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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

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 × 10(5) 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

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

Microsoft Academic Search

Among many reports investigating microbial diversity from environmental samples with denaturing gradient gel electrophoresis\\u000a (DGGE), limited attention has been given to the effects of universal primers and DNA extraction on the outcome of DGGE analysis.\\u000a In this study, these effects were tested with 16S rRNA gene-based DGGE on a bacterial community from farming water samples.\\u000a The results indicate that the

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

2007-01-01

3

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

NASA Astrophysics Data System (ADS)

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

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

2007-07-01

4

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

5

Quantifying bacterial population dynamics in compost using 16S rRNA gene probes  

Microsoft Academic Search

Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time. This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors. Bacterial 16S rRNA gene targets accounted for only 37% of

Patrick D. Schloss; Anthony G. Hay; David B. Wilson; James M. Gossett; Larry P. Walker

2005-01-01

6

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 ...

7

Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses  

Microsoft Academic Search

Bacterial diversity associated with Baer Soda Lake in Inner Mongolia of China was investigated using a culture-independent method. Bacterial 16S rRNA gene libraries were generated using bacterial oligonucleotide primers, and 16S rRNA gene sequences of 58 clones were analyzed phylogenetically. The library was dominated by 16S rDNAs of Gram-negative bacteria (24% a-Proteobacteria, 31% ß-Proteobacteria, 33% ?-Proteobacteria, and 2% d-Proteobacteria), with

Yanhe Ma; Weizhou Zhang; Yanfen Xue; Peijin Zhou; Antonio Ventosa; William D. Grant

2004-01-01

8

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

EPA Science Inventory

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

9

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

EPA Science Inventory

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

10

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

Microsoft Academic Search

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

Randy P. Revetta; Robin S. Matlib

2011-01-01

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

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

2015-04-01

13

Complete Bacterial Proteome Extraction Kit  

E-print Network

....................................................................................7 7. Extraction of proteins from gram-negative bacteria.................................................................8 8. Extraction of proteins from gram-positive bacteria by centrifugation and sample preparation with was performed. 200 µg proteins of the whole cell extract were

Lebendiker, Mario

14

Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes  

Microsoft Academic Search

rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose

Jeremy A. Frank; Claudia I. Reich; Shobha Sharma; Jon S. Weisbaum; Brenda A. Wilson; Gary J. Olsen

2008-01-01

15

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

16

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

PubMed Central

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

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

2004-01-01

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

PCR-DGGE detection of the bacterial community structure in the Inner Mongolia steppe with two different DNA extraction methods  

Microsoft Academic Search

Compared with conventional methods, molecular biological technique, such as PCR-DGGE (denaturing gradient gel electrophoresis), is informative in examining the structure of the soil bacterial community through the extraction of microbial DNA from soil and generation of bacterial community profiles by PCR amplification of 16S rRNA genes. Extraction efficiency of soil microbial DNA is the most important step in these methods.

Zhou Xiaoqi; Wang Yanfen; Cai Ying; Huang Xiangzhong; Hao Yanbin; Tian Jianqing; Chai Tuanyao

2007-01-01

19

Taxonomic Classification of Bacterial 16S rRNA Genes Using Short Sequencing Reads: Evaluation of Effective Study Designs  

PubMed Central

Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ?8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution. PMID:23308262

Mizrahi-Man, Orna; Davenport, Emily R.; Gilad, Yoav

2013-01-01

20

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA  

Microsoft Academic Search

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully comple- mentary RNAs and selectivity against single nucle- otide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences.

Adam P. Silverman; Eric T. Kool

2005-01-01

21

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

PubMed

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

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

2015-03-01

22

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

23

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

24

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

PubMed

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

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

2015-03-01

25

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

Microsoft Academic Search

Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water. To identify members of the domain Bacteria which inhabit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow Bay, located on the Savannah River Site, near Aiken, S.C.

MARK G. WISE; L. J. Shimkets; J. V. McArthur

1997-01-01

26

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

PubMed Central

Objective: To identify the prevalence of acidogenic and nonacidogenic bacteria in patients with polycaries lesions, and to ascertain caries specific bacterial prevalence in relation to noncaries controls. Materials and Methods: Total genomic DNA extracted from saliva of three adults and four children from the same family were subjected to 16S rRNA gene sequencing analysis on a next generation sequencer, the PGS-Ion Torrent. Those bacterial genera with read counts > 1000 were considered as significant in each of the subject and used to associate the occurrence with caries. Results and Conclusion: Sequencing analysis indicated a higher prevalence of Streptococcus, Rothia, Granulicatella, Gemella, Actinomyces, Selenomonas, Haemophilus and Veillonella in the caries group relative to controls. While higher prevalence of Streptococcus, Rothia and Granulicatella were observed in all caries samples, the prevalence of others was observable in 29–57% of samples. Interestingly, Rothia and Selenomonas, which are known to occur within anaerobic environments of dentinal caries and subgingival plaque biofilms, were seen in the saliva of these caries patients. Taken together, the study has identified for the first time a unique co-prevalence pattern of bacteria in caries patients that may be explored as distinct caries specific bacterial signature to predict cariogenesis in high-risk primary and mixed dentition age groups. PMID:25713496

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

2015-01-01

27

Influence of Particle Size on Bacterial Community Structure in Aquatic Sediments as Revealed by 16S rRNA Gene Sequence Analysis  

Microsoft Academic Search

Bacterial communities associated with sediment particles were examined using PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing. Particle size influenced community structure, with attached bacterial assemblages separating into 63- to 125-, 125- to 1,000-, and 1,000- to 2,000-m fractions. Differences were particularly pronounced for the Verrucomicrobia-Planctomycetes, whose numbers were significantly re- duced on coarser particles. Bacterial communities associated with

Colin R. Jackson

2008-01-01

28

Adhesion molecules in antibacterial defenses: effects of bacterial extracts.  

PubMed

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

Marchant, A; Duchow, J; Goldman, M

1992-01-01

29

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

PubMed

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

30

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

31

Evaluation of 16SpathDB 2.0, an automated 16S rRNA gene sequence database, using 689 complete bacterial genomes.  

PubMed

Interpretation of 16S rRNA sequences is a difficult problem faced by clinical microbiologists and technicians. In this study, we evaluated the updated 16SpathDB 2.0 database, using 689 16S rRNA sequences from 689 complete genomes of medically important bacteria. Among these 689 16S rRNA sequences, none was wrongly identified, with 35.8% reported as a single bacterial species having >98% identity with the query sequence (category 1), 63.9% reported as more than 1 bacterial species having >98% identity with the query sequence (category 2), 0.3% reported to the genus level (category 3), and none reported as no match (category 4). For the 16S rRNA sequences of non-duplicated bacterial species reported as category 1 or 2, the percentage of bacterial species reported as category 1 was significantly higher for anaerobic Gram-positive/Gram-negative bacteria than aerobic/facultative anaerobic Gram-positive/Gram-negative bacteria. 16SpathDB 2.0 is a user-friendly and accurate database for 16S rRNA sequence interpretation in clinical laboratories. PMID:24295571

Teng, Jade L L; Ho, Tom C C; Yeung, Ronald S Y; Wong, Annette Y P; Wang, Haiyin; Chen, Chen; Fung, Kitty S C; Lau, Susanna K P; Woo, Patrick C Y

2014-02-01

32

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...

