing of rRNA gene amplicons. Conventional culturing techniques recovered a substantial proportion of the community, with isolates representing 22% of 98,557 total pyrotags. Growth characteristics of isolates indicated that ecological traits were associated with relative abundances of corresponding pyrotag operational taxonomic units. PMID:23315727

VanInsberghe, David; Hartmann, Martin; Stewart, Gordon R.

2013-01-01

284

Isolation of a substantial proportion of forest soil bacterial communities detected via pyrotag sequencing.  

PubMed

We isolated 1,264 bacterial strains from forest soils previously surveyed via pyrosequencing of rRNA gene amplicons. Conventional culturing techniques recovered a substantial proportion of the community, with isolates representing 22% of 98,557 total pyrotags. Growth characteristics of isolates indicated that ecological traits were associated with relative abundances of corresponding pyrotag operational taxonomic units. PMID:23315727

VanInsberghe, David; Hartmann, Martin; Stewart, Gordon R; Mohn, William W

2013-03-01

285

Identification of polybacterial communities in patients with postoperative, posttraumatic, and endogenous endophthalmitis through 16S rRNA gene libraries.  

PubMed

Endophthalmitis is a potential vision-threatening complication following surgical procedures (postoperative endophthalmitis [POE]), trauma (posttraumatic endophthalmitis [PTE]), and bacteremic seeding of the eye from a distant infection site (endogenous endophthalmitis [EE]). Several studies have revealed the polybacterial characteristics of endophthalmitis, which make the administration of antibiotics to treat the disease challenging. However, until now, the polybacterial communities of POE, PTE, and EE have not been precisely studied. Hence, the present study was designed to identify the bacterial community of endophthalmitis through 16S rRNA gene libraries. Of the 40 intraocular samples tested, 30 libraries were constructed with bacterial nested-PCR-positive samples. The obtained recombinant clones were screened through amplified rRNA gene restriction analysis (ARDRA) to identify unique clones. The multiple types of ARDRA patterns (P=0.345) and diverse bacterial sequences (P=0.277) within the libraries revealed the polybacterial nature of infection in POE, PTE, and EE. Moreover, to the best of our knowledge, this is the first report on polybacterial infection in EE. Gram-positive bacteria, including Bacillus spp. (n=19), Streptococcus spp. (n=18), Staphylococcus spp. (n=6), Exiguobacterium spp. (n=3), Gemella spp. (n=2), Enterococcus spp. (n=2), a Lysinibacillus sp. (n=1), a Clostridium sp. (n=1), and a Nocardia sp. (n=1), and Gram-negative bacteria, including Serratia spp. (n=18), Pseudomonas spp. (n=10), Enterobacter spp. (n=8), Acinetobacter spp. (n=3), Pantoea spp. (n=3), a Haemophilus sp. (n=1), and a Massilia sp. (n=1), were identified. Interestingly, among them, 10 bacterial species were not previously reported to be associated with endophthalmitis or other ocular infections. Besides, the presence of 4 unidentifiable clones suggests the possibility of novel organisms that might cause eye infections. Therefore, it is recommended that, in addition to the polybacterial nature of POE, PTE, and EE infections, the spectrum of the pathogenic bacterial community identified in this work should be considered while administering antibiotic therapy in suspected endophthalmitis cases. PMID:24574297

Jayasudha, Rajagopalaboopathi; Narendran, Venkatapathy; Manikandan, Palanisamy; Prabagaran, Solai Ramatchandirane

2014-05-01

286

Identification of Polybacterial Communities in Patients with Postoperative, Posttraumatic, and Endogenous Endophthalmitis through 16S rRNA Gene Libraries  

PubMed Central

Endophthalmitis is a potential vision-threatening complication following surgical procedures (postoperative endophthalmitis [POE]), trauma (posttraumatic endophthalmitis [PTE]), and bacteremic seeding of the eye from a distant infection site (endogenous endophthalmitis [EE]). Several studies have revealed the polybacterial characteristics of endophthalmitis, which make the administration of antibiotics to treat the disease challenging. However, until now, the polybacterial communities of POE, PTE, and EE have not been precisely studied. Hence, the present study was designed to identify the bacterial community of endophthalmitis through 16S rRNA gene libraries. Of the 40 intraocular samples tested, 30 libraries were constructed with bacterial nested-PCR-positive samples. The obtained recombinant clones were screened through amplified rRNA gene restriction analysis (ARDRA) to identify unique clones. The multiple types of ARDRA patterns (P = 0.345) and diverse bacterial sequences (P = 0.277) within the libraries revealed the polybacterial nature of infection in POE, PTE, and EE. Moreover, to the best of our knowledge, this is the first report on polybacterial infection in EE. Gram-positive bacteria, including Bacillus spp. (n = 19), Streptococcus spp. (n = 18), Staphylococcus spp. (n = 6), Exiguobacterium spp. (n = 3), Gemella spp. (n = 2), Enterococcus spp. (n = 2), a Lysinibacillus sp. (n = 1), a Clostridium sp. (n = 1), and a Nocardia sp. (n = 1), and Gram-negative bacteria, including Serratia spp. (n = 18), Pseudomonas spp. (n = 10), Enterobacter spp. (n = 8), Acinetobacter spp. (n = 3), Pantoea spp. (n = 3), a Haemophilus sp. (n = 1), and a Massilia sp. (n = 1), were identified. Interestingly, among them, 10 bacterial species were not previously reported to be associated with endophthalmitis or other ocular infections. Besides, the presence of 4 unidentifiable clones suggests the possibility of novel organisms that might cause eye infections. Therefore, it is recommended that, in addition to the polybacterial nature of POE, PTE, and EE infections, the spectrum of the pathogenic bacterial community identified in this work should be considered while administering antibiotic therapy in suspected endophthalmitis cases. PMID:24574297

Jayasudha, Rajagopalaboopathi; Narendran, Venkatapathy; Manikandan, Palanisamy

2014-01-01

287

Response and resilience of soil biocrust bacterial communities to chronic physical disturbance in arid shrublands  

PubMed Central

The impact of 10 years of annual foot trampling on soil biocrusts was examined in replicated field experiments at three cold desert sites of the Colorado Plateau, USA. Trampling detrimentally impacted lichens and mosses, and the keystone cyanobacterium, Microcoleus vaginatus, resulting in increased soil erosion and reduced C and N concentrations in surface soils. Trampled biocrusts contained approximately half as much extractable DNA and 20–52% less chlorophyll a when compared with intact biocrusts at each site. Two of the three sites also showed a decline in scytonemin-containing, diazotrophic cyanobacteria in trampled biocrusts. 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses of soil bacteria from untrampled and trampled biocrusts demonstrated a reduced proportion (23–65% reduction) of M. vaginatus and other Cyanobacteria in trampled plots. In parallel, other soil bacterial species that are natural residents of biocrusts, specifically members of the Actinobacteria, Chloroflexi and Bacteroidetes, became more readily detected in trampled than in untrampled biocrusts. Replicate 16S rRNA T-RFLP profiles from trampled biocrusts at all three sites contained significantly more fragments (n=17) than those of untrampled biocrusts (n?6) and exhibited much higher variability among field replicates, indicating transition to an unstable disturbed state. Despite the dramatic negative impacts of trampling on biocrust physical structure and composition, M. vaginatus could still be detected in surface soils after 10 years of annual trampling, suggesting the potential for biocrust re-formation over time. Physical damage of biocrusts, in concert with changing temperature and precipitation patterns, has potential to alter performance of dryland ecosystems for decades. PMID:22113374

Kuske, Cheryl R; Yeager, Chris M; Johnson, Shannon; Ticknor, Lawrence O; Belnap, Jayne

2012-01-01

288

Extremely acidophilic protists from acid mine drainage host Rickettsiales-lineage endosymbionts that have intervening sequences in their 16S rRNA genes.  

PubMed

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the protist Acanthamoeba. Oligonucleotide 16S rRNA probes were designed and used to observe members of this group within acidophilic protists. To improve visualization of eukaryotic populations in the acid mine drainage samples, broad-specificity probes for eukaryotes were redesigned and combined to highlight this component of the acid mine drainage community. Approximately 4% of protists in the acid mine drainage samples contained endosymbionts. Measurements of internal pH of the protists showed that their cytosol is close to neutral, indicating that the endosymbionts may be neutrophilic. The endosymbionts had a conserved 273-nucleotide intervening sequence (IVS) in variable region V1 of their 16S rRNA genes. The IVS does not match any sequence in current databases, but the predicted secondary structure forms well-defined stem loops. IVSs are uncommon in rRNA genes and appear to be confined to bacteria living in close association with eukaryotes. Based on the phylogenetic novelty of the endosymbiont sequences and initial culture-independent characterization, we propose the name "Candidatus Captivus acidiprotistae." To our knowledge, this is the first report of an endosymbiotic relationship in an extremely acidic habitat. PMID:12957940

Baker, Brett J; Hugenholtz, Philip; Dawson, Scott C; Banfield, Jillian F

2003-09-01

289

A small protein unique to bacteria organizes rRNA tertiary structure over an extensive region of the 50 S ribosomal subunit.  

PubMed

A number of small, basic proteins penetrate into the structure of the large subunit of the ribosome. While these proteins presumably aid in the folding of the rRNA, the extent of their contribution to the stability or function of the ribosome is unknown. One of these small, basic proteins is L36, which is highly conserved in Bacteria, but is not present in Archaea or Eucarya. Comparison of ribosome crystal structures shows that the space occupied by L36 in a bacterial ribosome is empty in an archaeal ribosome. To ask what L36 contributes to ribosome stability and function, we have constructed an Escherichia coli strain lacking ribosomal protein L36; cell growth is slowed by 40-50% between 30 degrees C and 42 degrees C. Ribosomes from this deletion strain sediment normally and have a full complement of proteins, other than L36. Chemical protection experiments comparing rRNA from wild-type and L36-deficient ribosomes show the expected increase in reagent accessibility in the immediate vicinity of the L36 binding site, but suggest that a cooperative network of rRNA tertiary interactions has been disrupted along a path extending 60 A deep into the ribosome. These data argue that L36 plays a significant role in organizing 23 S rRNA structure. Perhaps the Archaea and Eucarya have compensated for their lack of L36 by maintaining more stable rRNA tertiary contacts or by adopting alternative protein-RNA interactions elsewhere in the ribosome. PMID:16246363

Maeder, Corina; Draper, David E

2005-11-25

290

Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae.  

