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1

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

PubMed Central

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

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

2014-01-01

2

Affinity capillary electrophoresis with magnetic beads for multiplex quantitative analysis of bacterial 16S rRNA  

Microsoft Academic Search

We have developed a novel method for microbial community analysis of bacterial 16S rRNAs based on affinity capillary electrophoresis using 16S rRNA-conjugated magnetic beads. We called this method magnetic beads affinity capillary electrophoresis (MB-ACE) which can be used for sequential and quantitative analysis of 16S rRNA. In this method, RNA extracted from a microbial community is biotin-modified and mixed with

Ken Adachi; Masahiro Yamaguchi; Makoto Nakashige; Takahiro Kanagawa; Masaki Torimura; Satoshi Tsuneda; Yuji Sekiguchi; Naohiro Noda

2009-01-01

3

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

PubMed Central

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 bacterial isolates and 801 16S rRNA genes amplified from extracted soil DNA. Phylotypes were defined by performing a restriction fragment length polymorphism analysis of 16S rRNA gene sequences with the enzymes RsaI and BstUI. The average level of 16S rRNA gene sequence similarity of members of a phylotype was 86.6% based on an analysis of partial sequences. A total of 498 phylotypes were identified among the 16S ribosomal DNA (rDNA) clones, while 34 phylotypes occurred among the cultivated isolates. Analysis of sequences from a subset of the phylotypes showed that at least seven bacterial divisions were represented in the clone libraries, whereas the isolates represented only three. The phylotype richness, frequency distribution (evenness), and composition of the four culture collections and the four clone libraries were investigated by using a variety of diversity indices. Although cultivation and 16S rRNA cloning analyses gave contradictory descriptions of the relative phylotype richness for one of the four environments, the two methods identified qualitatively consistent relationships when levels of evenness were compared. The levels of phylotype similarity between communities were uniformly low (15 to 31%). Both methods consistently indicated that one environment was distinct from the other three. Our data illustrate that while 16S rDNA cloning and cultivation generally describe similar relationships between soil microbial communities, significant discrepancies can occur. PMID:10103265

Dunbar, John; Takala, Shannon; Barns, Susan M.; Davis, Jody A.; Kuske, Cheryl R.

1999-01-01

4

Bacterial metabarcoding by 16S rRNA gene ion torrent amplicon sequencing.  

PubMed

Ion Torrent is a next generation sequencing technology based on the detection of hydrogen ions produced during DNA chain elongation; this technology allows analyzing and characterizing genomes, genes, and species. Here, we describe an Ion Torrent procedure applied to the metagenomic analysis of 16S rRNA gene amplicons to study the bacterial diversity in food and environmental samples. PMID:25343859

Fantini, Elio; Gianese, Giulio; Giuliano, Giovanni; Fiore, Alessia

2015-01-01

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

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 ...

7

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 ...

8

Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis  

E-print Network

Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis Anita- chemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected and high pH.13-15 Careful decontami- nation together with assay standardization or automation can mitigate

Santiago, Juan G.

9

Spatiotemporal Analysis of Bacterial Diversity in Sediments of Sundarbans Using Parallel 16S rRNA Gene Tag Sequencing.  

PubMed

The influence of temporal and spatial variations on the microbial community composition was assessed in the unique coastal mangrove of Sundarbans using parallel 16S rRNA gene pyrosequencing. The total sediment DNA was extracted and subjected to the 16S rRNA gene pyrosequencing, which resulted in 117 Mbp of data from three experimental stations. The taxonomic analysis of the pyrosequencing data was grouped into 24 different phyla. In general, Proteobacteria were the most dominant phyla with predominance of Deltaproteobacteria, Alphaproteobacteria, and Gammaproteobacteria within the sediments. Besides Proteobacteria, there are a number of sequences affiliated to the following major phyla detected in all three stations in both the sampling seasons: Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Chloroflexi, Cyanobacteria, Nitrospira, and Firmicutes. Further taxonomic analysis revealed abundance of micro-aerophilic and anaerobic microbial population in the surface layers, suggesting anaerobic nature of the sediments in Sundarbans. The results of this study add valuable information about the composition of microbial communities in Sundarbans mangrove and shed light on possible transformations promoted by bacterial communities in the sediments. PMID:25256302

Basak, Pijush; Majumder, Niladri Shekhar; Nag, Sudip; Bhattacharyya, Anish; Roy, Debojyoti; Chakraborty, Arpita; SenGupta, Sohan; Roy, Arunava; Mukherjee, Arghya; Pattanayak, Rudradip; Ghosh, Abhrajyoti; Chattopadhyay, Dhrubajyoti; Bhattacharyya, Maitree

2014-09-26

10

Analysis of transduction in wastewater bacterial populations by targeting the phage-derived 16S rRNA gene sequences.  

PubMed

Bacterial 16S rRNA genes transduced by bacteriophages were identified and analyzed in order to estimate the extent of the bacteriophage-mediated horizontal gene transfer in the wastewater environment. For this purpose, phage and bacterial DNA was isolated from the oxidation tank of a municipal wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequences cloned from a phage metagenome revealed that bacteriophages transduce genetic material in several major groups of bacteria. The groups identified were as follows: Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Actinomycetales and Firmicutes. Analysis of the 16S rRNA gene sequences in the total bacterial DNA from the same sample revealed that several bacterial groups found in the oxidation tank were not present in the phage metagenome (e.g. Deltaproteobacteria, Nitrospira, Planctomycetes and many Actinobacteria genera). These results suggest that transduction in a wastewater environment occurs in several bacterial groups; however, not all species are equally involved into this process. The data also showed that a number of distinctive bacterial strains participate in transduction-mediated gene transfer within identified bacterial groupings. Denaturing gradient gel electrophoresis analysis confirmed that profiles of the transduced 16S rRNA gene sequences and those present in the whole microbial community show significant differences. PMID:21223328

Del Casale, Antonio; Flanagan, Paul V; Larkin, Michael J; Allen, Christopher C R; Kulakov, Leonid A

2011-04-01

11

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

12

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

13

Influence of DNA extraction on oral microbial profiles obtained via 16S rRNA gene sequencing  

PubMed Central

Background and objective The advent of next-generation sequencing has significantly facilitated characterization of the oral microbiome. Despite great efforts in streamlining the processes of sequencing and data curation, upstream steps required for amplicon library generation could still influence 16S rRNA gene-based microbial profiles. Among upstream processes, DNA extraction is a critical step that could represent a great source of bias. Accounting for bias introduced by extraction procedures is important when comparing studies that use different methods. Identifying the method that best portrays communities is also desirable. Accordingly, the aim of this study was to evaluate bias introduced by different DNA extraction procedures on oral microbiome profiles. Design Four DNA extraction methods were tested on mock communities consisting of seven representative oral bacteria. Additionally, supragingival plaque samples were collected from seven individuals and divided equally to test two commonly used DNA extraction procedures. Amplicon libraries of the 16S rRNA gene were generated and sequenced via 454-pyrosequencing. Results Evaluation of mock communities revealed that DNA yield and bacterial species representation varied with DNA extraction methods. Despite producing the lowest yield of DNA, a method that included bead beating was the only protocol capable of detecting all seven species in the mock community. Comparison of the performance of two commonly used methods (crude lysis and a chemical/enzymatic lysis+column-based DNA isolation) on plaque samples showed no effect of extraction protocols on taxa prevalence but global community structure and relative abundance of individual taxa were affected. At the phylum level, the latter method improved the recovery of Actinobacteria, Bacteroidetes, and Spirochaetes over crude lysis. Conclusion DNA extraction distorts microbial profiles in simulated and clinical oral samples, reinforcing the importance of careful selection of a DNA extraction protocol to improve species recovery and facilitate data comparison across oral microbiology studies. PMID:24778776

Abusleme, Loreto; Hong, Bo-Young; Dupuy, Amanda K.; Strausbaugh, Linda D.; Diaz, Patricia I.

2014-01-01

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

Extent and variation of phage-borne bacterial 16S rRNA gene sequences in wastewater environments.  

PubMed

Phage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains of Proteobacteria, Bacteroidetes, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community. PMID:21666016

Del Casale, Antonio; Flanagan, Paul V; Larkin, Michael J; Allen, Christopher C R; Kulakov, Leonid A

2011-08-01

16

Extent and Variation of Phage-Borne Bacterial 16S rRNA Gene Sequences in Wastewater Environments ?  

PubMed Central

Phage metagenomes isolated from wastewater over a 12-month period were analyzed. The results suggested that various strains of Proteobacteria, Bacteroidetes, and other phyla are likely to participate in transduction. The patterns of 16S rRNA sequences found in phage metagenomes did not follow changes in the total bacterial community. PMID:21666016

Del Casale, Antonio; Flanagan, Paul V.; Larkin, Michael J.; Allen, Christopher C. R.; Kulakov, Leonid A.

2011-01-01

17

Distinct Ectomycorrhizospheres Share Similar Bacterial Communities as Revealed by Pyrosequencing-Based Analysis of 16S rRNA Genes  

PubMed Central

Analysis of the 16S rRNA gene sequences generated from Xerocomus pruinatus and Scleroderma citrinum ectomycorrhizospheres revealed that similar bacterial communities inhabited the two ectomycorrhizospheres in terms of phyla and genera, with an enrichment of the Burkholderia genus. Compared to the bulk soil habitat, ectomycorrhizospheres hosted significantly more Alpha-, Beta-, and Gammaproteobacteria. PMID:22307291

Oger, P.; Morin, E.; Frey-Klett, P.

2012-01-01

18

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

PubMed Central

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

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

1998-01-01

19

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

PubMed Central

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

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

2013-01-01

20

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

PubMed

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

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

2014-12-01

21

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

PubMed Central

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

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

2014-01-01

22

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

23

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

PubMed Central

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

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

2015-01-01

24

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

EPA Science Inventory

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

25

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

PubMed

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

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

2012-11-01

26

Macroalgal Extracts Induce Bacterial Assemblage Shifts and Sublethal Tissue Stress in Caribbean Corals  

PubMed Central

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

27

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; Höltje, Claudia; Westphal, Petra; Bötel, Julia; Brettar, Ingrid; Mehling, Arndt; Höfle, Manfred G.

2006-01-01

28

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

29

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

30

Bacterial community composition of anthropogenic biochar and Amazonian anthrosols assessed by 16S rRNA gene 454 pyrosequencing.  

PubMed

Biochar (BC) is a common minor constituent of soils and is usually derived from the burning of wood materials. In the case of Amazonian dark earth (ADE) soils, the increased amount of this material is believed to be due to anthropogenic action by ancient indigenous populations. In this study, we use 16S rRNA gene pyrosequencing to assess the bacterial diversity observed in the BC found in ADEs as well as in the dark earth itself and the adjacent Acrisol. Samples were taken from two sites, one cultivated with manioc and one with secondary forest cover. Analyses revealed that the community structure found in each sample had unique features. At a coarse phylogenetic resolution, the most abundant phyla in all sequence libraries were Actinobacteria, Acidobacteria, Verrucomicrobia and Proteobacteria that were present in similar relative abundance across all samples. However, the class composition varied between them highlighting the difference between the Acrisol and the remaining samples. This result was also corroborated by the comparison of the OTU composition (at 97 % identity). Also, soil coverage has shown an effect over the community structure observed in all samples. This pattern was found to be significant through unweighted UniFrac as well as P tests. These results indicate that, although the ADEs are found in patches within the Acrisols, the contrasting characteristics found between them led to the development of significantly different communities. PMID:23743632

Taketani, Rodrigo Gouvêa; Lima, Amanda Barbosa; da Conceição Jesus, Ederson; Teixeira, Wenceslau Geraldes; Tiedje, James M; Tsai, Siu Mui

2013-08-01

31

Diversity analysis of the bacterial community in tobacco waste extract during reconstituted tobacco process.  

PubMed

Reconstituted tobacco sheet process has been developed to treat and reuse tobacco wastes in the industry. During this process, microorganisms in original and concentrated tobacco waste extract (TWE) might play important roles in the final quality of the reconstituted tobacco. However, microbial communities in TWE remain largely unknown. In the present study, the Roche 454 bar-coded pyrosequencing was applied to analyze the bacterial community structure in samples. Comparison based on 16S rRNA gene sequences showed that the original and concentrated solutions of TWE harbored abundant bacteria probably resistant to the acid, high nicotine concentration, and high osmotic pressure environment. The dominant phyla were Firmicutes and Proteobacteria. Lactobacillus and Lysinibacillus were the dominant genera of Firmicutes. The most interesting genus of Proteobacteria was Pseudomonas. It is the first time to reveal the bacterial diversities on the TWE samples from the process of reconstituted tobacco sheets. PMID:25142693

Liu, Huagui; He, Houlong; Cheng, Changhe; Liu, Jinli; Shu, Ming; Jiao, Yang; Tao, Feng; Zhong, Weihong

2015-01-01

32

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

33

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

34

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

35

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

PubMed

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

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

2004-06-01

36

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

37

Comparison between MICRO–CARD–FISH and 16S rRNA gene clone libraries to assess the active versus total bacterial community in the coastal Arctic.  

PubMed

We collected surface- and deep-water samples (maximum depth 300 m) during the spring–summer transition in the coastal Arctic along a transect in the Kongsfjorden (Ny-Ålesund, Spitsbergen, Norway) to determine the structure of the active versus total marine bacterioplankton community using different approaches. Catalysed reporter deposition– fluorescence in situ hybridization combined with microautoradiography (MICRO–CARD–FISH) was used to determine the abundance and activity of different bacterial groups. The bacterial communities were dominated by members of Alphaproteobacteria followed by Bacteroidetes, whereas Gammaproteobacteria were present at low abundance but exhibited a high percentage of active cells taking up leucine. The clone libraries of 16S rRNA genes (16S rDNA) and 16S rRNA from two different depths were used to decipher the bacterial community structure. Independently of the type of clone libraries analysed (16S rDNA- or 16S rRNA-based), four major and four minor taxonomic groups were detected. The bacterioplankton community was mainly dominated at both the DNA and the RNA levels by Alphaproteobacteria followed by Gammaproteobacteria. The Rhodobacteriaceae were the most abundant members of the Alphaproteobacteria in both DNA and RNA clone libraries, followed by the SAR11 clade, which was only detectable at the 16S PMID:23565124

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

2013-04-01

38

Bacterial diversity from the source to the tap: a comparative study based on 16S rRNA gene-DGGE and culture-dependent methods.  

PubMed

This study aimed to assess the influence of water treatment and distribution on the bacterial communities with particular emphasis on tap water. Samples from the water treatment plant, the bulk supply distribution system and household taps, supplied by the same drinking water treatment plant, were analyzed using culture-dependent and culture-independent methods. Water treatment imposed alterations in the composition of the bacterial community, although this effect was more evident in the cultivable bacteria rather than among the total community assessed by 16S rRNA gene-denaturing gradient gel electrophoresis (DGGE) profiling. Water disinfection, mainly chlorination, promoted a reduction on bacterial diversity and cultivability, with a shift in the pattern of cultivable bacteria from predominantly Gram-negative to predominately Gram-positive and acid-fast. Downstream of the chlorination stages, tap water, in comparison with raw water, presented higher diversity indices and cultivability percentages. From the source to the tap, members of the Alpha-, Beta- and Gammaproteobacteria were the predominant lineages identified using 16S rRNA gene-DGGE analysis. Although with a lower coverage, the DGGE-based lineage identifications were in agreement with those found using 454-pyrosequencing analysis. Despite the effectiveness of water treatment to eliminate or inactivate most of the bacteria, Proteobacteria such as Acinetobacter, Bosea and Sphingomonadaceae may successfully colonize tap water. PMID:22938591

Vaz-Moreira, Ivone; Egas, Conceição; Nunes, Olga C; Manaia, Célia M

2013-02-01

39

Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments coding for 16S rRNA  

Microsoft Academic Search

The effect of developing chrysanthemum roots on the presence and activity of bacterial populations in the rhizosphere was examined by using culture-independent methods. Nucleic acids were extracted from rhizosphere soil samples associated with the bases of roots or root tips of plants harvested at different stages of development. PCR and reverse transcriptase (RT) PCR were used to amplify 16S ribosomal

BERNADETTE M. DUINEVELD; GEORGE A. KOWALCHUK; ANNEKE KEIJZER; JAN DIRK VAN ELSAS; JOHANNES A. VAN VEEN

2001-01-01

40

CpG motifs: the active ingredient in bacterial extracts?  

Microsoft Academic Search

The use of bacteria and bacterial extracts for immunotherapy has a checkered past. Recent developments in immunology reveal that these nonspecific immune activators actually work by triggering specific receptors that are expressed by subsets of immune cells. Identification of these receptors and the molecular signaling pathways that they activate has enabled a new era of specific targeted immunotherapy using chemically

Arthur M Krieg

2003-01-01

41

Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing? †  

PubMed Central

The bacterial diversity associated with citrus leaf midribs was characterized for 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 rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for 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 in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. “Candidatus Liberibacter asiaticus” was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of “Candidatus Liberibacter asiaticus” in symptomatic leaves. These data implicate “Candidatus Liberibacter asiaticus” as the pathogen responsible for HLB disease. PMID:19151177

Sagaram, Uma Shankar; DeAngelis, Kristen M.; Trivedi, Pankaj; Andersen, Gary L.; Lu, Shi-En; Wang, Nian

2009-01-01

42

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

43

Characterization of bacterial community shift in human Ulcerative Colitis patients revealed by Illumina based 16S rRNA gene amplicon sequencing  

PubMed Central

Background The healthy human intestine is represented by the presence of bacterial communities predominantly belonging to obligate anaerobes; however disparity and dysanaerobiosis in intestinal microflora may lead to the progression of ulcerative colitis (UC). The foremost aim of this study is to consider and compare the gut microbiota composition in patients suffering from different stages of UC. Methods This study represents data from the biopsy samples of six individuals suffering from UC. The samples were collected by colonoscopy and were processed immediately for isolation of DNA. Mucosal microbiota was analyzed by means of 16S rRNA gene-based Illumina high throughput sequencing. Quantitative real-time PCR (qPCR) was performed to determine total bacterial abundances. Results Analysis of 23,927 OTUs demonstrated a significant reduction of bacterial diversity consistently from phylum to species level (p?bacterial count was detected in patients suffering from severe inflammatory stage (2.98 +/-0.49 E?+?09/ml) when compared with patients with moderate (1.03+/-0.29 E?+?08/ml) and mild (1.76 +/-0.34 E?+?08/ml) stages of inflammation. Conclusion The reduction of bacterial diversity with an increase in the total bacterial count indicates a shift of bacterial communities which signifies dysbiosis and dysanaerobiosis at the mucosal level for patients suffering from UC. PMID:25018784

2014-01-01

44

Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness and translation initiation  

PubMed Central

The functional centers of the ribosome in all organisms contain ribosomal RNA (rRNA) modifications, which are introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none of the modifications tested so far was essential for growth and hence the functional role of modifications is largely unknown. Here, we show that the methyl groups of nucleosides m2G966 and m5C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m2G966/m5C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNAfMet to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m2G966 and m5C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates that determine the fate of a given messenger RNA (mRNA) at early checkpoints of mRNA selection. PMID:22649054

Burakovsky, Dmitry E.; Prokhorova, Irina V.; Sergiev, Petr V.; Milón, Pohl; Sergeeva, Olga V.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.

2012-01-01

45

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

46

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing.  

PubMed

This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol-chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae. PMID:25393530

Saraithong, Prakaimuk; Li, Yihong; Saenphet, Kanokporn; Chen, Zhou; Chantawannakul, Panuwan

2014-11-13

47

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

PubMed Central

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

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

2008-01-01

48

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

49

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

50

Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies.  

PubMed

A cultivation-independent approach based on polymerase chain reaction (PCR)-amplified partial small subunit rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP) was used to characterize the bacterial diversity inhabiting the rhizosphere of maize plants grown on an agricultural field. The community structures of two cultivars, a genetically engineered and a nonengineered variety, different herbicide regimes and soil tillage were compared with each other at two sampling dates. SSCP-profiles were generated with DNA from bacterial cell consortia with primers hybridizing to evolutionarily highly conserved rRNA gene regions. On silver-stained gels, each profile consisted of approx. 50 distinguishable bands. Similarity analyses of patterns recorded by digital image analyses could not detect any difference between cultivars or treatments that was greater than the variability between replicates. A total of 54 sequences recovered from different bands were identified and grouped into operational taxonomical units (OTUs). Surprisingly, only five of 40 OTUs contained sequences of both samplings. Three different bands from a profile were selected to test whether this small overlap was due to an incomplete recovery of sequences. From a faint band, two different OTUs were found when 12 clones were analysed, and from two strong bands 24 and 22 OTUs were detected from a total of 26 and 36 clones, respectively. The OTUs belonged to phylogenetically different groups of bacteria. Gene probes that were developed to target different bands of the profiles, however, indicated in Southern blot analyses that patterns between treatments, replicates and samplings, and even from two different growing seasons were highly conserved. Our study demonstrates that community profiles can consist of more sequences than detectable by staining and that gene probes in Southern blot can be a useful control to investigate the composition of microbial communities by genetic profiles. PMID:12492893

Schmalenberger, Achim; Tebbe, Christoph C

2003-01-01

51

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

52

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

PubMed

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

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

2015-01-01

53

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

PubMed Central

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

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

2013-01-01

54

Isolation of new bacterial species from drinking water biofilms and proof of their in situ dominance with highly specific 16S rRNA probes.  

PubMed Central

A polyphasic approach involving cultivation, direct viable counts, rRNA-based phylogenetic classification, and in situ probing was applied for the characterization of the dominant microbial population in a municipal drinking water distribution system. A total of 234 bacterial strains cultivated on R2A medium were screened for bacteria affiliated with the in situ dominating beta subclass of Proteobacteria. The isolates were grouped according to common features of their cell and colony morphologies, and eight representative strains were used for 16S rRNA sequencing and the development of a suite of strain-specific oligonucleotide probes. Phylogenetic analysis indicated that all of the isolates were hitherto unknown bacteria. Three of them, strains B4, B6, and B8, formed a separate cluster of closely related organisms within the beta 1 subclass of Proteobacteria. In situ probing revealed that (i) 67 to 72% of total bacteria, corresponding to more than 80% of beta-subclass bacteria, could be encompassed with the strain-specific probes and (ii) the dominating bacterial species were culturable on R2A medium. Additionally, two-thirds of the autochthonous drinking water population could be shown to be in a viable but nonculturable (VBNC) state by using a direct viable count approach. The comparison of isolation frequencies with the in situ abundances of the eight investigated strains revealed differences in their culturability, indicating variable ratios of culturable to VBNC cells among the strains. The further characterization of biofilms throughout the distribution network demonstrated strains B6 and B8 to be dominant bacterial strains in groundwater and distribution system biofilms. The other strains could be found at various frequencies in the different parts of the distribution system; several strains appeared exclusively in drinking water biofilms obtained from a house installation system. PMID:9361400

Kalmbach, S; Manz, W; Szewzyk, U

1997-01-01

55

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

56

Bacterial Cellulose Production from the Litchi Extract by Gluconacetobacter Xylinus.  

PubMed

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

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

2014-09-01

57

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

PubMed

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

Po?ka, Justyna; Rebecchi, Annalisa; Pisacane, Vincenza; Morelli, Lorenzo; Puglisi, Edoardo

2015-04-01

58

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

59

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

60

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

61

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

PubMed

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

2012-12-01

62

Infectious Diseases and Tropical Disease Pathology: SC16-1 rRNA SEQUENCING IN MOLECULAR MICROBIOLOGICAL DIAGNOSIS OF BACTERIAL INFECTIONS IN THE AUTOPSY SETTING.  

PubMed

Diagnosing the aetiology of infectious diseases at autopsy, such as pneumonia, meningitis, sepsis or SUDI, is complicated due to issues including post mortem contamination, difficulty culturing fastidious organisms and subjective interpretation of polymicrobial cultures. Death of organisms may also occur post mortem, especially if antibiotics were given to the patient, but residual DNA from non-viable organisms, amenable to molecular detection, may remain. The 16S rRNA gene is present in all bacteria with conserved and hyper-variable regions along its length, allowing amplification and sequencing of all bacterial 16S sequences present in a sample. 16S sequencing offers potential advantages over culture-based diagnostics and is increasingly used in clinical practice. It has been used to identify bacteria in formalin fixed paraffin embedded (FFPE) surgical pathology specimens but its use has not been reported in autopsy diagnosis. This talk will summarise a study aimed to assess the utility of 16S sequencing as an adjunctive microbiological test in the autopsy. Our preliminary work has used post mortem lung tissue samples from children dying with pneumonia as part of the Pneumonia Etiology Research for Child Health (PERCH) project. The technique has identified known pathogens in some cases and provided additional diagnostic information in others. The presentation will discuss the technical aspects of 16S sequencing from FFPE and autopsy material, and the issues surrounding its application to diagnosis in comparison with standard culture based diagnostics on surgical/autopsy material. PMID:25188113

Hart, John D; Street, Teresa; Wrightson, John M; Moore, David P; Scott, Anthony G; Crook, Derrick W; Turner, Gareth D H

2014-10-01

63

The 26S rRNA binding ribosomal protein equivalent to bacterial protein L11 is encoded by unspliced duplicated genes in Saccharomyces cerevisiae.  

PubMed Central

Transformant phages expressing L15, a yeast ribosomal protein which binds to 26S rRNA and interacts with the acidic ribosomal proteins, were isolated by screening a yeast cDNA expression library in lambda gt11 with specific monoclonal antibodies. Using yeast DNA HindIII fragments that hybridize with the cDNA insert from the L15-expressing clones, minilibraries were prepared in pUC18, which were afterward screened with the same cDNA probe. In this way, plasmids carrying two different types of genomic DNA inserts were obtained. The inserts were subcloned and sequenced and we found a similar coding sequence in both cases flanked by 5' and 3' regions with very low homology. Sequences homologous to the consensus TUF-binding UAS boxes are present in the 5' flanking regions of both genes. Southern analysis revealed the presence of two copies of the L15 gene in the Saccharomyces cerevisiae genome, which are located in different chromosomes. The encoded amino acid sequence corresponds, as expected, to protein L15 and shows a high similarity to bacterial ribosomal protein L11. Images PMID:2167467

Pucciarelli, M G; Remacha, M; Vilella, M D; Ballesta, J P

1990-01-01

64

A Computer-Simulated Restriction Fragment Length Polymorphism Analysis of Bacterial Small-Subunit rRNA Genes: Efficacy of Selected Tetrameric Restriction Enzymes for Studies of Microbial Diversity in Nature  

Microsoft Academic Search

An assessment of 10 tetrameric restriction enzymes (TREs) was conducted by using a computer-simulated restriction fragment length polymorphism (RFLP) analysis for over 100 proximally and distally related bacterial small-subunit (SSU) rRNA gene sequences. Screening SSU rDNA clone libraries with TREs has become an effective strategy because of logistic simplicity, commercial availability, and economy. However, the rationale for selecting the type

CRAIG L. MOYER; JAMES M. TIEDJE; FRED C. DOBBS; ANDDAVID M. KARL

65

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

66

Extraction of the metagenomic DNA and assessment of the bacterial diversity from the petroleum-polluted sites.  