33

Bacterial communities in two Antarctic ice cores analyzed by 16S rRNA gene sequencing analysis  

NASA Astrophysics Data System (ADS)

Antarctic ice cores could preserve ancient airborne microorganisms. We examined bacteria in two Antarctic ice core samples, an interglacial age sample from Mizuho Base and a glacial age sample from the Yamato Mountains, by 16S rRNA gene sequencing analysis. Bacterial density, the number of bacterial OTUs and Simpson’s diversity index was larger in the Mizuho sample than in the Yamato sample. The 16S rDNA clone library from the Mizuho sample was dominated by the phylum Firmicutes, while the large part of that from the Yamato sample was composed of the Gamma proteobacteria group. Major sources of these identified bacteria estimated from their database records also differed between the samples: in the Mizuho sample bacterial species recorded from animals were higher than that of the Yamato sample, while in the Yamato sample bacteria from aquatic and snow-ice environments were higher than that of the Mizuho sample. The results suggest that these bacteria were past airborne bacteria that would vary in density, diversity and species composition depending on global environmental change. Our results imply that bacteria in Antarctic ice cores could be used as new environmental markers for past environmental studies.

Segawa, Takahiro; Ushida, Kazunari; Narita, Hideki; Kanda, Hiroshi; Kohshima, Shiro

2010-08-01

34

Extraction of Bacterial RNA from Soil: Challenges and Solutions  

PubMed Central

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

Wang, Yong; Hayatsu, Masahito; Fujii, Takeshi

2012-01-01

35

Detection of bacterial 16S rRNA using a molecular beacon-based X sensor.  

PubMed

We demonstrate how a long structurally constrained RNA can be analyzed in homogeneous solution at ambient temperatures with high specificity using a sophisticated biosensor. The sensor consists of a molecular beacon probe as a signal reporter and two DNA adaptor strands, which have fragments complementary to the reporter and to the analyzed RNA. One adaptor strand uses its long RNA-binding arm to unwind the RNA secondary structure. Second adaptor strand with a short RNA-binding arm hybridizes only to a completely complementary site, thus providing high recognition specificity. Overall the three-component sensor and the target RNA form a four-stranded DNA crossover (X) structure. Using this sensor, Escherichia coli16S rRNA was detected in real time with the detection limit of ~0.17 nM. The high specificity of the analysis was proven by differentiating Bacillus subtilis from E. coli 16S rRNA sequences. The sensor responds to the presence of the analyte within seconds. PMID:23021850

Gerasimova, Yulia V; Kolpashchikov, Dmitry M

2013-03-15

36

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

PubMed Central

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

2012-01-01

37

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

PubMed Central

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

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

1997-01-01

38

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

PubMed

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

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

1997-07-01

39

Use of 16S rRNA Gene Terminal Restriction Fragment Analysis To Assess the Impact of Solids Retention Time on the Bacterial Diversity of Activated Sludge  

Microsoft Academic Search

Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to investigate the reproducibility and stability in the bacterial community structure of laboratory-scale sequenc- ing batch bioreactors (SBR) and to assess the impact of solids retention time (SRT) on bacterial diversity. Two experiments were performed. In each experiment two sets of replicate SBRs were operated for a

Pascal E. Saikaly; Peter G. Stroot; Daniel B. Oerther

2005-01-01

40

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

Microsoft Academic Search

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

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

1997-01-01

41

Bacterial and Archaeal 16S rRNA Genes in Late Pleistocene to Holocene Muddy Sediments from the Kanto Plain of Japan  

Microsoft Academic Search

Microbial communities in ancient marine sediments composed of clay and silt obtained from the terrestrial subsurface were phylogenetically analyzed based on their 16S rRNA gene sequences. Chloroflexi and Miscellaneous Crenarchaeotic Group were predominant in bacterial and archaeal clone libraries, respectively. Of 44 operational taxonomic units (OTUs) that had close relatives in the database, 30 were close to sequences obtained from

Mio Takeuchi; Takeshi Komai; Satoshi Hanada; Hideyuki Tamaki; Susumu Tanabe; Yoshinori Miyachi; Mieko Uchiyama; Tsutomu Nakazawa; Katsumi Kimura; Yoichi Kamagata

2009-01-01

42

Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities  

PubMed Central

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes. PMID:11133445

Dunbar, John; Ticknor, Lawrence O.; Kuske, Cheryl R.

2001-01-01

43

Adhesion Molecules in Antibacterial Defenses: Effects of Bacterial Extracts  

Microsoft Academic Search

Adhesion of polymorphonuclear leukocytes (PMN) to vascular endothelium is one of the first events in their response against local bacterial infection. Different adhesion molecules sequentially mediate PMN adherence to endothelium and extravasation into inflamed tissues. We show that bacterial extracts OM-85 BV and OM-89 increase the expression of adhesion molecules at the surface of PMN and we suggest that this

A. Marchant; J. Duchow; M. Goldman

1992-01-01

44

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

PubMed Central

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

2013-01-01

45

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

46

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

PubMed Central

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

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

1992-01-01

47

16S rRNA PCR followed by restriction endonuclease digestion: a rapid approach for genus level identification of important enteric bacterial pathogens.  

PubMed

The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated. PMID:24140577

Vergis, J; Negi, M; Poharkar, K; Das, D P; Malik, S V S; Kumar, A; Doijad, S P; Barbuddhe, S B; Rawool, D B

2013-12-01

48

Use of quantitative 16S rRNA PCR to determine bacterial load does not augment conventional cerebrospinal fluid (CSF) cultures among children undergoing treatment for CSF shunt infection.  

PubMed

The aim of this study was to develop a quantitative 16S rRNA assay for determination of bacterial nucleic acid load in cerebrospinal fluid (CSF) shunt infection and to compare quantitative 16S rRNA polymerase chain reaction (PCR) findings to those of conventional bacterial culture in patients treated for CSF shunt infection. We developed a quantitative 16S rRNA PCR assay that detected bacterial load across a range of 2.5 × 10(9) down to 2.5 × 10(4) 16S copies/mL CSF under experimental conditions for numerous Gram-positive and Gram-negative organisms. However, when applied to archived CSF samples from 25 shunt infection episodes, correlations between positive bacterial culture and 16S rRNA levels were seen in only half of infections, and 16S rRNA levels dropped precipitously after an initial peak on the first day of sample collection. Bacterial load measured using 16S rRNA PCR does not provide sufficient information beyond bacterial culture to inform CSF shunt infection treatment. PMID:23953744

Simon, Tamara D; Van Yserloo, Brian; Nelson, Kevin; Gillespie, David; Jensen, Randy; McAllister, James P; Riva-Cambrin, Jay; Stockmann, Chris; Daly, Judy A; Blaschke, Anne J

2014-02-01

49

Use of quantitative 16S rRNA PCR to determine bacterial load does not augment conventional cerebrospinal fluid (CSF) cultures among children undergoing treatment for CSF shunt infection?,??  