PubMed

Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus-Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves. PMID:15388716

Christensen, Henrik; Kuhnert, Peter; Olsen, John Elmerdahl; Bisgaard, Magne

2004-09-01

291

Methylation of 23S rRNA Nucleotide G748 by RlmAII Methyltransferase Renders Streptococcus pneumoniae Telithromycin Susceptible  

PubMed Central

Several posttranscriptional modifications of bacterial rRNAs are important in determining antibiotic resistance or sensitivity. In all Gram-positive bacteria, dimethylation of nucleotide A2058, located in domain V of 23S rRNA, by the dimethyltransferase Erm(B) results in low susceptibility and resistance to telithromycin (TEL). However, this is insufficient to produce high-level resistance to TEL in Streptococcus pneumoniae. Inactivation of the methyltransferase RlmAII, which methylates the N-1 position of nucleotide G748, located in hairpin 35 of domain II of 23S rRNA, results in increased resistance to TEL in erm(B)-carrying S. pneumoniae. Sixteen TEL-resistant mutants (MICs, 16 to 32 ?g/ml) were obtained from a clinically isolated S. pneumoniae strain showing low TEL susceptibility (MIC, 2 ?g/ml), with mutation resulting in constitutive dimethylation of A2058 because of nucleotide differences in the regulatory region of erm(B) mRNA. Primer extension analysis showed that the degree of methylation at G748 in all TEL-resistant mutants was significantly reduced by a mutation in the gene encoding RlmAII to create a stop codon or change an amino acid residue. Furthermore, RNA footprinting with dimethyl sulfate and a molecular modeling study suggested that methylation of G748 may contribute to the stable interaction of TEL with domain II of 23S rRNA, even after dimethylation of A2058 by Erm(B). This novel finding shows that methylation of G748 by RlmAII renders S. pneumoniae TEL susceptible. PMID:23716046

Sato, Yoshiharu; Shoji, Tatsuma; Yamamoto, Tomoko

2013-01-01

292

Detection of Gallibacterium spp. in chickens by fluorescent 16S rRNA in situ hybridization.  

PubMed

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, "Actinobacillus salpingitidis," and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens. PMID:14605154

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-11-01

293

The Importance of Temperature and Nitrogen Speciation on Bacterial Diversity in Stream Sediments in the McMurdo Dry Valleys  

NASA Astrophysics Data System (ADS)

Once called the Valleys of the Dead, the McMurdo Dry Valleys in Antarctica have been shown to harbor life that can withstand some of the coldest, windiest, driest and extreme conditions on Earth. The Dry Valleys are often referred to as `ecosystems waiting for water' because of the rapid response of biological activity when liquid water appears. Although our understanding of life in the Valleys is progressing, there are numerous unanswered questions about how the organisms survive there. Most of the attention in this delicate ecosystem has been focused on nematodes and lake and stream algal mat communities. Microbe abundances in stream sediments not associated with mats are only an order of a magnitude lower than those found in temporal streams. Yet very little is known about the metabolic capabilities, energy demands, nutrient requirements and genomes of these organisms. In December of 2004, sediment and water quality samples were collected from 19 streams in Taylor Valley. Bacterial DNA was extracted from the sediments and 16S rRNA amplified using 8f and 926r bacterial primers. Terminal Restriction Length Polymorphism (tRFLP) analysis was used to obtain community diversity fingerprints for all stream sites. The Bonney Basin had the most diverse bacterial assemblages on the basis of such fingerprints. There was no correlation between bacterial diversity and algal mat presence or absence, indicating that bacterial diversity does not depend on mats. Statistical analysis comparing water chemistry data and diversity indicates that temperature and nitrogen speciation and concentration are important factors contributing to diversity in these oligotrophic streams. Five clone libraries were sequenced and used to determine the major bacteria present in the streams. Approximately 15% of the sequences had less than a 97% similarity to any known bacterial sequence present in GenBank, suggesting a high incidence of bacterial species unique to the Dry Valleys. This research is the first step toward understanding the microbial communities in this ecosystem and will provide the foundation for studies on biogeochemical function and microbe survival. These will not only be important for better understanding the dry valleys but will likely give insights into new anti-freeze proteins, desiccation mechanisms and UV-damaged DNA repair strategies which are of societal importance.

Baeseman, J. L.; Ward, B. B.

2006-12-01

294

Immunostimulatory activities of specific bacterial secondary metabolite of Anoxybacillus flavithermus strain SX-4 on carp, Cyprinus carpio.  

PubMed

Aims:? To determine the capacity of secondary metabolite of strain SX-4, to enhance the nonspecific immunity and survival of carp (Cyprinus carpio), and to identify the constituents that are responsible. Methods and Results:? A thermophilic strain SX-4 that is able to produce immunostimulatory metabolite was isolated from sludge sample of hot spring and identified by comparison with 16S rRNA sequences (99% of homology) as Anoxybacillus flavithermus. Bioactivity-guided fractionation of methanol extract from its cell-free culture, one bacterial peptide with the capacity of improving the nonspecific immune responses and disease resistance (relative per cent survival?=?66·67%) was obtained and the compound was characterized as cyclo-(L-Pro-Gly) by IR, ESI-MS, (1) H NMR and (13) C NMR spectroscopic analyses. After intraperitoneal administration of this peptide, selected innate immune parameters including phagocytic activity, superoxide anion production, serum lysozyme activity and serum SOD activity, along with immune-related genes expression (i.e. interleukin-1? and inducible nitric oxide synthase), in the blood were found to be significantly increased. Conclusions:? The bacterial peptide cyclo-(L-Pro-Gly) significantly enhances nonspecific immunity and survival of carp. Significance and Impact of the Study:? There is a possibility of using cyclo-(L-Pro-Gly) as a better natural immunostimulant, which could have a promising role in aquaculture to prevent diseases and disease outbreaks. PMID:21294820

Liu, J; Lei, Y; Wang, F; Yi, Y; Liu, Y; Wang, G

2011-02-01

295

Time between Collection and Storage Significantly Influences Bacterial Sequence Composition in Sputum Samples from Cystic Fibrosis Respiratory Infections  

PubMed Central

Spontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. While guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means that it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before being frozen at ?80°C for increasing intervals, up to a 72-h period. Samples were treated with propidium monoazide to distinguish live from dead cells prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterize their bacterial compositions. Substantial variation was observed in samples with high-diversity bacterial communities over time, whereas little variation was observed in low-diversity communities dominated by recognized CF pathogens, regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 h at room temperature (P = 0.008). Failure to stabilize samples at ?80°C within 12 h of collection results in significant changes in the detected community composition. PMID:24920767

Cuthbertson, Leah; Rogers, Geraint B.; Walker, Alan W.; Oliver, Anna; Hafiz, Tarana; Hoffman, Lucas R.; Carroll, Mary P.; Parkhill, Julian; Bruce, Kenneth D.

2014-01-01

296

Bacterial biodiversity from anthropogenic extreme environments: a hyper-alkaline and hyper-saline industrial residue contaminated by chromium and iron.  

PubMed

Anthropogenic extreme environments are among the most interesting sites for the bioprospection of extremophiles since the selection pressures may favor the presence of microorganisms of great interest for taxonomical and astrobiological research as well as for bioremediation technologies and industrial applications. In this work, T-RFLP and 16S rRNA gene library analyses were carried out to describe the autochthonous bacterial populations from an industrial waste characterized as hyper-alkaline (pH between 9 and 14), hyper-saline (around 100 PSU) and highly contaminated with metals, mainly chromium (from 5 to 18 g kg(-1)) and iron (from 2 to 108 g kg(-1)). Due to matrix interference with DNA extraction, a protocol optimization step was required in order to carry out molecular analyses. The most abundant populations, as evaluated by both T-RFLP and 16S rRNA gene library analyses, were affiliated to Bacillus and Lysobacter genera. Lysobacter related sequences were present in the three samples: solid residue and lixiviate sediments from both dry and wet seasons. Sequences related to Thiobacillus were also found; although strains affiliated to this genus are known to have tolerance to metals, they have not previously been detected in alkaline environments. Together with Bacillus (already described as a metal reducer), such organisms could be of use in bioremediation technologies for reducing chromium, as well as for the prospection of enzymes of biotechnological interest. PMID:22350256

Brito, Elcia M S; Piñón-Castillo, Hilda A; Guyoneaud, Rémy; Caretta, César A; Gutiérrez-Corona, J Félix; Duran, Robert; Reyna-López, Georgina E; Nevárez-Moorillón, G Virginia; Fahy, Anne; Goñi-Urriza, Marisol

2013-01-01

297

A Markovian analysis of bacterial genome sequence constraints  

PubMed Central

The arrangement of nucleotides within a bacterial chromosome is influenced by numerous factors. The degeneracy of the third codon within each reading frame allows some flexibility of nucleotide selection; however, the third nucleotide in the triplet of each codon is at least partly determined by the preceding two. This is most evident in organisms with a strong G + C bias, as the degenerate codon must contribute disproportionately to maintaining that bias. Therefore, a correlation exists between the first two nucleotides and the third in all open reading frames. If the arrangement of nucleotides in a bacterial chromosome is represented as a Markov process, we would expect that the correlation would be completely captured by a second-order Markov model and an increase in the order of the model (e.g., third-, fourth-…order) would not capture any additional uncertainty in the process. In this manuscript, we present the results of a comprehensive study of the Markov property that exists in the DNA sequences of 906 bacterial chromosomes. All of the 906 bacterial chromosomes studied exhibit a statistically significant Markov property that extends beyond second-order, and therefore cannot be fully explained by codon usage. An unrooted tree containing all 906 bacterial chromosomes based on their transition probability matrices of third-order shares ?25% similarity to a tree based on sequence homologies of 16S rRNA sequences. This congruence to the 16S rRNA tree is greater than for trees based on lower-order models (e.g., second-order), and higher-order models result in diminishing improvements in congruence. A nucleotide correlation most likely exists within every bacterial chromosome that extends past three nucleotides. This correlation places significant limits on the number of nucleotide sequences that can represent probable bacterial chromosomes. Transition matrix usage is largely conserved by taxa, indicating that this property is likely inherited, however some important exceptions exist that may indicate the convergent evolution of some bacteria. PMID:24010012

Skewes, Aaron D.

2013-01-01

298

YhiQ is RsmJ, the methyltransferase responsible for methylation of G1516 in 16S rRNA of E. coli  

PubMed Central

Ten methyltransferases and one pseudouridine synthase are required for complete modification of the small ribosomal subunit in E. coli. Nine methyltransferases, as well as the pseudouridine synthase, are already known. Here we identify RsmJ, the last unknown methyltransferase required for methylation of m2G1516 in 16S rRNA, as the protein encoded by yhiQ. Reverse transcription primer extension analysis reveals that rRNA extracted from a yhiQ deletion strain is not methylated at G1516. Moreover, methylation is restored upon gene complementation. Also, purified recombinant YhiQ specifically methylates 30S subunits extracted from the deletion strain. The absence of the yhiQ gene leads to a cold sensitive phenotype. Based on these data we propose that the yhiQ gene be renamed rsmJ. PMID:22079366

Basturea, Georgeta N.; Dague, Darryl R.; Deutscher, Murray P.; Rudd, Kenneth E.