PubMed

The assessment of the microbial diversity of the entire community of a given habitat requires the extraction of the total environmental DNA. Metagenomic investigations of a petroleum-polluted habitat have its unique challenges. The specific methods were developed for the extraction of high-quality metagenome in good quantity from the petroleum-polluted saline and non-saline sites in Gujarat (India). The soil samples were washed to remove the toxic, hazardous organic pollutants which might interfere with the recovery of the metagenomic DNA. The metagenomic DNA extraction results were encouraging with the mechanical bead beating, soft lysis, and combination of both. The extracted DNA was assessed for its purity and yield followed by its application in the amplification of the 16S rRNA region. The amplicons were used for judging the molecular diversity by the denaturing gradient gel electrophoresis (DGGE). The microbial diversity was also analyzed statistically by calculating various diversity indices and principal component analysis (PCA). The results on the metagenomic diversity of the bacterial population among the three cohorts based on the culture-independent technique exhibited significant difference among the PAH sites and Okha-Madhi and Porbandar Madhavpur habitats. PMID:24869956

Akbari, Viral G; Pandya, Rupal D; Singh, Satya P

2014-10-01

67

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...

68

Characterization of Chemoautotrophic Bacterial Symbionts in a Gutless Marine Worm (Oligochaeta, Annelida) by Phylogenetic 16S rRNA Sequence Analysis and In Situ Hybridization  

Microsoft Academic Search

ThephylogeneticrelationshipsofchemoautotrophicendosymbiontsinthegutlessmarineoligochaeteInanid- rilus leukodermatus to chemoautotrophic ecto- and endosymbionts from other host phyla and to free-living bacteria were determined by comparative 16S rRNA sequence analysis. Fluorescent in situ hybridizations confirmed that the 16S rRNA sequence obtained from these worms originated from the symbionts. The symbiontsequenceisuniquetoI.leukodermatus.Inphylogenetictreesinferredbybothdistanceandparsimony methods, the oligochaete symbiont is peripherally associated with one of two clusters of chemoautotrophic symbionts that

NICOLE DUBILIER; OLAV GIERE; DANIEL L. DISTEL; ANDCOLLEEN M. CAVANAUGH

1995-01-01

69

Bacterial diversity in worker adults of Apis mellifera capensis and Apis mellifera scutellata (Insecta: Hymenoptera) assessed using 16S rRNA sequences  

Microsoft Academic Search

High-fidelity PCR of 16S rRNA sequences was used to identify bacteria associated with worker adults of the honeybee subspecies Apis mellifera capensis and Apis mellifera scutellata. An expected ?1.5-kb DNA band, representing almost the entire length of the 16S rRNA gene, was amplified from both subspecies and cloned. Ten unique sequences were obtained: one sequence each clustered with Bifidobacterium (Gram-positive

Ayyamperumal Jeyaprakash; Marjorie A. Hoy; Michael H. Allsopp

2003-01-01

70

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

71

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

PubMed Central

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

72

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

PubMed

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

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

2013-01-01

73

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

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; Skvarc, Miha; Stubljar, David; Kofol, Romina; Kaasch, Achim

2014-01-01

74

16S rRNA gene-based identification of cultured bacterial flora from host-seeking Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks, vectors of vertebrate pathogens.  

PubMed

A total of 151 bacterial isolates were recovered from different developmental stages (larvae, nymphs and adults) of field-collected ticks (67 strains from Ixodes ricinus, 38 from Dermacentor reticulatus, 46 from Haemaphysalis concinna). Microorganisms were identified by means of 16S rRNA gene sequencing. Almost 87 % of the strains belonged to G(+) bacteria with predominantly occurring genera Bacillus and Paenibacillus. Other G(+) strains included Arthrobacter, Corynebacterium, Frigoribacterium, Kocuria, Microbacterium, Micrococcus, Plantibacter, Rhodococcus, Rothia, and Staphylococcus. G(-) strains occurred less frequently, comprising genera Advenella, Pseudomonas, Rahnella, Stenotrophomonas, and Xanthomonas. Several strains of medical importance were found, namely Advenella incenata, Corynebacterium aurimucosum, Microbacterium oxydans, M. schleiferi, Staphylococcus spp., and Stenotrophomonas maltophilia. Data on cultivable microbial diversity in Eurasian tick species D. reticulatus and H. concinna are given, along with the extension of present knowledge concerning bacterial flora of I. ricinus. PMID:19937215

Rudolf, I; Mendel, J; Sikutová, S; Svec, P; Masaríková, J; Nováková, D; Bunková, L; Sedlácek, I; Hubálek, Z

2009-09-01

75

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

PubMed

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

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

2010-08-01

76

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

77

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

PubMed Central

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

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

2014-01-01

78

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. PMID:25438017

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

2014-01-01

79

Wild mushroom extracts as inhibitors of bacterial biofilm formation.  

PubMed

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

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

2014-01-01

80

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

2011-01-01

81

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

82

Extraction of total nucleic acids from ticks for the detection of bacterial and viral pathogens.  

PubMed

Ticks harbor numerous bacterial, protozoal, and viral pathogens that can cause serious infections in humans and domestic animals. Active surveillance of the tick vector can provide insight into the frequency and distribution of important pathogens in the environment. Nucleic-acid based detection of tick-borne bacterial, protozoan, and viral pathogens requires the extraction of both DNA and RNA (total nucleic acids) from ticks. Traditional methods for nucleic acid extraction are limited to extraction of either DNA or the RNA from a sample. Here we present a simple bead-beating based protocol for extraction of DNA and RNA from a single tick and show detection of Borrelia burgdorferi and Powassan virus from individual, infected Ixodes scapularis ticks. We determined expected yields for total nucleic acids by this protocol for a variety of adult tick species. The method is applicable to a variety of arthropod vectors, including fleas and mosquitoes, and was partially automated on a liquid handling robot. PMID:20180313

Crowder, Chris D; Rounds, Megan A; Phillipson, Curtis A; Picuri, John M; Matthews, Heather E; Halverson, Justina; Schutzer, Steven E; Ecker, David J; Eshoo, Mark W

2010-01-01

83

The Inhibiting Effect of Aqueous Azadirachta indica (Neem) Extract Upon Bacterial Properties Influencing in vitro Plaque Formation  

Microsoft Academic Search

The purpose of this investigation was to examine the inhibitory effects of aqueous extracts derived from the bark-containing sticks (Neem stick) of Azadirachta indica upon bacterial aggregation, growth, adhesion to hydroxyapatite, and production of insoluble glucan, which may affect in vitro plaque formation. Neem stick extracts were screened for minimal bacterial growth inhibition (MIC) against a panel of streptococci by

L. E. Wolinsky; S. Mania; S. Nachnani; S. Ling

1996-01-01

84

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

85

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

86

Bacterial and fungal DNA extraction from blood samples: automated protocols.  

PubMed

Automation in DNA isolation is a necessity for routine practice employing molecular diagnosis of infectious agents. To this end, the development of automated systems for the molecular diagnosis of microorganisms directly in blood samples is at its beginning. Important characteristics of systems demanded for routine use include high recovery of microbial DNA, DNA-free containment for the reduction of DNA contamination from exogenous sources, DNA-free reagents and consumables, ideally a walkaway system, and economical pricing of the equipment and consumables. Such full automation of DNA extraction evaluated and in use for sepsis diagnostics is yet not available. Here, we present protocols for the semiautomated isolation of microbial DNA from blood culture and low- and high-volume blood samples. The protocols include a manual pretreatment step followed by automated extraction and purification of microbial DNA. PMID:25319785

Lorenz, Michael G; Disqué, Claudia; Mühl, Helge

2015-01-01

87

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

88

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

PubMed Central

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

2014-01-01

89

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis.  

PubMed

Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, to our knowledge, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16S rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16S rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction. PMID:22902729

Harrington, Catriona; Del Casale, Antonio; Kennedy, Jonathan; Neve, Horst; Picton, Bernard E; Mooij, Marlies J; O'Gara, Fergal; Kulakov, Leonid A; Larkin, Michael J; Dobson, Alan D W

2012-11-01

90

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

Microsoft Academic Search

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

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

2006-01-01

91

Detection of Prosthetic Hip Infection at Revision Arthroplasty by Immunofluorescence Microscopy and PCR Amplification of the Bacterial 16S rRNA Gene  

Microsoft Academic Search

Received 19 January 1999\\/Returned for modification 2 May 1999\\/Accepted 26 June 1999 In this study the detection rates of bacterial infection of hip prostheses by culture and nonculture methods were compared for 120 patients with total hip revision surgery. By use of strict anaerobic bacteriological practice during the processing of samples and without enrichment, the incidence of infection by culture

MICHAEL M. TUNNEY; SHEILA PATRICK; MARTIN D. CURRAN; GORDON RAMAGE; DONNA HANNA; JAMES R. NIXON; SEAN P. GORMAN; RICHARD I. DAVIS; NEIL ANDERSON

1999-01-01

92

Methanogen and bacterial diversity and distribution in deep gas hydrate sediments from the Cascadia Margin as revealed by 16S rRNA molecular analysis  

Microsoft Academic Search

The microbial community of a deep (to 234 m below the sea floor) sediment gas hydrate deposit (Cascadia Margin Ocean Drilling Program Site 889\\/890, Leg 146) was analysed for the first time by molecular genetic techniques. Both bacterial and methanogen diversity were determined by phylogenetic analysis of ribosomal DNA sequences. High molecular mass DNA, indicative of active bacteria, was present

Julian R Marchesi; Andrew J Weightman; Barry A Cragg; R. John Parkes; John C Fry

2001-01-01

93

Bacterial Communities in the Rhizosphere of Biofuel Crops Grown on Marginal Lands as Evaluated by 16S rRNA Gene Pyrosequences  

Microsoft Academic Search

Microbes are key components of the soil environment and are important contributors to the sustainability of agricultural systems,\\u000a which is especially significant for biofuel crops growing on marginal lands. We studied bacterial communities in the rhizosphere\\u000a of five biofuel crops cultivated in four locations in Michigan to determine which factors were correlated to changes in the\\u000a structure of those communities.

Ederson da C. Jesus; Endang Susilawati; Stephanie L. Smith; Qiong Wang; Benli Chai; Ryan Farris; Jorge L. M. Rodrigues; Kurt D. Thelen; James M. Tiedje

2010-01-01

94

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

95

Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species.  

PubMed Central

In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification. The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T. thermophilus." The values for the pair of Bacillus subtilis and "T. thermophilus" showed greater deviations from the predicted value. The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present. PMID:7618894

Farrelly, V; Rainey, F A; Stackebrandt, E

1995-01-01

96

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

PubMed

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

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

2013-01-01

97

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

PubMed Central

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

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

2013-01-01

98

Antimicrobial potential of Ricinus communis leaf extracts in different solvents against pathogenic bacterial and fungal strains  

PubMed Central

Objective To investigate the in vitro antimicrobial activities of the leaf extract in different solvents viz., methanol, ethanol and water extracts of the selected plant Ricinus communis. Methods Agar well diffusion method and agar tube dilution method were carried out to perform the antibacterial and antifungal activity of methanol, ethanol and aqueous extracts. Results Methanol leaf extracts were found to be more active against Gram positive bacteria (Bacillus subtilis: ATCC 6059 and Staphylococcus aureus: ATCC 6538) as well as Gram negative bacteria (Pseudomonas aeruginosa: ATCC 7221 and Klebsiella pneumoniae) than ethanol and aqueous leaf extracts. Antifungal activity of methanol and aqueous leaf extracts were also carried out against selected fungal strains as Aspergillus fumigatus and Aspergillus flavus. Methanolic as well as aqueous leaf extracts of Ricinus communis were effective in inhibiting the fungal growth. Conclusions The efficient antibacterial and antifungal activity of Ricinus communis from the present investigation revealed that the methanol leaf extracts of the selected plant have significant potential to inhibit the growth of pathogenic bacterial and fungal strains than ethanol and aqueous leaf extracts. PMID:23593573

Naz, Rabia; Bano, Asghari

2012-01-01

99

Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis  

Microsoft Academic Search

Bacterial community structure was studied in a Flevo silt loam (FSL) soil microplot, as well as in 15 other soils, by using DNA extraction followed by molecular fingerprinting. Total community DNA was extracted and purified by a direct method, which yielded amplifiable DNA of high molecular weight for all soils. A variable region of the 16S rRNA gene was then

Antonio Gelsomino; Anneke C Keijzer-Wolters; Giovanni Cacco; Jan Dirk van Elsas

1999-01-01

100

Bacterial diversity and community structure in the East China Sea by 454 sequencing of the 16S rRNA gene  

NASA Astrophysics Data System (ADS)

The 454 sequencing method was used to detect bacterial diversity and community structure in the East China Sea. Overall, 149 067 optimized reads with an average length of 454 nucleotides were obtained from 17 seawater samples and five sediment samples sourced in May 2011. A total of 22 phyla, 34 classes, 74 orders, 146 families, and 333 genera were identified in this study. Some of them were detected for the first time from the East China Sea. The estimated richness and diversity indices were both higher in the sediment samples compared with in the seawater samples. All the samples were divided by their diversity indices into four regions. Similarity analysis showed that the seawater samples could be classified into six groups. The groups differed from each other and had unique community structure characteristics. It was found that different water masses in the sampling areas may have had some influence on the bacterial community structure. A canonical correspondence analysis revealed that seawater samples in different areas and at different depths were affected by different environmental parameters. This study will lay the foundation for future research on microbiology in the East China Sea.

Dong, Yi; Zhao, Yuan; Zhang, Wenyan; Li, Yan; Zhou, Feng; Liu, Chenggang; Wu, Ying; Liu, Sumei; Zhang, Wuchang; Xiao, Tian

2014-05-01

101

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

102

Anti-bacterial action of onion (Allium cepa L.) extracts against oral pathogenic bacteria.  

PubMed

In this study, the focus was on the antibacterial activity of onions. This study researched the activities of onion extracts on Streptococcus mutans and Streptococcus sobrinus, the main causal bacteria for dental caries, and Porphyromonas gingivalis and Prevotella intermedia, considered to be the main causal bacteria of adult periodontitis. The results showed that the onion extracts possess an effect on all test bacterial strains (S.mutans JC-2, S. sobrinus OMZ176, P. gingivalis ATCC 33277 and P. intermedia ATCC 25611), and the effects were bactericidal against cultured and resting bacterial cells. The activity of the onion extracts was stable even after 48 hours in the culture medium. This result suggests that no decomposition or volatility of onion extracts occurred in the culture medium. The antibacterial activity of onion extracts was not markedly influenced by cysteine (10 mM) treatment. However, activity significantly decreased with alkali treatment. Grated onion left to stand at 37 degrees C for 48 hours did not show antibacterial activity. Also, activity of steam treated (100 degrees C, 10 min.) onion was not observed. Using avicel plate by thin layer chromatography with the solvent of n-butanol:acetic acid:water (3:3:1), the main component of the substance (the substance which develops color with ninhydrin) was observed at an Rf value of about 0.9. PMID:9354029

Kim, J H

1997-09-01

103

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

104

Comparison of DNA extraction methods in analysis of salivary bacterial communities.  

PubMed

Culture-independent high-throughput sequencing-based methods are widely used to study bacterial communities. Although these approaches are superior to traditional culture-based methods, they introduce bias at the experimental and bioinformatics levels. We assessed the diversity of the human salivary microbiome by pyrosequencing of the 16S rDNA V1-3 amplicons using metagenomic DNA extracted by two different protocols: a simple proteinase K digestion without a subsequent DNA clean-up step, and a bead-beating mechanical lysis protocol followed by column DNA purification. A high degree of congruence was found between the two extraction methods, most notably in regard to the microbial community composition. The results showed that for a given bioinformatics pipeline, all the taxa with an average proportion >0.12% in samples processed using one extraction method were also detected in samples extracted using the other method. The same taxa tended to be abundant and frequent for both extraction methods. The relative abundance of sequence reads assigned to the phyla Actinobacteria, Spirochaetes, TM7, Synergistetes, and Tenericutes was significantly higher in the mechanically-treated samples than in the enzymatically-treated samples, whereas the phylum Firmicutes showed the opposite pattern. No significant differences in diversity indices were found between the extraction methods, although the mechanical lysis method revealed higher operational taxonomic unit richness. Differences between the extraction procedures outweighed the variations due to the bioinformatics analysis pipelines used. PMID:23844068

Lazarevic, Vladimir; Gaïa, Nadia; Girard, Myriam; François, Patrice; Schrenzel, Jacques

2013-01-01

105

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

Microsoft Academic Search

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

Darrell P. Chandler; Ann E. Jarrell

2004-01-01

106

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

107

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

PubMed Central

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

Setterington, Emma B.; Alocilja, Evangelyn C.

2012-01-01

108

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

109

Archaeal and bacterial communities in geochemically diverse hot springs of Yellowstone National Park, USA  

Microsoft Academic Search

Microbiological and geochemical surveys were conducted at three hot springs (Obsidian Pool, Sylvan Spring, and 'Bison Pool') in Yellowstone National Park (Wyoming, USA). Microbial community structure was investi- gated by polymerase chain reaction (PCR) amplification of 16S rRNA gene sequences from DNA extracted from sediments of each hot spring, followed by molecular cloning. Both bacterial and archaeal DNA was retrieved

D. R. MEYER-DOMBARD; E. L. SHOCK; J. P. AMEND

2005-01-01

110

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

PubMed Central

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

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

2014-01-01

111

Antibiofilm activity, compound characterization, and acute toxicity of extract from a novel bacterial species of paenibacillus.  

PubMed

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

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

2014-01-01

112

Induction of sister chromatid exchanges and bacterial revertants by organic extracts of airborne particles. [Humans  

SciTech Connect

The genotoxicities of organic extracts of airborne particles have been studied extensively in the Salmonella/mammalian microsome (Ames) test, but in few other bioassays. In these studies, we tested benzene-acetone extracts of particulate pollutants collected in Lexington, Kentucky, for capacity to induce increases in sister chromatid exchanges (SCE) in human lumphocytes and V79 cells, as well as in the Ames assay. Extracts induced linear dose-related increases in SCE in human lumphocytes and in bacterial revertants.However, variable responses were observed in SCE assays in V79 cells with and without activation by rat liver S9 or feeder layers of irradiated Syrian hamster fetal cells. We conclude that the SCE assay in human lumphocytes may be a useful indicator of the potential risks to humans of airborne particulate pollutants, as it utilizes human cells recently taken from the host, is rapid and economical, and requires small quantities of test materials. However, thorough studies of the quantitative relationships between SCE induction and mutagenicity in human cells are needed.

Lockard, J.M. (Thomas Hunt Morgan School of Biological Sciences, Lexington, KY); Viau, C.J.; Lee-Stephens, C.; Caldwell, J.C.; Wojciechowski, J.P.; Enoch, H.G.; Sabharwal, P.S.

1981-01-01

113

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

114

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

PubMed

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

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

2014-07-01

115

Use of microcapsules with electrostatically immobilized bacterial cells or enzyme extract to remove nonylphenol in wastewater sludge.  

PubMed

We investigated the use of a high-voltage electrostatic system to immobilize bacterial cells or enzyme extract in alginate microcapsules for removing nonylphenol (NP) from wastewater sludge. With applied potential increased from 0 to 12kV, the gel bead diameter decreased from 950 to 250 ?m. The amount of bacterial cells or enzyme extract immobilized in alginate microcapsules was greater than that in suspension, for improved tolerance to environmental loadings. Removal of NP at 2.0-20.0 mg L(-1) was greater with extract- than cell-containing microcapsules. The percentage of toxic chemicals (2.0 mg L(-1)) removed with alginate microcapsules, in descending order of magnitude, was bisphenol-F>bisphenol-A>NP>oxytetracycline>chlortetracycline>tetracycline>dibromodiphenyl ethers>tetrabromobisphenol-A>decabromodiphenyl ether. PMID:23499222

Hsu, Fu-Yin; Wang, Zheng-Yi; Chang, Bea-Ven

2013-05-01

116

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

117

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

PubMed

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

2014-04-01

118

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

119

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

120

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

121

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

122

Choice of bacterial DNA extraction method from fecal material influences community structure as evaluated by metagenomic analysis  

PubMed Central

Background In recent years, studies on the human intestinal microbiota have attracted tremendous attention. Application of next generation sequencing for mapping of bacterial phylogeny and function has opened new doors to this field of research. However, little attention has been given to the effects of choice of methodology on the output resulting from such studies. Results In this study we conducted a systematic comparison of the DNA extraction methods used by the two major collaborative efforts: The European MetaHIT and the American Human Microbiome Project (HMP). Additionally, effects of homogenizing the samples before extraction were addressed. We observed significant differences in distribution of bacterial taxa depending on the method. While eukaryotic DNA was most efficiently extracted by the MetaHIT protocol, DNA from bacteria within the Bacteroidetes phylum was most efficiently extracted by the HMP protocol. Conclusions Whereas it is comforting that the inter-individual variation clearly exceeded the variation resulting from choice of extraction method, our data highlight the challenge of comparing data across studies applying different methodologies. PMID:24949196

2014-01-01

123

Life History Implications of rRNA Gene Copy Number in Escherichia coli  

Microsoft Academic Search

The role of the rRNA gene copy number as a central component of bacterial life histories was studied by using strains of Escherichia coli in which one or two of the seven rRNA operons (rrnA and\\/or rrnB) were deleted. The relative fitness of these strains was determined in competition experiments in both batch and chemostat cultures. In batch cultures, the

Bradley S. Stevenson; Thomas M. Schmidt

2004-01-01

124

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

125

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

PubMed Central

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

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

2015-01-01

126

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

PubMed

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

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

2015-01-01

127

Reverse transcription and polymerase chain reaction amplification of rRNA for detection of Helicobacter species.  

PubMed Central

Sequence data on Helicobacter pylori 16S rRNA were used to select two 22-base oligonucleotide primers for use in a polymerase chain reaction (PCR) for detection of H. pylori. H. pylori cells were treated with lysis buffer, boiled, and chloroform extracted. Reverse transcription of rRNA was followed by PCR amplification (RT-PCR) of the synthesized cDNA and 16S rRNA gene. The amplified PCR products were analyzed by agarose gel electrophoresis and Southern blotting. Using ethidium bromide-stained agarose gels, we were able to detect the expected 500-bp DNA fragment from as few as two H. pylori organisms per reaction. The specificity of the RT-PCR assay was tested with 27 clinical isolates and related reference strains; although the number of bacterial cells used per reaction was 10(5)-fold greater than the number of H. pylori organisms used, amplification was detected only with bacteria in the same genus, H. cinaedi and H. mustelae. Ten H. pylori organisms per biopsy specimen were detected on agarose gels when organisms were added to samples prepared from a processed colon biopsy sample. RT-PCR results were consistent with urea breath test and culture results in 14 of 15 gastric biopsy specimens; the specificity was 100%. RT-PCR of rRNA from H. pylori increased the sensitivity of pathogen detection at least 25- to 50-fold compared with that of previous PCR assays. This low level of detection by RT-PCR assay may prove to be well suited for verifying eradication following therapy. Images PMID:1383268

Engstrand, L; Nguyen, A M; Graham, D Y; el-Zaatari, F A

1992-01-01

128

Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR.  

PubMed

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps of sampling and sample processing. We used quantitative real-time polymerase chain reaction (qrtPCR) for Escherichia coli and Staphylococcus aureus to measure the recovery of DNA from defined numbers of bacterial cells that were subjected to three different DNA extraction methods: the QIAamp DNA Mini Kit, Reischl et al.'s method and FTA Elute. FTA Elute significantly showed the highest median DNA extraction efficiency of 76.9% for E. coli and 108.9% for S. aureus. The Reischl et al. method and QIAamp DNA Mini Kit inhibited the E. coli qrtPCR assay with a 10-fold decrease of detectable DNA. None of the methods inhibited the S. aureus qrtPCR assay. The FTA Elute applicability was demonstrated with swab samples taken from the International Space Station (ISS) interior. Overall, the FTA Elute method was found to be the most suitable to selected criteria in terms of rapidity, easiness of use, DNA extraction efficiency, toxicity, and transport and storage conditions. PMID:21311936

van Tongeren, S P; Degener, J E; Harmsen, H J M

2011-09-01

129

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

PubMed

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

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

2010-01-01

130

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

131

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

PubMed Central

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

2014-01-01

132

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

133

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

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; Lovejoy, Connie

2014-04-01

134

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

PubMed Central

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

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

2014-01-01

135

Acinetobacter diversity in environmental samples assessed by 16S rRNA gene PCR–DGGE fingerprinting  

Microsoft Academic Search

A primer pair was designed to selectively amplify a fragment of the Acinetobacter 16S rRNA gene from environmental samples by PCR. 16S rRNA gene products were only obtained in PCRs with DNA from members of the genus Acinetobacter and not with DNA from other bacterial species. Denaturing gradient gel electrophoresis (DGGE) of the Acinetobacter 16S rRNA gene amplicons enabled discrimination

Karolien Vanbroekhoven; Annemie Ryngaert; Pierre Wattiau; René De Mot; Dirk Springael

2004-01-01

136

Bacterial biodegradation of extractives and patterns of bordered pit membrane attack in pine wood.  

PubMed

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

Burnes, T A; Blanchette, R A; Farrell, R L

2000-12-01

137

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

PubMed Central

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

2014-01-01

138

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

139

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

2013-01-01

140

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

Microsoft Academic Search

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

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

141

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

142

Phytochemical and anti-bacterial activity of epidermal glands extract of Christella parasitica (L.) H. Lev.  