PubMed Central

The aim of this study was to develop a quantitative 16S rRNA assay for determination of bacterial nucleic acid load in cerebrospinal fluid (CSF) shunt infection and to compare quantitative 16S rRNA polymerase chain reaction (PCR) findings to those of conventional bacterial culture in patients treated for CSF shunt infection. We developed a quantitative 16S rRNA PCR assay that detected bacterial load across a range of 2.5 × 109 down to 2.5 × 104 16S copies/mL CSF under experimental conditions for numerous Gram-positive and Gram-negative organisms. However, when applied to archived CSF samples from 25 shunt infection episodes, correlations between positive bacterial culture and 16S rRNA levels were seen in only half of infections, and 16S rRNA levels dropped precipitously after an initial peak on the first day of sample collection. Bacterial load measured using 16S rRNA PCR does not provide sufficient information beyond bacterial culture to inform CSF shunt infection treatment. PMID:23953744

Simon, Tamara D.; Van Yserloo, Brian; Nelson, Kevin; Gillespie, David; Jensen, Randy; McAllister, James P.; Riva-Cambrin, Jay; Stockmann, Chris; Daly, Judy A.; Blaschke, Anne J.

2013-01-01

50

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

PubMed Central

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

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

2001-01-01

51

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

PubMed Central

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

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

2004-01-01

52

Lactic acid bacterial extract as a biogenic mineral growth modifier  

NASA Astrophysics Data System (ADS)

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

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

2009-04-01

53

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

PubMed

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

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

2015-05-01

54

Comprehensive phylogenetic analysis of evolutionarily conserved rRNA adenine dimethyltransferase suggests diverse bacterial contributions to the nucleus-encoded plastid proteome.  

PubMed

The KsgA/Dim1 protein family of rRNA adenine dimethyltransferase (rAD) is well conserved throughout evolution. This protein family has been recognized to play multiple additional roles: as a mitochondrial transcription factor (mtTFB); as a yeast pre-rRNA cleavage enzyme (Dim1p); and as a chloroplast developmental protein (PFC1). Comprehensive phylogenetic analysis of rAD led to three main findings. First, rAD sequences were grouped by three domains of life, bacteria, archaea, and eukaryotes. Second, mtTFB shows alpha-proteobacterial endosymbiotic ancestry. Third, plastid-targeted rADs do not show cyanobacterial affiliation. Instead, plastid-targeted homologs from chlorophytes/land plants were clustered with chlamydiae, while those from rhodophytes/red algal lineage grouped with the bacteroides/chlorobi group. These unusual two-bacterial ancestries of plastid-targeted rAD suggest that bacterial genes influenced the evolution of the proteome of eukaryotic plastids in a complex way, involving more diverse bacterial groups than previously believed, and underscoring the importance of eukaryotic acquisition of bacterial functions. PMID:19017544

Park, Ae Kyung; Kim, Ho; Jin, Hyung Jong

2009-02-01

55

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

PubMed Central

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

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

2008-01-01

56

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

PubMed

The bacterial population of a graywater treatment system was monitored over the course of 100 days, along with several wastewater biochemical parameters. The graywater treatment system employed an 1,800-liter membrane bioreactor (MBR) to process the waste, with essentially 100% recycling of the biomass. Graywater feed consisting of 10% galley water and 90% laundry water, selected to approximate the graywater composition on board U.S. Navy ships, was collected offsite. Five-day biological oxygen demand (BOD(5)), oils and greases (O/G), nitrogen, and phosphorus were monitored in the feed and were found to vary greatly day to day. Changes in the bacterial population were monitored by PCR amplification of region 332 to 518 (Escherichia coli numbering) of the 16S rRNA gene and denaturing gradient gel electrophoresis (DGGE) analysis of the resultant PCR products. DGGE analysis indicated a diverse and unstable bacterial population throughout the 100-day period, with spikes in feed strength causing significant changes in community structure. Long-term similarity between the communities was 0 to 25%, depending on the method of analysis. In spite of the unstable bacterial population, the MBR system was able to meet effluent quality parameters approximately 90% of the time. PMID:12571004

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

2003-02-01

57

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

Microsoft Academic Search

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

Myungjin Kim; Jongsik Chun

2005-01-01

58

Impact of training sets on classification of high-throughput bacterial 16s rRNA gene surveys  

Microsoft Academic Search

Taxonomic classification of the thousands–millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of

Jeffrey J Werner; Omry Koren; Philip Hugenholtz; Todd Z DeSantis; William A Walters; J Gregory Caporaso; Largus T Angenent; Rob Knight; Ruth E Ley

2012-01-01

59

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

Microsoft Academic Search

The aim of this study was to describe the microbial communities in the distal gut of wild wolves (Canis lupus). Fecal samples were collected from three healthy unrelated adult wolves captured at the nearby of Dalai Lake Nature Reserve\\u000a in Inner Mongolia of China. The diversity of fecal bacteria was investigated by constructing PCR-amplified 16S rRNA gene clone\\u000a libraries using

Honghai Zhang; Lei Chen

2010-01-01

60

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

61

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

62

Bacterial community composition in Central European running waters examined by temperature gradient gel electrophoresis and sequence analysis of 16S rRNA genes.  

PubMed

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

63

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

PubMed Central

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 of material dislodged from retrieved prostheses after ultrasonication (sonicate) was 22%. Bacteria were observed by immunofluorescence microscopy in 63% of sonicate samples with a monoclonal antibody specific for Propionibacterium acnes and polyclonal antiserum specific for Staphylococcus spp. The bacteria were present either as single cells or in aggregates of up to 300 bacterial cells. These aggregates were not observed without sonication to dislodge the biofilm. Bacteria were observed in all of the culture-positive samples, and in some cases in which only one type of bacterium was identified by culture, both coccoid and coryneform bacteria were observed by immunofluorescence microscopy. Bacteria from skin-flake contamination were readily distinguishable from infecting bacteria by immunofluorescence microscopy. Examination of skin scrapings did not reveal large aggregates of bacteria but did reveal skin cells. These were not observed in the sonicates. Bacterial DNA was detected in 72% of sonicate samples by PCR amplification of a region of the bacterial 16S rRNA gene with universal primers. All of the culture-positive samples were also positive for bacterial DNA. Evidence of high-level infiltration either of neutrophils or of lymphocytes or macrophages into associated tissue was observed in 73% of patients. Our results indicate that the incidence of prosthetic joint infection is grossly underestimated by current culture detection methods. It is therefore imperative that current clinical practice with regard to the detection and subsequent treatment of prosthetic joint infection be reassessed in the light of these results. PMID:10488193