2011-01-01

299

16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances.  

PubMed

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances. PMID:24191230

Piwat, S; Teanpaisan, R

2013-01-01

300

16S rRNA PCR-Denaturing Gradient Gel Electrophoresis of Oral Lactobacillus casei Group and Their Phenotypic Appearances  

PubMed Central

This study aimed to develop a 16S rRNA PCR-denaturing gradient gel electrophoresis (DGGE) to identify the species level of Lactobacillus casei group and to investigate their characteristics of acid production and inhibitory effect. PCR-DGGE has been developed based on the 16S rRNA gene, and a set of HDA-1-GC and HDA-2, designed at V2-V3 region, and another set of CARP-1-GC and CARP-2, designed at V1 region, have been used. The bacterial strains included L. casei ATCC 393, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515, and 46 clinical strains of L. casei/paracasei/rhamnosus. Inhibitory effect against Streptococcus mutans and acid production were examined. Results revealed that each type species strain and identified clinical isolate showed its own unique DGGE pattern using CARP1-GC and CARP2 primers. HDA1-GC and HDA2 primers could distinguish the strains of L. paracasei from L. casei. It was found that inhibitory effect of L. paracasei was stronger than L. casei and L. rhamnosus. The acid production of L. paracasei was lower than L. casei and L. rhamnosus. In conclusion, the technique has been proven to be able to differentiate between closely related species in L. casei group and thus provide reliable information of their phenotypic appearances. PMID:24191230

Piwat, S.; Teanpaisan, R.

2013-01-01

301

Higher-order structure of rRNA  

NASA Technical Reports Server (NTRS)

A comparative search for phylogenetically covarying basepair replacements within potential helices has been the only reliable method to determine the correct secondary structure of the 3 rRNAs, 5S, 16S, and 23S. The analysis of 16S from a wide phylogenetic spectrum, that includes various branches of the eubacteria, archaebacteria, eucaryotes, in addition to the mitochondria and chloroplast, is beginning to reveal the constraints on the secondary structures of these rRNAs. Based on the success of this analysis, and the assumption that higher order structure will also be phylogenetically conserved, a comparative search was initiated for positions that show co-variation not involved in secondary structure helices. From a list of potential higher order interactions within 16S rRNA, two higher-order interactions are presented. The first of these interactions involves positions 570 and 866. Based on the extent of phylogenetic covariation between these positions while maintaining Watson-Crick pairing, this higher-order interaction is considered proven. The other interaction involves a minimum of six positions between the 1400 and 1500 regions of the 16S rRNA. Although these patterns of covariation are not as striking as the 570/866 interaction, the fact that they all exist in an anti-parallel fashion and that experimental methods previously implicated these two regions of the molecule in tRNA function suggests that these interactions be given serious consideration.

Gutell, R. R.; Woese, C. R.

1986-01-01

302

The rRNA evolution and procaryotic phylogeny  

NASA Technical Reports Server (NTRS)

Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

Fox, G. E.

1986-01-01

303

Different bacterial communities in ectomycorrhizae and surrounding soil  

PubMed Central

Several eukaryotic symbioses have shown to host a rich diversity of prokaryotes that interact with their hosts. Here, we study bacterial communities associated with ectomycorrhizal root systems of Bistorta vivipara compared to bacterial communities in bulk soil using pyrosequencing of 16S rRNA amplicons. A high richness of Operational Taxonomic Units (OTUs) was found in plant roots (3,571?OTUs) and surrounding soil (3,476?OTUs). The community composition differed markedly between these two environments. Actinobacteria, Armatimonadetes, Chloroflexi and OTUs unclassified at phylum level were significantly more abundant in plant roots than in soil. A large proportion of the OTUs, especially those in plant roots, presented low similarity to Sanger 16S rRNA reference sequences, suggesting novel bacterial diversity in ectomycorrhizae. Furthermore, the bacterial communities of the plant roots were spatially structured up to a distance of 60?cm, which may be explained by bacteria using fungal hyphae as a transport vector. The analyzed ectomycorrhizae presents a distinct microbiome, which likely influence the functioning of the plant-fungus symbiosis. PMID:24326907

Vik, Unni; Logares, Ramiro; Blaalid, Rakel; Halvorsen, Rune; Carlsen, Tor; Bakke, Ingrid; Kolst?, Anne-Brit; ?kstad, Ole Andreas; Kauserud, Havard

2013-01-01

304

[Diversity of 16S rRNA genes in metagenomic community of the freshwater sponge Lubomirskia baicalensis].  

PubMed

Phylogenetic analysis of the nucleotide sequences of 16S rRNA genes in the metagenomic community of Lubomirskia baicalensis has revealed taxonomic diversity of bacteria associated with the endemic freshwater sponge. Fifty-four operational taxonomic units (OTUs) belonging to six bacterial phyla (Actinobacteria, alpha-Proteobacteria, beta-Proteobacteria, Verrucomicrobia, Bacteroidetes, Cyanobacteria, and Nitrospiracea) have been identified. Actinobacteria, whose representatives are known as antibiotic producers, is the dominant phylum of the community (37%, 20 OTUs). All sequences detected shared the maximal homology with unculturable microorganisms from freshwater habitats. The wide diversity of bacteria closely coexisting with the Baikal sponge indicate the complex ecological relationships in the community formed under the unique conditions of Lake Baikal. PMID:23035553

Kaliuzhnaia, O V; Krivich, A A; Itskovich, V B

2012-08-01

305

Cell size distributions of soil bacterial and archaeal taxa.  

PubMed

Cell size is a key ecological trait of soil microorganisms that determines a wide range of life history attributes, including the efficiency of nutrient acquisition. However, because of the methodological issues associated with determining cell sizes in situ, we have a limited understanding of how cell abundances vary across cell size fractions and whether certain microbial taxa have consistently smaller cells than other taxa. In this study, we extracted cells from three distinct soils and fractionated them into seven size ranges (5 ?m to 0.2 ?m) by filtration. Cell abundances in each size fraction were determined by direct microscopy, with the taxonomic composition of each size fraction determined by high-throughput sequencing of the 16S rRNA gene. Most of the cells were smaller than cells typically grown in culture, with 59 to 67% of cells <1.2 ?m in diameter. Furthermore, each size fraction harbored distinct bacterial and archaeal communities in each of the three soils, and many of the taxa exhibited distinct size distribution patterns, with the smaller size fractions having higher relative abundances of taxa that are rare or poorly characterized (including Acidobacteria, Gemmatimonadetes, Crenarchaeota, Verrucomicrobia, and Elusimicrobia). In general, there was a direct relationship between average cell size and culturability, with those soil taxa that are poorly represented in culture collections tending to be smaller. Size fractionation not only provides important insight into the life history strategies of soil microbial taxa but also is a useful tool to enable more focused investigations into those taxa that remain poorly characterized. PMID:24077710

Portillo, Maria C; Leff, Jonathan W; Lauber, Christian L; Fierer, Noah

2013-12-01

306

Object extraction Object extraction  

E-print Network

Object extraction #12;Object extraction · Extracting topographic objects from images · the main goal of aerial photogrammetry · object extraction consists of two steps · image interpretation extraction · Extracting topographic objects from images · identify all objects of a certain class · measure

Giger, Christine

307

Characterization of the Vaginal Microbiota among Sexual Risk Behavior Groups of Women with Bacterial Vaginosis  

PubMed Central

Background The pathogenesis of bacterial vaginosis (BV) remains elusive. BV may be more common among women who have sex with women (WSW). The objective of this study was to use 454 pyrosequencing to investigate the vaginal microbiome of WSW, women who have sex with women and men (WSWM), and women who have sex with men (WSM) with BV to determine if there are differences in organism composition between groups that may inform new hypotheses regarding the pathogenesis of BV. Methods Vaginal swab specimens from eligible women with BV at the Mississippi State Department of Health STD Clinic were used. After DNA extraction, 454 pyrosequencing of PCR-amplified 16S rRNA gene sequences was performed. Sequence data was classified using the Ribosomal Database Program classifer. Complete linkage clustering analysis was performed to compare bacterial community composition among samples. Differences in operational taxonomic units with an abundance of ?2% between risk behavior groups were determined. Alpha and beta diversity were measured using Shannon’s Index implemented in QIIME and Unifrac analysis, respectively. Results 33 WSW, 35 WSWM, and 44 WSM were included. The vaginal bacterial communities of all women clustered into four taxonomic groups with the dominant taxonomic group in each being Lactobacillus, Lachnospiraceae, Prevotella, and Sneathia. Regarding differences in organism composition between risk behavior groups, the abundance of Atopobium (relative ratio (RR)=0.24; 95%CI 0.11-0.54) and Parvimonas (RR=0.33; 95%CI 0.11-0.93) were significantly lower in WSW than WSM, the abundance of Prevotella was significantly higher in WSW than WSWM (RR=1.77; 95%CI 1.10-2.86), and the abundance of Atopobium (RR=0.41; 95%CI 0.18-0.88) was significantly lower in WSWM than WSM. Overall, WSM had the highest diversity of bacterial taxa. Conclusion The microbiology of BV among women in different risk behavior groups is heterogeneous. WSM in this study had the highest diversity of bacterial taxa. Additional studies are needed to better understand these differences. PMID:24236175

Muzny, Christina A.; Sunesara, Imran R.; Kumar, Ranjit; Mena, Leandro A.; Griswold, Michael E.; Martin, David H.; Lefkowitz, Elliot J.; Schwebke, Jane R.

2013-01-01

308

The bacterial communities in plant phloem-sap-feeding insects.  

PubMed

The resident microbiota of animals represents an important contribution to the global microbial diversity, but it is poorly known in many animals. This study investigated the bacterial diversity in plant phloem-sap-feeding whiteflies, aphids and psyllids by pyrosequencing bacterial 16S rRNA gene amplicons. After correction for sequencing error, just 3-7 bacterial operational taxonomic units were recovered from each insect sample sequenced to sufficient depth for saturation of rarefaction curves. Most samples were dominated by primary and secondary symbionts, which are localized to insect cells or the body cavity, indicative of a dearth of bacterial colonists of the gut lumen. Diversity indices of the bacterial communities (Shannon's index: 0.40-1.46, Simpson's index: 0.15-0.74) did not differ significantly between laboratory and field samples of the phloem-feeding insects, but were significantly lower than in drosophilid flies quantified by the same methods. Both the low bacterial content of the phloem sap diet and biological processes in the insect may contribute to the apparently low bacterial diversity in these phloem-feeding insects. PMID:24350573

Jing, Xiangfeng; Wong, Adam C-N; Chaston, John M; Colvin, John; McKenzie, Cindy L; Douglas, Angela E

2014-03-01

309

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

SciTech Connect

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

Jansson, Janet; Edlund, Anna; Hardeman, Fredrik; Jansson, Janet K.; Sjoling, Sara

2008-05-15

310

Seasonal changes in nitrogen-cycle gene abundances and in bacterial communities in acidic forest soils.  