PubMed Central

Objective To study the morphology, biochemistry and bioactivity of the epidermal glands of the glandular morphotype of Christella parasitica (C. parasitica) (L.) H. Lev. Methods Morphological studies on epidermal glands were carried out by using light microscope and scanning electron microscope. To prepare the extract, the shade-dried fronds of glandular morphotype were soaked in acetone. For antibacterial studies paper disc method was followed by using various pathogenic bacteria. Results Detailed micromorphological, phytochemical and bioactivity studies on a medicinal fern C. parasitica (L.) H. Lev. showed its intraspecific variation in antibacterial activity. The presence or absence of the epidermal glands was the key factor for antibacterial activity in the morphovariants of this species. The epidermal glands were orange-coloured, stalked and elongated ones of about 84.2 µm × 45 µm, and distributed on the undersurface of costa, costules and veins in croziers, young and mature leaves. Frequency of glands varied from 15/cm on costa in mature leaves to 140/cm on costules in croziers. The acetone extract of the glands showed antibacterial activities and also toxic effect against mosquito larvae and tadpoles of frog. Preliminary phytochemical analysis and HPLC studies of the gland extract showed the presence of various kinds of terpenoids, alkaloids, tannins, saponins and flavonoids in it. Conclusions The present study shows that epidermal glands of the glandular morphotype of C. parasitica (L.) H. Lev. have several bioactive compounds and such rare morphovariant should be conserved in nature. The next step is to isolate the pure compounds and to screen the bioactivity of individual compounds of the epidermal glands. PMID:23569716

Paul, Raj K; Irudayaraj, V; Johnson, M; Patric, Raja D

2011-01-01

143

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

PubMed Central

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

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

2011-01-01

144

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

145

Comparison of the Rhizosphere Bacterial Communities of Zigongdongdou Soybean and a High-Methionine Transgenic Line of This Cultivar  

PubMed Central

Previous studies have shown that methionine from root exudates affects the rhizosphere bacterial population involved in soil nitrogen fixation. A transgenic line of Zigongdongdou soybean cultivar (ZD91) that expresses Arabidopsis cystathionine ?-synthase resulting in an increased methionine production was examined for its influence to the rhizosphere bacterial population. Using 16S rRNA gene-based pyrosequencing analysis of the V4 region and DNA extracted from bacterial consortia collected from the rhizosphere of soybean plants grown in an agricultural field at the pod-setting stage, we characterized the populational structure of the bacterial community involved. In total, 87,267 sequences (approximately 10,908 per sample) were analyzed. We found that Acidobacteria, Proteobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Planctomycetes, Gemmatimonadetes, Firmicutes, and Verrucomicrobia constitute the dominant taxonomic groups in either the ZD91 transgenic line or parental cultivar ZD, and that there was no statistically significant difference in the rhizosphere bacterial community structure between the two cultivars. PMID:25079947

Ji, Jun; Wu, Haiying; Meng, Fang; Zhang, Mingrong; Zheng, Xiaobo; Wu, Cunxiang; Zhang, Zhengguang

2014-01-01

146

The responses of the taxa composition of particle-attached bacterial community to the decomposition of Microcystis blooms.  

PubMed

The changes of taxa within the particle-attached bacterial assemblage during the decomposition of Microcystis blooms were investigated under darkness and anoxic condition in mesocosm experiments. During 14 days of darkness incubation, chlorophyll-a (Ch-a) concentration decreased from 2000 ?g/L to 5 ?g/L. Samples were collected on days 0, 2 and 14 for bacterial 16S rRNA analysis, based on rapid decreases in the Chl-a concentration of water column. The total bacterial community DNA was extracted and 16S rRNA genes were amplified by polymerase chain reaction, cloned and sequenced of selected samples. The results showed that the abundance of attached bacteria increased significantly, and the composition of the particle-attached bacterial communities varied temporally during the decomposition of Microcystis blooms. The bacterial assemblage appeared to be dominated by members of Bacteroidetes, Alphaproteobacteria and Betaproteobacteria. Shift of some genera of Alphaproteobacteria and Alphaproteobacteria was also observed. Additionally, we found that the family Sphingomonas, affiliated with Alphaproteobacteria, identified as a microcystin-degrading bacterium, dominated the particle-attached bacterial communities. The results from the present study, together with previously published data highlighted the need for more studies concerning the bacterial degradation process in order to trace the environmental fate of microcystins in field conditions. PMID:24836132

Shao, Keqiang; Zhang, Lei; Wang, Yongping; Yao, Xin; Tang, Xiangming; Qin, Boqiang; Gao, Guang

2014-08-01

147

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

148

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

149

Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells  

Microsoft Academic Search

Crude juices of eight Brassica vegetables as well as juices and extracts of spices and phytopharmaceutical preparations from cruciferous vegetables were tested for induction of point mutations in Salmonella TA98 and TA100, repairable DNA damage in E.coli K-12 cells and clastogenic effects in mammalian cells. In bacterial assays, all juices caused genotoxic effects in the absence of metabolic activation, the

Fekadu Kassie; Wolfram Parzefall; Stephen Musk; Ian Johnson; Günther Lamprecht; Gerhard Sontag; Siegfried Knasmüller

1996-01-01

150

Towards complete biodiversity assessment: an evaluation of the subterranean bacterial communities in the Oklo region of the sole surviving natural nuclear reactor  

Microsoft Academic Search

Groundwater bacterial rRNA sequences extracted from the natural nuclear reactor region of Gabon are used to demonstrate the application of phylogenetic methods to biodiversity assessment. Clones were provisionally placed in `genera' using either the genus of the closest named EMBL entry, or by grouping clones at least 97.5% identical. The community is small, with 24 putative genera under the `closest-match'

R. H. Crozier; P.-M. Agapow; K. Pedersen

1999-01-01

151

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

152

Benthic Bacterial Diversity in Submerged Sinkhole Ecosystems? †  

PubMed Central

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

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

2010-01-01

153

Different bulk and active bacterial communities in cryoconite from the margin and interior of the Greenland ice sheet.  

PubMed

Biological processes in the supraglacial ecosystem, including cryoconite, contribute to nutrient cycling within the cryosphere and may affect surface melting, yet little is known of the diversity of the active microbes in these environments. We examined the bacterial abundance and community composition of cryoconite over a melt season at two contrasting sites at the margin and in the interior of the Greenland ice sheet, using sequence analysis and quantitative PCR of co-extracted 16S rDNA and rRNA. Significant differences were found between bulk (rDNA) and potentially active (rRNA) communities, and between communities sampled from the two sites. Higher concentrations of rRNA than rDNA were detected at the interior site, whereas at the margin several orders of magnitude less rRNA was found compared to rDNA, which may be explained by a lower proportion of active bacteria at the margin site. The rRNA communities at both sites were dominated by a few taxa of Cyanobacteria and Alpha- and/or Betaproteobacteria. The bulk alpha diversity was higher in the margin site community, suggesting that local sources may be contributing towards the gene pool in addition to long distance transport. PMID:25405749

Stibal, Marek; Schostag, Morten; Cameron, Karen A; Hansen, Lars H; Chandler, David M; Wadham, Jemma L; Jacobsen, Carsten S

2014-11-18

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

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).  

PubMed

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 of the middle ring of norathyriol, this study was designed to determine phenolic cleavage products in the feces of pigs fed an extract of C genistoides. Pigs received 74 mg mangiferin per kilogram of body weight daily for 11 days; feces fractions were collected on day 11 and on the first 2 days after the last intake of extract. Several phenolic acids were detected in the feces samples, including 3-hydroxyphenylacetic acid; 4-hydroxybenzoic acid; 3,4-dihydroxybenzoic acid; 3,4-dihydroxyphenylacetic acid; 2,4,6-trihydroxybenzoic acid; 3,4,5-trihydroxybenzoic acid (gallic acid); and phloroglucinol. However, in vivo formation was likely only for 3,4-dihydroxybenzoic acid; 3,4-dihydroxyphenylacetic acid; 2,4,6-trihydroxybenzoic acid; and 3,4,5-trihydroxybenzoic acid because these were not present in the blank feces, in the animals' normal diet, or in the C genistoides extract. The fact that these amounts were very low suggests further degradation of the metabolites by intestinal microflora or absorption of the cleavage products by the colon. PMID:20579527

Bock, Constance; Ternes, Waldemar

2010-05-01

156

rRNA subtraction protocol Stewart, Ottesen, and DeLong (2010) rRNA subtraction protocol for metatranscriptomics  

E-print Network

rRNA subtraction protocol ­ Stewart, Ottesen, and DeLong (2010) 1 rRNA subtraction protocol the availability of all downstream reagents before proceeding to the next step. #12;rRNA subtraction protocol ­ Stewart, Ottesen, and DeLong (2010) 2 rRNA subtraction A. PCR amplification of rRNA genes This step

Stewart, Frank

157

Phylogenetic Analysis of Pasteuria penetrans by 16S rRNA Gene Cloning and Sequencing.  

PubMed

Pasteuria penetrans is an endospore-forming bacterial parasite of Meloidogyne spp. This organism is among the most promising agents for the biological control of root-knot nematodes. In order to establish the phylogenetic position of this species relative to other endospore-forming bacteria, the 16S ribosomal genes from two isolates of P. penetrans, P-20, which preferentially infects M. arenaria race 1, and P-100, which preferentially infects M. incognita and M. javanica, were PCR-amplified from a purified endospore extraction. Universal primers for the 16S rRNA gene were used to amplify DNA which was cloned, and a nucleotide sequence was obtained for 92% of the gene (1,390 base pairs) encoding the 16S rDNA from each isolate. Comparison of both isolates showed identical sequences that were compared to 16S rDNA sequences of 30 other endospore-forming bacteria obtained from GenBank. Parsimony analyses indicated that P. penetrans is a species within a clade that includes Alicyclobacillus acidocaldarius, A. cycloheptanicus, Sulfobacillus sp., Bacillus tusciae, B. schlegelii, and P. ramosa. Its closest neighbor is P. ramosa, a parasite of Daphnia spp. (water fleas). This study provided a genomic basis for the relationship of species assigned to the genus Pasteuria, and for comparison of species that are parasites of different phytopathogenic nematodes. PMID:19270903

Anderson, J M; Preston, J F; Dickson, D W; Hewlett, T E; Williams, N H; Maruniak, J E

1999-09-01

158

Simultaneous Extraction from Bacterioplankton of Total RNA and DNA Suitable for Quantitative Structure and Function Analyses  

PubMed Central

The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (nir gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample. PMID:11872453

Weinbauer, Markus G.; Fritz, Ingo; Wenderoth, Dirk F.; Höfle, Manfred G.

2002-01-01

159

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

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

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

162

Rapid Antimicrobial Susceptibility Testing with Electrokinetics Enhanced Biosensors for Diagnosis of Acute Bacterial Infections  

E-print Network

of bacterial 16S rRNA. The assay determines the susceptibility of pathogens by detecting bacterial growth under,26 Conventional cul- ture-based analysis, however, requires at least 2­3 days for bacterial growth and could of Acute Bacterial Infections TINGTING LIU,1 YI LU,1 VINCENT GAU,2 JOSEPH C. LIAO,3,4 and PAK KIN WONG 1 1

Wong, Pak Kin

163

Bacterial diversity in hyperarid Atacama Desert soils  

NASA Astrophysics Data System (ADS)

Surface and subsurface soil samples analyzed for this investigation were collected from the hyperarid Yungay region in the Atacama Desert, Chile. This report details the bacterial diversity derived from DNA and PLFA extracted directly from these extremely desiccated soils. Actinobacteria, Proteobacteria, Firmicutes and TM7 division bacteria were detected. Ninety-four percent of the 16S rRNA genes cloned from these soils belong to the Actinobacteria phylum, and the majority of these were most closely related to the genus Frankia. A 24-hour water activity (aw) time course showed a diurnal cycle that peaked at 0.52 in the early predawn hours, and ranged from 0.01-0.08 during the day. All measured water activity values were below the levels required for microbial growth or enzyme activity. Total organic carbon (TOC) concentrations were above the limit of detection and below the limit of quantification (i.e., 200 ?g/g < TOC < 1000 ?g/g), and phospholipid fatty acid (PLFA) concentrations ranged from 2 × 105 to 7 × 106 cell equivalents per gram of soil. Soil extracts analyzed for culturable biomass yielded mostly no growth on R2A media; the highest single extract yielded 47 colony forming units (CFU) per gram of soil.

Connon, Stephanie A.; Lester, Elizabeth D.; Shafaat, Hannah S.; Obenhuber, Donald C.; Ponce, Adrian

2007-12-01

164

Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3'-terminal domain.  

PubMed

Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3'-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA-protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed. PMID:25462191

Gopanenko, Alexander V; Malygin, Alexey A; Karpova, Galina G

2015-02-01

165

The intestinal bacterial community in the food waste-reducing larvae of Hermetia illucens.  

PubMed

As it is known that food waste can be reduced by the larvae of Hermetia illucens (Black soldier fly, BSF), the scientific and commercial value of BSF larvae has increased recently. We hypothesised that the ability of catabolic degradation by BSF larvae might be due to intestinal microorganisms. Herein, we analysed the bacterial communities in the gut of BSF larvae by pyrosequencing of extracting intestinal metagenomic DNA from larvae that had been fed three different diets. The 16S rRNA sequencing results produced 9737, 9723 and 5985 PCR products from larval samples fed food waste, cooked rice and calf forage, respectively. A BLAST search using the EzTaxon program showed that the bacterial community in the gut of larvae fed three different diets was mainly composed of the four phyla with dissimilar proportions. Although the composition of the bacterial communities depended on the different nutrient sources, the identified bacterial strains in the gut of BSF larvae represented unique bacterial species that were unlike the intestinal microflora of other insects. Thus, our study analysed the structure of the bacterial communities in the gut of BSF larvae after three different feedings and assessed the application of particular bacteria for the efficient degradation of organic compounds. PMID:21267722

Jeon, Hyunbum; Park, Soyoung; Choi, Jiyoung; Jeong, Gilsang; Lee, Sang-Beom; Choi, Youngcheol; Lee, Sung-Jae

2011-05-01

166

Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower respiratory tract infections.  

PubMed

In this paper, we describe the development of an automated sample preparation procedure for etiological agents of community-acquired lower respiratory tract infections (CA-LRTI). The consecutive assay steps, including sample re-suspension, pre-treatment, lysis, nucleic acid purification, and concentration, were integrated into a microfluidic lab-on-a-chip (LOC) cassette that is operated hands-free by a demonstrator setup, providing fluidic and valve actuation. The performance of the assay was evaluated on viral and Gram-positive and Gram-negative bacterial broth cultures previously sampled using a nasopharyngeal swab. Sample preparation on the microfluidic cassette resulted in higher or similar concentrations of pure bacterial DNA or viral RNA compared to manual benchtop experiments. The miniaturization and integration of the complete sample preparation procedure, to extract purified nucleic acids from real samples of CA-LRTI pathogens to, and above, lab quality and efficiency, represent important steps towards its application in a point-of-care test (POCT) for rapid diagnosis of CA-LRTI. PMID:24615272

Van Heirstraeten, Liesbet; Spang, Peter; Schwind, Carmen; Drese, Klaus S; Ritzi-Lehnert, Marion; Nieto, Benjamin; Camps, Marta; Landgraf, Bryan; Guasch, Francesc; Corbera, Antoni Homs; Samitier, Josep; Goossens, Herman; Malhotra-Kumar, Surbhi; Roeser, Tina

2014-05-01

167

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

168

Rapid identification of bacterial isolates from wheat roots by high resolution whole cell MALDI-TOF MS analysis.  

PubMed

Whole-cell mass spectrometry analysis is a powerful tool to rapidly identify microorganisms. Several studies reported the successful application of this technique to identify a variety of bacterial species with a discriminatory power at the strain level, mainly for bacteria of clinical importance. In this study we used matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to assess the diversity of wheat-associated bacterial isolates. Wheat plants cultivated in non-sterile vermiculite, under greenhouse conditions were used for bacterial isolation. Total cellular extracts of 138 isolates were analyzed by MALDI-TOF MS and the mass spectra were used to cluster the isolates. Taxonomic identification and phylogenetic reconstruction based on 16S rRNA gene sequences showed the presence of Pseudomonas, Pantoea, Acinetobacter, Enterobacter and Curtobacterium. The 16S rRNA gene sequence analyses were congruent with the clusterization from mass spectra profile. Moreover, MALDI-TOF whole cell mass profiling allowed a finer discrimination of the isolates, suggesting that this technique has the potential of differentiating bacterial isolates at the strain level. PMID:23591594

Stets, Maria Isabel; Pinto, Artur Soares; Huergo, Luciano Fernandes; de Souza, Emanuel Maltempi; Guimarães, Vandeir Francisco; Alves, Alexessander Couto; Steffens, Maria Berenice Reynaud; Monteiro, Rose Adele; Pedrosa, Fábio de Oliveira; Cruz, Leonardo Magalhães

2013-06-10

169

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

170

[Preliminary research on bacterial diversity of Parece Vela Basin, Pacific Ocean by culture-independent method].  

PubMed

The environmental DNA was directly extracted from the sediment in Parece Vela Basin, Pacific Ocean, at a depth of 5010 m. Bacterial 16S rRNA gene library of 32 clones was generated using bacterial universal primers and 16S rDNA sequences were analyzed phylogenetically. 17 phylotypes were obtained. The library was dominated by gamma-Proteobacteria, alpha-Proteobacteria and marine uncultured bacteria. Sixty-two percent of the cloned sequences was highly related to the known bacteria in the genus Halomonas, Alcanivorax, Pseudomonas, Acinetobacter, Pseudoalteromonas (> 96% sequence similarity), while some of the cloned sequences showed less affiliation with known taxa (< 94% sequence similarity) and may represent novel taxa. PMID:15847151

Xie, Hua; Xue, Yan-fen; Zhao, Ai-min; Li, Tie-gang; Ma, Yan-he

2005-02-01

171

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

172

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.; Büchler, Andrea C.; Eich, Gerhard; Wanner, Roger M.; Speck, Roberto F.; Böttger, Erik C.; Bloemberg, Guido V.

2010-01-01

173

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

174

Czech ethanol-free propolis extract displays inhibitory activity against a broad spectrum of bacterial and fungal pathogens.  

PubMed

Propolis acts primarily as a biocide against invasive bacteria and fungi in the hive, suggesting its potential for industrial applications. In food application, propolis is considered as a chemical preservative in meat products, extending shelf life of frozen meat and other food. The mechanism of action is still unclear due to the synergy of multiple compounds contained in propolis and due to parallel targeting of multiple pathways within each affected organism. Here, we examined the antimicrobial properties of dimethylsulfoxide (DMSO) Czech propolis extract. Until recently, DMSO was only rarely used in the propolis studies, although the other solvents tested (mostly ethanol) may significantly affect the observed inhibitory effects, notwithstanding the antimicrobial effects of ethanol itself. Here, we provide results of zone inhibition tests against Aspergillus fumigatus, Microsporum gypseum, Microsporum canis, Candida albicans, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis. Although we determined inhibitory effects against all the microorganisms tested, the dose-dependent response curves were not similar to each other. While inhibitory effects against C. albicans or S. aureus were strictly dose-dependent, responses of M. gypseum and E. faecalis displayed plateau across the broad range of concentrations tested. Interestingly, response of E. coli revealed the double-peak dose-dependent curve, and responses of M. canis and L. monocytogenes decreased at the highest concentrations tested. Suggested is evaluation of DMSO propolis extracts in experimental treatment of human and veterinary infections, preferably in multitherapy with antibiotics. PMID:23915150

Netíková, Ladislava; Bogusch, Petr; Heneberg, Petr

2013-09-01

175

The Majority of In Vitro Macrophage Activation Exhibited by Extracts of Some Immune Enhancing Botanicals is Due to Bacterial Lipoproteins and Lipopolysaccharides  

Technology Transfer Automated Retrieval System (TEKTRAN)

We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacteriall...

176

Bacterial diversity in a deep-subsurface clay environment.  

PubMed Central

The presence of bacteria in a deep clay sediment was analyzed in a 20-m-long core horizontally drilled from a mine gallery at a depth of 224 m in the Boom clay formation (Mol, Belgium). This clay deposit is the result of a marine sedimentary process that occurred 35 million years ago. Bacterial activities were estimated by measuring respiration on [14C]glucose. Using the same samples, universal primers for the genes coding for eubacterial 16S rRNA were used to amplify extracted DNA. PCR products were then cloned, sequenced, and analyzed by molecular phylogeny. Our data showed a decrease in bacterial densities as a function of distance from the gallery, with few bacteria detectable by culture at more than 80 cm from the gallery wall. PCR experiments showed the presence of bacteria in all samples, and phylogenetic analyses were then used to tentatively identify these organisms. Because of low bacterial densities in deep clay samples, direct counts and enumeration of viable bacteria on diverse culture media remained negative. All experiments, both cultures and PCR, demonstrated the difficulty of analyzing samples that contain only a few poorly active bacteria as it is difficult to avoid a small contamination by active bacteria during sampling. Since the porosity of the Boom clay formation is less than the expected size of bacteria, it is possible that some of the bacteria present in this 35-million-year-old deep clay deposit derive from cells initially trapped during the sedimentation process. PMID:8795233

Boivin-Jahns, V; Ruimy, R; Bianchi, A; Daumas, S; Christen, R

1996-01-01

177

The impact of transcriptional tuning on in vitro integrated rRNA transcription and ribosome construction  

PubMed Central

In vitro ribosome construction could enable studies of ribosome assembly and function, provide a route toward constructing minimal cells for synthetic biology, and permit the construction of ribosome variants with new functions. Toward these long-term goals, we recently reported on an integrated, one-pot ribosomal RNA synthesis (rRNA), ribosome assembly, and translation technology (termed iSAT) for the construction of Escherichia coli ribosomes in crude ribosome-free S150 extracts. Here, we aimed to improve the activity of iSAT through transcriptional tuning. Specifically, we increased transcriptional efficiency through 3? modifications to the rRNA gene sequences, optimized plasmid and polymerase concentrations, and demonstrated the use of a T7-promoted rRNA operon for stoichiometrically balanced rRNA synthesis and native rRNA processing. Our modifications produced a 45-fold improvement in iSAT protein synthesis activity, enabling synthesis of 429 ± 15 nmol/l green fluorescent protein in 6 h batch reactions. Further, we show that the translational activity of ribosomes purified from iSAT reactions is about 20% the activity of native ribosomes purified directly from E. coli cells. Looking forward, we believe iSAT will enable unique studies to unravel the systems biology of ribosome biogenesis and open the way to new methods for making and studying ribosomal variants. PMID:24792158

Fritz, Brian R.; Jewett, Michael C.

2014-01-01

178

Biases in community structures of ammonia/ammonium-oxidizing microorganisms caused by insufficient DNA extractions from Baijiang soil revealed by comparative analysis of coastal wetland sediment and rice paddy soil.  

PubMed

Repetitive extraction of DNAs from surface sediments of a coastal wetland in Mai Po Nature Reserve (MP) of Hong Kong and surface Baijiang soils from a rice paddy (RP) in Northeast China was conducted to compare the microbial diversity in this study. Community structures of ammonia/ammonium-oxidizing microorganisms in these samples were analyzed by PCR-DGGE technique. The diversity and abundance of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and anaerobic ammonium-oxidizing (anammox) bacteria were also analyzed based on archaeal and bacterial ammonia monooxygenase subunit A encoding (amoA) and anammox bacterial 16S rRNA genes, respectively. DGGE profiles of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes showed a similar pattern among all five repetitively extracted DNA fractions from both MP and RP, except the anammox bacteria in RP, indicating a more diverse anammox community retrieved in the second to the fifth fractions than the first one. Both soil and marine group AOA were detected while soil and coastal group AOB and Scalindua-anammox bacteria were dominant in MP. Soil group AOA and marine group AOB were dominant in RP, while both Scalindua and Kuenenia species were detected in RP. Pearson correlation analysis showed that the abundance of archaeal and bacterial amoA and anammox bacterial 16S rRNA genes was significantly correlated with the DNA concentrations of the five DNA fractions from MP, but not from RP (except the archaeal amoA gene). Results suggest that anammox bacteria diversity may be biased by insufficient DNA extraction of rice paddy soil samples. PMID:23974369

Han, Ping; Li, Meng; Gu, Ji-Dong

2013-10-01

179

Mulberry leaf extract mediated synthesis of gold nanoparticles and its anti-bacterial activity against human pathogens  

NASA Astrophysics Data System (ADS)

Gold nanoparticles (Au-NPs) were synthesized at room temperature using Morus alba (mulberry) leaf extract as reducing and stabilizing agent. The development of plant mediated synthesis of nanoparticles is gaining importance due to its simplicity, low cost, non-toxicity, eco-friendliness, long term stability and reproducible aqueous synthesis method to obtain a self-assembly of nearly monodispersed Au-NPs. The formation and morphology of biosynthesized nanoparticles are investigated with the help of UV-Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), x-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FT-IR) techniques. Au-NPs formation was screened by UV-Vis spectroscopy through color conversion due to surface plasmon resonance band at 538 nm for Au-NPs. DLS studies revealed that the average size of Au-NPs was 50 nm. TEM studies showed the particles to be nearly spherical with few irregular shapes and particle size ranges 15?53 nm. The AFM image clearly shows the surface morphology of the well-dispersed Au-NPs with less than 50 nm. The high crystallinity of nanoparticles is evident from bright circular spots in the selected area electron diffraction (SAED) pattern. X-ray diffraction pattern showed high purity and face-centered cubic structure of Au-NPs. The FT-IR results indicate the presence of different functional groups present in the biomolecule capping the nanoparticles. Further, biosynthesized Au-NPs show strong zone of inhibition against Vibrio cholera (gram-negative) and Staphylococcus aureus (gram-positive) whereas, chemically synthesized Au-NPs and mulberry leaf extract exhibit a fair zone of inhibition.

Adavallan, K.; Krishnakumar, N.

2014-06-01

180

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

181

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

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

2013-01-01

182

Patterns of bacterial diversity across a range of Antarctic terrestrial habitats.  

PubMed

Although soil-borne bacteria represent the world's greatest source of biological diversity, it is not well understood whether extreme environmental conditions, such as those found in Antarctic habitats, result in reduced soil-borne microbial diversity. To address this issue, patterns of bacterial diversity were studied in soils sampled along a > 3200 km southern polar transect spanning a gradient of increased climate severity over 27 degrees of latitude. Vegetated and fell-field plots were sampled at the Falkland (51 degrees S), South Georgia (54 degrees S), Signy (60 degrees S) and Anchorage Islands (67 degrees S), while bare frost-sorted soil polygons were examined at Fossil Bluff (71 degrees S), Mars Oasis (72 degrees S), Coal Nunatak (72 degrees S) and the Ellsworth Mountains (78 degrees S). Bacterial 16S rRNA gene sequences were recovered subsequent to direct DNA extraction from soil, polymerase chain reaction amplification and cloning. Although bacterial diversity was observed to decline with increased latitude, habitat-specific patterns appeared to also be important. Namely, a negative relationship was found between bacterial diversity and latitude for fell-field soils, but no such pattern was observed for vegetated sites. The Mars Oasis site, previously identified as a biodiversity hotspot within this region, proved exceptional within the study transect, with unusually high bacterial diversity. In independent analyses, geographical distance and vegetation cover were found to significantly influence bacterial community composition. These results provide insight into the factors shaping the composition of bacterial communities in Antarctic terrestrial habitats and support the notion that bacterial diversity declines with increased climatic severity. PMID:17922752

Yergeau, Etienne; Newsham, Kevin K; Pearce, David A; Kowalchuk, George A

2007-11-01

183

Eukaryotic 5S rRNA biogenesis  

PubMed Central

The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. PMID:21957041

Ciganda, Martin; Williams, Noreen

2012-01-01

184

Bacterial and archaeal populations associated with freshwater ferromanganous micronodules and sediments.  