Tunney, Michael M.; Patrick, Sheila; Curran, Martin D.; Ramage, Gordon; Hanna, Donna; Nixon, James R.; Gorman, Sean P.; Davis, Richard I.; Anderson, Neil

1999-01-01

64

Profiling of biodegradation and bacterial 16S rRNA genes in diverse contaminated ecosystems using 60-mer oligonucleotide microarray  

Microsoft Academic Search

We have developed an oligonucleotide microarray for the detection of biodegradative genes and bacterial diversity and tested\\u000a it in five contaminated ecosystems. The array has 60-mer oligonucleotide probes comprising 14,327 unique probes derived from\\u000a 1,057 biodegradative genes and 880 probes representing 110 phylogenetic genes from diverse bacterial communities, and we named\\u000a it as BiodegPhyloChip. The biodegradative genes are involved in

Ashutosh Pathak; Rishi Shanker; Satyendra Kumar Garg; Natesan Manickam

2011-01-01

65

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

66

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

67

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

68

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  

Microsoft Academic Search

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

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

2009-01-01

69

Bacterial diversity of soils assessed by DGGE, T-RFLP and SSCP fingerprints of PCR-amplified 16S rRNA gene fragments: Do the different methods provide similar results?  

Microsoft Academic Search

Bacterial communities of four arable soils – pelosol, gley, para brown soil, and podsol brown soil – were analysed by fingerprinting of 16S rRNA gene fragments amplified from total DNA of four replicate samples for each soil type. Fingerprints were generated in parallel by denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism

Kornelia Smalla; Miruna Oros-Sichler; Annett Milling; Holger Heuer; Susanne Baumgarte; Regina Becker; Gabriele Neuber; Siegfried Kropf; Andreas Ulrich; Christoph C. Tebbe

2007-01-01

70

International Journal of Pharma and Bio Sciences V1(2)2010 IDENTIFICATION OF MYCOBACTERIUM Sp. AS AN ALFALFA ROOT BACTERIAL ENDOPHYTES USING 16S rRNA GENE SEQUENCE ANALYSIS  

E-print Network

sp. is introduced as bacterial endophyte of alfalfa following investigation of alfalfa wilt disease. We isolated microorganisms from the vascular tissues of surface sterilized roots and identified them as Mycobacterium sp. using 16S rRNA gene full and partial sequence analysis.

Ali Deljou; Mirkave Mousaviehzade; Abolghasem Ghasemi; Heshmatollah Rahimian

71

Dual priming oligonucleotides for broad-range amplification of the bacterial 16S rRNA gene directly from human clinical specimens.  

PubMed

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens. PMID:22278843

Kommedal, Øyvind; Simmon, Keith; Karaca, Dilek; Langeland, Nina; Wiker, Harald G

2012-04-01

72

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

73

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

PubMed Central

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

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

2014-01-01

74

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

PubMed Central

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

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

2015-01-01

75

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

PubMed

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

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

2015-01-01

76

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

77

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

SciTech Connect

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

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

2009-09-01

78

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

79

Synthesis of bacterial cellulose using hot water extracted wood sugars.  

PubMed

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

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

2015-06-25

80

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

81

Bacterial diversity in the rumen of Gayals (Bos frontalis), Swamp buffaloes (Bubalus bubalis) and Holstein cow as revealed by cloned 16S rRNA gene sequences.  

PubMed

Libraries of rumen bacterial 16S rRNA gene sequences of Gayals (Bos frontalis) and Swamp buffaloes (Bubalus bubalis) were cloned and sequenced in the present work to compare the bacterial diversity with the third published library of Holstein cow. Sequence similarity of 97% was used as the definition of operational taxonomic unit (OTU). The majority of the 470 sequences retrieved fell into the phyla of low G + C subdivision (329 sequences) and Cytophaga-Flexibacter-Bacteroides (CFB, 123 sequences) with the percentages of 70 and 26.2, respectively. The remaining clones belonged to the phyla of Proteobacter, high G + C gram positive bacteria (HGCGPB) and Spirochaetes, accounting for 3.8% totally. Only 73 clones (25 OTUs, 15.5%) could be closely related to cultured representatives. However, a larger fraction was related to uncultured representatives. Holstein cow may have more representatives of cultural bacteria and there were more uncultured clones for Gayals. The percentage of cultural representatives was 24, 13.3 and 9.5 for Holstein cow, Swamp buffaloes and Gayals, respectively. Twenty-three OTUs of the 236 ones appeared in more than one library, five of which were cultural. Selenomonas ruminantium, Ruminococcus flavefaciens and Butyrivibrio fibrisolvens were found in two different libraries, while Succiniclasticum ruminis and Pseudobutyrivibrio ruminis were found in all three libraries. Some of the animal-specific bacteria that had not been described previously in the ruminal ecosystem, e.g. Allisonella histaminiformans for Gayals and Staphylococcus sciuri for Swamp buffaloes were also recovered. PMID:19662514

Yang, Shuli; Ma, Songcheng; Chen, Jing; Mao, Huaming; He, Yiduo; Xi, Dongmei; Yang, Liangyu; He, Tianbao; Deng, Weidong

2010-04-01

82

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

83

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

PubMed

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

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

2014-01-01

84

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

85

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

PubMed

In this study, the composition and diversity of the bacterial community in thermophilic H2-producing reactors fed with glucose were investigated using pyrosequencing. The H2-producing experiments in batch were conducted using 0.5 and 2.0gl(-1) glucose at 550 °C. Under the two conditions, the H2 production and yield were 1.3 and 1.6mol H2mol glucose(-1), respectively. Acetic, butyric, iso-butyric, lactic and propionic acids were detected in the two reactors. The increase in substrate concentration favored a high H2 yield. In this reactor, a predominance of acetic and iso-butyric acids, 27.7% and 40%, were measured, respectively. By means of pyrosequencing, a total of 323 and 247 operational taxonomic units were obtained, with a predominance of the phylum Firmicutes (68.73-67.61%) for reactors with 0.5 and 2.0gl(-1) glucose, respectively. Approximately 40.55% and 62.34% of sequences were affiliated with Thermoanaerobacterium and Thermohydrogenium, microorganisms that produce H2 under thermophilic conditions. PMID:25801966

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

2015-04-01

86

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

PubMed Central

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

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

2012-01-01

87

Sequencing-Independent Method To Generate Oligonucleotide Probes Targeting a Variable Region in Bacterial 16S rRNA by PCR with Detachable Primers  

Microsoft Academic Search

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer

Stefan Bertilsson; Colleen M. Cavanaugh; Martin F. Polz

2002-01-01

88

Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods  

Microsoft Academic Search

The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct\\u000a DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and\\u000a mutually compared. The DNA quantity and purity were determined using real-time PCR targeting the bacterial 16S rDNA gene.\\u000a Microbial community fingerprints were assessed by automated ribosomal

Louise Vanysacker; Steven A. J. Declerck; Bart Hellemans; Luc De Meester; Ivo Vankelecom; Priscilla Declerck

2010-01-01

89

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

Microsoft Academic Search

The long-term effects of methylparathion contamination on the diversity of soil microbial community was investigated by a culture-independent approach using small subunit ribosomal RNA (SSU rRNA) gene-based cloning. Microbial DNA extracted from both the control soil sample and methylparathion contaminated soil sample was subjected to PCR amplification with primers specific for bacterial 16S rRNA gene sequences. From the PCR amplification

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

2006-01-01

90

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...