PubMed

The abundance of genes related to the nitrogen biogeochemical cycle and the microbial community in forest soils (bacteria, archaea, fungi) were quantitatively analyzed via real-time PCR using 11 sets of specific primers amplifying nifH, bacterial amoA, archaeal amoA, narG, nirS, nirK, norB, nosZ, bacterial 16S rRNA gene, archaeal 16S rRNA gene, and the ITS sequence of fungi. Soils were sampled from Bukhan Mountain from September of 2010 to July of 2011 (7 times). Bacteria were the predominant microbial community in all samples. However, the abundance of archaeal amoA was greater than bacterial amoA throughout the year. The abundances of nifH, nirS, nirK, and norB genes changed in a similar pattern, while narG and nosZ appeared in sensitive to the environmental changes. Clone libraries of bacterial 16S rRNA genes were constructed from summer and winter soil samples and these revealed that Acidobacteria was the most predominant phylum in acidic forest soil environments in both samples. Although a specific correlation of environmental factor and gene abundance was not verified by principle component analysis, our data suggested that the combination of biological, physical, and chemical characteristics of forest soils created distinct conditions favoring the nitrogen biogeochemical cycle and that bacterial communities in undisturbed acidic forest soils were quite stable during seasonal change. PMID:22752898

Jung, Jaejoon; Yeom, Jinki; Han, Jiwon; Kim, Jisun; Park, Woojun

2012-06-01

311

Rapid Extraction from and Direct Identification in Clinical Samples of Methicillin-Resistant Staphylococci Using the PCR  

PubMed Central

Methicillin-resistant staphylococci (MRS) are one of the most common causes of nosocomial infections and bacteremia. Standard bacterial identification and susceptibility testing frequently require as long as 72 h to report results, and there may be difficulty in rapidly and accurately identifying methicillin resistance. The use of the PCR is a rapid and simple process for the amplification of target DNA sequences, which can be used to identify and test bacteria for antimicrobial resistance. However, many sample preparation methods are unsuitable for PCR utilization in the clinical laboratory because they either are not cost-effective, take too long to perform, or do not provide a satisfactory DNA template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis and therapy. In this report, we describe a rapid method for extraction of bacterial DNA directly from blood culture bottles that gave quality DNA for PCR in as little as 20 min. We compared this extraction method to the standard QIAGEN method for turnaround time (TAT), cost, purity, and use of template in PCR. Specific identification of MRS was determined using intragenic primer sets for bacterial and Staphylococcus 16S rRNA and mecA gene sequences. The PCR primer sets were validated with 416 isolates of staphylococci, including methicillin-resistant Staphylococcus aureus (n = 106), methicillin-sensitive S. aureus (n = 134), and coagulase-negative Staphylococcus (n = 176). The total supply cost of our extraction method and PCR was $2.15 per sample with a result TAT of less than 4 h. The methods described herein represent a rapid and accurate DNA extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may have utility in the clinical laboratory. PMID:10970392

Jaffe, Richard I.; Lane, Janae D.; Albury, Stephen V.; Niemeyer, Debra M.

2000-01-01

312

Influence of menstruation on the microbiota of healthy women's labia minora as analyzed using a 16S rRNA gene-based clone library method.  

PubMed

The aim of this study was to determine the influence of menstruation on the bacterial population of healthy Japanese women's vulvas, especially the labia minora. Labia minora swabs were obtained from 10 premenopausal, nonpregnant Japanese women at premenstruation and on day 2 of menstruation. Vaginal swabs were also obtained from 3 out of the 10 women. No significant difference was found in the average bacterial cell count between the menstruation and premenstruation samples. Molecular analysis using a 16S rRNA gene-based clone library method detected 22 genera from the labia minora swabs (total 20), with the genus Lactobacillus being predominant at both premenstruation and during menstruation in 7 out of the 10 women. Of the other 3 women, 2 showed various kinds of bacterial species, including oral and fecal bacteria, with Atopobium vaginae and Gardnerella vaginalis predominating in the remaining woman's vulva in both conditions. In total, 6 out of 10 cases (60%) showed significantly different microbiota of the labia minora between the two conditions. These results imply that menstruation may promote a distortion of the bacterial flora around the vulva, although it causes no significant increase of the bacterial count. PMID:21266747

Shiraishi, Tsukasa; Fukuda, Kazumasa; Morotomi, Nobuo; Imamura, Yuri; Mishima, Junko; Imai, Shigeo; Miyazawa, Kiyoshi; Taniguchi, Hatsumi

2011-01-01

313

Evolution of bacterial genomes  

Microsoft Academic Search

This review examines evolution of bacterial genomes with an emphasis on RNA based life, the transition to functional DNA and small evolving genomes (possibly plasmids) that led to larger, functional bacterial genomes.

J. T. Trevors

1997-01-01

314

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host  

Microsoft Academic Search

We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restric- tion fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of ter- mites. Both analyses revealed that the bacterial community

Yuichi Hongoh; Pinsurang Deevong; Tetsushi Inoue; Shigeharu Moriya; S. Trakulnaleamsai; M. Ohkuma; C. Vongkaluang; N. Noparatnaraporn; T. Kudo

2005-01-01

315

Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16S rRNA gene clone library analysis.  

PubMed

Bacteroides spp. represent a prominent bacterial group in human intestinal microbiota with roles in symbiosis and pathogenicity; however, the detailed composition of this group in human feces has yet to be comprehensively characterized. In this study, the molecular diversity of Bacteroides spp. in human fecal microbiota was analyzed from a seven-member, four-generation Chinese family using Bacteroides spp. group-specific 16S rRNA gene clone library analysis. A total of 549 partial 16S rRNA sequences amplified by Bacteroides spp.-specific primers were classified into 52 operational taxonomic units (OTUs) with a 99% sequence identity cut-off. Twenty-three OTUs, representing 83% of all clones, were related to 11 validly described Bacteroides species, dominated by Bacteroides coprocola, B. uniformis, and B. vulgatus. Most of the OTUs did not correspond to known species and represented hitherto uncharacterized bacteria. Relative to 16S rRNA gene universal libraries, the diversity of Bacteroides spp. detected by the group-specific libraries was much higher than previously described. Remarkable inter-individual differences were also observed in the composition of Bacteroides spp. in this family cohort. The comprehensive observation of molecular diversity of Bacteroides spp. provides new insights into potential contributions of various species in this group to human health and disease. PMID:19303731

Li, Min; Zhou, Haokui; Hua, Weiying; Wang, Baohong; Wang, Shengyue; Zhao, Guoping; Li, Lanjuan; Zhao, Liping; Pang, Xiaoyan

2009-05-01

316

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan  

PubMed Central

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome’s ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome. PMID:24137004

Martinez, Allyson K.; Gordon, Emily; Sengupta, Arnab; Shirole, Nitin; Klepacki, Dorota; Martinez-Garriga, Blanca; Brown, Lewis M.; Benedik, Michael J.; Yanofsky, Charles; Mankin, Alexander S.; Vazquez-Laslop, Nora; Sachs, Matthew S.; Cruz-Vera, Luis R.

2014-01-01

317

Effects of Abiotic Factors on the Phylogenetic Diversity of Bacterial Communities in Acidic Thermal Springs?  

PubMed Central

Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and Eh of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to Hydrogenobaculum spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of Hydrogenobaculum spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature. PMID:17220248

Mathur, Jayanti; Bizzoco, Richard W.; Ellis, Dean G.; Lipson, David A.; Poole, Alexander W.; Levine, Richard; Kelley, Scott T.

2007-01-01

318

Molecular Kinetics of Reviving Bacterial Spores  

PubMed Central

Bacterial spores can remain dormant for years, yet they possess a remarkable potential to rapidly resume a vegetative life form. Here, we identified a distinct phase at the onset of spore outgrowth, designated the ripening period. This transition phase is exploited by the germinating spore for molecular reorganization toward elongation and subsequent cell division. We have previously shown that spores of different ages, kept under various temperatures, harbor dissimilar molecular reservoirs (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139–149, 2012). Utilizing this phenomenon, we observed that the length of the ripening period can vary according to the spore molecular content. Importantly, the duration of the ripening period was found to correlate with the initial spore rRNA content and the kinetics of rRNA accumulation upon exiting dormancy. Further, the synthesis of the ribosomal protein RplA and the degradation of the spore-specific protein SspA also correlated with the duration of the ripening period. Our data suggest that the spore molecular cargo determines the extent of the ripening period, a potentially crucial phase for a germinating spore in obtaining limited resources during revival. PMID:23417486

Segev, Einat; Rosenberg, Alex; Mamou, Gideon; Sinai, Lior

2013-01-01

319

Phylogeny of theMycoplasma mycoidesCluster as Determined by Sequence Analysis of the 16S rRNA Genes from the Two rRNA Operons  

Microsoft Academic Search

The so-calledMycoplasma mycoidescluster consists of six species or subspecies of mycoplasmas (Mollicutes). These species are pathogenic for ruminants and some of them are of great concern in veterinary medicine. The members of theM. mycoidescluster have two rRNA operons (rrnAandrrnB). The nucleotide sequences of the 16S rRNA genes of 10 strains, representing all of the known species and subspecies of theM.

BERTIL PETTERSSON; THOMAS LEITNER; MOSTAFA RONAGHI; GORAN BOLSKE; MATHIAS UHLEN; ANDKARL-ERIK JOHANSSON

1996-01-01

320

Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens  

PubMed Central

Background The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean® Fecal DNA Isolation Kit, M; QIAamp® DNA Stool Mini Kit, Q; FastDNA® SPIN Kit, FSp; FastDNA® SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles. Method Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons. Results Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences. Conclusion We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial community fingerprints using PCR-DGGE technique. Subsequently, PCR-DGGE technique can be applied for studying variations in human intestinal microbial communities. PMID:20492702

2010-01-01

321

Identification of bacterial infection in neotropical primates.  