PubMed

Biology is believed to play a large role in the cycling of iron and manganese in many freshwater environments, but specific microbial groups indigenous to these systems have not been well characterized. To investigate the populations of Bacteria and Archaea associated with metal-rich sediments from Green Bay, WI, we extracted nucleic acids and analysed the phylogenetic relationships of cloned 16S rRNA genes. Because nucleic acids have not been routinely extracted from metal-rich samples, we investigated the bias inherent in DNA extraction and gene amplification from pure MnO2 using defined populations of whole cells or naked DNA. From the sediments, we screened for manganese-oxidizing bacteria using indicator media and found three isolates that were capable of manganese oxidation. In the phylogenetic analysis of bacterial 16S rRNA gene clones, we found two groups related to known metal-oxidizing genera, Leptothrix of the beta-Proteobacteria and Hyphomicrobium of the alpha-Proteobacteria, and a Fe(III)-reducing group related to the Magnetospirillum genus of the alpha-Proteobacteria. Groups related to the metal-reducing delta-Proteobacteria constituted 22% of the gene clones. In addition, gene sequences from one group of methanogens and a group of Crenarchaeota, identified in the archaeal gene clone library, were related to those found previously in Lake Michigan sediments. PMID:11225719

Stein, L Y; La Duc, M T; Grundl, T J; Nealson, K H

2001-01-01

185

The Ribosomal Database Project: improved alignments and new tools for rRNA analysis.  

PubMed

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/. PMID:19004872

Cole, J R; Wang, Q; Cardenas, E; Fish, J; Chai, B; Farris, R J; Kulam-Syed-Mohideen, A S; McGarrell, D M; Marsh, T; Garrity, G M; Tiedje, J M

2009-01-01

186

The Ribosomal Database Project: improved alignments and new tools for rRNA analysis  

PubMed Central

The Ribosomal Database Project (RDP) provides researchers with quality-controlled bacterial and archaeal small subunit rRNA alignments and analysis tools. An improved alignment strategy uses the Infernal secondary structure aware aligner to provide a more consistent higher quality alignment and faster processing of user sequences. Substantial new analysis features include a new Pyrosequencing Pipeline that provides tools to support analysis of ultra high-throughput rRNA sequencing data. This pipeline offers a collection of tools that automate the data processing and simplify the computationally intensive analysis of large sequencing libraries. In addition, a new Taxomatic visualization tool allows rapid visualization of taxonomic inconsistencies and suggests corrections, and a new class Assignment Generator provides instructors with a lesson plan and individualized teaching materials. Details about RDP data and analytical functions can be found at http://rdp.cme.msu.edu/. PMID:19004872

Cole, J. R.; Wang, Q.; Cardenas, E.; Fish, J.; Chai, B.; Farris, R. J.; Kulam-Syed-Mohideen, A. S.; McGarrell, D. M.; Marsh, T.; Garrity, G. M.; Tiedje, J. M.

2009-01-01

187

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

188

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100-bp insertion in helix 6  

SciTech Connect

The determination of variation in 16S rRNA gene sequences is perhaps the most common method for assessing microbial community diversity. However, the occurrence of multiple copies of 16S rRNA genes within some organisms can bias estimates of microbial diversity. During phylogenetic characterization of a metal-transforming, fermentative bacterium (strain UFO1) isolated from the Field Research Center (FRC) in Oak Ridge, TN, we detected an apparent 16S rRNA pseudogene. The putative 16S rRNA pseudogene was first detected in clone libraries constructed with 16S rRNA genes amplified from UFO1 genomic DNA. Sequencing revealed two distinct 16S rRNA genes, with one differing from the other by a 100 bp insert near the 5’ end. Ribosomal RNA was extracted from strain UFO1 and analyzed by RT-qPCR with insert and non-insert specific primers; however, only the non-insert 16S rRNA sequence was expressed. Reverse-transcribed rRNA from strain UFO1 was also used to construct a cDNA library. Of 190 clones screened by PCR, none contained the 16S rRNA gene with the 100 bp insert. Examination of GenBank 16S rRNA gene sequences revealed that the same insert sequence was present in other clones, including those from an environmental library constructed from FRC enrichments. These findings demonstrate the existence of widely disparate copies of the 16S rRNA gene in the same species and a putative 16S rRNA pseudogene, which may confound 16S rRNA-based methods for assessments of microbial diversity in environmental samples.

Allison E. Ray; Stephanie A. Connon; Peter P. Sheridan; Jeremy Gilbreath; Malcolm S. Shields; Deborah T. Newby; Yoshiko Fujita; Timothy S. Magnuson

2010-06-01

189

Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome  

PubMed Central

AIM: To design and validate broad-range 16S rRNA primers for use in high throughput sequencing to classify bacteria isolated from the human foregut microbiome. METHODS: A foregut microbiome dataset was constructed using 16S rRNA gene sequences obtained from oral, esophageal, and gastric microbiomes produced by Sanger sequencing in previous studies represented by 219 bacterial species. Candidate primers evaluated were from the European rRNA database. To assess the effect of sequence length on accuracy of classification, 16S rRNA genes of various lengths were created by trimming the full length sequences. Sequences spanning various hypervariable regions were selected to simulate the amplicons that would be obtained using possible primer pairs. The sequences were compared with full length 16S rRNA genes for accuracy in taxonomic classification using online software at the Ribosomal Database Project (RDP). The universality of the primer set was evaluated using the RDP 16S rRNA database which is comprised of 433 306 16S rRNA genes, represented by 36 phyla. RESULTS: Truncation to 100 nucleotides (nt) downstream from the position corresponding to base 28 in the Escherichia coli 16S rRNA gene caused misclassification of 87 (39.7%) of the 219 sequences, compared with misclassification of only 29 (13.2%) sequences with truncation to 350 nt. Among 350-nt sequence reads within various regions of the 16S rRNA gene, the reverse read of an amplicon generated using the 343F/798R primers had the least (8.2%) effect on classification. In comparison, truncation to 900 nt mimicking single pass Sanger reads misclassified 5.0% of the 219 sequences. The 343F/798R amplicon accurately assigned 91.8% of the 219 sequences at the species level. Weighted by abundance of the species in the esophageal dataset, the 343F/798R amplicon yielded similar classification accuracy without a significant loss in species coverage (92%). Modification of the 343F/798R primers to 347F/803R increased their universality among foregut species. Assuming that a typical polymerase chain reaction can tolerate 2 mismatches between a primer and a template, the modified 347F and 803R primers should be able to anneal 98% and 99.6% of all 16S rRNA genes in the RDP database. CONCLUSION: 347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes. PMID:20806429

Nossa, Carlos W; Oberdorf, William E; Yang, Liying; Aas, Jørn A; Paster, Bruce J; DeSantis, Todd Z; Brodie, Eoin L; Malamud, Daniel; Poles, Michael A; Pei, Zhiheng

2010-01-01

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

Phylogenetic analysis of a biofilm bacterial population in a water pipeline in the Gulf of Mexico.  

PubMed

The aim of this study was to assess the bacterial diversity associated with a corrosive biofilm in a steel pipeline from the Gulf of Mexico used to inject marine water into the oil reservoir. Several aerobic and heterotrophic bacteria were isolated and identified by 16S rRNA gene sequence analysis. Metagenomic DNA was also extracted to perform a denaturing gradient gel electrophoresis analysis of ribosomal genes and to construct a 16S rRNA gene metagenomic library. Denaturing gradient gel electrophoresis profiles and ribosomal libraries exhibited a limited bacterial diversity. Most of the species detected in the ribosomal library or isolated from the pipeline were assigned to Proteobacteria (Halomonas spp., Idiomarina spp., Marinobacter aquaeolei, Thalassospira sp., Silicibacter sp. and Chromohalobacter sp.) and Bacilli (Bacillus spp. and Exiguobacterium spp.). This is the first report that associates some of these bacteria with a corrosive biofilm. It is relevant that no sulfate-reducing bacteria were isolated or detected by a PCR-based method. The diversity and relative abundance of bacteria from water pipeline biofilms may contribute to an understanding of the complexity and mechanisms of metal corrosion during marine water injection in oil secondary recovery. PMID:16958915

López, Miguel A; Zavala-Díaz de la Serna, F Javier; Jan-Roblero, Janet; Romero, Juan M; Hernández-Rodríguez, César

2006-10-01

192

The feline oral microbiome: A provisional 16S rRNA gene based taxonomy with full-length reference sequences.  

PubMed

The human oral microbiome is known to play a significant role in human health and disease. While less well studied, the feline oral microbiome is thought to play a similarly important role. To determine roles oral bacteria play in health and disease, one first has to be able to accurately identify bacterial species present. 16S rRNA gene sequence information is widely used for molecular identification of bacteria and is also useful for establishing the taxonomy of novel species. The objective of this research was to obtain full 16S rRNA gene reference sequences for feline oral bacteria, place the sequences in species-level phylotypes, and create a curated 16S rRNA based taxonomy for common feline oral bacteria. Clone libraries were produced using "universal" and phylum-selective PCR primers and DNA from pooled subgingival plaque from healthy and periodontally diseased cats. Bacteria in subgingival samples were also cultivated to obtain isolates. Full-length 16S rDNA sequences were determined for clones and isolates that represent 171 feline oral taxa. A provisional curated taxonomy was developed based on the position of each taxon in 16S rRNA phylogenetic trees. The feline oral microbiome curated taxonomy and 16S rRNA gene reference set will allow investigators to refer to precisely defined bacterial taxa. A provisional name such as "Propionibacterium sp. feline oral taxon FOT-327" is an anchor to which clone, strain or GenBank names or accession numbers can point. Future next-generation-sequencing studies of feline oral bacteria will be able to map reads to taxonomically curated full-length 16S rRNA gene sequences. PMID:25523504

Dewhirst, Floyd E; Klein, Erin A; Bennett, Marie-Louise; Croft, Julie M; Harris, Stephen J; Marshall-Jones, Zoe V

2015-02-25

193

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; Mühlemann, Kathrin; Hilty, Markus

2012-01-01

194

Common 5S rRNA variants are likely to be accepted in many sequence contexts  

NASA Technical Reports Server (NTRS)

Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.

Zhang, Zhengdong; D'Souza, Lisa M.; Lee, Youn-Hyung; Fox, George E.

2003-01-01

195

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

2014-01-01

196

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

197

Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt  

Microsoft Academic Search

Prokaryotic diversity in Alpine salt sediments was investigated by polymerase chain reaction (PCR) amplification of 16S rRNA genes, sequencing of cloned products, and comparisons with culturable strains. DNA was extracted from the residue following filtration of dissolved Permo-Triassic rock salt. Fifty-four haloarchaeal sequences were obtained, which could be grouped into at least five distinct clusters. Similarity values of three clusters

Christian Radax; Claudia Gruber; Helga Stan-Lotter

2001-01-01

198

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

199

Detection of Live and Antibiotic-Killed Bacteria by Quantitative Real-Time PCR of Specific Fragments of rRNA  

Microsoft Academic Search

Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic- induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mech- anistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either

Steve Aellen; Yok-Ai Que; Bertrand Guignard; Marisa Haenni; Philippe Moreillon

2006-01-01

200

Characterization of bacterial community associated to biofilms of corroded oil pipelines from the southeast of Mexico.  

PubMed

Microbial communities associated to biofilms promote corrosion of oil pipelines. The community structure of bacteria in the biofilm formed in oil pipelines is the basic knowledge to understand the complexity and mechanisms of metal corrosion. To assess bacterial diversity, biofilm samples were obtained from X52 steel coupons corroded after 40 days of exposure to normal operation and flow conditions. The biofilm samples were directly used to extract metagenomic DNA, which was used as template to amplify 16S ribosomal gene by PCR. The PCR products of 16S ribosomal gene were also employed as template for sulfate-reducing bacteria (SRB) specific nested-PCR and both PCR products were utilized for the construction of gene libraries. The V3 region of the 16S rRNA gene was also amplified to analyse the bacterial diversity by analysis of denaturing gradient gel electrophoresis (DGGE). Ribosomal library and DGGE profiles exhibited limited bacterial diversity, basically including Citrobacter spp., Enterobacter spp. and Halanaerobium spp. while Desulfovibrio alaskensis and a novel clade within the genus Desulfonatronovibrio were detected from the nested PCR library. The biofilm samples were also taken for the isolation of SRB. Desulfovibrio alaskensis and Desulfovibrio capillatus, as well as some strains related to Citrobacter were isolated. SRB consists in a very small proportion of the community and Desulfovibrio spp. were the relatively abundant groups among the SRB. This is the first study directly exploring bacterial diversity in corrosive biofilms associated to steel pipelines subjected to normal operation conditions. PMID:16765858

Neria-González, Isabel; Wang, En Tao; Ramírez, Florina; Romero, Juan M; Hernández-Rodríguez, César

2006-06-01

201

Study of the bacterial ecosystem in tropical cooked and peeled shrimps using a polyphasic approach.  

PubMed

The characterization of the microbial ecosystem of cooked tropical shrimps was carried out using a polyphasic approach. First, culture-dependent methods were used for bacterial enumeration and the phenotypic and molecular identification of bacterial isolates. Then, culture-independent methods, including PCR-TTGE (V3 region of the 16S rRNA gene), provided a fingerprinting of bacterial DNA directly extracted from shrimps. Two batches of cooked and peeled tropical shrimps were stored at 5 and 15 degrees C for 5 and 3 weeks, respectively. Trained panelists carried out a sensory evaluation and microbiological enumerations were performed. When spoilage of samples was perceived, several colonies were isolated from the total viable count media. Thus, 137 bacterial strains were identified by phenotypic and molecular tests. Lactic acid bacteria (LAB) constituted the major group with the most represented genera being Carnobacterium (C. divergens, C. maltaromaticum and indiscernible C. alterfunditum/pleistocenium), Vagococcus (indiscernible V. carniphilus/fluvialis) and Enterococcus (E. faecalis and E. faecium). The other groups corresponded to Brochothrix thermosphacta and Enterobacteriaceae (Serratia liquefaciens). In PCR-TTGE profiles some of DNA fragments were assigned to those of standard strains (S. liquefaciens, B. thermosphacta, E. faecalis, C. divergens and C. maltaromaticum) or identified isolates from culture-dependent analysis (E. faecium). Other additional informations were provided by fragment cloning (Psychrobacter sp, Citrobacter gillenii and Firmicute). In conclusion, TTGE is an excellent tool to monitor the evolution of the microbial ecosystem in seafood products. PMID:18573557

Jaffrès, E; Sohier, D; Leroi, F; Pilet, M F; Prévost, H; Joffraud, J J; Dousset, X

2009-04-30

202

rRNA Stability in Heat-Killed and UV-Irradiated Enterotoxigenic Staphylococcus aureus and Escherichia coli O157:H7†  

PubMed Central

Differentiation of viable cells from nonviable cells is of considerable importance in the development of methods to detect foodborne pathogens. To study the suitability of 16S rRNA as an indicator of cell viability in nucleic acid-based detection assays, we examined rRNA stability in two representative foodborne pathogens, Escherichia coli O157:H7 and enterotoxigenic Staphylococcus aureus, which were inactivated by extreme heat, moderate heat, and UV irradiation. Cell death under all conditions was confirmed by a failure to grow in brain heart infusion broth after incubation for 48 h at 37°C. rRNA stability was monitored by a Northern blot analysis, and detection was evaluated by using reverse transcription (RT)-PCR performed with two primer sets (which produced 325- and 1,400-bp amplicons). rRNA of neither pathogen was detected by Northern blot analysis and RT-PCR after cells were killed by autoclaving at 121°C for 15 min. In contrast, intact rRNA of both pathogens were detected by Northern blotting and could be amplified by RT-PCR up to 48 h after cells were killed by heat treatment at 80°C and UV irradiation at 254 nm. rRNA was a suitable target molecule for monitoring bacterial viability under extreme heat conditions, but the presence of rRNA was not correlated with viability following moderate heat inactivation or UV irradiation of cells. PMID:9797275

McKillip, John L.; Jaykus, Lee-Ann; Drake, Maryanne

1998-01-01

203

Phylogenetic Diversity of Winter Bacterioplankton of Eutrophic Siberian Reservoirs as Revealed by 16S rRNA Gene Sequence  

Microsoft Academic Search

Using 16S rRNA gene sequence analyses we investigated the bacterial diversity of winter bacterioplankton of two eutrophic Siberian reservoirs. These reservoirs show similarity in phytoplankton community composition in spring and autumn but tend to differ in summer in exhibiting cyanobacterial bloom. Forty-eight unique partial 16S RNA gene sequences retrieved from two libraries were mostly affiliated with the class Actinobacteria, b

M. Yu. Trusova; M. I. Gladyshev

2002-01-01

204

Prostatitis - bacterial  

MedlinePLUS

Prostatitis is most often caused by a bacterial infection of the prostate gland. Any bacteria that can cause a urinary tract infection can cause acute bacterial prostatitis. Some sexually transmitted diseases (STDs) ...

205

[Rapid method to extract high-quality RNA from activated sludge].  

PubMed

An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield, purity, integrity, RT-PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism (T-RFLP) analysis. That is, the precipitated activated sludge was washed with TENP and PBS buffer, followed by using lysozyme and TRIzol to direct lysis of microbial cells, chloroform to remove protein and most of the DNA from bacterial lysate, isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA. To further purify RNA, RNA purifying column was utilized. The results demonstrated that the extraction method, with the aid of TRIzol and RNA purification kit, can effectively extract high-quality RNA. It not only means low degradability and high quantity, purity and diversity, but also the genes of 16S rRNA and amoA can be amplified by RT-PCR. Compared with other methods, it showed great advantage of low cost and high efficiency and can be applied to RNA extraction of activated sludge in a large number. Furthermore, T-RFLP results indicated that the community composition as well as the abundance of individual members was affected by the kind of RNA extraction methods. This work established a rapid and effective method to extract high-quality RNA from activated sludge and would show great potential for monitoring microbial changes and studying metabolism and community array of activated sludge. PMID:20329549

Jin, Min; Zhao, Zu-Guo; Qiu, Zhi-Gang; Wang, Jing-Feng; Chen, Zhao-Li; Shen, Zhi-Qiang; Li, Chao; Wang, Xin-Wei; Dong, Yan; Li, Jun-Wen

2010-01-01

206

Linkage between bacterial carbon processing and the structure of the active bacterial community at a coastal site in the NW Mediterranean Sea.  

PubMed

The temporal dynamics in bulk bacterial parameters and in the richness of the total and active bacterial community, determined from CE-SSCP fingerprints of 16S rRNA genes and 16S rRNA transcripts, respectively, were followed weekly to bimonthly at an oligotrophic coastal site in the NW Mediterranean Sea. Bacterial abundance, bacterial heterotrophic production, and bacterial and community respiration determined over two seasonal cycles displayed large short-term variability and no pronounced temporal pattern was detectable for these parameters. Concentrations in inorganic nutrients, salinity, or concentrations of chlorophyll a could not significantly explain the temporal variability of the bacterial parameters determined. By contrast, bacterial respiration and the bacterial carbon demand were both negatively correlated with the richness of the active bacterial community, while the bacterial parameters determined herein were not related to the richness of the total bacterial community present. Our results indicate that a reduced number of ribotypes is active when rates of bacteria-mediated carbon processes are high. Our approach, based on fingerprints of 16S rRNA transcripts, could represent an interesting tool to investigate the relationship between the structure and function of marine bacteria, in particular, on short temporal and spatial scales. PMID:19789909

Obernosterer, Ingrid; Lami, Raphael; Larcher, Mariele; Batailler, Nicole; Catala, Philippe; Lebaron, Philippe

2010-04-01

207

Assessment of microbial dynamics in the Pearl River Estuary by 16S rRNA terminal restriction fragment analysis  

NASA Astrophysics Data System (ADS)

We have evaluated the feasibility of using the terminal restriction fragment length polymorphism (T-RFLP) pattern of polymerase chain reaction (PCR) amplified 16S rRNA sequences to track the changes of the free-living bacterial community for the Pearl River Estuary surface waters. The suitability of specific PCR primers, PCR bias induced by thermal cycles, and field-sampling volumes were critically evaluated in laboratory tests. We established a workable protocol and obtained TRF patterns that reflected the changes in the bacterial population. The temporal dynamics over a 24 h period were examined at one anchored station, as well as the spatial distribution pattern of the bacterial community at several stations, covering the transects along the river discharge direction and across the river plume. The TRF pattern revealed 9 dominant bacterial groups. Changes in their relative abundance reflecting the changes in the bacterial community composition were documented. Many culturable species were isolated from each field sample and a portion of the 16S rRNA gene for each species was sequenced. The species was identified based on sequence data comparison. In this region, the dominant species belong to the ?-subdivision of proteobacteria and the Bacillus/Clostridium group of Firmicutes. We also detected the wide spread distribution of Acinetobacter spp.; many of these species are known nosocomial pathogen for humans.

Wu, Madeline; Song, Liansheng; Ren, Jianping; Kan, Jianjun; Qian, Pei-Yuan

2004-10-01

208

Bacterial Sialidase  

NASA Technical Reports Server (NTRS)

Data shows that elevated sialidase in bacterial vaginosis patients correlates to premature births in women. Bacterial sialidase also plays a significant role in the unusual colonization of Pseudomonas aeruginosa in cystic fibrosis patients. Crystals of Salmonella sialidase have been reproduced and are used for studying the inhibitor-enzyme complexes. These inhibitors may also be used to inhibit a trans-sialidase of Trypanosome cruzi, a very similar enzyme to bacterial sialidase, therefore preventing T. cruzi infection, the causitive agent of Chagas' disease. The Center for Macromolecular Crystallography suggests that inhibitors of bacterial sialidases can be used as prophylactic drugs to prevent bacterial infections in these critical cases.

2004-01-01

209

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

210

Succession of Microbial Communities during Hot Composting as Detected by PCR-Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes  

Microsoft Academic Search

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region

SABINE PETERS; STEFANIE KOSCHINSKY; FRANK SCHWIEGER; CHRISTOPH C. TEBBE

2000-01-01

211

Comparing the identification of Clostridium spp. by two Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) mass spectrometry platforms to 16S rRNA PCR sequencing as a reference standard: A detailed analysis of age of culture and sample preparation.  

PubMed

We compared the identification of Clostridium species using mass spectrometry by two different Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) platforms (Bruker MS and Vitek MS) against 16S rRNA sequencing as the reference standard. We then examined the impact of different sample preparations and (on one of those platforms) age of bacterial colonial growth on the performance of the MALDI-TOF MS systems. We identified 10 different species amongst the 52 isolates by 16S rRNA sequencing, with Clostridium perfringens the most prevalent (n = 30). Spectrometric analysis using Vitek MS correctly speciated 47/52 (90.4%) isolates and was not affected by the sample preparation used. Performance of the Bruker MS was dependent on sample preparation with correct speciation obtained for 36 of 52 (69.2%) isolates tested using the Direct Transfer [DT] protocol, but all 52 (100%) isolates were correctly speciated using either an Extended Direct Transfer [EDT] or a Full Formic Extraction [EX] protocol. We then examined the effect of bacterial colonial growth age on the performance of Bruker MS and found substantial agreement in speciation using DT (Kappa = 0.62, 95% CI: 0.46-0.75), almost perfect agreement for EDT (Kappa = 0.94, 95% CI: 0.86-1.00) and exact agreement for EX (Kappa = 1.00) between different days. PMID:25230331

Chean, Roy; Kotsanas, Despina; Francis, Michelle J; Palombo, Enzo A; Jadhav, Snehal R; Awad, Milena M; Lyras, Dena; Korman, Tony M; Jenkin, Grant A

2014-12-01

212

Bacterial community composition in Brazilian Anthrosols and adjacent soils characterized using culturing and molecular identification.  

PubMed

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols. PMID:19381712

O'Neill, B; Grossman, J; Tsai, M T; Gomes, J E; Lehmann, J; Peterson, J; Neves, E; Thies, J E

2009-07-01

213

Identification of bacterial populations in drinking water using 16S rRNA-based sequence analyses.  

PubMed

Intracellular RNA is rapidly degraded in stressed cells and is more unstable outside of the cell than DNA. As a result, RNA-based methods have been suggested to study the active microbial fraction in environmental matrices. The aim of this study was to identify bacterial populations in drinking water by analyzing 16S rRNA-based clone libraries. Hollow-fiber ultrafiltration was used to concentrate bacterial communities from 40l of tap water collected at 12 different times during three different summer months from a single point-of-use. Total RNA was extracted from the microbial concentrates and used to develop 16S rRNA-based clone libraries. Phylogenetic analyses of 1231 partial 16S rRNA gene sequences showed that difficult-to-classify bacterial sequences were the most predominant clones, representing 57.6% of the sequences analyzed. Within these unclassified clades, most sequences were closely related to sequences retrieved from previous DNA- and RNA-based drinking water studies. Other bacterial groups represented in this study included Proteobacteria, cyanobacteria, Actinobacteria, Bacteroidetes, and Planctomycetes. Overall, the results suggest that these bacterial groups are amongst potentially active bacteria in drinking water. Diversity analyses of clones generated show that while overall diversity is similar amongst the different months, membership changes with respect to time. The results from this study further improve our understanding of the molecular diversity and bacterial population dynamics of drinking water microbial communities. Moreover, these results provide the sequence foundation for the development of molecular assays that target active drinking water bacteria. PMID:19944442

Revetta, Randy P; Pemberton, Adin; Lamendella, Regina; Iker, Brandon; Santo Domingo, Jorge W

2010-03-01

214

Evaluation of Microbial Bacterial and Fungal Diversity in Cerebrospinal Fluid Shunt Infection  

PubMed Central

Background Cerebrospinal fluid shunt infection can be recalcitrant. Recurrence is common despite appropriate therapy for the pathogens identified by culture. Improved diagnostic and therapeutic approaches are required, and culture-independent molecular approaches to cerebrospinal fluid shunt infections have not been described. Objectives To identify the bacteria and fungi present in cerebrospinal fluid from children with cerebrospinal fluid shunt infection using a high-throughput sequencing approach, and to compare those results to those from negative controls and conventional culture. Methods This descriptive study included eight children ?18 years old undergoing treatment for culture-identified cerebrospinal fluid shunt infection. After routine aerobic culture of each cerebrospinal fluid sample, deoxyribonucleic acid (DNA) extraction was followed by amplification of the bacterial 16S rRNA gene and the fungal ITS DNA region tag-encoded FLX-Titanium amplicon pyrosequencing and microbial phylogenetic analysis. Results The microbiota analyses for the initial cerebrospinal fluid samples from all eight infections identified a variety of bacteria and fungi, many of which did not grow in conventional culture. Detection by conventional culture did not predict the relative abundance of an organism by pyrosequencing, but in all cases, at least one bacterial taxon was detected by both conventional culture and pyrosequencing. Individual bacterial species fluctuated in relative abundance but remained above the limits of detection during infection treatment. Conclusions Numerous bacterial and fungal organisms were detected in these cerebrospinal fluid shunt infections, even during and after treatment, indicating diverse and recalcitrant shunt microbiota. In evaluating cerebrospinal fluid shunt infection, fungal and anaerobic bacterial cultures should be considered in addition to aerobic bacterial cultures, and culture-independent approaches offer a promising alternative diagnostic approach. More effective treatment of cerebrospinal fluid shunt infections is needed to reduce unacceptably high rates of reinfection, and this work suggests that one effective strategy may be reduction of the diverse microbiota present in infection. PMID:24421877

Simon, Tamara D.; Pope, Christopher E.; Browd, Samuel R.; Ojemann, Jeffrey G.; Riva-Cambrin, Jay; Mayer-Hamblett, Nicole; Rosenfeld, Margaret; Zerr, Danielle M.; Hoffman, Lucas

2014-01-01

215

A mitochondrial rRNA dimethyladenosine methyltransferase in Arabidopsis  

PubMed Central

S-adenosyl-l-methionine-dependent rRNA dimethylases mediate the methylation of two conserved adenosines near the 3? end of the rRNA in the small ribosomal subunits of bacteria, archaea and eukaryotes. Proteins related to this family of dimethylases play an essential role as transcription factors (mtTFBs) in fungal and animal mitochondria. Human mitochondrial rRNA is methylated and human mitochondria contain two related mtTFBs, one proposed to act as rRNA dimethylase, the other as transcription factor. The nuclear genome of Arabidopsis thaliana encodes three dimethylase/mtTFB-like proteins, one of which, Dim1B, is shown here to be imported into mitochondria. Transcription initiation by mitochondrial RNA polymerases appears not to be stimulated by Dim1B in vitro. In line with this finding, phylogenetic analyses revealed Dim1B to be more closely related to a group of eukaryotic non-mitochondrial rRNA dimethylases (Dim1s) than to fungal and animal mtTFBs. We found that Dim1B was capable of substituting the E. coli rRNA dimethylase activity of KsgA. Moreover, we observed methylation of the conserved adenines in the 18S rRNA of Arabidopsis mitochondria; this modification was not detectable in a mutant lacking Dim1B. These data provide evidence: (i) for rRNA methylation in Arabidopsis mitochondria; and (ii) that Dim1B is the enzyme catalyzing this process. PMID:19929881

Richter, Uwe; Kühn, Kristina; Okada, Sachiko; Brennicke, Axel; Weihe, Andreas; Börner, Thomas

2010-01-01

216

Bead Array Direct rRNA Capture Assay (rCapA) for Amplification Free Speciation of Mycobacterium Cultures  

PubMed Central

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

217

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

218

Isolation and characteristics of a novel biphenyl-degrading bacterial strain, Dyella ginsengisoli LA-4.  