91

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...

92

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

93

Impact of DNA extraction method on bacterial community composition measured by denaturing gradient gel electrophoresis  

Microsoft Academic Search

The impact of DNA extraction protocol on soil DNA yield and bacterial community composition was evaluated. Three different procedures to physically disrupt cells were compared: sonication, grinding–freezing–thawing, and bead beating. The three protocols were applied to three different topsoils. For all soils, we found that each DNA extraction method resulted in unique community patterns as measured by denaturing gradient gel

Julia R. de Lipthay; Christiane Enzinger; Kaare Johnsen; Jens Aamand; Søren J. Sørensen

2004-01-01

94

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

PubMed Central

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

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

2012-01-01

95

INFLUENCE OF A BACTERIAL CELL EXTRACT UPON THE MORPHOGENESIS OF ARTHROBACTER UREAFACIENS  

PubMed Central

Blankenship, L. C. (University of Maryland, College Park) and R. N. Doetsch. Influence of a bacterial cell extract upon the morphogenesis of Arthrobacter ureafaciens. J. Bacteriol. 82:882–888. 1961—The effect of a bacterial extract on alleviating the abnormal morphological appearance of Arthrobacter ureafaciens when cultivated in Thiotone broth is described. Physical and chemical analyses of this extract revealed that the mineral components therein were largely responsible for the observed effects. Similar results were obtained using ashed Trypticase soy broth, known mineral mixtures, or magnesium ions. The role played by these materials in governing the morphology of A. ureafaciens is discussed. Images PMID:13869842

Blankenship, L. C.; Doetsch, R. N.

1961-01-01

96

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

PubMed

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

Witek, Marta A; Conn, Graeme L

2014-09-01

97

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

98

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

99

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

100

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

101

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

102

Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes.  

PubMed Central

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of chimeric molecule formation, mixed genomic DNAs from eight actinomycete species whose 16S rRNA sequences had been determined were used for PCR coamplification of 16S rRNA genes. A large number of cloned 16S ribosomal DNAs were examined by sequence analysis, and chimeric molecules were identified by multiple-sequence alignment with reference species. Here, we report that the level of occurrence of chimeric sequences after 30 cycles of PCR amplification was 32%. We also show that PCR-induced chimeras were formed between different rRNA gene copies from the same organism. Because of the wide use of PCR for direct isolation of 16S rRNA sequences from environmental DNA to assess microbial diversity, the extent of chimeric molecule formation deserves serious attention. PMID:9406382

Wang, G C; Wang, Y

1997-01-01

103

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

PubMed Central

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

Rogina, Petra; Kofol, Romina; Kaasch, Achim

2014-01-01

104

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

105

Frequency of Formation of Chimeric Molecules as a Consequence of PCR Coamplification of 16S rRNA Genes from Mixed Bacterial Genomes  

Microsoft Academic Search

PCR is routinely used in amplification and cloning of rRNA genes from environmental DNA samples for studies of microbial community structure and identification of novel organisms. There have been concerns about generation of chimeric sequences as a consequence of PCR coamplification of highly conserved genes, because such sequences may lead to reports of nonexistent organisms. To quantify the frequency of

GRACE C.-Y. WANG; YUE WANG

1997-01-01

106

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

107

Antibacterial activity of certain plant extracts against bacterial wilt of tomato  

Microsoft Academic Search

Five isolates of Ralstonia solanacearum were isolated from a naturally wilted root of tomato plants grown in Assiut governorate. The antibacterial activity of extract of Datura, Garlic and Nerium were tested in controlling R. solanacearum in vitro and in vivo. Garlic exhibited the strongest antibacterial activity against bacterial wilt in vitro and in vivo followed by Datura and then Nerium.

K. A. M. Abo-Elyousr; M. R. Asran

2009-01-01

108

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...

109

Diverse and Novel Oral Bacterial Species in Blood following Dental Procedures?  

PubMed Central

We identified oral bacterial species in blood cultures following single-tooth extraction and tooth brushing. Sequence analysis of 16S rRNA genes identified 98 different bacterial species recovered from 151 bacteremic subjects. Of interest, 48 of the isolates represented 19 novel species of Prevotella, Fusobacterium, Streptococcus, Actinomyces, Capnocytophaga, Selenomonas, and Veillonella. PMID:18434561

Bahrani-Mougeot, Farah K.; Paster, Bruce J.; Coleman, Shirley; Ashar, Jignya; Barbuto, Sara; Lockhart, Peter B.

2008-01-01

110

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

Microsoft Academic Search

BACKGROUND: The pathogenesis of bacterial vaginosis remains largely elusive, although some microorganisms, including Gardnerella vaginalis, are suspected of playing a role in the etiology of this disorder. Recently culture-independent analysis of microbial ecosystems has proven its efficacy in characterizing the diversity of bacterial populations. Here, we report on the results obtained by combining culture and PCR-based methods to characterize the

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

2004-01-01

111

Effect of DNA extraction and sample preservation method on rumen bacterial population.  

PubMed

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. PMID:24125910

Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kope?ný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

2014-10-01

112

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

PubMed Central

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

Dang, Hongyue; Lovell, Charles R.

2000-01-01

113

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

114

Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts  

Microsoft Academic Search

Direct and sensitive PCR detection of contaminant microflora in cocoa extracts is affected by the quality of the template\\u000a DNA. This study compares the efficacy of five different commercial DNA extraction methods, selective enrichment broths and\\u000a use of glycolitic enzymes to obtain quality DNA for PCR detection of both fungi and bacteria in artificially inoculated cocoa\\u000a extract samples. PCR-based methods

Marta Tortajada; Pedro Vicente Martínez-Culebras; Verónica Navarro; Honorato Monzó; Daniel Ramón

2009-01-01

115

Automated purification and suspension array detection of 16S rRNA from soil and sediment extracts by using tunable surface microparticles.  