PubMed

Emerging infectious diseases usually arise from wild animal populations. In the present work, we performed a screening for bacterial infection in natural populations of New World primates. The blood cell bulk DNAs from 181 individuals of four Platyrrhini genera were PCR screened for eubacterial 16S rRNA genes. Bacteria were detected and identified in 13 distinct individuals of Alouatta belzebul, Alouatta caraya, and Cebus apella monkeys from geographically distant regions in the states of Mato Grosso and Pará, Brazil. Sequence analyses showed that these Platyrrhini bacteria are closely related not only to human pathogens Pseudomonas spp. but also to Pseudomonas simiae and sheep-Acari infecting Pseudomonas spp. The identified Pseudomonas possibly represents a group of bacteria circulating in natural monkey populations. PMID:23797292

Menezes-Costa, Andre; Machado-Ferreira, Erik; Voloch, Carolina M; Bonvicino, Cibele R; Seuánez, Hector N; Leoncini, Orilio; Soares, Carlos A G

2013-08-01

322

Seasonal and Spatial Variability of Bacterial and Archaeal Assemblages in the Coastal Waters near Anvers Island, Antarctica  

Microsoft Academic Search

A previous report of high levels of members of the domain Archaea in Antarctic coastal waters prompted us to investigate the ecology of Antarctic planktonic prokaryotes. rRNA hybridization techniques and denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial V3 region were used to study variation in Antarctic picoplankton assemblages. In Anvers Island nearshore waters during late winter to early

A. E. MURRAY; C. M. PRESTON; R. MASSANA; L. T. TAYLOR; A. BLAKIS; K. WU; E. F. DELONG

1998-01-01

323

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains.  

PubMed

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species. PMID:17241238

Safronova, Vera; Chizhevskaya, Elena; Bullitta, Simonetta; Andronov, Evgeny; Belimov, Andrei; Charles, Trevor C; Lindström, Kristina

2007-04-01

324

Molecular epidemiology of Theileria annulata and identification of 18S rRNA gene and ITS regions sequences variants in apparently healthy buffaloes and cattle in Pakistan.  

PubMed

A molecular epidemiological survey was conducted to determine the prevalence of piroplasms in buffaloes and cattle from Sheikhupura and Okara districts of Punjab, Pakistan using reverse line blot (RLB) hybridization assay. The genetic diversity within 18S rRNA gene and ITS regions sequences of various obtained Theileria species (spp.) was also investigated. Briefly, 102 blood samples from buffaloes and cattle in the study districts were collected on blood collection cards and brought to the laboratory. DNA was extracted; the V4 hypervariable region of 18S rRNA was amplified and analyzed using RLB. Out of total samples analyzed, 61 (59.8%) were hybridized with Babesia/Theileria (B/T) genus-specific probe. Only one species of piroplasm was detected in buffaloes and cattle in study districts, i.e. Theileria (T.) annulata. Six samples only hybridized with B/T genus-specific and Theileria genus-specific probes but not with any species-specific probe indicating the presence of novel species or variants. The sequences of 18S rRNA gene and ITS regions of these six samples revealed the presence of T. annulata variants as confirmed through sequence identity estimation and phylogenetic analyses. Meanwhile, an unexpected sequence variation was observed within the 18S rRNA gene and ITS regions sequences of T. annulata identified in the present study. This is the first report on the simultaneous detection of species of piroplasms infecting buffaloes and cattle in Pakistan and molecular characterization of T. annulata 18S rRNA gene and ITS regions. The present study may address the new insights into the epidemiology of theileriosis which will help researches in designing control strategies and developing various molecular diagnostic tools at national level. PMID:23059196

Khan, Muhammad Kasib; He, Lan; Hussain, Altaf; Azam, Sabita; Zhang, Wen-Jie; Wang, Li-Xia; Zhang, Qing-Li; Hu, Min; Zhou, Yan-Qin; Zhao, Junlong

2013-01-01

325

Detection and identification of putative bacterial endosymbionts and endogenous viruses in tick cell lines?  

PubMed Central

As well as being vectors of many viral, bacterial, and protozoan pathogens of medical and veterinary importance, ticks harbour a variety of microorganisms which are not known to be pathogenic for vertebrate hosts. Continuous cell lines established from ixodid and argasid ticks could be infected with such endosymbiotic bacteria and endogenous viruses, but to date very few cell lines have been examined for their presence. DNA and RNA extracted from over 50 tick cell lines deposited in the Roslin Wellcome Trust Tick Cell Biobank (http://tickcells.roslin.ac.uk) were screened for presence of bacteria and RNA viruses, respectively. Sequencing of PCR products amplified using pan-16S rRNA primers revealed the presence of DNA sequences from bacterial endosymbionts in several cell lines derived from Amblyomma and Dermacentor spp. ticks. Identification to species level was attempted using Rickettsia- and Francisella-specific primers. Pan-Nairovirus primers amplified PCR products of uncertain specificity in cell lines derived from Rhipicephalus, Hyalomma, Ixodes, Carios, and Ornithodoros spp. ticks. Further characterisation attempted with primers specific for Crimean-Congo haemorrhagic fever virus segments confirmed the absence of this arbovirus in the cells. A set of pan-Flavivirus primers did not detect endogenous viruses in any of the cell lines. Transmission electron microscopy revealed the presence of endogenous reovirus-like viruses in many of the cell lines; only 4 of these lines gave positive results with primers specific for the tick Orbivirus St Croix River virus, indicating that there may be additional, as yet undescribed ‘tick-only’ viruses inhabiting tick cell lines. PMID:22743047

Alberdi, M. Pilar; Dalby, Matthew J.; Rodriguez-Andres, Julio; Fazakerley, John K.; Kohl, Alain; Bell-Sakyi, Lesley

2012-01-01

326

Use of Stable-Isotope Probing, Full-Cycle rRNA Analysis, and Fluorescence In Situ Hybridization-Microautoradiography To Study a Methanol-Fed Denitrifying Microbial Community  

PubMed Central

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO3?-N mg of mixed-liquor volatile suspended solids (MLVSS)?1 h?1 to a steady-state value of 0.06 mg of NO3?-N mg of MLVSS?1 h?1 over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [13C]methanol to biomark the DNA of the denitrifiers. The extracted [13C]DNA and [12C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [13C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [12C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [14C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges. PMID:14711691

Ginige, Maneesha P.; Hugenholtz, Philip; Daims, Holger; Wagner, Michael; Keller, Jurg; Blackall, Linda L.

2004-01-01

327

Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase  

PubMed Central

The ‘radical S-adenosyl-L-methionine (AdoMet)’ enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (? 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage. PMID:23861844

Challand, Martin R.; Salvadori, Enrico; Driesener, Rebecca C.; Kay, Christopher W. M.; Roach, Peter L.; Spencer, James

2013-01-01

328

Specific Detection of Pasteurella multocida in Chickens with Fowl Cholera and in Pig Lung Tissues Using Fluorescent rRNA In Situ Hybridization  

PubMed Central

A Pasteurella multocida species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated P. multocida from other bacterial species of the families Pasteurellaceae and Enterobacteriaceae and also from divergent species of the order Cytophagales (except biovar 2 strains of Pasteurella avium and Pasteurella canis, which have high 16S rRNA similarity to P. multocida). The potential of the probe for specific identification and differentiation of P. multocida was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected P. multocida was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida. PMID:11427580

Mbuthia, Paul Gichohi; Christensen, Henrik; Boye, Mette; Petersen, Kamille Majken Dumong; Bisgaard, Magne; Nyaga, Phillip Njeru; Olsen, John Elmerdahl

2001-01-01

329

Direct 16S rRNA Gene Sequencing from Clinical Specimens, with Special Focus on Polybacterial Samples and Interpretation of Mixed DNA Chromatograms ?  

PubMed Central

RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms. It opens the possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from polybacterial human clinical samples. In this study, we used direct 16S rRNA gene sequencing to investigate 264 samples from a wide range of suspected human bacterial infections. The sequence-based identification was compared with the results from routine culture-based identification. A total of 151 samples were positive by the first PCR, producing 85 pure and 66 mixed DNA chromatograms. All mixed chromatograms were analyzed by RipSeq, although seven were so complex that only the dominant bacterial sequences could be identified. In general, sequence-based identification detected a larger number of species than did culture for samples from patients who had received antibiotics prior to sample collection and for samples containing anaerobic bacteria. RipSeq made it possible to apply this supplementary diagnostic tool to typical polybacterial specimens, such as internal abscesses, pleural fluids, and bile. PMID:19741089

Kommedal, ?yvind; Kvello, Kristine; Skjastad, Rune; Langeland, Nina; Wiker, Harald G.

2009-01-01

330

Application of SmartGene IDNS Software to Partial 16S rRNA Gene Sequences for a Diverse Group of Bacteria in a Clinical Laboratory?  

PubMed Central

Laboratories often receive clinical isolates for bacterial identification that have ambiguous biochemical profiles by conventional testing. With the emergence of 16S rRNA gene sequencing as an identification tool, we evaluated the usefulness of SmartGene IDNS, a 16S rRNA sequence database and software program for microbial identification. Identification by conventional methods of a diverse group of bacterial clinical isolates was compared with gene sequences interrogated by the SmartGene and MicroSeq databases. Of 300 isolates, SmartGene identified 295 (98%) to the genus level and 262 (87%) to the species level, with 5 (2%) being inconclusive. MicroSeq identified 271 (90%) to the genus level and 223 (74%) to the species level, with 29 (10%) being inconclusive. SmartGene and MicroSeq agreed on the genus for 233 (78%) isolates and the species for 212 (71%) isolates. Conventional methods identified 291 (97%) isolates to the genus level and 208 (69%) to the species level, with 9 (3%) being inconclusive. SmartGene, MicroSeq, and conventional identifications agreed for 193 (64%) of the results. Twenty-seven microorganisms were not represented in MicroSeq, compared to only 2 not represented in SmartGene. Overall, SmartGene IDNS provides comprehensive and accurate identification of a diverse group of bacteria and has the added benefit of being a user-friendly program that can be modified to meet the unique needs of clinical laboratories. PMID:17050811

Simmon, Keith E.; Croft, Ann C.; Petti, Cathy A.

2006-01-01

331

Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in-vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis.  

PubMed

Summary Drug resistance in Mycobacteriumtuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug-resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in M. smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A-site of 16S rRNA that (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem-loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug-resistant tuberculosis disease. PMID:20545852

Shcherbakov, Dmitri; Akbergenov, Rashid; Matt, Tanja; Sander, Peter; Andersson, Dan I; Böttger, Erik C

2010-06-01

332

A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2  

PubMed Central

Background Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR. Methods Control (n?=?16) and COPD GOLD 2 (n?=?16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue. Results There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05). Conclusion Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay. PMID:25329701

Sze, Marc A.; Abbasi, Meysam; Hogg, James C.; Sin, Don D.