PubMed

A novel biphenyl-degrading bacterial strain LA-4 was isolated from activated sludge. It was identified as Dyella ginsengisoli according to phylogenetic similarity of 16S rRNA gene sequence. This isolate could utilize biphenyl as sole source of carbon and energy, which degraded over 95 mg/L biphenyl within 36 h. The major metabolites formed from biphenyl, such as 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) and benzoic acid, were identified by LC-MS. The crude cell extract of strain LA-4 exhibited the activity of 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD) and the kinetic parameters K(m) was 26.48 micromol/L and V(max) was 8.12 U/mg protein. A conserved region of the biphenyl dioxygenase gene bphA1 of strain LA-4 was amplified by PCR and confirmed by DNA sequencing. PMID:19402424

Li, Ang; Qu, Yuanyuan; Zhou, Jiti; Gou, Min

2009-01-01

219

Nonradioactive Method To Study Genetic Profiles of Natural Bacterial Communities by PCR-Single-Strand-Conformation Polymorphism  

Microsoft Academic Search

We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262

DONG-HUN LEE; YOUNG-GUN ZO; ANDSANG-JONG KIM

1996-01-01

220

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

221

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

222

Downregulation of rRNA Transcription Triggers Cell Differentiation  

PubMed Central

Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation. PMID:24879416

Hayashi, Yuki; Kuroda, Takao; Kishimoto, Hiroyuki; Wang, Changshan; Iwama, Atsushi; Kimura, Keiji

2014-01-01

223

Spatial Changes in the Bacterial Community Structure along a Vertical Oxygen Gradient in Flooded Paddy Soil Cores  

PubMed Central

Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Microsensor measurements showed that oxygen was depleted from 140 ?M at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below. Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-?m-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique. The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth. 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone. Comparative sequence analysis of these clones identified members of the ? and ? subclasses of Proteobacteria as the abundant populations in the oxic zone. In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil. The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis. This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined. PMID:10653747

Lüdemann, Heiner; Arth, Inko; Liesack, Werner

2000-01-01

224

The aminoglycoside resistance methyltransferase Sgm impedes RsmF methylation at an adjacent rRNA nucleotide in the ribosomal A site  

PubMed Central

Ribosome-targeting antibiotics block protein synthesis by binding at functionally important regions of the bacterial rRNA. Resistance is often conferred by addition of a methyl group at the antibiotic binding site within an rRNA region that is already highly modified with several nucleotide methylations. In bacterial rRNA, each methylation requires its own specific methyltransferase enzyme, and this raises the question as to how an extra methyltransferase conferring antibiotic resistance can be accommodated and how it can gain access to its nucleotide target within a short and functionally crowded stretch of the rRNA sequence. Here, we show that the Sgm methyltransferase confers resistance to 4,6-disubstituted deoxystreptamine aminoglycosides by introducing the 16S rRNA modification m7G1405 within the ribosomal A site. This region of Escherichia coli 16S rRNA already contains several methylated nucleotides including m4Cm1402 and m5C1407. Modification at m5C1407 by the methyltransferase RsmF is impeded as Sgm gains access to its adjacent G1405 target on the 30S ribosomal subunit. An Sgm mutant (G135A), which is impaired in S-adenosylmethionine binding and confers lower resistance, is less able to interfere with RsmF methylation on the 30S subunit. The two methylations at 16S rRNA nucleotide m4Cm1402 are unaffected by both the wild-type and the mutant versions of Sgm. The data indicate that interplay between resistance methyltransferases and the cell's own indigenous methyltransferases can play an important role in determining resistance levels. PMID:19509304

?ubrilo, Sonja; Babi?, Fedora; Douthwaite, Stephen; Maravi? Vlahovi?ek, Gordana

2009-01-01

225

Bacterial diversity of terra preta and pristine forest soil from the Western Amazon  

Microsoft Academic Search

The survey presented here describes the bacterial diversity and community structures of a pristine forest soil and an anthropogenic terra preta from the Western Amazon forest using molecular methods to identify the predominant phylogenetic groups. Bacterial community similarities and species diversity in the two soils were compared using oligonucleotide fingerprint grouping of 16S rRNA gene sequences for 1500 clones (OFRG)

Jong-Shik Kim; Gerd Sparovek; Regina M. Longo; Wanderley Jose De Melo; David Crowley

2007-01-01

226

Short communication Bacterial diversity of terra preta and pristine forest soil from the Western Amazon  

Microsoft Academic Search

The survey presented here describes the bacterial diversity and community structures of a pristine forest soil and an anthropogenic terra preta from the Western Amazon forest using molecular methods to identify the predominant phylogenetic groups. Bacterial community similarities and species diversity in the two soils were compared using oligonucleotide fingerprint grouping of 16S rRNA gene sequences for 1500 clones (OFRG)

Jong-Shik Kim; Gerd Sparovek; Regina M. Longo; Wanderley Jose De Melo; David Crowley

227

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

228

Analysis of the Dynamics of Bacterial Communities in the Rhizosphere of the Chrysanthemum via Denaturing Gradient Gel Electrophoresis and Substrate Utilization Patterns  

PubMed Central

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE patterns showed that the bacterial communities as determined from direct rhizosphere DNA extracts were largely stable along developing roots of the chrysanthemum, with very little change over time or between root parts of different ages. The patterns were also similar to those produced with DNA extracts obtained from bulk soil samples. The DGGE patterns obtained by using microbial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. Moreover, these patterns showed differences among plant replicates but also among replicate plates. Results obtained with the sole-carbon-source utilization tests indicated that the metabolic profile of the bacterial communities in the rhizosphere of the root tip did not change substantially during plant growth. This suggests selective development of specific bacterial populations by the presence of a root tip. On the other hand, the metabolic profile of bacterial communities in the rhizosphere of the root base changed during plant growth. With eight sole-carbon-source utilization tests, a significant effect of the development stage of the plant on the number of bacteria which were able to grow on these carbon sources was observed. PMID:9835588

Duineveld, Bernadette M.; Rosado, Alexandre S.; van Elsas, Jan Dirk; van Veen, Johannes A.

1998-01-01

229

The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods.  

PubMed

Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a Facklamia sp., Eubacterium tenue, and a Bifidobacterium sp. 16S rRNA gene sequencing identified the isolates as Enterococcus faecalis, Cardiobacterium valvarum, and Streptococcus mutans, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors. PMID:16333109

Petti, C A; Polage, C R; Schreckenberger, P

2005-12-01

230

Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans  

NASA Technical Reports Server (NTRS)

A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.

Jurtshuk, R. J.; Blick, M.; Bresser, J.; Fox, G. E.; Jurtshuk, P. Jr

1992-01-01

231

Biodegradability of bacterial surfactants.  

PubMed

This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO(2) evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO(2) emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production. PMID:21053055

Lima, Tânia M S; Procópio, Lorena C; Brandão, Felipe D; Carvalho, André M X; Tótola, Marcos R; Borges, Arnaldo C

2011-06-01

232

Identification of bacteria in a biodegraded wall painting by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA.  

PubMed Central

Medieval wall paintings are often affected by biodecay. An inventory of the existing microorganisms associated with the damage to the paintings is not yet an integral part of the restoration process. This stems from the lack of effective means for such a stocktaking. Nevertheless, fungi and bacteria cause severe damage through mechanical processes from growth into the painting and its grounding and through their metabolism. Detailed information on the bacterial colonization of ancient wall paintings is essential for the protection of the paintings. We used a molecular approach based on the detection and identification of DNA sequences encoding rRNA (rDNA) to identify bacteria present on an ancient wall painting without prior cultivation of the organisms, since it has been shown that most of these bacteria cannot be cultivated under laboratory conditions. To trace the noncultivated fraction of bacteria, total DNA from a biodegraded wall painting sample from a 13th century fresco was extracted and 194-bp fragments of the 16S rDNA were amplified with eubacterial primers. The 16S rDNA fragments of uniform length obtained from the different bacterial species were separated according to their sequence differences by denaturing gradient gel electrophoresis (DGGE). By sequencing excised and reamplified individual DNA bands, we characterized the phylogenetic affiliation of the corresponding bacteria. Using this approach, we identified members or close relatives of the genera Halomonas, Clostridium, and Frankia. To our knowledge, these groups of bacteria have not yet been isolated and implicated by conventional microbiological techniques as contributing to the biodegradation of wall paintings. PMID:8787403

Rölleke, S; Muyzer, G; Wawer, C; Wanner, G; Lubitz, W

1996-01-01

233

Novel bacterial phylotypes associated with the healthy feline oral cavity and feline chronic gingivostomatitis.  

PubMed

Feline chronic gingivostomatitis (FCGS) is a painful inflammatory disease of the oral cavity. Treatment options for FCGS are very limited and little is known regarding its aetiology. The aim of this study was to investigate the presence of putative novel species in the oral cavity of cats with and without FCGS. Bacterial DNA was extracted from oral swabs and identified by 16S rRNA gene sequencing. The 16S rRNA genes of 54 clones representing distinct potentially novel species were sequenced (1202-1325 base pairs). Obtained sequences were compared to the BLAST database, aligned using the ClustalW2 alignment tool and a phylogenetic tree created. Twenty-two clones (18 from control and four from FCGS samples) had a similarity of less than 97% and were considered novel. The proportion of novel phylotypes in each group was 19.6% (control) and 2.3% (FCGS). In the derived phylogenetic tree, 15 novel phylotypes clustered together and branched away from known species and phyla. This suggests the presence of a group of novel, previously unidentified bacteria that are associated with the feline oral cavity in both health and disease. PMID:23261159

Dolieslager, Sanne M J; Bennett, David; Johnston, Norman; Riggio, Marcello P

2013-06-01

234

The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis  

PubMed Central

The Ribosomal Database Project (RDP-II) provides the research community with aligned and annotated rRNA gene sequences, along with analysis services and a phylogenetically consistent taxonomic framework for these data. Updated monthly, these services are made available through the RDP-II website (http://rdp.cme.msu.edu/). RDP-II release 9.21 (August 2004) contains 101?632 bacterial small subunit rRNA gene sequences in aligned and annotated format. High-throughput tools for initial taxonomic placement, identification of related sequences, probe and primer testing, data navigation and subalignment download are provided. The RDP-II email address for questions or comments is rdpstaff@msu.edu. PMID:15608200

Cole, J. R.; Chai, B.; Farris, R. J.; Wang, Q.; Kulam, S. A.; McGarrell, D. M.; Garrity, G. M.; Tiedje, J. M.

2005-01-01

235

Chronic bacterial prostatitis: efficacy of short-lasting antibiotic therapy with prulifloxacin (Unidrox®) in association with saw palmetto extract, lactobacillus sporogens and arbutin (Lactorepens®)  

PubMed Central

Background Bacterial prostatitis (BP) is a common condition accounting responsible for about 5-10% of all prostatitis cases; chronic bacterial prostatitis (CBP) classified as type II, are less common but is a condition that significantly hampers the quality of life, (QoL) because not only is it a physical condition but also a psychological distress. Commonly patients are treated with antibiotics alone, and in particular fluoroquinolones are suggested by the European Urology guidelines. This approach, although recommended, may not be enough. Thus, a multimodal approach to the prolonged antibiotic therapy may be helpful. Methods 210 patients affected by chronic bacterial prostatitis were enrolled in the study. All patients were positive to Meares-Stamey test and symptoms duration was?>?3 months. The purpose of the study was to evaluate the efficacy of a long lasting therapy with a fluoroquinolone in association with a nutraceutical supplement (prulifloxacin 600 mg for 21 days and an association of Serenoa repens 320 mg, Lactobacillus Sporogens 200 mg, Arbutin 100 mg for 30 days). Patients were randomized in two groups (A and B) receiving respectively antibiotic alone and an association of antibiotic plus supplement. Results Biological recurrence at 2 months in Group A was observed in 21 patients (27.6%) and in Group B in 6 patients (7.8%). Uropathogens found at the first follow-up were for the majority Gram – (E. coli and Enterobacter spp.). A statistically significant difference was found at the time of the follow-up between Group A and B in the NIH-CPSI questionnaire score, symptoms evidence and serum PSA. Conclusions Broad band, short-lasting antibiotic therapy in association with a nutritional supplement (serenoa repens, lactobacillus sporogens and arbutin) show better control and recurrence rate on patients affected by chronic bacterial prostatitits in comparison with antibiotic treatment alone. Trial registration NCT02130713 Date of trial Registration: 30/04/2014 PMID:25038794

2014-01-01

236

The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase.  

PubMed Central

The ErmE methyltransferase confers resistance to MLS antibiotics by specifically dimethylating adenine 2058 (A2058, Escherichia coli numbering) in bacterial 23S rRNA. To define nucleotides in the rRNA that are part of the motif recognized by ErmE, we investigated both in vivo and in vitro the effects of mutations around position A2058 on methylation. Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE. No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058. Breaking the neighboring G2057-C2611 Watson-Crick base pair by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents and it was shown that the A2057 and U2611 mutations alone and in combination alter the reactivity of A2058 and adjacent bases. However, mutagenizing position G-->A2032 in an adjacent loop, which has been implicated to interact with A2058, alters neither the ErmE methylation at A2058 nor the accessibility of this region to the chemical reagents. The data indicate that a less-exposed conformation at A2058 leads to reduction in methylation by ErmE. Nucleotide G2057 and its interaction with C2611 maintain the conformation at A2058, and are thus important in forming the structural motif that is recognized by the ErmE methyltransferase. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 FIGURE 6 PMID:7489511

Vester, B; Hansen, L H; Douthwaite, S

1995-01-01

237

Development of a novel long-range 16S rRNA universal primer set for metagenomic analysis of gastrointestinal microbiota in newborn infants.  

PubMed

Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias. PMID:24722376

Ku, Hye-Jin; Lee, Ju-Hoon

2014-06-28

238

Phylogenetic Relationship of Phosphate Solubilizing Bacteria according to 16S rRNA Genes  

PubMed Central

Phosphate solubilizing bacteria (PSB) can convert insoluble form of phosphorous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by plant in the field. In this study, isolation and precise identification of PSB were carried out in Malaysian (Serdang) oil palm field (University Putra Malaysia). Identification and phylogenetic analysis of 8 better isolates were carried out by 16S rRNA gene sequencing in which as a result five isolates belong to the Beta subdivision of Proteobacteria, one isolate was related to the Gama subdivision of Proteobacteria, and two isolates were related to the Firmicutes. Bacterial isolates of 6upmr, 2upmr, 19upmnr, 10upmr, and 24upmr were identified as Alcaligenes faecalis. Also, bacterial isolates of 20upmnr and 17upmnr were identified as Bacillus cereus and Vagococcus carniphilus, respectively, and bacterial isolates of 31upmr were identified as Serratia plymuthica. Molecular identification and characterization of oil palm strains as the specific phosphate solubilizer can reduce the time and cost of producing effective inoculate (biofertilizer) in an oil palm field. PMID:25632387

Javadi Nobandegani, Mohammad Bagher; Saud, Halimi Mohd; Yun, Wong Mui

2015-01-01

239

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

240

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

241

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

PubMed Central

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

2014-01-01

242

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

243

Bacterial conjunctivitis  

PubMed Central

Introduction Most cases of conjunctivitis in adults are probably due to viral infection, but children are more likely to develop bacterial conjunctivitis than they are viral forms. The main bacterial pathogens are Haemophilus influenzae and Streptococcus pneumoniae in adults and children, and Moraxella catarrhalis in children. Contact lens wearers may be more likely to develop gram-negative infections. Bacterial keratitis occurs in up to 30 per 100,000 contact lens wearers. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of empirical treatment in adults and children with suspected bacterial conjunctivitis? What are the effects of treatment in adults and children with bacteriologically confirmed bacterial conjunctivitis? What are the effects of treatment in adults and children with clinically confirmed gonococcal conjunctivitis? We searched: Medline, Embase, The Cochrane Library, and other important databases up to July 2009 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 40 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review we present information relating to the effectiveness and safety of the following interventions: ocular decongestants; oral antibiotics; parenteral antibiotics; saline; topical antibiotics; and warm compresses. PMID:21718563

2010-01-01

244

Bacterial conjunctivitis  

PubMed Central

Introduction Most cases of conjunctivitis in adults are probably due to viral infection, but children are more likely to develop bacterial conjunctivitis than they are viral forms. The main bacterial pathogens are Haemophilus influenzae and Streptococcus pneumoniae in adults and children, and Moraxella catarrhalis in children. Contact lens wearers may be more likely to develop gram-negative infections. Bacterial keratitis occurs in up to 30 per 100,000 contact lens wearers. Methods and outcomes We conducted a systematic review and aimed to answer the following clinical questions: What are the effects of empirical treatment in adults and children with suspected bacterial conjunctivitis? What are the effects of treatment in adults and children with bacteriologically confirmed bacterial conjunctivitis? What are the effects of treatment in adults and children with clinically confirmed gonococcal conjunctivitis? We searched: Medline, Embase, The Cochrane Library, and other important databases up to July 2011 (Clinical Evidence reviews are updated periodically; please check our website for the most up-to-date version of this review). We included harms alerts from relevant organisations such as the US Food and Drug Administration (FDA) and the UK Medicines and Healthcare products Regulatory Agency (MHRA). Results We found 44 systematic reviews, RCTs, or observational studies that met our inclusion criteria. We performed a GRADE evaluation of the quality of evidence for interventions. Conclusions In this systematic review we present information relating to the effectiveness and safety of the following interventions: ocular decongestants, oral antibiotics, parenteral antibiotics, saline, topical antibiotics, and warm compresses. PMID:22348418

2012-01-01

245

Characterization of redox-related soil microbial communities along a river floodplain continuum by fatty acid methyl ester (FAME) and 16S rRNA genes  

Microsoft Academic Search

Redox states affect substrate availability and energy transformation, and, thus, play a crucial role in regulating soil microbial abundance, diversity, and community structure. We evaluated microbial communities in soils under oxic, intermittent, and anoxic conditions along a river floodplain continuum using fatty acid methyl ester (FAME) and 16S rRNA genes-based terminal-restriction fragment length polymorphism (T-RFLP) bacterial fingerprints. In all the

Y. Song; S. P. Deng; V. Acosta-Martínez; E. Katsalirou

2008-01-01

246

Small-Scale Vertical Distribution of Bacterial Biomass and Diversity in Biological Soil Crusts from Arid Lands in the Colorado Plateau  

USGS Publications Warehouse

We characterized, at millimeter resolution, bacterial biomass, diversity, and vertical stratification of biological soil crusts in arid lands from the Colorado Plateau. Microscopic counts, extractable DNA, and plate counts of viable aerobic copiotrophs (VAC) revealed that the top centimeter of crusted soils contained atypically large bacterial populations, tenfold larger than those in uncrusted, deeper soils. The plate counts were not always consistent with more direct estimates of microbial biomass. Bacterial populations peaked at the immediate subsurface (1-2 mm) in light-appearing, young crusts, and at the surface (0-1 mm) in well-developed, dark crusts, which corresponds to the location of cyanobacterial populations. Bacterial abundance decreased with depth below these horizons. Spatially resolved DGGE fingerprints of Bacterial 16S rRNA genes demonstrated the presence of highly diverse natural communities, but we could detect neither trends with depth in bacterial richness or diversity, nor a difference in diversity indices between crust types. Fingerprints, however, revealed the presence of marked stratification in the structure of the microbial communities, probably a result of vertical gradients in physicochemical parameters. Sequencing and phylogenetic analyses indicated that most of the naturally occurring bacteria are novel types, with low sequence similarity (83-93%) to those available in public databases. DGGE analyses of the VAC populations indicated communities of lower diversity, with most types having sequences more than 94% similar to those in public databases. Our study indicates that soil crusts represent small-scale mantles of fertility in arid ecosystems, harboring vertically structured, little-known bacterial populations that are not well represented by standard cultivation methods.

Garcia-Pichel, F.; Johnson, S.L.; Youngkin, D.; Belnap, J.

2003-01-01

247

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

248

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

E-print Network

reasonable to work with prokaryotic cells with their single chromosome, well-defined genome of manageable when applied to biology is the potential to extract and analyze the contents of a single cell the ultimate challenge to the single cell project is a human eukaryotic cell, at this stage of work it is more

249

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

250

In situ analysis of the bacterial community associated with the reindeer lichen Cladonia arbuscula reveals predominance of Alphaproteobacteria.  

PubMed

The diversity and spatial pattern of the bacterial community hosted by the shrub-like reindeer lichen Cladonia arbuscula were investigated by general DNA staining and FISH, coupled with confocal laser scanning microscopy (CLSM). Using an optimized protocol for FISH using cryosections of small lichen fragments, we found about 6 x 10(7) bacteria g(-1) of C. arbuscula. Approximately 86% of acridine orange-stained cells were also stained by the universal FISH probe EUB338. Using group-specific FISH probes, we detected a dominance of Alphaproteobacteria (more than 60% of all bacteria), while the abundance of Actinobacteria and Betaproteobacteria was much lower (<10%). Firmicutes were rarely detected, and no Gammaproteobacteria were present. Bacterial cells of different taxonomic groups are embedded in a biofilm-like, continuous layer on the internal surface of the C. arbuscula podetia, mainly occurring in small colonies of a few to a few hundred cells. The other parts of the lichen showed a lower bacterial colonization. alpha-proteobacterial 16S rRNA genes were amplified using total DNA extracts from C. arbuscula and separated by single-strand conformation polymorphism (SSCP). Sequencing of excised bands revealed the dominance of Acetobacteraceae. PMID:18631179

Cardinale, Massimiliano; Vieira de Castro, João; Müller, Henry; Berg, Gabriele; Grube, Martin

2008-10-01

251

Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis.  

PubMed Central

The synthesis of rRNA was unbalanced by the introduction of plasmids containing rRNA operons with large internal deletions. Significant unbalanced synthesis was achieved only when the deletions affected both 16S and 23S RNA genes or when the deletions affected the 23S RNA gene alone. Although large imbalances in rRNA synthesis resulted from deletions affecting 16S and 23S RNA genes or only 23S RNA genes, excess 16S RNA and defective rRNA species were rapidly degraded. Large imbalances in the synthesis of regions of rRNA did not result in significantly unbalanced synthesis of ribosomal proteins. It therefore is probable that excess intact 16S RNA is degraded because ribosomal proteins are not available for packaging the RNA into ribosomes. Defective RNA species also may be degraded for this reason or because proper ribosome assembly is prevented by the defects in RNA structure. We propose two possible explanations for the finding that unbalanced overproduction of binding sites for feedback ribosomal protein does not result in significant unbalanced translational feedback depression of ribosomal protein mRNAs. Images PMID:3894326

Siehnel, R J; Morgan, E A

1985-01-01

252

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

253

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

254

Field-scale transplantation experiment to investigate structures of soil bacterial communities at pioneering sites.  

PubMed

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, NO(3)(-) showed a seasonal pattern, increasing from 1 to 3 ?g NO(3)(-) (g soil dry weight)(-1) after the melting of snow but decreasing to <1 ?g NO(3)(-) (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

2011-12-01

255

Bacterial pathogenomics  

Microsoft Academic Search

Genomes from all of the crucial bacterial pathogens of humans, plants and animals have now been sequenced, as have genomes from many of the important commensal, symbiotic and environmental microorganisms. Analysis of these sequences has revealed the forces that shape pathogen evolution and has brought to light unexpected aspects of pathogen biology. The finding that horizontal gene transfer and genome

Mark J. Pallen; Brendan W. Wren

2007-01-01

256

Bacterial Biofertilizers  

Microsoft Academic Search

Many bacteria and fungi can enhance plant growth. The present review is limited to plant growth promoting rhizobacteria (PGPR). However, it includes endophytic bacteria that show plant growth enhancing activity as well. Also the best studied bacterial mechanisms of plant growth promotion are discussed, with a special emphasis on biological nitrogen fixation and synthesis of phytohormones, including less understood mechanisms

LUIS E. FUENTES-RAMIREZ; Jesus Caballero-Mellado

257

Bacterial concrete  

Microsoft Academic Search

Cracks in concrete are inevitable and are one of the inherent weaknesses of concrete. Water and other salts seep through these cracks, corrosion initiates, and thus reduces the life of concrete. So there was a need to develop an inherent biomaterial, a self-repairing material which can remediate the cracks and fissures in concrete. Bacterial concrete is a material, which can

Venkataswamy Ramakrishnan; K. P. Ramesh; S. S. Bang

2001-01-01

258

Phylogenetic analysis of the bacterial communities in marine sediments.  