PubMed

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 ( approximately 10(4) cell equivalents) in 15-min test tube hybridizations and 10 ng of total RNA ( approximately 10(5) cell equivalents) in hybridizations with the automated system (30-s contact time). RNA fragmentation was essential for achieving tunable surface suspension array specificity. Chaperone probes reduced but did not completely eliminate cross-hybridization, even with probes sharing <50% identity to target sequences. Nonpurified environmental extracts did not irreparably affect our ability to classify color-coded microparticles, but residual environmental constituents significantly quenched the Alexa-532 reporter fluor. Modulating surface charge did not influence the interaction of soluble environmental contaminants with conjugated beads. The automated system greatly reduced the effects of fluorescence quenching, especially in the soil background. The automated system was as efficacious as manual methods for simultaneous sample purification, hybridization, and washing prior to flow cytometry detection. The implications of unexpected target cross-hybridization and fluorescence quenching are discussed relative to the design and implementation of an integrated microbial monitoring system. PMID:15128511

Chandler, Darrell P; Jarrell, Ann E

2004-05-01

116

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

117

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

118

Optimising Bacterial DNA Extraction from Faecal Samples: Comparison of Three Methods  

PubMed Central

Culture independent methods are used widely in diagnostic laboratories for infectious disease Isolation of genomic DNA from clinical samples is the first and important step in the procedure. Several procedures for extracting DNA from faecal samples have been described, including different mechanical cell disruptors. To our knowledge, the use of TissueLyser as a mechanical disruptor on faecal samples before DNA extraction has not been previously described. The purpose of the study was to implement a method for preparing faecal samples for optimal DNA extraction. Thus, three different procedures for extracting DNA from human faeces were compared. This was done either by using the mechanical disrupter by Mini BeadBeater 8, or the TissueLyser both followed by DNA purification using QIAamp DNA stool MiniKit, in comparison with DNA extractions using QIAamp DNA stool MiniKit without any prior mechanical disruption, according to manufacturer’s instructions. The obtained DNA from the three procedures was analysed by DGGE, and the number of bands was compared between each procedure. There was no significant difference between the numbers of bacterial bands obtained from DGGE when using a TissueLyser or Mini BeadBeater 8, so the two different mechanical cell disruptors can be used comparably when isolating bacterial DNA from faecal samples. The QIAamp DNA stool MiniKit alone resulted in a reduced number of bands compared to the two mechanical disruption methods. PMID:21643498

Smith, Birgitte; Li, Nan; Andersen, Anders Schou; Slotved, Hans Christian; Krogfelt, Karen Angeliki

2011-01-01

119

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

120

Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies  

PubMed Central

Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519–539, E969–983, E1063–1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies. PMID:19816594

Wang, Yong; Qian, Pei-Yuan

2009-01-01

121

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

122

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

123

Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes  

Microsoft Academic Search

Endophytic bacteria are ubiquitous in most plants and colonise plants without exhibiting pathogenicity. Studies on the diversity of bacterial endophytes have been mainly approached by characterisation of isolates obtained from internal tissues. Despite the broad application of culture-independent techniques for the analysis of microbial communities in a wide range of natural habitats, little information is available on the species diversity

Angela Sessitsch; Birgit Reiter; Ulrike Pfeifer; Eva Wilhelm

2002-01-01

124

Use of Species-Directed 16S rRNA Gene PCR Primers for Detection of Atopobium vaginae in Patients with Bacterial Vaginosis  

Microsoft Academic Search

Received 24 May 2004\\/Returned for modification 21 June 2004\\/Accepted 23 August 2004 Recent studies suggest that the association between a metronidazole-resistant anaerobe, Atopobium vaginae, and bacterial vaginosis (BV) warrants further investigation. In the present study, specific primers enhanced detection of A. vaginae and provided additional evidence that this bacterium is prevalent among patients with BV but absent among patients with

Michael J. Ferris; Alicia Masztal; David H. Martin

2004-01-01

125

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

PubMed Central

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

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

2012-01-01

126

Comparison of DNA Extraction Methods in Analysis of Salivary Bacterial Communities  

PubMed Central

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

Girard, Myriam; François, Patrice; Schrenzel, Jacques

2013-01-01

127

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

128

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

129

Isolation of New Bacterial Species from Drinking Water Biofilms and Proof of Their In Situ Dominance with Highly Specific 16S rRNA Probes  

Microsoft Academic Search

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

SIBYLLE KALMBACH; WERNER MANZ; ULRICH SZEWZYK

1997-01-01

130

Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies  

Microsoft Academic Search

Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown.

Yong Wang; Pei-Yuan Qian; Dawn Field

2009-01-01

131

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

132

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

PubMed Central

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

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

2015-01-01

133

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

PubMed

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

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

2011-01-01

134

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

135

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

136

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

PubMed

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

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

2014-03-01

137

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

PubMed

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

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

2013-03-01

138

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

PubMed

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

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

2015-04-15

139

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

PubMed

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

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

2013-10-01

140

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

PubMed

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

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

2013-08-01

141

Bacterial diversity of soils assessed by DGGE, T-RFLP and SSCP fingerprints of PCR-amplified 16S rRNA gene fragments: do the different methods provide similar results?  

PubMed

Bacterial communities of four arable soils--pelosol, gley, para brown soil, and podsol brown soil--were analysed by fingerprinting of 16S rRNA gene fragments amplified from total DNA of four replicate samples for each soil type. Fingerprints were generated in parallel by denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP) to test whether these commonly applied techniques are interchangeable. PCR amplicons could be separated with all three methods resulting in complex ribotype patterns. Although the fragments amplified comprised different variable regions and lengths, DGGE, T-RFLP and SSCP analyses led to similar findings: (a) a clustering of fingerprints which correlated with soil physico-chemical properties, (b) little variability between the four replicates of the same soil, (c) the patterns of the two brown soils were more similar to each other than to those of the other two soils, and (d) the fingerprints of the different soil types revealed significant differences in a permutation test, which was recently developed for this purpose. PMID:17407797

Smalla, Kornelia; Oros-Sichler, Miruna; Milling, Annett; Heuer, Holger; Baumgarte, Susanne; Becker, Regina; Neuber, Gabriele; Kropf, Siegfried; Ulrich, Andreas; Tebbe, Christoph C

2007-06-01

142

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

143

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

144

Mutagenicity spectra in bacterial strains of airborne and engine exhaust particulate extracts.  