2014-01-01

333

Plaque Bacterial Microbiome Diversity in Children Younger than 30 Months with or without Caries Prior to Eruption of Second Primary Molars  

PubMed Central

Objective Our primary objective is to phylogenetically characterize the supragingival plaque bacterial microbiome of children prior to eruption of second primary molars by pyrosequencing method for studying etiology of early childhood caries. Methods Supragingival plaque samples were collected from 10 caries children and 9 caries-free children. Plaque DNA was extracted, used to generate DNA amplicons of the V1–V3 hypervariable region of the bacterial 16S rRNA gene, and subjected to 454-pyrosequencing. Results On average, over 22,000 sequences per sample were generated. High bacterial diversity was noted in the plaque of children with caries [170 operational taxonomical units (OTU) at 3% divergence] and caries-free children (201 OTU at 3% divergence) with no significant difference. A total of 8 phyla, 15 classes, 21 orders, 30 families, 41 genera and 99 species were represented. In addition, five predominant phyla (Firmicute, Fusobacteria, Proteobacteria, Bacteroidetes and Actinobacteria) and seven genera (Leptotrichia, Streptococcus, Actinomyces, Prevotella, Porphyromonas, Neisseria, and Veillonella) constituted a majority of contents of the total microbiota, independent of the presence or absence of caries. Principal Component Analysis (PCA) presented that caries-related genera included Streptococcus and Veillonella; while Leptotrichia, Selenomonas, Fusobacterium, Capnocytophaga and Porphyromonas were more related to the caries-free samples. Neisseria and Prevotella presented approximately in between. In both groups, the degree of shared organism lineages (as defined by species-level OTUs) among individual supragingival plaque microbiomes was minimal. Conclusion Our study represented for the first time using pyrosequencing to elucidate and monitor supragingival plaque bacterial diversity at such young age with second primary molar unerrupted. Distinctions were revealed between caries and caries-free microbiomes in terms of microbial community structure. We observed differences in abundance for several microbial groups between the caries and caries-free host populations, which were consistent with the ecological plaque hypothesis. Our approach and findings could be extended to correlating microbiomic changes after occlusion establishment and caries treatment. PMID:24586647

Xu, He; Hao, Wenjing; Zhou, Qiong; Wang, Wenhong; Xia, Zhongkui; Liu, Chuan; Chen, Xiaochi; Qin, Man; Chen, Feng

2014-01-01

334

Biodecolorization of Reactive Black-5 by a metal and salt tolerant bacterial strain Pseudomonas sp. RA20 isolated from Paharang drain effluents in Pakistan.  

PubMed

Discharge of untreated azo dyes contaminated textile wastewater into soil and water bodies causes severe contamination. The present study was conducted to isolate dye degrading bacterial strains from a textile industry wastewater carrying drain in the neighborhood of Faisalabad, Pakistan. Seventy six bacterial strains were initially isolated and was screened using liquid mineral salts medium spiked with Reactive Black-5 azo dye. The strain RA20 was found to be the most efficient azo dye degrading bacterial isolate and was identified by amplifying and sequencing its 16S rRNA. Analysis indicated that this strain belonged to genus Pseudomonas and was designated as Pseudomonas sp. RA20. It had the highest decolorization activity at pH 8 and 25 °C incubation temperature under static conditions using yeast extract as an additional C source. This strain was also effective in decolorizing structurally related other reactive dyes including Reactive Orange 16, Reactive Yellow 2 and Reactive Red 120 but with varying efficacy. RA20 decolorized Reactive Black-5 significantly in the presence of up to 30 g L?¹ NaCl; however, the decolorization rate was significantly (p?0.05) reduced beyond this salt concentration. Moreover, this bacterial strain also exhibited moderate tolerance to different heavy metals including zinc (Zn), cadmium (Cd), chromium (Cr), lead (Pb) and copper (Cu). RA20 also decolorized Reactive Black-5 in the presence of a mixture of the selected heavy metals depending upon their concentrations. This study highlights the importance of Pseudomonas sp. RA20 as a prospective biological resource for bioremediation of water and soils contaminated with azo dyes. PMID:24138897

Hussain, Sabir; Maqbool, Zahid; Ali, Shafaqat; Yasmeen, Tahira; Imran, Muhammad; Mahmood, Faisal; Abbas, Farhat

2013-12-01

335

Targeting Species-Specific Low-Affinity 16S rRNA Binding Sites by Using Peptide Nucleic Acids for Detection of Legionellae in Biofilms  

PubMed Central

Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state. PMID:16885298

Wilks, Sandra A.; Keevil, C. William

2006-01-01

336

Evidence for the Presence of 5S rRNA in Mammalian Mitochondria  

PubMed Central

Mammalian mitochondrial ribosomes contain two prokaryotic-like rRNAs, 12S and 16S, both encoded by mitochondrial DNA. As opposed to cytosolic ribosomes, however, these ribosomes are not thought to contain 5S rRNA. For this reason, it has been unclear whether 5S rRNA, which can be detected in mitochondrial preparations, is an authentic organellar species imported from the cytosol or is merely a copurifying cytosol-derived contaminant. We now show that 5S rRNA is tightly associated with highly purified mitochondrial fractions of human and rat cells and that 5S rRNA transcripts derived from a synthetic gene transfected transiently into human cells are both expressed in vivo and present in highly purified mitochondria and mitoplasts. We conclude that 5S rRNA is imported into mammalian mitochondria, but its function there still remains to be clarified. PMID:9725900

Magalhaes, Paolo J.; Andreu, Antonio L.; Schon, Eric A.

1998-01-01

337

PyroTRF-ID: a novel bioinformatics methodology for the affiliation of terminal-restriction fragments using 16S rRNA gene pyrosequencing data  

PubMed Central

Background In molecular microbial ecology, massive sequencing is gradually replacing classical fingerprinting techniques such as terminal-restriction fragment length polymorphism (T-RFLP) combined with cloning-sequencing for the characterization of microbiomes. Here, a bioinformatics methodology for pyrosequencing-based T-RF identification (PyroTRF-ID) was developed to combine pyrosequencing and T-RFLP approaches for the description of microbial communities. The strength of this methodology relies on the identification of T-RFs by comparison of experimental and digital T-RFLP profiles obtained from the same samples. DNA extracts were subjected to amplification of the 16S rRNA gene pool, T-RFLP with the HaeIII restriction enzyme, 454 tag encoded FLX amplicon pyrosequencing, and PyroTRF-ID analysis. Digital T-RFLP profiles were generated from the denoised full pyrosequencing datasets, and the sequences contributing to each digital T-RF were classified to taxonomic bins using the Greengenes reference database. The method was tested both on bacterial communities found in chloroethene-contaminated groundwater samples and in aerobic granular sludge biofilms originating from wastewater treatment systems. Results PyroTRF-ID was efficient for high-throughput mapping and digital T-RFLP profiling of pyrosequencing datasets. After denoising, a dataset comprising ca. 10?000 reads of 300 to 500 bp was typically processed within ca. 20 minutes on a high-performance computing cluster, running on a Linux-related CentOS 5.5 operating system, enabling parallel processing of multiple samples. Both digital and experimental T-RFLP profiles were aligned with maximum cross-correlation coefficients of 0.71 and 0.92 for high- and low-complexity environments, respectively. On average, 63±18% of all experimental T-RFs (30 to 93 peaks per sample) were affiliated to phylotypes. Conclusions PyroTRF-ID profits from complementary advantages of pyrosequencing and T-RFLP and is particularly adapted for optimizing laboratory and computational efforts to describe microbial communities and their dynamics in any biological system. The high resolution of the microbial community composition is provided by pyrosequencing, which can be performed on a restricted set of selected samples, whereas T-RFLP enables simultaneous fingerprinting of numerous samples at relatively low cost and is especially adapted for routine analysis and follow-up of microbial communities on the long run. PMID:23270314

2012-01-01

338

Characteristic archaebacterial 16S rRNA oligonucleotides  

NASA Technical Reports Server (NTRS)

A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

1986-01-01

339

Impacts of Poultry House Environment on Poultry Litter Bacterial Community Composition  

PubMed Central

Viral and bacterial pathogens are a significant economic concern to the US broiler industry and the ecological epicenter for poultry pathogens is the mixture of bedding material, chicken excrement and feathers that comprises the litter of a poultry house. This study used high-throughput sequencing to assess the richness and diversity of poultry litter bacterial communities, and to look for connections between these communities and the environmental characteristics of a poultry house including its history of gangrenous dermatitis (GD). Cluster analysis of 16S rRNA gene sequences revealed differences in the distribution of bacterial phylotypes between Wet and Dry litter samples and between houses. Wet litter contained greater diversity with 90% of total bacterial abundance occurring within the top 214 OTU clusters. In contrast, only 50 clusters accounted for 90% of Dry litter bacterial abundance. The sixth largest OTU cluster across all samples classified as an Arcobacter sp., an emerging human pathogen, occurring in only the Wet litter samples of a house with a modern evaporative cooling system. Ironically, the primary pathogenic clostridial and staphylococcal species associated with GD were not found in any house; however, there were thirteen 16S rRNA gene phylotypes of mostly Gram-positive phyla that were unique to GD-affected houses and primarily occurred in Wet litter samples. Overall, the poultry house environment appeared to substantially impact the composition of litter bacterial communities and may play a key role in the emergence of food-borne pathogens. PMID:21949751

Dumas, Michael D.; Polson, Shawn W.; Ritter, Don; Ravel, Jacques; Gelb, Jack; Morgan, Robin; Wommack, K. Eric

2011-01-01

340

Investigation of the Bacterial Communities Associated with Females of Lutzomyia Sand Fly Species from South America  

PubMed Central

Phlebotomine sand flies are vectors of Leishmania that are acquired by the female sand fly during blood feeding on an infected mammal. Leishmania parasites develop exclusively in the gut lumen during their residence in the insect before transmission to a suitable host during the next blood feed. Female phlebotomine sand flies are blood feeding insects but their life style of visiting plants as well as animals, and the propensity for larvae to feed on detritus including animal faeces means that the insect host and parasite are exposed to a range of microorganisms. Thus, the sand fly microbiota may interact with the developing Leishmania population in the gut. The aim of the study was to investigate and identify the bacterial diversity associated with wild adult female Lutzomyia sand flies from different geographical locations in the New World. The bacterial phylotypes recovered from 16S rRNA gene clone libraries obtained from wild caught adult female Lutzomyia sand flies were estimated from direct band sequencing after denaturing gradient gel electrophoresis of bacterial 16 rRNA gene fragments. These results confirm that the Lutzomyia sand flies contain a limited array of bacterial phylotypes across several divisions. Several potential plant-related bacterial sequences were detected including Erwinia sp. and putative Ralstonia sp. from two sand fly species sampled from 3 geographically separated regions in Brazil. Identification of putative human pathogens also demonstrated the potential for sand flies to act as vectors of bacterial pathogens of medical importance in addition to their role in Leishmania transmission. PMID:22880020

Sant'Anna, Mauricio R. V.; Darby, Alistair C.; Brazil, Reginaldo P.; Montoya-Lerma, James; Dillon, Viv M.; Bates, Paul A.; Dillon, Rod J.