PubMed Central

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

1996-01-01

259

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

260

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

261

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 ...

262

Efficacy of bacterial consortium-AIE2 for contemporaneous Cr(VI) and azo dye bioremediation in batch and continuous bioreactor systems, monitoring steady-state bacterial dynamics using qPCR assays  

Microsoft Academic Search

Bacterial consortium-AIE2 with a capability of contemporaneous Cr(VI) reduction and azo dye RV5 decolourization was developed\\u000a from industrial wastewaters by enrichment culture technique. The 16S rRNA gene based molecular analyses revealed that the\\u000a consortium bacterial community structure consisted of four bacterial strains namely, Alcaligenes sp. DMA, Bacillus sp. DMB, Stenotrophomonas sp. DMS and Enterococcus sp. DME. Cumulative mechanism of Cr(VI)

Chirayu Desai; Kunal Jain; Bharat Patel; Datta Madamwar

2009-01-01

263

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

264

Pyrosequencing 16S rRNA genes of bacteria associated with wild tiger mosquito Aedes albopictus: a pilot study  

PubMed Central

The Asian tiger mosquito Aedes (Stegomya) albopictus is an invasive species that has spread across the world in the last two decades, showing a great capacity to adapt to contrasting climates and environments. While demonstrated in many insects, the contribution of bacterial symbionts in Aedes ecology is a challenging aspect that needs to be investigated. Also some bacterial species have already been identified in Ae. albopictus using classical methods, but a more accurate survey of mosquito-associated bacterial diversity is needed to decipher the potential biological functions of bacterial symbionts in mediating or constraining insect adaptation. We surveyed the bacteria associated with field populations of Ae. albopictus from Madagascar by pyrosequencing 16S rRNA gene amplicons. Different aspects of amplicon preparation and sequencing depth were tested to optimize the breadth of bacterial diversity identified. The results revealed that all mosquitoes collected from different sites have a bacterial microbiota dominated by a single taxon, Wolbachia pipientis, which accounted for about 99% of all 92,615 sequences obtained. As Ae. albopictus is known to harbor two Wolbachia strains (wAlbA and wAlbB), a quantitative PCR was used to estimate the relative densities, (i.e., the bacteria-to-host gene ratios) of each strains in individual mosquitoes. Relative densities were between 6.25 × 100.01 and 5.47 × 100.1 for wAlbA and between 2.03 × 100.1 and 1.4 × 101 for wAlbB. Apart from Wolbachia, a total of 31 bacterial taxa were identified at the genus level using different method variations. Diversity index values were low and probably underestimated the true diversity due to the high abundance of Wolbachia sequences vastly outnumbering sequences from other taxa. Further studies should implement alternative strategies to specifically discard from analysis any sequences from Wolbachia, the dominant endosymbiotic bacterium in Ae. albopictus from this area. PMID:24860790

Minard, Guillaume; Tran, Florence-Hélène; Dubost, Audrey; Tran-Van, Van; Mavingui, Patrick; Valiente Moro, Claire

2014-01-01

265

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

266

Salinity and Bacterial Diversity: To What Extent Does the Concentration of Salt Affect the Bacterial Community in a Saline Soil?  

PubMed Central

In this study, the evaluation of soil characteristics was coupled with a pyrosequencing analysis of the V2-V3 16S rRNA gene region in order to investigate the bacterial community structure and diversity in the A horizon of a natural saline soil located in Sicily (Italy). The main aim of the research was to assess the organisation and diversity of microbial taxa using a spatial scale that revealed physical and chemical heterogeneity of the habitat under investigation. The results provided information on the type of distribution of different bacterial groups as a function of spatial gradients of soil salinity and pH. The analysis of bacterial 16S rRNA showed differences in bacterial composition and diversity due to a variable salt concentration in the soil. The bacterial community showed a statistically significant spatial variability. Some bacterial phyla appeared spread in the whole area, whatever the salinity gradient. It emerged therefore that a patchy saline soil can not contain just a single microbial community selected to withstand extreme osmotic phenomena, but many communities that can be variously correlated to one or more environmental parameters. Sequences have been deposited to the SRA database and can be accessed on ID Project PRJNA241061. PMID:25188357

Canfora, Loredana; Bacci, Giovanni; Pinzari, Flavia; Lo Papa, Giuseppe; Dazzi, Carmelo; Benedetti, Anna

2014-01-01

267

Salinity and bacterial diversity: to what extent does the concentration of salt affect the bacterial community in a saline soil?  

PubMed

In this study, the evaluation of soil characteristics was coupled with a pyrosequencing analysis of the V2-V3 16S rRNA gene region in order to investigate the bacterial community structure and diversity in the A horizon of a natural saline soil located in Sicily (Italy). The main aim of the research was to assess the organisation and diversity of microbial taxa using a spatial scale that revealed physical and chemical heterogeneity of the habitat under investigation. The results provided information on the type of distribution of different bacterial groups as a function of spatial gradients of soil salinity and pH. The analysis of bacterial 16S rRNA showed differences in bacterial composition and diversity due to a variable salt concentration in the soil. The bacterial community showed a statistically significant spatial variability. Some bacterial phyla appeared spread in the whole area, whatever the salinity gradient. It emerged therefore that a patchy saline soil can not contain just a single microbial community selected to withstand extreme osmotic phenomena, but many communities that can be variously correlated to one or more environmental parameters. Sequences have been deposited to the SRA database and can be accessed on ID Project PRJNA241061. PMID:25188357

Canfora, Loredana; Bacci, Giovanni; Pinzari, Flavia; Lo Papa, Giuseppe; Dazzi, Carmelo; Benedetti, Anna

2014-01-01

268

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

269

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

270

Identification and classification of seafood-borne pathogenic and spoilage bacteria: 16S rRNA sequencing versus MALDI-TOF MS fingerprinting.  

PubMed

The present study aims to compare two molecular technologies, 16S rRNA sequencing and MALDI-TOF MS, for bacterial species identification in seafood. With this aim, 70 reference strains from culture collections, including important seafood-borne pathogenic and spoilage bacterial species, and 50 strains isolated from commercial seafood products, were analysed by both techniques. Genomic analysis only identified the species of 50% of the isolated strains, proving to be particularly poor at identifying members of the Pseudomonas and Bacillus genera. In contrast, MALDI-TOF MS fingerprinting identified 76% of the strains at the species level. The mass spectral data were submitted to the SpectraBank database (http://www.spectrabank.org), making this information available to other researchers. Furthermore, cluster analysis of the peak mass lists was carried out with the web application SPECLUST and the calculated groupings were consistent with results determined by a phylogenetic approach that is based on the 16S rRNA sequences. However, the MALDI-TOF MS analysis demonstrated more discriminating potential that allowed for better classification, especially for the Pseudomonas and Bacillus genera. This is of importance with respect to the varying pathogenic and spoilage character at the intragenus and intraspecies level. In this sense, MALDI-TOF MS demonstrated to be a competent bacterial typing tool that extends phenotypic and genotypic approaches, allowing a more ample classification of bacterial strains. PMID:23334977

Böhme, Karola; Fernández-No, Inmaculada C; Pazos, Manuel; Gallardo, José M; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

2013-03-01

271

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

272

Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species.  

PubMed

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains. PMID:24020769

Mechergui, Arij; Achour, Wafa; Hassen, Assia Ben

2014-05-01

273

An efficient RNA extraction method for estimating gut microbial diversity by polymerase chain reaction.  

PubMed

An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis. PMID:19159975

Kang, Seungha; Denman, Stuart E; Morrison, Mark; Yu, Zhongtang; McSweeney, Chris S

2009-05-01

274

Serenoa repens associated with Urtica dioica (ProstaMEV) and curcumin and quercitin (FlogMEV) extracts are able to improve the efficacy of prulifloxacin in bacterial prostatitis patients: results from a prospective randomised study.  

PubMed

We report the results of a prospective randomised study to evaluate the therapeutic effect of Serenoa repens, Urtica dioica (ProstaMEV), quercitin and curcumin (FlogMEV) extracts associated with prulifloxacin in patients affected by chronic bacterial prostatitis (CBP). From a whole population of 284 patients, 143 patients affected by CBP [National Institutes of Health (NIH) class II prostatitis] were enrolled. All patients received prulifloxacin 600 mg daily for 14 days, in accordance with antibiogram results. Patients were split into two groups: Group A received prulifloxacin associated with ProstaMEV and FlogMEV; Group B received only antibiotic therapy. Microbiological and clinical efficacies were tested by two follow-up visits at 1 month and 6 months, respectively. Quality of life (QoL) was measured using the NIH Chronic Prostatitis Symptom Index (CPSI) and International Prostatic Symptom Score (IPSS) questionnaires. Group A comprised 106 patients and Group B comprised 37 patients. One month after treatment, 89.6% of patients who had received prulifloxacin associated with ProstaMEV and FlogMEV did not report any symptoms related to CBP, whilst only 27% of patients who received antibiotic therapy alone were recurrence-free (P < 0.0001). Significant differences were found between groups in terms of symptoms and QoL (P < 0.0001 for both). Six months after treatment, no patients in Group A had recurrence of disease whilst two patients in Group B did. Questionnaire results demonstrated statistically significant differences between groups (all P < 0.001). The association of S. repens, U. dioica (ProstaMEV), quercitin and curcumin (FlogMEV) extracts is able to improve the clinical efficacy of prulifloxacin in patients affected by CBP. PMID:19181486

Cai, Tommaso; Mazzoli, Sandra; Bechi, Adriano; Addonisio, Patrizia; Mondaini, Nicola; Pagliai, Roberto Castricchi; Bartoletti, Riccardo

2009-06-01

275

Dynamic bacterial communities on reverse-osmosis membranes in a full-scale desalination plant  

Microsoft Academic Search

To better understand biofouling of seawater reverse osmosis (SWRO) membranes, bacterial diversity was characterized in the intake water, in subsequently pretreated water and on SWRO membranes from a full-scale desalination plant (FSDP) during a 9 month period. 16S rRNA gene fingerprinting and sequencing revealed that bacterial communities in the water samples and on the SWRO membranes were very different. For

C.-L. de O. Manes; S. Rapenne; P. Lebaron

2010-01-01

276

Loss of Bacterial Diversity during Antibiotic Treatment of Intubated Patients Colonized with Pseudomonas aeruginosa  

Microsoft Academic Search

Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic

J. L. Flanagan; E. L. Brodie; L. Weng; S. V. Lynch; O. Garcia; R. Brown; P. Hugenholtz; T. Z. DeSantis; G. L. Andersen; J. P. Wiener-Kronish; J. Bristow

2007-01-01

277

Bacterial Community Structure and Diversity in a Century-Old Manure-Treated Agroecosystem  

Microsoft Academic Search

Changes in soil microbial community structure and diversity may reflect environmental impact. We exam- ined 16S rRNA gene fingerprints of bacterial communities in six agroecosystems by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. These soils were treated with manure for over a century or different fertilizers for over 70 years. Bacterial community structure and diversity were affected by

H. Y. Sun; S. P. Deng; W. R. Raun

2004-01-01

278

Rapid detection and identification of pathogens in patients with continuous ambulatory peritoneal dialysis (CAPD) associated peritonitis by 16s rRNA gene sequencing.  

PubMed

Peritonitis still remains a serious complication with high rate of morbidity and mortality in patients on CAPD. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early and optimal treatment. The aim of this study was to use 16S rRNA and ITS gene sequencing to identify common bacterial and fungal pathogens directly from the peritoneal fluid without culturing. Ninety one peritoneal fluids obtained from 91 different patients on CAPD suspected for peritonitis were investigated for etiological agents by 16S rRNA and ITS gene sequencing. Data obtained by molecular method was compared with the results obtained by culture method. Among the 45 patients confirmed for peritonitis based on international society of peritoneal dialysis (ISPD) guidelines, the etiological agents were identified in 37(82.2%) samples by culture method, while molecular method identified the etiological agents in 40(88.9%) samples. Despite the high potential application of the 16S rRNA and ITS gene sequencing in comparison to culture method to detect the vast majority of etiological agents directly from peritoneal fluids; it could not be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in Gene Bank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis. PMID:24522129

Ahmadi, S H; Neela, V; Hamat, R A; Goh, B L; Syafinaz, A N

2013-12-01

279

Divergent members of the bacterial division Verrucomicrobiales in a temperate freshwater lake 1 Publication 2291 of the Netherlands Institute of Ecology, Centre for Limnology, Nieuwersluis, The Netherlands. 1  

Microsoft Academic Search

Bacterial diversity in the water column of a freshwater lake in the Netherlands was investigated by analysis of 16S rRNA gene sequences recovered through PCR amplification from total community DNA. Among 23 unique cloned sequences, two appeared to belong to the recently described bacterial division Verrucomicrobiales. One of the two sequences was most similar to a group of environmental clones

Gabriël Zwart; Raymond Huismans; Miranda P van Agterveld; Yves Van de Peer; Peter De Rijk; Hugo Eenhoorn; Gerard Muyzer; Erik J van Hannen; Herman J Gons; Hendrikus J Laanbroek

1998-01-01

280

Characterization of Halophilic Bacterial Communities in Turda Salt Mine (Romania)  

NASA Astrophysics Data System (ADS)

Halophilic organisms are having adaptations to extreme salinity, the majority of them being Archaean, which have the ability to grow at extremely high salt concentrations, (from 3 % to 35 %). Level of salinity causes natural fluctuations in the halophilic populations that inhabit this particular habitat, raising problems in maintaining homeostasis of the osmotic pressure. Samples such as salt and water taken from Turda Salt Mine were analyzed in order to identify the eco-physiological bacterial groups. Considering the number of bacteria of each eco-physiological group, the bacterial indicators of salt quality (BISQ) were calculated and studied for each sample. The phosphatase, catalase and dehydrogenases enzymatic activities were quantitatively determined and the enzymatic indicators of salt quality (EISQ) were calculated. Bacterial isolates were analyzed using 16S rRNA gene sequence analysis. Universal bacterial primers, targeting the consensus region of the bacterial 16S rRNA gene were used. Analysis of a large fragment, of 1499 bp was performed to improve discrimination at the species level.

Carpa, Rahela; Keul, Anca; Muntean, Vasile; Dobrot?, Cristina

2014-09-01

281

Characterization of Halophilic Bacterial Communities in Turda Salt Mine (Romania).  

PubMed

Halophilic organisms are having adaptations to extreme salinity, the majority of them being Archaean, which have the ability to grow at extremely high salt concentrations, (from 3 % to 35 %). Level of salinity causes natural fluctuations in the halophilic populations that inhabit this particular habitat, raising problems in maintaining homeostasis of the osmotic pressure. Samples such as salt and water taken from Turda Salt Mine were analyzed in order to identify the eco-physiological bacterial groups. Considering the number of bacteria of each eco-physiological group, the bacterial indicators of salt quality (BISQ) were calculated and studied for each sample. The phosphatase, catalase and dehydrogenases enzymatic activities were quantitatively determined and the enzymatic indicators of salt quality (EISQ) were calculated. Bacterial isolates were analyzed using 16S rRNA gene sequence analysis. Universal bacterial primers, targeting the consensus region of the bacterial 16S rRNA gene were used. Analysis of a large fragment, of 1499 bp was performed to improve discrimination at the species level. PMID:25476992

Carpa, Rahela; Keul, Anca; Muntean, Vasile; Dobrot?, Cristina

2014-12-01

282

Changes in Bacterial Communities of the Marine Sponge Mycale laxissima on Transfer into Aquaculture? †‡  

PubMed Central

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.

2008-01-01

283

Nucleolar Assembly of the Rrna Processing Machinery in Living Cells  

Microsoft Academic Search

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions (NORs)) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time- lapse microscopy was

Tulia Maria Savino; J. Gebrane-Younes; Jan De Mey; Jean-Baptiste Sibarita; Danièle Hernandez-Verdun

2001-01-01

284

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

Microsoft Academic Search

Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae. These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been

WAYNE L. MILLER; KANTI PABBARAJU; KENNETH E. SANDERSON

2000-01-01

285

Drivers shaping the diversity and biogeography of total and active bacterial communities in the South China Sea  

PubMed Central

To test the hypothesis that different drivers shape the diversity and biogeography of the total and active bacterial community, we examined the bacterial community composition along two transects, one from the inner Pearl River estuary to the open waters of the South China Sea (SCS) and the other from the Luzon Strait to the SCS basin, using 454 pyrosequencing of the 16S rRNA and 16S rRNA gene (V1-3 regions) and thereby characterizing the active and total bacterial community, respectively. The diversity and biogeographic patterns differed substantially between the active and total bacterial communities. Although the composition of both the total and active bacterial community was strongly correlated with environmental factors and weakly correlated with geographic distance, the active bacterial community displayed higher environmental sensitivity than the total community and particularly a greater distance effect largely caused by the active assemblage from deep waters. The 16S rRNA vs. rDNA relationships indicated that the active bacteria were low in relative abundance in the SCS. This might be due to a high competition between active bacterial taxa as indicated by our community network models. Based on these analyses, we speculate that high competition could cause some dispersal limitation of the active bacterial community resulting in a distinct distance-decay relationship. Altogether, our results indicated that the biogeographic distribution of bacteria in the SCS is the result of both environmental control and distance decay. PMID:24684298

Zhang, Yao; Zhao, Zihao; Dai, Minhan; Jiao, Nianzhi; Herndl, Gerhard J

2014-01-01

286

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

287

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

288

No evidence for an endosymbiotic bacterial origin of tetrodotoxin in the newt Taricha granulosa  

E-print Network

No evidence for an endosymbiotic bacterial origin of tetrodotoxin in the newt Taricha granulosa Tetrodotoxin (TTX) is a potent neurotoxin which is known to occur in numerous taxa, including newts. The origin that specifically amplify 16S rRNA genes of bacteria, we examined tissues from rough-skin newts, Taricha granulosa

Brodie III, Edmund D.

289

High-resolution melt analysis for rapid comparison of bacterial community compositions.  

PubMed

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition. PMID:24610853

Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Baelum, Jacob; Feld, Louise; Holben, William E; Jacobsen, Carsten Suhr

2014-06-01

290

Metagenomic Evaluation of Bacterial and Archaeal Diversity in the Geothermal Hot Springs of Manikaran, India  

PubMed Central

Bacterial and archaeal diversity in geothermal spring water were investigated using 16S rRNA gene amplicon metagenomic sequencing. This revealed the dominance of Firmicutes, Aquificae, and the Deinococcus-Thermus group in this thermophilic environment. A number of sequences remained taxonomically unresolved, indicating the presence of potentially novel microbes in this unique habitat.

Pathak, Ashish; Green, Stefan J.; Joshi, Amit; Chauhan, Ashvini

2015-01-01

291

Comparison of midgut bacterial diversity in tropical caterpillars (Lepidoptera: Saturniidae) fed on different diets.  

PubMed

As primary consumers of foliage, caterpillars play essential roles in shaping the trophic structure of tropical forests. The caterpillar midgut is specialized in plant tissue processing; its pH is exceptionally alkaline and contains high concentrations of toxic compounds derived from the ingested plant material (secondary compounds or allelochemicals) and from the insect itself. The midgut, therefore, represents an extreme environment for microbial life. Isolates from different bacterial taxa have been recovered from caterpillar midguts, but little is known about the impact of these microorganisms on caterpillar biology. Our long-term goals are to identify midgut symbionts and to investigate their functions. As a first step, different diet formulations were evaluated for rearing two species of tropical saturniid caterpillars. Using the polymerase chain reaction (PCR) with primers hybridizing broadly to sequences from the bacterial domain, 16S rRNA gene libraries were constructed with midgut DNA extracted from caterpillars reared on different diets. Amplified rDNA restriction analysis indicated that bacterial sequences recovered from the midguts of caterpillars fed on foliage were more diverse than those from caterpillars fed on artificial diet. Sequences related to Methylobacterium sp., Bradyrhizobium sp., and Propionibacterium sp. were detected in all caterpillar libraries regardless of diet, but were not detected in a library constructed from the diet itself. Furthermore, libraries constructed with DNA recovered from surface-sterilized eggs indicated potential for vertical transmission of midgut symbionts. Taken together, these results suggest that microorganisms associated with the tropical caterpillar midgut may engage in symbiotic interactions with these ecologically important insects. PMID:22251723

Pinto-Tomás, Adrián A; Sittenfeld, Ana; Uribe-Lorío, Lorena; Chavarría, Felipe; Mora, Marielos; Janzen, Daniel H; Goodman, Robert M; Simon, Holly M

2011-10-01

292

Spatial heterogeneity and stability of bacterial community in the gastrointestinal tracts of broiler chickens.  

PubMed

Bacterial communities in the different regions of gastrointestinal tract (GIT) of broiler chickens were analyzed by pyrosequencing approach to understand microbial composition and diversity. The DNA samples extracted from 7 different regions along the GIT were subjected to bacterial-community analysis by pyrosequencing of the V1-V3 region of 16S rRNA gene. Major bacterial phyla in the chicken-gut microbiota included Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Acidobacteria, but Firmicutes were mostly dominant (67.3 ± 16.1% of the total sequence reads identified). Among Firmicutes, Lactobacillales, including the genera Lactobacillus and Enterococcus, were the most dominant (51.8 ± 34.5% of the total sequence reads identified) from the crop to ileum. In contrast, in the cecum and large intestine, those genera were rarely detected, and Clostridiales were dominant (55.9 ± 31.4%). Fast UniFrac analysis showed that microbial communities from the crop to jejunum of the same individual chicken were grouped together, and those from ileum, cecum, and large intestine were clustered in a more GIT-specific manner. The numbers of shared operational taxonomic units between the neighboring segments of GIT were low, ranging from 2.9 to 20.3%. However, the abundance of shared operational taxonomic units in each segment was relatively high, ranging from 61.7 to 85.0%, suggesting that substantial proportions of microbial communities were shared between each segment and its neighboring segments, comprising a core microbiota. Our results suggested that the microbial communities of 7 main segments in the chicken GIT were distinctive according to both individuals and the different segments of GIT, but their stability was maintained along the GIT. PMID:24931967

Choi, J H; Kim, G B; Cha, C J

2014-08-01

293

Beyond Streptococcus mutans: Dental Caries Onset Linked to Multiple Species by 16S rRNA Community Analysis  

PubMed Central

Dental caries in very young children may be severe, result in serious infection, and require general anesthesia for treatment. Dental caries results from a shift within the biofilm community specific to the tooth surface, and acidogenic species are responsible for caries. Streptococcus mutans, the most common acid producer in caries, is not always present and occurs as part of a complex microbial community. Understanding the degree to which multiple acidogenic species provide functional redundancy and resilience to caries-associated communities will be important for developing biologic interventions. In addition, microbial community interactions in health and caries pathogenesis are not well understood. The purpose of this study was to investigate bacterial community profiles associated with the onset of caries in the primary dentition. In a combination cross-sectional and longitudinal design, bacterial community profiles at progressive stages of caries and over time were examined and compared to those of health. 16S rRNA gene sequencing was used for bacterial community analysis. Streptococcus mutans was the dominant species in many, but not all, subjects with caries. Elevated levels of S. salivarius, S. sobrinus, and S. parasanguinis were also associated with caries, especially in subjects with no or low levels of S. mutans, suggesting these species are alternative pathogens, and that multiple species may need to be targeted for interventions. Veillonella, which metabolizes lactate, was associated with caries and was highly correlated with total acid producing species. Among children without previous history of caries, Veillonella, but not S. mutans or other acid-producing species, predicted future caries. Bacterial community diversity was reduced in caries as compared to health, as many species appeared to occur at lower levels or be lost as caries advanced, including the Streptococcus mitis group, Neisseria, and Streptococcus sanguinis. This may have implications for bacterial community resilience and the restoration of oral health. PMID:23091642

Gross, Erin L.; Beall, Clifford J.; Kutsch, Stacey R.; Firestone, Noah D.; Leys, Eugene J.; Griffen, Ann L.

2012-01-01

294

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

295

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

2011-04-01

296

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

297

The phylogenetic position of Siboglinidae (Annelida) inferred from 18S rRNA, 28S rRNA and morphological data  

E-print Network

The phylogenetic position of Siboglinidae (Annelida) inferred from 18S rRNA, 28S r and Vestimentifera, but now referred to Annelida) in parsimony analyses of 1100 bp from 18S rRNA, 320 bp from the D1). For a while, they were regarded as not being closely related to Annelida, but referred to as deuterostomes

Siddall, Mark E.

298

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

299

Detection of Gallibacterium spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization  

PubMed Central

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

2003-01-01

300

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

301

The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.  

PubMed Central

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA. Images PMID:7010313

Branlant, C; Krol, A; Machatt, A; Ebel, J P

1981-01-01

302

Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity  

PubMed Central

Aminoglycoside ototoxicity has been related to a surprisingly large number of cellular structures and metabolic pathways. The finding that patients with mutations in mitochondrial rRNA are hypersusceptible to aminoglycoside-induced hearing loss has indicated a possible role for mitochondrial protein synthesis. To study the molecular interaction of aminoglycosides with eukaryotic ribosomes, we made use of the observation that the drug binding site is a distinct domain defined by the small subunit rRNA, and investigated drug susceptibility of bacterial hybrid ribosomes carrying various alleles of the eukaryotic decoding site. Compared to hybrid ribosomes with the A site of human cytosolic ribosomes, susceptibility of mitochondrial hybrid ribosomes to various aminoglycosides correlated with the relative cochleotoxicity of these drugs. Sequence alterations that correspond to the mitochondrial deafness mutations A1555G and C1494T increased drug-binding and rendered the ribosomal decoding site hypersusceptible to aminoglycoside-induced mistranslation and inhibition of protein synthesis. Our results provide experimental support for aminoglycoside-induced dysfunction of the mitochondrial ribosome. We propose a pathogenic mechanism in which interference of aminoglycosides with mitochondrial protein synthesis exacerbates the drugs' cochlear toxicity, playing a key role in sporadic dose-dependent and genetically inherited, aminoglycoside-induced deafness. PMID:19104050

Hobbie, Sven N.; Akshay, Subramanian; Kalapala, Sarath K.; Bruell, Christian M.; Shcherbakov, Dmitry; Böttger, Erik C.

2008-01-01

303

Biomineralization and formulation of endosulfan degrading bacterial and fungal consortiums.  