PubMed

The mutagenicity spectra of the organic extracts of both airborne particulate matter and diesel and gasoline soot particles were determined using a battery of 9 bacterial strains of different genetic specificity. The assays with crude extracts and with fractionated acidic, neutral and basic components revealed striking differences in the patterns of mutagenic responses produced by each of the complex mixtures investigated. The mutagenicity of air particulate matter was shown to depend mainly on direct-acting acidic and neutral compounds, with a lesser contribution of basic promutagens which required exogenous metabolic activation by liver S9. The assays with a diesel soot extract indicated the prevailing contribution of direct-acting acidic and neutral compounds, and suggested an important role also for nitro derivatives other than nitropyrenes. The gasoline exhaust was characterized by powerful promutagenic compounds, belonging to either the acidic, neutral or basic fractions. The implications of these results are discussed with respect to the contribution of engine exhausts to air pollution, and the possible use of mutagenicity spectra in the analysis of environmental complex mixtures. PMID:1722279

Crebelli, R; Fuselli, S; Conti, G; Conti, L; Carere, A

1991-12-01

145

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

146

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

PubMed

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

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

2015-05-01

147

Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils.  

PubMed

Molecular characterization of the microbial populations of soils and sediments contaminated with polycyclic aromatic hydrocarbons (PAHs) is often a first step in assessing intrinsic biodegradation potential. However, soils are problematic for molecular analysis owing to the presence of organic matter, such as humic acids. Furthermore, the presence of contaminants, such as PAHs, can cause further challenges to DNA extraction, quantification, and amplification. The goal of our study was to compare the effectiveness of four commercial soil DNA extraction kits (UltraClean Soil DNA Isolation kit, PowerSoil DNA Isolation kit, PowerMax Soil DNA Isolation kit, and FastDNA SPIN kit) to extract pure, high-quality bacterial and eukaryotic DNA from PAH-contaminated soils. Six different contaminated soils were used to determine if there were any biases among the kits due to soil properties or level of contamination. Extracted DNA was used as a template for bacterial 16S rDNA and eukaryotic 18S rDNA amplifications, and PCR products were subsequently analyzed using denaturing gel gradient electrophoresis (DGGE). We found that the FastDNA SPIN kit provided significantly higher DNA yields for all soils; however, it also resulted in the highest levels of humic acid contamination. Soil texture and organic carbon content of the soil did not affect the DNA yield of any kit. Moreover, a liquid-liquid extraction of the DNA extracts found no residual PAHs, indicating that all kits were effective at removing contaminants in the extraction process. Although the PowerSoil DNA Isolation kit gave relatively low DNA yields, it provided the highest quality DNA based on successful amplification of both bacterial and eukaryotic DNA for all six soils. DGGE fingerprints among the kits were dramatically different for both bacterial and eukaryotic DNA. The PowerSoil DNA Isolation kit revealed multiple bands for each soil and provided the most consistent DGGE profiles among replicates for both bacterial and eukaryotic DNA. PMID:21815819

Mahmoudi, Nagissa; Slater, Greg F; Fulthorpe, Roberta R

2011-08-01

148

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

SciTech Connect

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

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

2009-03-01

149

Mite-Specifi c Immunotherapy Using Allergen and\\/or Bacterial Extracts in Atopic Patients in Brazil  

Microsoft Academic Search

? Abstract Objective: This study aimed to evaluate the clinical effi cacy and antibody response changes after specifi c immunotherapy (SIT) using Dermatophagoides pteronyssinus (Dpt) allergens with or without bacterial extracts in Brazilian mite-atopic patients. Methods: One-hundred patients with allergic rhinitis were selected for a randomized double-blind, placebo-controlled trial and distributed into 4 groups: Dpt (Dpt allergen extract), Dpt +

M Guimarães; Junqueira de Queirós; Oliveira Silva; R Alves; H Fukuhara Chiba; Soares de Amaral; KC de Almeida; R de Oliveira Resende; M Camargo Sopelete; G Rodrigues Silva Segundo

150

Leuconostoc pseudomesenteroides WCFur3 partial 16S rRNA gene  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

151

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

152

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

ERIC Educational Resources Information Center

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

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

1986-01-01

153

Inhibitory effects of Olea ferruginea crude leaves extract against some bacterial and fungal pathogen.  

PubMed

This work aimed to evaluate the inhibitory effects of Olea ferruginea crude leaves extract that are commonly used as remedy to cure infections in the tribal (Khyber Agency) areas of Pakistan against some of bacterial and fungal pathogens. The crude n-hexane fraction was appreciably active against both gram positive and negative microorganisms (MIC ranged from 7.5 to 15 mg/ml) followed by butanol fraction (MIC 15 to 30 mg/ml). Conversely least biological activity was shown by chloroform (30mg/ml) and methanol (15 to 30mg/ml) crude fractions. The MBC observed for all crude fractions was same or 2 times higher when compared with MIC for all crude extract fractions. Likewise all the fractions showed activity against Aspergillus niger and maximum zones of inhibition were shown by the n-hexane fraction (14 ± (0.02), butanol (13 ± (0.02) followed by methanol (9 ± (0.05) and chloroform fractions (7 ± (0.02). These results clearly imitate the antibacterial and antifungal potential of Olea ferruginea and hence we recommend the whole plant for further futuristic studies. PMID:23455192

Amin, Adnan; Khan, Muhammad Ayaz; Shah, Swahid; Ahmad, Mushatq; Zafar, Muhammad; Hameed, Abdul

2013-03-01

154

Antibacterial efficacy of the seed extracts of Melia azedarach against some hospital isolated human pathogenic bacterial strains  

PubMed Central

Objective To investigate the antibacterial potential of the polar and non-polar extracts of the seeds of Melia azedarach (M. azedarach) L. (Meliaceae) against eighteen hospital isolated human pathogenic bacterial strains. Methods Petrol, benzene, ethyl acetate, methanol, and aqueous extracts at five different concentrations (1, 2, 5, 10 and 15 mg/mL) were evaluated. Disk diffusion method was followed to evaluate the antibacterial efficacy. Results All extracts of the seeds demonstrated significant antibacterial activity against tested pathogens. Among all extracts, ethyl acetate extract revealed the highest inhibition comparatively. The present study also favored the traditional uses reported earlier. Conclusions Results of this study strongly confirm that the seed extracts of M. azedarach could be effective antibiotics, both in controlling gram-positive and gram-negative human pathogenic infections. PMID:23569812

Khan, Abdul Viqar; Ahmed, Qamar Uddin; Mir, M Ramzan; Shukla, Indu; Khan, Athar Ali

2011-01-01

155

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

156

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

157

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

158

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

PubMed

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

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

2010-01-01

159

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

160

Bacterial diversity of the rhizosphere of maize (Zea mays) grown in tropical soil studied by temperature gradient gel electrophoresis  

Microsoft Academic Search

The bacterial diversity and population dynamics in the rhizosphere of two maize cultivars (Nitroflint and Nitrodent) grown in tropical soils was studied, by traditional cultivation techniques and 16S rRNA gene-based molecular analysis of DNA directly extracted from soil and rhizosphere samples. Rhizosphere and soil samples were taken at three different plant growth stages. Total aerobic bacterial counts were determined. Fingerprints

N. C. M. Gomes; H. Heuer; J. Schönfeld; R. Costa; L. Mendonça-Hagler; K. Smalla

2001-01-01

161

Colony-Forming Analysis of Bacterial Community Succession in Deglaciated Soils Indicates Pioneer Stress-Tolerant Opportunists  