2012-01-01

341

Mineral composition and charcoal determine the bacterial community structure in artificial soils.  

PubMed

To study the influence of the clay minerals montmorillonite (M) and illite (I), the metal oxides ferrihydrite (F) and aluminum hydroxide (A), and charcoal (C) on soil bacterial communities, seven artificial soils with identical texture provided by quartz (Q) were mixed with sterilized manure as organic carbon source before adding a microbial inoculant derived from a Cambisol. Bacterial communities established in artificial soils after 90 days of incubation were compared by DGGE analysis of bacterial and taxon-specific 16S rRNA gene amplicons. The bacterial community structure of charcoal-containing soils highly differed from the other soils at all taxonomic levels studied. Effects of montmorillonite and illite were observed for Bacteria and Betaproteobacteria, but not for Actinobacteria or Alphaproteobacteria. A weak influence of metal oxides on Betaproteobacteria was found. Barcoded pyrosequencing of 16S rRNA gene amplicons done for QM, QI, QIF, and QMC revealed a high bacterial diversity in the artificial soils. The composition of the artificial soils was different from the inoculant, and the structure of the bacterial communities established in QMC soil was most different from the other soils, suggesting that charcoal provided distinct microenvironments and biogeochemical interfaces formed. Several populations with discriminative relative abundance between artificial soils were identified. PMID:23289489

Ding, Guo-Chun; Pronk, Geertje Johanna; Babin, Doreen; Heuer, Holger; Heister, Katja; Kögel-Knabner, Ingrid; Smalla, Kornelia

2013-10-01

342

Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques.  

PubMed

Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34/38 (89.5%) of total bacterial numbers consisting of 12 genera and included Staphylococcus (most common genus isolated), Arthrobacter (2nd most common genus isolated), Brachybacterium, Exiguobacterium, Lactococcus, Microbacterium and Sporosarcina (next most common genera isolated) and finally, Bacillus, Brevibacterium, Frigoribacterium, Mycoplana and Pseudoclavibacter. Gram-negative organisms accounted for only 4/38 (10.5%) bacterial isolates and included the following genera, Brevundimonas, Lysobacter, Psychrobacter and Rhizobium. No gastrointestinal pathogens were detected. Although this study demonstrated a high diversity of the microorganisms present, only a few have been shown to be opportunistically pathogenic to humans and none of these organisms described have been described previously as having an inhalational route of infection and therefore we do not believe that the species of organisms identified pose a significant health and safety threat for immunocompetant individuals. PMID:20018377

Murayama, Mayumi; Kakinuma, Yuki; Maeda, Yasunori; Rao, Juluri R; Matsuda, Motoo; Xu, Jiru; Moore, Peter J A; Millar, B Cherie; Rooney, Paul J; Goldsmith, Colin E; Loughrey, Anne; McMahon, M Ann S; McDowell, David A; Moore, John E

2010-03-01

343

Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: Application to the rapid screening of 5S rRNA from ovarian cancer cells.  

PubMed

RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (Mavg=4,000,000Da) as a sieving matrix for total RNA separation. Ethidium bromide (?gmL(-1)) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100pg?L(-1) with a 6min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps. PMID:25261903

Shih, Ya-Chu; Liao, Ching-Ru; Chung, I-Che; Chang, Yu-Sun; Chang, Po-Ling

2014-10-17

344

Variation in 16S-23S rRNA Intergenic Spacer Regions in Photobacterium damselae: a Mosaic-Like Structure  

PubMed Central

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNAGlu(UUC), tRNALys(UUU), tRNAVal(UAC), and tRNAAla(GGC). Five amplicons contained tRNAGlu(UUC) combined with two additional tRNA genes, including tRNALys(UUU), tRNAVal(UAC), or tRNAAla(UGC). Five amplicons contained tRNAIle(GAU) and tRNAAla(UGC). Two amplicons contained tRNAGlu(UUC) and tRNAAla(UGC). Two different isoacceptor tRNAAla genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNAGlu(UUC)-tRNAVal(UAC)-tRNAAla(UGC) and tRNAGlu(UUC)-tRNAAla(UGC) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure. PMID:15691912

Osorio, Carlos R.; Collins, Matthew D.; Romalde, Jesus L.; Toranzo, Alicia E.

2005-01-01

345

Identification of pathogenic Nocardia species by reverse line blot hybridization targeting the 16S rRNA and 16S-23S rRNA gene spacer regions.  

PubMed

Although 16S rRNA gene sequence analysis is employed most often for the definitive identification of Nocardia species, alternate molecular methods and polymorphisms in other gene targets have also enabled species determinations. We evaluated a combined Nocardia PCR-based reverse line blot (RLB) hybridization assay based on 16S and 16S-23S rRNA gene spacer region polymorphisms to identify 12 American Type Culture Collection and 123 clinical Nocardia isolates representing 14 species; results were compared with results from 16S rRNA gene sequencing. Thirteen 16S rRNA gene-based (two group-specific and 11 species-specific) and five 16S-23S spacer-targeted (two taxon-specific and three species-specific) probes were utilized. 16S rRNA gene-based probes correctly identified 124 of 135 isolates (sensitivity, 92%) but were unable to identify Nocardia paucivorans strains (n = 10 strains) and a Nocardia asteroides isolate with a novel 16S rRNA gene sequence. Nocardia farcinica and Nocardia cyriacigeorgica strains were identified by the sequential use of an N. farcinica-"negative" probe and a combined N. farcinica/N. cyriacigeorgica probe. The assay specificity was high (99%) except for weak cross-reactivity between the Nocardia brasiliensis probe with the Nocardia thailandica DNA product; however, cross-hybridization with closely related nontarget species may occur. The incorporation of 16S-23S rRNA gene spacer-based probes enabled the identification of all N. paucivorans strains. The overall sensitivity using both probe sets was >99%. Both N. farcinica-specific 16S-23S rRNA gene spacer-directed probes were required to identify all N. farcinica stains by using this probe set. The study demonstrates the utility of a combined PCR/RLB assay for the identification of clinically relevant Nocardia species and its potential for studying subtypes of N. farcinica. Where species assignment is ambiguous or not possible, 16S rRNA gene sequencing is recommended. PMID:19955277

Xiao, Meng; Kong, Fanrong; Sorrell, Tania C; Cao, Yongyan; Lee, Ok Cha; Liu, Ying; Sintchenko, Vitali; Chen, Sharon C A

2010-02-01

346

Airborne Bacterial Communities in Residences: Similarities and Differences with Fungi  

PubMed Central

Genetic analysis of indoor air has uncovered a rich microbial presence, but rarely have both the bacterial and fungal components been examined in the same samples. Here we present a study that examined the bacterial component of passively settled microbes from both indoor and outdoor air over a discrete time period and for which the fungal component has already been reported. Dust was allowed to passively settle in five common locations around a home ? living room, bedroom, bathroom, kitchen, and balcony ? at different dwellings within a university-housing complex for a one-month period at two time points, once in summer and again in winter. We amplified the bacterial 16S rRNA gene in these samples and analyzed them with high-throughput sequencing. Like fungal OTU-richness, bacterial OTU-richness was higher outdoors then indoors and was invariant across different indoor room types. While fungal composition was structured largely by season and residential unit, bacterial composition varied by residential unit and room type. Bacteria from putative outdoor sources, such as Sphingomonas and Deinococcus, comprised a large percentage of the balcony samples, while human-associated taxa comprised a large percentage of the indoor samples. Abundant outdoor bacterial taxa were also observed indoors, but the reverse was not true; this is unlike fungi, in which the taxa abundant indoors were also well-represented outdoors. Moreover, there was a partial association of bacterial composition and geographic distance, such that samples separated by even a few hundred meters tended have greater compositional differences than samples closer together in space, a pattern also observed for fungi. These data show that while the outdoor source for indoor bacteria and fungi varies in both space and time, humans provide a strong and homogenizing effect on indoor bacterial bioaerosols, a pattern not observed in fungi. PMID:24603548

Adams, Rachel I.; Miletto, Marzia; Lindow, Steven E.; Taylor, John W.; Bruns, Thomas D.

2014-01-01

347

Demonstrating Bacterial Flagella.  

ERIC Educational Resources Information Center

Describes an effective laboratory method for demonstrating bacterial flagella that utilizes the Proteus mirabilis organism and a special harvesting technique. Includes safety considerations for the laboratory exercise. (MDH)

Porter, John R.; And Others

1992-01-01

348

Stability of a biogas-producing bacterial, archaeal and fungal community degrading food residues.  

PubMed

The resident microbiota was analyzed in a mesophilic, continuously operating biogas plant predominantly utilizing food residues, stale bread, and other waste cosubstrates together with pig manure and maize silage. The dominating bacterial, archaeal, and eukaryotic community members were characterized by two different 16S/18S rRNA gene culture-independent approaches. Prokaryotic 16S rRNA gene and eukaryotic 18S rRNA gene clone libraries were constructed and further analyzed by restriction fragment length polymorphism (RFLP), 16S/18S rRNA gene sequencing, and phylogenetic tree reconstruction. The most dominant bacteria belonged to the phyla Bacteriodetes, Chloroflexus, and Firmicutes. On the family level, the bacterial composition confirmed high differences among biogas plants studied so fare. In contrast, the methanogenic archaeal community was similar to that of other studied biogas plants. Furthermore, it was possible to identify fungi at the genus level, namely Saccharomyces and Mucor. Both genera, which are important for microbial degradation of complex compounds, were up to now not found in biogas plants. The results revealed their long-term presence as indicated by denaturating gradient gel electrophoresis (DGGE). The DGGE method confirmed that the main members of the microbial community were constantly present over more than one-year period. PMID:23228065

Bengelsdorf, Frank R; Gerischer, Ulrike; Langer, Susanne; Zak, Manuel; Kazda, Marian

2013-04-01

349

Evidence for successional development in Antarctic hypolithic bacterial communities.  