PubMed

Microbial degradation offers an effective approach to remove toxicants and in this study, a microbial consortium consisting of bacterial strains and fungal strains were originally obtained from endosulfan contaminated agricultural soils. Identification of the bacterial isolates by 16S rRNA sequences revealed the isolates to be Halophilic bacterium JAS4, Klebsiella pneumoniae JAS8, Enterobacter asburiae JAS5, and Enterobacter cloacae JAS7, whereas the fungal isolates were identified by 18S rRNA sequences and the isolates were Botryosphaeria laricina JAS6, Aspergillus tamarii JAS9 and Lasiodiplodia sp. JAS12. The biodegradation of endosulfan was monitored by using HPLC and FTIR analysis. The bacterial and fungal consortium could degrade 1000?mg l(-1) of endosulfan efficiently in aqueous medium and in soil. The infrared spectrum of endosulfan degraded samples in the aqueous medium by bacterial and fungal consortium showed bands at 1400 and 950?cm(-1) which are the characteristics of COOH group and acid dimer band respectively. In the present investigation, low cost solid materials such as sawdust, soil, fly ash, molasses and nutrients were used for the formulation of microbial consortium and to achieve greater multiplication and survival of the microbial strains. PMID:25454517

Abraham, Jayanthi; Silambarasan, Sivagnanam

2014-11-01

304

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

305

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

306

Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR.  

PubMed

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, R I; Lane, J D; Albury, S V; Niemeyer, D M

2000-09-01

307

Initial insights into bacterial succession during human decomposition.  

PubMed

Decomposition is a dynamic ecological process dependent upon many factors such as environment, climate, and bacterial, insect, and vertebrate activity in addition to intrinsic properties inherent to individual cadavers. Although largely attributed to microbial metabolism, very little is known about the bacterial basis of human decomposition. To assess the change in bacterial community structure through time, bacterial samples were collected from several sites across two cadavers placed outdoors to decompose and analyzed through 454 pyrosequencing and analysis of variable regions 3-5 of the bacterial 16S ribosomal RNA (16S rRNA) gene. Each cadaver was characterized by a change in bacterial community structure for all sites sampled as time, and decomposition, progressed. Bacteria community structure is variable at placement and before purge for all body sites. At bloat and purge and until tissues began to dehydrate or were removed, bacteria associated with flies, such as Ignatzschineria and Wohlfahrtimonas, were common. After dehydration and skeletonization, bacteria associated with soil, such as Acinetobacter, were common at most body sites sampled. However, more cadavers sampled through multiple seasons are necessary to assess major trends in bacterial succession. PMID:25431049

Hyde, Embriette R; Haarmann, Daniel P; Petrosino, Joseph F; Lynne, Aaron M; Bucheli, Sibyl R

2014-11-28

308

Bacterial concrete  

NASA Astrophysics Data System (ADS)

Cracks in concrete are inevitable and are one of the inherent weaknesses of concrete. Water and other salts seep through these cracks, corrosion initiates, and thus reduces the life of concrete. So there was a need to develop an inherent biomaterial, a self-repairing material which can remediate the cracks and fissures in concrete. Bacterial concrete is a material, which can successfully remediate cracks in concrete. This technique is highly desirable because the mineral precipitation induced as a result of microbial activities is pollution free and natural. As the cell wall of bacteria is anionic, metal accumulation (calcite) on the surface of the wall is substantial, thus the entire cell becomes crystalline and they eventually plug the pores and cracks in concrete. This paper discusses the plugging of artificially cracked cement mortar using Bacillus Pasteurii and Sporosarcina bacteria combined with sand as a filling material in artificially made cuts in cement mortar which was cured in urea and CaCl2 medium. The effect on the compressive strength and stiffness of the cement mortar cubes due to the mixing of bacteria is also discussed in this paper. It was found that use of bacteria improves the stiffness and compressive strength of concrete. Scanning electron microscope (SEM) is used to document the role of bacteria in microbiologically induced mineral precipitation. Rod like impressions were found on the face of calcite crystals indicating the presence of bacteria in those places. Energy- dispersive X-ray (EDX) spectra of the microbial precipitation on the surface of the crack indicated the abundance of calcium and the precipitation was inferred to be calcite (CaCO3).

Ramakrishnan, Venkataswamy; Ramesh, K. P.; Bang, S. S.

2001-04-01

309

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

310

Linking soil bacterial biodiversity and soil carbon stability.  

PubMed

Native soil carbon (C) can be lost in response to fresh C inputs, a phenomenon observed for decades yet still not understood. Using dual-stable isotope probing, we show that changes in the diversity and composition of two functional bacterial groups occur with this 'priming' effect. A single-substrate pulse suppressed native soil C loss and reduced bacterial diversity, whereas repeated substrate pulses stimulated native soil C loss and increased diversity. Increased diversity after repeated C amendments contrasts with resource competition theory, and may be explained by increased predation as evidenced by a decrease in bacterial 16S rRNA gene copies. Our results suggest that biodiversity and composition of the soil microbial community change in concert with its functioning, with consequences for native soil C stability. PMID:25350158

Mau, Rebecca L; Liu, Cindy M; Aziz, Maliha; Schwartz, Egbert; Dijkstra, Paul; Marks, Jane C; Price, Lance B; Keim, Paul; Hungate, Bruce A

2014-10-28

311

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

PubMed

The combination of flow cytometry (FCM) and 16S rRNA gene pyrosequencing data was investigated for the purpose of monitoring and characterizing microbial changes in drinking water distribution systems. High frequency sampling (5 min intervals for 1 h) was performed at the outlet of a treatment plant and at one location in the full-scale distribution network. In total, 52 bulk water samples were analysed with FCM, pyrosequencing and conventional methods (adenosine-triphosphate, ATP; heterotrophic plate count, HPC). FCM and pyrosequencing results individually showed that changes in the microbial community occurred in the water distribution system, which was not detected with conventional monitoring. FCM data showed an increase in the total bacterial cell concentrations (from 345 ± 15 × 10(3) to 425 ± 35 × 10(3) cells mL(-1)) and in the percentage of intact bacterial cells (from 39 ± 3.5% to 53 ± 4.4%) during water distribution. This shift was also observed in the FCM fluorescence fingerprints, which are characteristic of each water sample. A similar shift was detected in the microbial community composition as characterized with pyrosequencing, showing that FCM and genetic fingerprints are congruent. FCM and pyrosequencing data were subsequently combined for the calculation of cell concentration changes for each bacterial phylum. The results revealed an increase in cell concentrations of specific bacterial phyla (e.g., Proteobacteria), along with a decrease in other phyla (e.g., Actinobacteria), which could not be concluded from the two methods individually. The combination of FCM and pyrosequencing methods is a promising approach for future drinking water quality monitoring and for advanced studies on drinking water distribution pipeline ecology. PMID:25000200

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

2014-10-15

312

Culture independent molecular analysis of bacterial communities in the mangrove sediment of Sundarban, India  

PubMed Central

Background Sundarban is the world's largest coastal sediment comprising of mangrove forest which covers about one million hectares in the south-eastern parts of India and southern parts of Bangladesh. The microbial diversity in this sediment is largely unknown till date. In the present study an attempt has been made to understand the microbial diversity in this sediment using a cultivation-independent molecular approach. Results Two 16 S rRNA gene libraries were constructed and partial sequencing of the selected clones was carried out to identify bacterial strains present in the sediment. Phylogenetic analysis of partially sequenced 16 S rRNA gene sequences revealed the diversity of bacterial strains in the Sundarban sediment. At least 8 different bacterial phyla were detected. The major divisions of detected bacterial phyla were Proteobacteria (alpha, beta, gamma, and delta), Flexibacteria (CFB group), Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, Planctomycetes and Gammatimonadates. Conclusion The gammaproteobacteria were found to be the most abundant bacterial group in Sundarban sediment. Many clones showed similarity with previously reported bacterial lineages recovered from various marine sediments. The present study indicates a probable hydrocarbon and oil contamination in this sediment. In the present study, a number of clones were identified that have shown similarity with bacterial clones or isolates responsible for the maintenance of the S-cycle in the saline environment. PMID:20163727

2010-01-01

313

Clone-based comparative sequence analysis of 16S rRNA genes retrieved from biodeteriorating brick buildings of the former Auschwitz II-Birkenau concentration and extermination camp.  

PubMed

The aim of this work was to analyze the bacterial communities in four samples of historical materials (plaster, brick, and wood) derived from buildings located in the former Auschwitz II-Birkenau concentration and extermination camp in Brzezinka, Poland. For this purpose a molecular strategy based on the construction of 16S rRNA clone libraries was used. In total, 138 partial 16S rRNA gene sequences (?600bp) were obtained and compared. The clones belonged to phyla Proteobacteria (classes: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes. The plaster samples predominantly contained clones closely related to Actinobacteria and Alphaproteobacteria, brick samples contained Gammaproteobacteria, while wood samples had Actinobacteria clones. Interestingly, the historic plaster and brick samples contained the following bacteria with known and described biodeterioration potential: chemoorganotrophic Streptomyces sp. and Pseudonocardia sp., halotolerant or halophilic Rubrobacter sp., Salinisphaera sp. and Halomonas sp. Principal component analysis (PCA) showed that amongst the bacterial species detected and identified none occurred on all the tested historical materials. The 16S rRNA clone library construction method was successfully used for the detection and diversity determination of bacterial communities inhabiting brick barracks located in the former Auschwitz II-Birkenau concentration and extermination camp in Brzezinka. PMID:25458608

Otlewska, Anna; Adamiak, Justyna; Gutarowska, Beata

2014-10-13

314

A Multiphasic Approach for the Identification of Endophytic Bacterial in Strawberry Fruit and their Potential for Plant Growth Promotion  

Microsoft Academic Search

This study used a multiphasic approach, characterized by the simultaneous use of culture-dependent and culture-independent\\u000a methods, to investigate endophytic bacterial communities in strawberry (Fragaria ananassa) fruit. A total of 92 bacterial endophytes were isolated and initially grouped by their repetitive extragenic palindromic\\u000a (rep)-PCR banding pattern and biochemical features. Phylogenetic analysis of the 16S rRNA gene sequences of 45 representatives\\u000a showed

Gilberto Vinícius de Melo Pereira; Karina Teixeira Magalhães; Emi Rainildes Lorenzetii; Thiago Pereira Souza; Rosane Freitas Schwan

315

Epigenetic Programming of the rRNA Promoter by MBD3  

Microsoft Academic Search

Within the human genome there are hundreds of copies of the rRNA gene, but only a fraction of these genes are active. Silencing through epigenetics has been extensively studied; however, it is essential to understand how active rRNA genes are maintained. Here, we propose a role for the methyl-CpG binding domain protein MBD3 in epigenetically maintaining active rRNA promoters. We

Shelley E. Brown; Moshe Szyf

2007-01-01

316

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing  

Microsoft Academic Search

Background Massively-parallel pyrosequencing of hypervariable regions of small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy

Susan M. Huse; Les Dethlefsen; Julie A. Huber; David Mark Welch; David A. Relman; Mitchell L. Sogin

2008-01-01

317

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

PubMed

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

Martínez, 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

318

[Bacterial community structure in intestine of the white shrimp, Litopenaeus vannamei].  

PubMed

The composition of bacterial community in the intestine of the white shrimp, Litopenaeus vannamei under laboratory culture condition was determined using the 16S rDNA clone library. 16s rRNA gene was amplified and a library was constructed by using the genomic DNA extracted from the bacteria in the shrimp intestine as template. 12 different RFLP patterns of the clones were obtained by restriction fragment length polymorphism analysis using Afa I and Msp I. Compared with the published sequences in GenBank database, sequencing results of cloned 16S rDNA amplicons revealed a diverse community including gamma-proteobacteria and Firmicutes in the intestine of artificial diet-fed shrimp. Results showed that the Firmicutes group can be a dominant component (75.4%) in the shrimp intestinal microflora and other clones belong to gamma-proteobacteria (24.6%) which were identified as Shewanella sp., Pantoea sp., Aranicola sp., Pseudomonas sp. and Vibrio sp., respectively. These results provide the first comprehensive description of microbial diversity of the white shrimp intestine and suggest that most of the bacteria associated with shrimp intestine are uncultured and novel species. PMID:17944366

Li, Ke; Zheng, Tian-ling; Tian, Yun; Yuan, Jian-jun

2007-08-01

319

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

320

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

321

Consensus structure and evolution of 5S rRNA.  

PubMed Central

A consensus structure model of 5S rRNA presenting all conserved nucleotides in fixed positions has been deduced from the primary and secondary structure of 71 eubacterial, archaebacterial, eukaryotic cytosolic and organellar molecules. Phylogenetically related groups of molecules are characterized by nucleotide deletions in helices III, IV and V, and by potential base pair interactions in helix IV. The group-specific deletions are correlated with the early branching pattern of a dendrogram calculated from nucleotide substitution data: the first major division separates the group of eubacterial and organellar molecules from a second group containing the common ancestors of archaebacterial and eukaryotic/cytosolic molecules. The earliest diverging branch of the eubacterial/organellar group includes molecules from Thermus thermophilus, T. aquaticus, Rhodospirillum rubrum, Paracoccus denitrificans and wheat mitochondria. PMID:6835839

Küntzel, H; Piechulla, B; Hahn, U

1983-01-01

322

Novel Microarray Design Strategy To Study Complex Bacterial Communities?  

PubMed Central

Assessing bacterial flora composition appears to be of increasing importance to fields as diverse as physiology, development, medicine, epidemiology, the environment, and the food industry. We report here the development and validation of an original microarray strategy that allows analysis of the phylogenic composition of complex bacterial mixtures. The microarray contains ?9,500 feature elements targeting 16S rRNA gene-specific regions. Probe design was performed by selecting oligonucleotide sequences specific to each node of the seven levels of the bacterial phylogenetic tree (domain, phylum, class, order, family, genus, and species). This approach, based on sequence information, allows analysis of the bacterial contents of complex bacterial mixtures to detect both known and unknown microorganisms. The presence of unknown organisms can be suspected and mapped on the phylogenetic tree, indicating where to refine analysis. Initial proof-of-concept experiments were performed on oral bacterial communities. Our results show that this hierarchical approach can reveal minor changes (?1%) in gingival flora content when samples collected in individuals from similar geographical origins are compared. PMID:18203854

Huyghe, Antoine; Francois, Patrice; Charbonnier, Yvan; Tangomo-Bento, Manuela; Bonetti, Eve-Julie; Paster, Bruce J.; Bolivar, Ignacio; Baratti-Mayer, Denise; Pittet, Didier; Schrenzel, Jacques

2008-01-01

323

Bacterial Functional Redundancy along a Soil Reclamation Gradient  

PubMed Central

A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU). BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site. The number and diversity of bacterial groups responding to four carbon substrates (l-serine, l-threonine, sodium citrate, and ?-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning. PMID:11010883

Yin, Bei; Crowley, David; Sparovek, Gerd; De Melo, Wanderley Jose; Borneman, James

2000-01-01

324

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

325

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

326

Bacterial community composition in the rhizosphere of a transgenic, herbicide-resistant maize (Zea mays) and comparison to its non-transgenic cultivar Bosphore.  

PubMed

Bacterial communities in rhizospheres of transgenic maize (Zea mays, with the pat-gene conferring resistance to the herbicide glufosinate; syn. l-phosphinothricin) were compared to its isogenic, non-transgenic cultivar. Total DNA was extracted from bacterial cell consortia collected from rhizospheres of plants grown in an agricultural field. With the use of three different primer pairs binding to evolutionarily conserved regions of the bacterial 16S rRNA gene, partial sequences were amplified by polymerase chain reaction (PCR). The PCR products were subjected to single-strand conformation polymorphism (SSCP) to generate genetic profiles which corresponded to the diversity of the amplified sequences. Genetic profiles of rhizospheres consisted of 40-60 distinguishable bands depending on the chosen primer pairs, and the variability between independent replicates was very low. Neither the genetic modification nor the use of the herbicide Liberty (syn. Basta; active ingredient: glufosinate) affected the SSCP profiles as investigated with digital image analysis. In contrast, PCR-SSCP profiles of bacterial communities from rhizospheres of sugar beet, grown in the same field as a control crop, were clearly different. A less pronounced but significant difference was also observed with rhizosphere samples from fine roots of maize plants collected 35 and 70 days after sowing. Sequencing of the dominant 30 products from one typical SSCP profile generated from transgenic maize rhizospheres indicated the presence of typical soil and rhizosphere bacteria: half of the bands could be attributed to Proteobacteria, mainly of the alpha- and beta-subgroups. Other SSCP bands could be assigned to members of the following phylogenetic groups: Cytophaga-Flavobacterium-Bacteroides, Chlamydiales-Verrucomicrobium, Planctomyces, Holophaga and to Gram-positive bacteria with a high G+C DNA content. PMID:19709208

Schmalenberger, Achim; Tebbe, Christoph C

2002-04-01

327

Bacterial Skin Infections  

MedlinePLUS

... Disorders Pigment Disorders Blistering Diseases Parasitic Skin Infections Bacterial Skin Infections Fungal Skin Infections Viral Skin Infections Sunlight and Skin Damage Noncancerous Skin Growths Skin Cancers Nail Disorders Topics in Bacterial Skin ...

328

Bacterial Gene Transfer  

NSDL National Science Digital Library

This resource provides detailed instructions for carrying out several laboratory exercises relating to bacterial transformation and conjugation. In this multi-session experiment, students are exposed to various techniques in microbiology, including bacterial transformation and assay and sterile techniques.

Roberta Ellington (Northwestern University; )

1991-01-01

329

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

PubMed

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

2012-06-01

330

Bacterial Communities in Polluted Seabed Sediments: A Molecular Biology Assay in Leghorn Harbor  

PubMed Central

Seabed sediments of commercial ports are often characterized by high pollution levels. Differences in number and distribution of bacteria in such areas can be related to distribution of pollutants in the port and to sediment conditions. In this study, the bacterial communities of five sites from Leghorn Harbor seabed were characterized, and the main bacterial groups were identified. T-RFLP was used for all samples; two 16S rRNA libraries and in silico digestion of clones were used to identify fingerprint profiles. Library data, phylogenetic analysis, and T-RFLP coupled with in silico digestion of the obtained sequences evidenced the dominance of Proteobacteria and the high percentage of Bacteroidetes in all sites. The approach highlighted similar bacterial communities between samples coming from the five sites, suggesting a modest differentiation among bacterial communities of different harbor seabed sediments and hence the capacity of bacterial communities to adapt to different levels and types of pollution. PMID:24227997

Verni, Franco; Petroni, Giulio

2013-01-01

331

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

332

Correlation between protection against sepsis by probiotic therapy and stimulation of a novel bacterial phylotype.  

PubMed

Prophylactic probiotic therapy has shown beneficial effects in an experimental rat model for acute pancreatitis on the health status of the animals. Mechanisms by which probiotic therapy interferes with severity of acute pancreatitis and associated sepsis, however, are poorly understood. The aims of this study were to identify the probiotic-induced changes in the gut microbiota and to correlate these changes to disease outcome. Duodenum and ileum samples were obtained from healthy and diseased rats subjected to pancreatitis for 7 days and prophylactically treated with either a multispecies probiotic mixture or a placebo. Intestinal microbiota was characterized by terminal-restriction fragment length polymorphism (T-RFLP) analyses of PCR-amplified 16S rRNA gene fragments. These analyses showed that during acute pancreatitis the host-specific ileal microbiota was replaced by an "acute pancreatitis-associated microbiota." This replacement was not reversed by administration of the probiotic mixture. An increase, however, was observed in the relative abundance of a novel bacterial phylotype most closely related to Clostridium lituseburense and referred to as commensal rat ileum bacterium (CRIB). Specific primers targeting the CRIB 16S rRNA gene sequence were developed to detect this phylotype by quantitative PCR. An ileal abundance of CRIB 16S rRNA genes of more than 7.5% of the total bacterial 16S rRNA gene pool was correlated with reduced duodenal bacterial overgrowth, reduced bacterial translocation to remote organs, improved pancreas pathology, and reduced proinflammatory cytokine levels in plasma. Our current findings and future studies involving this uncharacterized bacterial phylotype will contribute to unraveling one of the potential mechanisms of probiotic therapy. PMID:21926217

Gerritsen, Jacoline; Timmerman, Harro M; Fuentes, Susana; van Minnen, L Paul; Panneman, Henk; Konstantinov, Sergey R; Rombouts, Frans M; Gooszen, Hein G; Akkermans, Louis M A; Smidt, Hauke; Rijkers, Ger T

2011-11-01

333

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

334

Bacterial populations in the vaginas of healthy adolescent women.  

PubMed

Given that the microbiota of the healthy vagina plays an important role in the maintenance of health, it follows that an understanding of its composition and development may offer insights into the etiology and prevention of disease. In contrast to previous studies, this study exclusively investigated the structure and composition of adolescent vaginal bacterial communities. In this report, the vaginal bacterial communities of 90 menarcheal adolescents, ages 13-18y, were characterized using terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes. Further characterization involved cluster analysis of the T-RFLP data to identify the number of different kinds of microbial communities found among the adolescents sampled, and phylogenetic analysis of 16S rRNA gene sequences cloned from samples representative of each cluster. We report the identification of four major clusters that accounted for 96.7% of the cohort. In general, these clusters could be divided into those dominated by Lactobacillus spp. and those dominated by a variety of lactic acid producing, anaerobic bacterial types such as Atopobium vaginae and Streptococcus spp. The compositional and structural similarity of the vaginal microbiota of menarcheal adolescents and adults suggests that the vaginal microbiota does not change significantly after the onset of menarche. PMID:19232297

Yamamoto, Ted; Zhou, Xia; Williams, Chris J; Hochwalt, Anne; Forney, Larry J

2009-02-01

335

Functional profiling and distribution of the forest soil bacterial communities along the soil mycorrhizosphere continuum.  

PubMed

An ectomycorrhiza is a multitrophic association between a tree root, an ectomycorrhizal fungus, free-living fungi and the associated bacterial communities. Enzymatic activities of ectomycorrhizal root tips are therefore result of the contribution from different partners of the symbiotic organ. However, the functional potential of the fungus-associated bacterial communities remains unknown. In this study, a collection of 80 bacterial strains randomly selected and isolated from a soil-ectomycorrhiza continuum (oak-Scleroderma citrinum ectomycorrhizas, the ectomycorrhizosphere and the surrounding bulk soil) were characterized. All the bacterial isolates were identified by partial 16S rRNA gene sequences as members of the genera Burkholderia, Collimonas, Dyella, Mesorhizobium, Pseudomonas, Rhizobium and Sphingomonas. The bacterial strains were then assayed for ?-xylosidase, ?-glucosidase, N-acetyl-hexosaminidase, ?-glucuronidase, cellobiohydrolase, phosphomonoesterase, leucine-aminopeptidase and laccase activities, chitin solubilization and auxin production. Using these bioassays, we demonstrated significant differences in the functional distribution of the bacterial communities living in the different compartments of the soil-ectomycorrhiza continuum. The surrounding bulk soil was significantly enriched in bacterial isolates capable of hydrolysing cellobiose and N-acetylglucosamine. In contrast, the ectomycorrhizosphere appeared significantly enriched in bacterial isolates capable of hydrolysing glucopyranoside and chitin. Notably, chitinase and laccase activities were found only in bacterial isolates belonging to the Collimonas and Pseudomonas genera. Overall, the results suggest that the ectomycorrhizal fungi favour specific bacterial communities with contrasting functional characteristics from the surrounding soil. PMID:23455431

Uroz, S; Courty, P E; Pierrat, J C; Peter, M; Buée, M; Turpault, M P; Garbaye, J; Frey-Klett, P

2013-08-01

336

Diversity of arsenite oxidizing bacterial communities in arsenic-rich deltaic aquifers in West Bengal, India.  

PubMed

High arsenic (As) concentration in groundwater has affected human health, particularly in South-East Asia putting millions of people at risk. Biogeochemical cycling of As carried out by different bacterial groups are suggested to control the As fluxes in aquifers. A functional diversity approach in link with As precipitation was adopted to study bacterial community structures and their variation within the As contaminated Bengal Delta Plain (BDP) aquifers of India. Groundwater samples collected from two shallow aquifers in Karimpur II (West Bengal, India), during years 2010 and 2011, were investigated to trace the effects immediately after monsoon period (precipitation) on community structure and diversity of bacterial assemblages with a focus on arsenite oxidizing bacterial phyla for two successive years. The study focused on amplification, clone library generation and sequencing of the arsenite oxidase large sub-unit gene aioA and 16S rRNA marker, with respect to changes in elemental concentrations. New set of primers were designed to amplify the aioA gene as a phylogenetic marker to study taxonomically diverse arsenite oxidizing bacterial groups in these aquifers. The overall narrow distribution of bacterial communities based on aioA and 16S rRNA sequences observed was due to poor nutrient status and anoxic conditions in these As contaminated aquifers. Proteobacteria was the dominant phylum detected, within which Acidovorax, Hydrogenophaga, Albidiferax, Bosea, and Polymorphum were the major arsenite oxidizing bacterial genera based on the number of clones sequenced. The structure of bacterial assemblages including those of arsenite oxidizing bacteria seems to have been affected by increase in major elemental concentrations (e.g., As, Fe, S, and Si) within two sampling sessions, which was supported by statistical analyses. One of the significant findings of this study is detection of novel lineages of 16S rRNA-like bacterial sequences indicating presence of indigenous bacterial communities BDP wells that can play important role in biogeochemical cycling of elements including As. PMID:25484877

Ghosh, Devanita; Bhadury, Punyasloke; Routh, Joyanto

2014-01-01

337

Diversity of arsenite oxidizing bacterial communities in arsenic-rich deltaic aquifers in West Bengal, India  

PubMed Central

High arsenic (As) concentration in groundwater has affected human health, particularly in South-East Asia putting millions of people at risk. Biogeochemical cycling of As carried out by different bacterial groups are suggested to control the As fluxes in aquifers. A functional diversity approach in link with As precipitation was adopted to study bacterial community structures and their variation within the As contaminated Bengal Delta Plain (BDP) aquifers of India. Groundwater samples collected from two shallow aquifers in Karimpur II (West Bengal, India), during years 2010 and 2011, were investigated to trace the effects immediately after monsoon period (precipitation) on community structure and diversity of bacterial assemblages with a focus on arsenite oxidizing bacterial phyla for two successive years. The study focused on amplification, clone library generation and sequencing of the arsenite oxidase large sub-unit gene aioA and 16S rRNA marker, with respect to changes in elemental concentrations. New set of primers were designed to amplify the aioA gene as a phylogenetic marker to study taxonomically diverse arsenite oxidizing bacterial groups in these aquifers. The overall narrow distribution of bacterial communities based on aioA and 16S rRNA sequences observed was due to poor nutrient status and anoxic conditions in these As contaminated aquifers. Proteobacteria was the dominant phylum detected, within which Acidovorax, Hydrogenophaga, Albidiferax, Bosea, and Polymorphum were the major arsenite oxidizing bacterial genera based on the number of clones sequenced. The structure of bacterial assemblages including those of arsenite oxidizing bacteria seems to have been affected by increase in major elemental concentrations (e.g., As, Fe, S, and Si) within two sampling sessions, which was supported by statistical analyses. One of the significant findings of this study is detection of novel lineages of 16S rRNA-like bacterial sequences indicating presence of indigenous bacterial communities BDP wells that can play important role in biogeochemical cycling of elements including As. PMID:25484877

Ghosh, Devanita; Bhadury, Punyasloke; Routh, Joyanto

2014-01-01

338

Sequence specific detection of bacterial 23S ribosomal RNA by TLR13  

PubMed Central

Toll-like receptors (TLRs) detect microbial infections and trigger innate immune responses. Among vertebrate TLRs, the role of TLR13 and its ligand are unknown. Here we show that TLR13 detects the 23S ribosomal RNA of both gram-positive and gram-negative bacteria. A sequence containing 13 nucleotides near the active site of 23S rRNA ribozyme, which catalyzes peptide bond synthesis, was both necessary and sufficient to trigger TLR13-dependent interleukin-1? production. Single point mutations within this sequence destroyed the ability of the 23S rRNA to stimulate the TLR13 pathway. Knockout of TLR13 in mice abolished the induction of interleukin-1? and other cytokines by the 23S rRNA sequence. Thus, TLR13 detects bacterial RNA with exquisite sequence specificity. DOI: http://dx.doi.org/10.7554/eLife.00102.001 PMID:23110254

Li, Xiao-Dong; Chen, Zhijian J

2012-01-01

339

MICROBIAL DIVERSITY AND COMMUNITY STRUCTURE DETERMINATIONS THROUGH ANALYSES OF SSU rRNA GENE  

E-print Network

MICROBIAL DIVERSITY AND COMMUNITY STRUCTURE DETERMINATIONS THROUGH ANALYSES OF SSU rRNA GENE of diversity and community stnlcture through an examination of small-subunit (SSU) rRNA genes from the microbial mat assemblage located at an active, hydrothermal vent system, Loihi Seamount, Hawaii. The habitat

Luther, Douglas S.

340

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

341

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

342

Bacterial moonlighting proteins and bacterial virulence.  

PubMed

Implicit in the central dogma is the hypothesis that each protein gene product has but one function. However, over the past decade, it has become clear that many proteins have one or more unique functions, over-and-above the principal biological action of the specific protein. This phenomenon is now known as protein moonlighting and many well-known proteins such as metabolic enzymes and molecular chaperones are now recognised as moonlighting proteins. A growing number of bacterial species are being found to have moonlighting proteins and the moonlighting activities of such proteins can contribute to bacterial virulence behaviour. The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPD) and enolase, and the cell stress proteins: chaperonin 60, Hsp70 and peptidyl prolyl isomerase, are among the most common of the bacterial moonlighting proteins which play a role in bacterial virulence. Moonlighting activities include adhesion and modulation of cell signalling processes. It is likely that only the tip of the bacterial moonlighting iceberg has been sighted and the next decade will bring with it many new discoveries of bacterial moonlighting proteins with a role in bacterial virulence. PMID:22143554

Henderson, Brian; Martin, Andrew

2013-01-01

343

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

344

Bacterial communities associated with the ctenophores Mnemiopsis leidyi and Beroe ovata.  

PubMed

Residing in a phylum of their own, ctenophores are gelatinous zooplankton that drift through the ocean's water column. Although ctenophores are known to be parasitized by a variety of eukaryotes, no studies have examined their bacterial associates. This study describes the bacterial communities associated with the lobate ctenophore Mnemiopsis leidyi and its natural predator Beroe ovata in Tampa Bay, Florida, USA. Investigations using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of 16S rRNA genes demonstrated that ctenophore bacterial communities were distinct from the surrounding water. In addition, each ctenophore genus contained a unique microbiota. Ctenophore samples contained fewer bacterial operational taxonomic units (OTUs) by T-RFLP and lower diversity communities by 16S rRNA gene sequencing than the water column. Both ctenophore genera contained sequences related to bacteria previously described in marine invertebrates, and sequences similar to a sea anemone pathogen were abundant in B. ovata. Temporal sampling revealed that the ctenophore-associated bacterial communities varied over time, with no single OTU detected at all time points. This is the first report of distinct and dynamic bacterial communities associated with ctenophores, suggesting that these microbial consortia may play important roles in ctenophore ecology. Future work needs to elucidate the functional roles and mode of acquisition of these bacteria. PMID:22571334

Daniels, Camille; Breitbart, Mya

2012-10-01

345

In vitro transcription of two rRNA genes of the archaebacterium Sulfolobus sp. B12 indicates a factor requirement for specific initiation.  

PubMed Central

We describe a cell-free transcription system for the archaebacterium Sulfolobus sp. B12 that specifically initiates transcription at the 5S rRNA-encoding DNA and the 16S/23S rRNA-encoding DNA promoters of the same species. With this crude extract system, specific initiation was absolutely dependent on the box A motif, a highly conserved promoter element in archaebacteria located approximately 25 base pairs upstream of transcription initiation sites. In vitro transcription of the rRNA genes by purified RNA polymerase, however, resulted in semi-specific, box A-independent initiation, indicating that factor(s) in the crude extract were necessary for the highly specific box A-dependent transcription. Fractionation of the cell-free extract by sucrose-gradient centrifugation resulted in the identification of a low molecular weight fraction complementing purified RNA polymerase to an extract-like specificity. Images PMID:2116009

Hüdepohl, U; Reiter, W D; Zillig, W

1990-01-01

346

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

347

Nucleolar Assembly of the Rrna Processing Machinery in Living Cells  

PubMed Central

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein–tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories. PMID:11381093

Savino, Tulia Maria; Gébrane-Younès, Jeannine; De Mey, Jan; Sibarita, Jean-Baptiste; Hernandez-Verdun, Danièle

2001-01-01

348

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, Jesús L.; Toranzo, Alicia E.

2005-01-01

349

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

350

An Assessment of Urea-Formaldehyde Fertilizer on the Diversity of Bacterial Communities in Onion and Sugar Beet  

PubMed Central

The impact of a urea-formaldehyde (UF) fertilizer on bacterial diversity in onion bulbs and main roots of sugar beet were examined using a 16S rRNA gene clone library. The UF fertilizer markedly increased bacterial diversity in both plants. The results of principal coordinates analysis (PCoA) revealed that nearly 30% of the variance observed in bacterial diversity in both the onion and sugar beet was attributed to the fertilization conditions and also that the community structures in both plants shifted unidirectionally in response to the UF fertilizer. PMID:24882062

Ikeda, Seishi; Suzuki, Keijiro; Kawahara, Makoto; Noshiro, Masao; Takahashi, Naokazu

2014-01-01

351

Distinct Bacterial Communities Exist beneath a High Arctic Polythermal Glacier†  

PubMed Central

Bacterial communities reside in basal ice, sediment, and meltwater in the supra-, sub-, and proglacial environments of John Evans Glacier, Nunavut, Canada. We examined whether the subglacial bacterial community shares common members with the pro- and supraglacial communities, and by inference, whether it could be derived from communities in either of these environments (e.g., by ice overriding proglacial sediments or by in-wash of surface meltwaters). Terminal restriction fragment length polymorphism analysis of bacterial 16S rRNA genes amplified from these environments revealed that the subglacial water, basal ice, and sediment communities were distinct from those detected in supraglacial meltwater and proglacial sediments, with 60 of 142 unique terminal restriction fragments (T-RFs) detected exclusively in subglacial samples and only 8 T-RFs detected in all three environments. Supraglacial waters shared some T-RFs with subglacial water and ice, likely reflecting the seasonal flow of surface meltwater into the subglacial drainage system, whereas supraglacial and proglacial communities shared the fewest T-RFs. Thus, the subglacial community at John Evans Glacier appears to be predominantly autochthonous rather than allochthonous, and it may be adapted to subglacial conditions. Chemical analysis of water and melted ice also revealed differences between the supraglacial and proglacial environments, particularly regarding electrical conductivity and nitrate, sulfate, and dissolved organic carbon concentrations. Whereas the potential exists for common bacterial types to be broadly distributed throughout the glacial system, we have observed distinct bacterial communities in physically and chemically different glacial environments. PMID:16957202

Bhatia, Maya; Sharp, Martin; Foght, Julia

2006-01-01

352

Bacterial diversity characterization in petroleum samples from Brazilian reservoirs  

PubMed Central

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

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

2008-01-01

353

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

PubMed Central

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

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

2012-01-01

354

18S rRNA degradation is not accompanied by altered rRNA transport at early times following irradiation of HeLa cells  

SciTech Connect

In recent investigations on the effects of radiation on rRNA processing in HeLa S3 cells, the authors pulse-labeled the cells with uridine immediately prior to irradiation. The 45 S rRNA precursor, which undergoes nuclear processing to form one each of its major daughter species, 28S and 18S rRNA, was separated from the daughter species by gel electrophoresis and the radiolabel in each species determined at various times after irradiation. By pulse-labeling the cells prior to irradiation, superimposed effects caused by radiation-induced alterations of rRNA transcription and Refs. therein were minimized, permitting selective analysis of the processing of that fraction of 45S precursor that had been synthesized (radiolabeled) predominantly prior to irradiation. They now report more detailed studies on 45S rRNA processing within the first 2 h following irradiation in which they have found a maximum 28 S:18 S ratio of 2:1 that is observed about 1 h following irradiation of 5 or 10 Gy.

Fuchs, P.; Krolak, J.M.; McClain, D.; Minton, K.W.

1990-01-01

355

Metagenomics Reveals Planktonic Bacterial Community Shifts across a Natural CO2 Gradient in the Mediterranean Sea  

PubMed Central

Bacterial communities at a CO2 vent (pH 6.7) were compared with those at control (pH 8.0) and transition sites (pH 7.6) using 16S rRNA metagenomics. Firmicutes and unclassified bacteria dominated across all sites, Proteobacteria, especially Gammaproteobacteria, declined, and Epsilonproteobacteria increased in the vent with an increase in Bacteroidetes at both the vent and transition sites. PMID:25676760

Pathak, Ashish; Rodolfo-Metalpa, Riccardo; Milazzo, Marco; Green, Stefan J.; Hall-Spencer, Jason M.

2015-01-01

356

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

357

Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors  

Microsoft Academic Search

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were moni- tored using 16S rRNA slot blot

Cesar Mota; Melanie A. Head; Jennifer A. Ridenoure; Jay J. Cheng; F. L. de los Reyes

2005-01-01

358

Bacterial Diversity Associated with Blood Falls, a Subglacial Outflow from the Taylor Glacier, Antarctica  

Microsoft Academic Search

Blood Falls is the surface manifestation of brine released from below the Taylor Glacier, McMurdo Dry Valleys, Antarctica. Geochemical analyses of Blood Falls show that this brine is of a marine origin. The discovery that 74% of clones and isolates from Blood Falls share high 16S rRNA gene sequence homology with phylotypes from marine systems supports this contention. The bacterial

Jill A. Mikucki; John C. Priscu

2007-01-01

359

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

360

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin  

Microsoft Academic Search

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a\\u000a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods.\\u000a Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant\\u000a differences between the

Lara D. Sette; Karen C. M. Simioni; Suzan P. Vasconcellos; Lucia J. Dussan; Eugênio V. S. Neto; Valéria M. Oliveira

2007-01-01

361

Global Patterns of Bacterial Beta-Diversity in Seafloor and Seawater Ecosystems  

Microsoft Academic Search

BackgroundMarine microbial communities have been essential contributors to global biomass, nutrient cycling, and biodiversity since the early history of Earth, but so far their community distribution patterns remain unknown in most marine ecosystems.Methodology\\/Principal FindingsThe synthesis of 9.6 million bacterial V6-rRNA amplicons for 509 samples that span the global ocean's surface to the deep-sea floor shows that pelagic and benthic communities

Lucie Zinger; Linda A. Amaral-Zettler; Jed A. Fuhrman; M. Claire Horner-Devine; Susan M. Huse; David B. Mark Welch; Jennifer B. H. Martiny; Mitchell Sogin; Antje Boetius; Alban Ramette; Jack Anthony Gilbert

2011-01-01

362

Metagenomics Reveals Planktonic Bacterial Community Shifts across a Natural CO2 Gradient in the Mediterranean Sea.  

PubMed

Bacterial communities at a CO2 vent (pH 6.7) were compared with those at control (pH 8.0) and transition sites (pH 7.6) using 16S rRNA metagenomics. Firmicutes and unclassified bacteria dominated across all sites, Proteobacteria, especially Gammaproteobacteria, declined, and Epsilonproteobacteria increased in the vent with an increase in Bacteroidetes at both the vent and transition sites. PMID:25676760

Chauhan, Ashvini; Pathak, Ashish; Rodolfo-Metalpa, Riccardo; Milazzo, Marco; Green, Stefan J; Hall-Spencer, Jason M

2015-01-01

363

Characterization of Bacterial Community Structure in Rhizosphere Soil of Grain Legumes  

Microsoft Academic Search

Molecular techniques were used to characterize bacterial community structure, diversity (16S rDNA), and activity (16S rRNA) in rhizospheres of three grain legumes: faba beans (Vicia faba L., cv. Scirocco), peas (Pisum sativum L., cv. Duel) and white lupin (Lupinus albus L., cv. Amiga). All plants were grown in the same soil under controlled conditions in a greenhouse and sampled after

S. Sharma; M. K. Aneja; J. Mayer; J. C. Munch; M. Schloter

2005-01-01

364

Bacterial diversity and White Plague Disease-associated community changes in the Caribbean coral Montastraea faveolata  

Microsoft Academic Search

Increasing evidence confirms the crucial role bacteria and archaea play within the coral holobiont, that is, the coral host and its associated microbial community. The bacterial component constitutes a community of high diversity, which appears to change in structure in response to disease events. In this study, we highlight the limitation of 16S rRNA gene (16S rDNA) clone library sequencing

Shinichi Sunagawa; Todd Z DeSantis; Yvette M Piceno; Eoin L Brodie; Michael K DeSalvo; Christian R Voolstra; Ernesto Weil; Gary L Andersen; Mónica Medina

2009-01-01

365

Effects of FIS Overexpression on Cell Growth, rRNA Synthesis, and Ribosome Content in Escherichia coli  

E-print Network

are sufficient to modulate rRNA synthesis (6). FIS is a 11.2 kDa, heat stable, DNA binding protein in Escherichia in enhanced rRNA promoter activity and recombinant protein synthesis (14). However, the precise mechanismEffects of FIS Overexpression on Cell Growth, rRNA Synthesis, and Ribosome Content in Escherichia

Chen, Wilfred

366

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

367

Pyrene effects on rhizoplane bacterial communities.  

PubMed

Certain plant species promote biodegradation of polycyclic aromatic hydrocarbons (PAHs), but few studies have examined the microbial populations that are associated with the rhizoplane of these plants. In this study, the bacterial composition of the rhizoplane were characterized for four plant species during in soils with different histories of exposure to PAH and in the presence or absence of a pyrene spike at 100 mg kg(-1) pyrene. Three of the plant species including Andropogon gerrardii, Panicum coloratum and Melilotus officinalis were known to stimulate PAH degradation. Wheat (Triticum aestivum) was used as a reference species. Results showed that after 90 days, approximately 45% of the pyrene spike disappeared from soil without plants. In contrast, cultivation of plants resulted in 95% disappearance of pyrene. There were no significant differences in the extent of pyrene disappearance for different plants. In all cases, 16S rRNA gene profiles of the rhizoplane were less complex in the pyrene-spiked soils, suggesting that richness and evenness of the predominant bacteria were reduced. Our results show that pyrene contamination results in significant shifts in the composition of rhizosphere bacterial communities that are still further influenced by the plant species and prior exposure history to PAH contamination. PMID:19810358

Balcom, Ian N; Crowley, David E

2009-09-01

368

Global dispersion of bacterial cells on Asian dust  

PubMed Central

The atmospheric dispersion of bacteria over long distances is an important facet of microbial ecology. Certain groups of dispersed bacteria can adapt to their new location and affect established ecosystems. Aeolian dust particles are known to be carriers of microbes but further research is needed to expand our understanding of this field of microbiology. Here we showed the potential of aeolian dust to global migration of bacterial cells. We demonstrated the presence of microbial cells on dust particles directly by bio-imaging. Bacterial abundance on dust particles declined from 105 to less than 103 cells/m3 as the dust event subsided. Taxonomically diverse bacteria were identified by 16S rRNA gene sequencing and some of these bacteria retained growth potential. Our results confirm that bacteria can attach to aeolian dust particles and they have the potential to migrate globally during dust events and thus can contribute to the diversity of downwind ecosystems. PMID:22826803

Yamaguchi, Nobuyasu; Ichijo, Tomoaki; Sakotani, Akiko; Baba, Takashi; Nasu, Masao

2012-01-01

369

Analysis of the dynamics of bacterial communities in the rhizosphere of the Chrysanthemum via denaturing gradient gel eletrophoresis and substrate utilization patterns  

Microsoft Academic Search

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE

BERNADETTE M. DUINEVELD; ALEXANDRE S. ROSADO; JAN DIRK VAN ELSAS; JOHANNES A. VAN VEEN

1998-01-01

370

Characterization of Bacterial Community Diversity in Cystic Fibrosis Lung Infections by Use of 16S Ribosomal DNA Terminal Restriction Fragment Length Polymorphism Profiling  

Microsoft Academic Search

Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data

G. B. Rogers; M. P. Carroll; D. J. Serisier; P. M. Hockey; G. Jones; K. D. Bruce

2004-01-01

371

Use of Real-Time qPCR to Quantify Members of the Unculturable Heterotrophic Bacterial Community in a Deep Sea Marine Sponge, Vetulina sp  

Microsoft Academic Search

In this report, real-time quantitative PCR (TaqMan® qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic\\u000a marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable,\\u000a microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean\\u000a Lithistid sponges have shown a wide diversity of

M. Cassler; C. L. Peterson; A. Ledger; S. A. Pomponi; A. E. Wright; R. Winegar; P. J. McCarthy; J. V. Lopez

2008-01-01

372

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine.  

PubMed

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37 % of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study. PMID:23963670

Lucheta, A R; Otero, X L; Macías, F; Lambais, M R

2013-11-01

373

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

374

Bacterial diversity in Cr(VI) and Cr(III)-contaminated industrial wastewaters.  

PubMed

The bacterial community structure of a chromium water bath, a chromium drainage waste system, a chromium pretreatment tank, and a trivalent chromium precipitation tank from the Hellenic Aerospace Industry S.A. was assessed using 16S rRNA libraries and a high-density DNA microarray (PhyloChip). 16S rRNA libraries revealed a bacterial diversity consisting of 14 distinct operational taxonomic units belonging to five bacterial phyla: Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, and Bacteroidetes. However, employing a novel microarray-based approach (PhyloChip), a high bacterial diversity consisting of 30 different phyla was revealed, with representatives of 181 different families. This made it possible to identify a core set of genera present in all wastewater treatment stages examined, consisting of members of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, and Bacteroidetes. In the chromium pretreatment tank, where the concentration of Cr(VI) is high (2.3 mg/l), we identified the presence of Pseudomonadales, Actinomycetales, and Enterobacteriales in abundance. In the chromium precipitation tank, where the concentration of Cr(III) is high, the dominant bacteria consortia were replaced by members of Rhodocyclales and Chloroflexi. The bacterial community structure changed significantly with changes in the chromium concentration. This in-depth analysis should prove useful for the design and development of improved bioremediation strategies. PMID:22258276

Katsaveli, Katerina; Vayenas, Dimitris; Tsiamis, George; Bourtzis, Kostas

2012-03-01

375

Soil-Borne Bacterial Structure and Diversity Does Not Reflect Community Activity in Pampa Biome  

PubMed Central

The Pampa biome is considered one of the main hotspots of the world’s biodiversity and it is estimated that half of its original vegetation was removed and converted to agricultural land and tree plantations. Although an increasing amount of knowledge is being assembled regarding the response of soil bacterial communities to land use change, to the associated plant community and to soil properties, our understanding about how these interactions affect the microbial community from the Brazilian Pampa is still poor and incomplete. In this study, we hypothesized that the same soil type from the same geographic region but under distinct land use present dissimilar soil bacterial communities. To test this hypothesis, we assessed the soil bacterial communities from four land-uses within the same soil type by 454-pyrosequencing of 16S rRNA gene and by soil microbial activity analyzes. We found that the same soil type under different land uses harbor similar (but not equal) bacterial communities and the differences were controlled by many microbial taxa. No differences regarding diversity and richness between natural areas and areas under anthropogenic disturbance were detected. However, the measures of microbial activity did not converge with the 16S rRNA data supporting the idea that the coupling between functioning and composition of bacterial communities is not necessarily correlated. PMID:24146873

Lupatini, Manoeli; Suleiman, Afnan Khalil Ahmad; Jacques, Rodrigo Josemar Seminoti; Antoniolli, Zaida Inês; Kuramae, Eiko Eurya; de Oliveira Camargo, Flávio Anastácio; Roesch, Luiz Fernando Würdig

2013-01-01

376

Shift of Bacterial Community in Synanthropic Mite Tyrophagus putrescentiae Induced by Fusarium Fungal Diet  

PubMed Central

Background Tyrophagus putrescentiae (Acari: Astigmata) and Fusarium sp. co-occur in poorly managed grain. In a laboratory experiment, mite grazing resulted in significant reduction of fungal mycelium on cultivation plates. The destruction of mycelium appeared to be a result of an interaction between the mites, fungi and associated bacteria. Methodology and Principal Findings A laboratory experiment was performed to simulate a situation of grain multiinfested by mites and Fusarium fungi. Changes of mite-associated bacterial community in T. putrescentiae were described in 3 habitats: (i) T. putrescentiae mites from a rearing diet prior to their transfer to fungal diet; (ii) fungal mycelium before mite introduction; (iii) mites after 7 day diet of each Fusarium avenaceum, F. culmorum, F. poae and F. verticillioides. Bacterial communities were characterized by 16 S rRNA gene sequencing. In total, 157 nearly full-length 16 S rRNA gene sequences from 9 samples representing selected habitats were analyzed. In the mites, the shift from rearing to fungal diet caused changes in mite associated bacterial community. A diverse bacterial community was associated with mites feeding on F. avenaceum, while feeding on the other three Fusarium spp. led to selection of a community dominated by Bacillaceae. Conclusions/Significance The work demonstrated changes of bacterial community associated with T. putrescentiae after shift to fungal diets suggesting selection for Bacillaceae species known as chitinase producers, which might participate in the fungal mycelium hydrolysis. PMID:23119013

Hubert, Jan; Nesvorná, Marta; Ságová-Mare?ková, Markéta; Kopecký, Jan

2012-01-01

377

Leptospira spp. strain identification by MALDI TOF MS is an equivalent tool to 16S rRNA gene sequencing and multi locus sequence typing (MLST)  

PubMed Central

Background In this study mass spectrometry was used for evaluating extracted leptospiral protein samples and results were compared with molecular typing methods. For this, an extraction protocol for Leptospira spp. was independently established in two separate laboratories. Reference spectra were created with 28 leptospiral strains, including pathogenic, non-pathogenic and intermediate strains. This set of spectra was then evaluated on the basis of measurements with well-defined, cultured leptospiral strains and with 16 field isolates of veterinary or human origin. To verify discriminating peaks for the applied pathogenic strains, statistical analysis of the protein spectra was performed using the software tool ClinProTools. In addition, a dendrogram of the reference spectra was compared with phylogenetic trees of the 16S rRNA gene sequences and multi locus sequence typing (MLST) analysis. Results Defined and reproducible protein spectra using MALDI-TOF MS were obtained for all leptospiral strains. Evaluation of the newly-built reference spectra database allowed reproducible identification at the species level for the defined leptospiral strains and the field isolates. Statistical analysis of three pathogenic genomospecies revealed peak differences at the species level and for certain serovars analyzed in this study. Specific peak patterns were reproducibly detected for the serovars Tarassovi, Saxkoebing, Pomona, Copenhageni, Australis, Icterohaemorrhagiae and Grippotyphosa. Analysis of the dendrograms of the MLST data, the 16S rRNA sequencing, and the MALDI-TOF MS reference spectra showed comparable clustering. Conclusions MALDI-TOF MS analysis is a fast and reliable method for species identification, although Leptospira organisms need to be produced in a time-consuming culture process. All leptospiral strains were identified, at least at the species level, using our described extraction protocol. Statistical analysis of the three genomospecies L. borgpetersenii, L. interrogans and L. kirschneri revealed distinctive, reproducible differentiating peaks for seven leptospiral strains which represent seven serovars. Results obtained by MALDI-TOF MS were confirmed by MLST and 16S rRNA gene sequencing. PMID:22925589

2012-01-01

378

Bacterial diversity at different stages of the composting process  

PubMed Central

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

2010-01-01

379

An Archaea 5S rRNA analog is stably expressed in Escherichia coli  

NASA Technical Reports Server (NTRS)

Mini-genes for 5S-like rRNA were constructed. These genes had a sequence which largely resembles that of the naturally occurring 5S rRNA of a bacterium, Halococcus morrhuae, which phylogenetically belongs to the Archaea. Plasmids carrying the mini-genes were transformed into Escherichia coli (Ec). Ribosomal incorporation was not a prerequisite for stable accumulation of the RNA product. However, only those constructs with a well-base-paired helix I accumulated RNA product. This result strongly implies that this aspect of the structure is likely to be an important condition for stabilizing 5S rRNA-like products. The results are consistent with our current understanding of 5S rRNA processing in Ec. When used in conjunction with rRNA probe technology, the resulting chimeric RNA may be useful as a monitoring tool for genetically engineered microorganisms or naturally occurring organisms that are released into the environment.

Yang, Y.; Fox, G. E.

1996-01-01

380

Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.  

PubMed

There is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (? 1 positive sample for strict pathogens and ? 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis. PMID:25056331

Bémer, Pascale; Plouzeau, Chloé; Tande, Didier; Léger, Julie; Giraudeau, Bruno; Valentin, Anne Sophie; Jolivet-Gougeon, Anne; Vincent, Pascal; Corvec, Stéphane; Gibaud, Sophie; Juvin, Marie Emmanuelle; Héry-Arnaud, Genevieve; Lemarié, Carole; Kempf, Marie; Bret, Laurent; Quentin, Roland; Coffre, Carine; de Pinieux, Gonzague; Bernard, Louis; Burucoa, Christophe

2014-10-01

381

Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance  

SciTech Connect

Soils are immensely diverse microbial habitats with thousands of co-existing bacterial, archaeal, and fungal species. Across broad spatial scales, factors such as pH and soil moisture appear to determine the diversity and structure of soil bacterial communities. Within any one site however, bacterial taxon diversity is high and factors maintaining this diversity are poorly resolved. Candidate factors include organic substrate availability and chemical recalcitrance, and given that they appear to structure bacterial communities at the phylum level, we examine whether these factors might structure bacterial communities at finer levels of taxonomic resolution. Analyzing 16S rRNA gene composition of nucleotide analog-labeled DNA by PhyloChip microarrays, w