PubMed

Hypoliths (cryptic microbial assemblages that develop on the undersides of translucent rocks) are significant contributors to regional C and N budgets in both hot and cold deserts. Previous studies in the Dry Valleys of Eastern Antarctica have reported three morphologically distinct hypolithic community types: cyanobacteria dominated (type I), fungus dominated (type II) and moss dominated (type III). Here we present terminal-restriction fragment length polymorphism analyses to elucidate the bacterial community structure in hypolithons and the surrounding soils. We show clear and robust distinction in bacterial composition between bulk surface soils and hypolithons. Moreover, the bacterial assemblages were similar in types II and III hypolithons and clearly distinct from those found in type I. Through 16S rRNA gene 454 pyrosequencing, we show that Proteobacteria dominated all three types of hypolithic communities. As expected, Cyanobacteria were more abundant in type I hypolithons, whereas Actinobacteria were relatively more abundant in types II and III hypolithons, and were the dominant group in soils. Using a probabilistic dissimilarity metric and random sampling, we demonstrate that deterministic processes are more important in shaping the structure of the bacterial community found in types II and III hypolithons. Most notably, the data presented in this study suggest that hypolithic bacterial communities establish via a successional model, with the type I hypolithons acting as the basal development state. PMID:23765099

Makhalanyane, Thulani P; Valverde, Angel; Birkeland, Nils-Kåre; Cary, Stephen C; Tuffin, I Marla; Cowan, Don A

2013-11-01

350

New Perspective on Uncultured Bacterial Phylogenetic Division OP11  

PubMed Central

Organisms belonging to the OP11 candidate phylogenetic division of Bacteria have been detected only in rRNA-based sequence surveys of environmental samples. Preliminary studies indicated that such organisms represented by the sequences are abundant and widespread in nature and highly diverse phylogenetically. In order to document more thoroughly the phylogenetic breadth and environmental distribution of this diverse group of organisms, we conducted further molecular analyses on environmental DNAs. Using PCR techniques and primers directed toward each of the five described subdivisions of OP11, we surveyed 17 environmental DNAs and analyzed rRNA gene sequences in 27 clonal libraries from 14 environments. Ninety-nine new and unique sequences were determined completely, and approximately 200 additional clones were subjected to partial sequencing. Extensive phylogenetic comparisons of the new sequences to those representing other bacterial divisions further resolved the phylogeny of the bacterial candidate division OP11 and identified two new candidate bacterial divisions, OP11-derived 1 (OD1) and Sulphur River 1 (SR1). The widespread environmental distribution of representatives of the bacterial divisions OD1, OP11, and SR1 suggests potentially conspicuous biogeochemical roles for these organisms in their respective environments. The information on environmental distribution offers clues for attempts to culture landmark representatives of these novel bacterial divisions, and the sequences are specific molecular signatures that provide for their identification in other contexts. PMID:14766563

Harris, J. Kirk; Kelley, Scott T.; Pace, Norman R.

2004-01-01

351

Evolutionary dynamics of rRNA gene clusters in cichlid fish  

PubMed Central

Background Among multigene families, ribosomal RNA (rRNA) genes are the most frequently studied and have been explored as cytogenetic markers to study the evolutionary history of karyotypes among animals and plants. In this report, we applied cytogenetic and genomic methods to investigate the organization of rRNA genes among cichlid fishes. Cichlids are a group of fishes that are of increasing scientific interest due to their rapid and convergent adaptive radiation, which has led to extensive ecological diversity. Results The present paper reports the cytogenetic mapping of the 5S rRNA genes from 18 South American, 22 African and one Asian species and the 18S rRNA genes from 3 African species. The data obtained were comparatively analyzed with previously published information related to the mapping of rRNA genes in cichlids. The number of 5S rRNA clusters per diploid genome ranged from 2 to 15, with the most common pattern being the presence of 2 chromosomes bearing a 5S rDNA cluster. Regarding 18S rDNA mapping, the number of sites ranged from 2 to 6, with the most common pattern being the presence of 2 sites per diploid genome. Furthermore, searching the Oreochromis niloticus genome database led to the identification of a total of 59 copies of 5S rRNA and 38 copies of 18S rRNA genes that were distributed in several genomic scaffolds. The rRNA genes were frequently flanked by transposable elements (TEs) and spread throughout the genome, complementing the FISH analysis that detect only clustered copies of rRNA genes. Conclusions The organization of rRNA gene clusters seems to reflect their intense and particular evolutionary pathway and not the evolutionary history of the associated taxa. The possible role of TEs as one source of rRNA gene movement, that could generates the spreading of ribosomal clusters/copies, is discussed. The present paper reinforces the notion that the integration of cytogenetic data and genomic analysis provides a more complete picture for understanding the organization of repeated sequences in the genome. PMID:23035959

2012-01-01

352

Bacterial diversity in relation to secondary production and succession on surfaces of the kelp Laminaria hyperborea  

PubMed Central

Kelp forests worldwide are known as hotspots for macroscopic biodiversity and primary production, yet very little is known about the biodiversity and roles of microorganisms in these ecosystems. Secondary production by heterotrophic bacteria associated to kelp is important in the food web as a link between kelp primary production and kelp forest consumers. The aim of this study was to investigate the relationship between bacterial diversity and two important processes in this ecosystem; bacterial secondary production and primary succession on kelp surfaces. To address this, kelp, Laminaria hyperborea, from southwestern Norway was sampled at different geographical locations and during an annual cycle. Pyrosequencing (454-sequencing) of amplicons of the 16S rRNA gene of bacteria was used to study bacterial diversity. Incorporation of tritiated thymidine was used as a measure of bacterial production. Our data show that bacterial diversity (richness and evenness) increases with the age of the kelp surface, which corresponds to the primary succession of its bacterial communities. Higher evenness of bacterial operational taxonomical units (OTUs) is linked to higher bacterial production. Owing to the dominance of a few abundant OTUs, kelp surface biofilm communities may be characterized as low-diversity habitats. This is the first detailed study of kelp-associated bacterial communities using high-throughput sequencing and it extends current knowledge on microbial community assembly and dynamics on living surfaces. PMID:22763650

Bengtsson, Mia M; Sj?tun, Kjersti; Lanzen, Anders; ?vreas, Lise

2012-01-01

353

The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil  

Microsoft Academic Search

The cell content of 12 bacterial phospholipid fatty acids (PLFA) was determined in bacteria extracted from soil by homogenization\\/centrifugation. The bacteria were enumerated using acridine orange direct counts. An average of 1.40×10-17 mol bacterial PLFA cell-1 was found in bacteria extracted from 15 soils covering a wide range of pH and organic matter contents. With this factor, the bacterial biomass

A. Frostegård; E. Bååth

1996-01-01

354

Feature extraction Feature extraction  

E-print Network

(hyperspectral sensors) Meteosat thermal IR channel hyperspectral "image cube" #12;Raw intensities · ProsFeature extraction #12;Feature extraction · Image interpretation: extract information from images · but the desired information may not be explicit in the raw observed pixel intensities · Transform image to make

Giger, Christine

355

Feature extraction Feature extraction  

E-print Network

(hyperspectral sensors) Meteosat thermal IR channel hyperspectral "image cube" #12;Raw intensities ! � ProsFeature extraction #12;Feature extraction ! � Image interpretation: extract information from images � but the desired information may not be explicit in the raw observed pixel intensities � Transform image to make

Giger, Christine

356

Use of 16S rRNA, 23S rRNA, and gyrB Gene Sequence Analysis To Determine Phylogenetic Relationships of Bacillus cereus Group Microorganisms  

PubMed Central

In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group. PMID:15297521

Bavykin, Sergei G.; Lysov, Yuri P.; Zakhariev, Vladimir; Kelly, John J.; Jackman, Joany; Stahl, David A.; Cherni, Alexey

2004-01-01

357

Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group.  

SciTech Connect

In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.

Bayvkin, S. G.; Lysov, Y. P.; Zakhariev, V.; Kelly, J. J.; Jackman, J.; Stahl, D. A.; Cherni, A.; Engelhardt Inst. of Molecular Biology; Loyola Univ.; Johns Hopkins Univ.; Univ. of Washington

2004-08-01

358

Analysis of a change in bacterial community in different environments with addition of chitin or chitosan  

Microsoft Academic Search

The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands

Kazuaki Sato; Yasuhito Azama; Masahiro Nogawa; Goro Taguchi; Makoto Shimosaka

2010-01-01

359

Phylogenetic Analysis of Bacterial Communities in Mesophilic and Thermophilic Bioreactors Treating Pharmaceutical Wastewater  

Microsoft Academic Search

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32°C) and thermophilic (50 to 58°C) temperatures. Community fingerprint analysis by denaturing gradient gel electro- phoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that

TIMOTHY M. LAPARA; CINDY H. NAKATSU; LISA PANTEA; JAMES E. ALLEMAN

2000-01-01

360

Bile tolerant Lactobacillus reuteri isolated from pig feces inhibits enteric bacterial pathogens and porcine rotavirus  

Microsoft Academic Search

Lactic acid producing bacterial strain Probio-16 was isolated from the swine excrements under anaerobic conditions and characterized\\u000a by morphology and biochemical characteristics. The strain was further identified by 16S rRNA gene sequencing and phylogeneitc\\u000a analysis. The antimicrobial activity of the strain was assayed by testing for growth inhibition of thirteen pathogenic microorganisms.\\u000a The strain was tested for antiviral activity against

Byeong Joo Seo; Mi Ran Mun; Rejish Kumar V. J; Chul-Joong Kim; Insun Lee; Young-Hyo Chang; Yong-Ha Park

2010-01-01

361

Endogeic earthworms differentially influence bacterial communities associated with different soil aggregate size fractions  

Microsoft Academic Search

Endogeic earthworm activities can strongly influence soil structure. Although soil microorganisms are thought to be central to earthworm-facilitated aggregate formation, how and where within the soil matrix earthworm-facilitated influences on soil microbial communities are manifested is poorly defined. In this study we used 16S rRNA gene-based terminal restriction fragment polymorphism (T-RFLP) analyses to examine bacterial communities associated with different aggregate

Daniel L. Mummey; Matthias C. Rillig; Johan Six

2006-01-01

362

Endophytic Bacterial Communities in Ginseng and their Antifungal Activity Against Pathogens  

Microsoft Academic Search

Plant roots are associated with diverse communities of endophytic bacteria which do not exert adverse effects. The diversity\\u000a of bacterial endophytes associated with ginseng roots cultivated in three different areas in Korea was investigated. Sixty-three\\u000a colonies were isolated from the interior of ginseng roots. Phylogenetic analysis based on 16S rRNA gene sequences showed that\\u000a the isolates belonged to three major

Kye Man Cho; Su Young Hong; Sun Mi Lee; Yong Hee Kim; Goon Gjung Kahng; Yong Pyo Lim; Hoon Kim; Han Dae Yun

2007-01-01<