Microsoft Academic Search

We investigated the response of bacterial communities inhabiting two deglaciated soils (10 and 100 years post-deglaciation) to two stimuli: (i) physical disruption (mixing), and (ii) disruption plus nutrient addition. PCR\\/DGGE analysis of 16S rRNA genes extracted from soil during a 168-h incubation period following the stimuli revealed that more bacterial phylotypes were stimulated in the 10-y soil than in the

W. V. Sigler; J. Zeyer

2004-01-01

162

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

PubMed Central

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

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

2013-01-01

163

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

PubMed Central

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

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

2001-01-01

164

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

165

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

166

Bacterial and Archaeal DNA Extracted from Inoculated Experiments: Implication for the Optimization of DNA Extraction from Deep-Sea Basalts  

Microsoft Academic Search

Difficulties in efficient DNA extraction from deep-sea volcanic basalt, due to high metal concentration, complex organic matter, or sometimes the low biomass, have hampered the understanding of the significant biosphere both at and below the sea floor. In order to optimize the DNA extraction from basaltic rocks, sterilized basalts with different particle sizes and chemically synthesized goethite were inoculated with

Hongmei Wang; Katrina J. Edwards

2009-01-01

167

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

PubMed Central

While mutans streptococci have long been assumed to be the specific pathogen responsible for human dental caries, the concept of a complex dental caries-associated microbiota has received significant attention in recent years. Molecular analyses revealed the complexity of the microbiota with the predominance of Lactobacillus and Prevotella in carious dentine lesions. However, characterization of the dentin caries-associated microbiota has not been extensively explored in different ethnicities and races. In the present study, the bacterial communities in the carious dentin of Japanese subjects were analyzed comprehensively with molecular approaches using the16S rRNA gene. Carious dentin lesion samples were collected from 32 subjects aged 4–76 years, and the 16S rRNA genes, amplified from the extracted DNA with universal primers, were sequenced with a pyrosequencer. The bacterial composition was classified into clusters I, II, and III according to the relative abundance (high, middle, low) of Lactobacillus. The bacterial composition in cluster II was composed of relatively high proportions of Olsenella and Propionibacterium or subdominated by heterogeneous genera. The bacterial communities in cluster III were characterized by the predominance of Atopobium, Prevotella, or Propionibacterium with Streptococcus or Actinomyces. Some samples in clusters II and III, mainly related to Atopobium and Propionibacterium, were novel combinations of microbiota in carious dentin lesions and may be characteristic of the Japanese population. Clone library analysis revealed that Atopobium sp. HOT-416 and P. acidifaciens were specific species associated with dentinal caries among these genera in a Japanese population. We summarized the bacterial composition of dentinal carious lesions in a Japanese population using next-generation sequencing and found typical Japanese types with Atopobium or Propionibacterium predominating. PMID:25083880

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

2014-01-01

168

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

Microsoft Academic Search

Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts

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

2010-01-01

169

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

170

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

171

Evaluation of a Fluorescence-Labelled Oligonucleotide Probe Targeting 23S rRNA for In Situ Detection of Salmonella Serovars in Paraffin-Embedded Tissue Sections and Their Rapid Identification in Bacterial Smears  

Microsoft Academic Search

A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin- embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all of the S. enterica subspecies and S. bongori, an 18-mer oligonucleotide probe was selected.

STEEN NORDENTOFT; HENRIK CHRISTENSEN; HENRIK CASPAR WEGENER

172

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

EPA Science Inventory

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

173

A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material  

Microsoft Academic Search

This study investigated the various commercially available kits and ‘in-house’ methods to extract DNA from Gram-negative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads\\/sonication and wash\\/alkali\\/heat lysis. The results indicated that

Beverley C. Millar; Xu Jiru; John E. Moore; John A. P. Earle

2000-01-01

174

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

PubMed Central

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

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

2014-01-01

175

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

PubMed

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

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

2014-11-01

176

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

177

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

PubMed

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

Nigro, A; Nicastro, A; Trodella, R

2014-01-01

178

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

PubMed Central

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

2000-01-01

179

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

PubMed

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

Rafiee, Mohammad; Jahangiri-Rad, Mahsa; Hajjaran, Homa; Mesdaghinia, Alireza; Hajaghazadeh, Mohammad

2014-01-01

180

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

181

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

SciTech Connect

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

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

1987-01-01

182

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

PubMed

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

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

2013-09-01

183

Seasonal variation in detection of bacterial DNA in arthritic stifle joints of dogs with cranial cruciate ligament rupture using PCR amplification of the 16S rRNA gene  

Microsoft Academic Search

An underappreciated cause and effect relationship between environmental bacteria and arthritis may exist. Previously, we found that stifle arthritis in dogs was associated with the presence of environmental bacteria within synovium. Cranial cruciate ligament rupture (CCLR) is often associated with stifle arthritis in dogs. We now wished to determine whether seasonal variation in detection of bacterial material may exist in

Peter Muir; Robin Fox; Qiang Wu; Theresa A. Baker; Nina C. Zitzer; Alan P. Hudson; Paul A. Manley; Susan L. Schaefer; Zhengling Hao

2010-01-01

184

High aromatic ring-cleavage diversity in birch rhizosphere: PAH treatment-specific changes of I.E.3 group extradiol dioxygenases and 16S rRNA bacterial communities in soil  

Microsoft Academic Search

Genes encoding key enzymes of catabolic pathways can be targeted by DNA fingerprinting to explore genetic degradation potential in pristine and polluted soils. We performed a greenhouse microcosm experiment to elucidate structural and functional bacterial diversity in polyaromatic hydrocarbon (PAH)-polluted soil and to test the suitability of birch (Betula pendula) for remediation. Degradation of PAHs was analysed by high-performance liquid

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

2008-01-01

185

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

186

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

PubMed Central

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

Mohit, Vani; Archambault, Philippe; Toupoint, Nicolas

2014-01-01

187

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

188

Vacuum-extraction of peatlands disturbs bacterial population and microbial biomass carbon  

Microsoft Academic Search

Knowledge concerning the microbial characteristics of natural and post-vacuum extracted ombrotrophic peatlands, as well as peatlands under restoration is limited. In one experiment, microbial comparisons of paired neighboring natural and post-vacuum peatlands in eastern Québec (Canada) were studied to assess the effects of peat mining on microbial indicators and nitrogen (N) cycling. Microbial counts, microbial biomass carbon (MB-C) and N

Manon Croft; Line Rochefort; Chantal J. Beauchamp

2001-01-01

189

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

190

Protective effect of Polygonum orientale L. extracts against Clavibater michiganense subsp. sepedonicum, the causal agent of bacterial ring rot of potato.  

PubMed

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 L(27)3(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

191

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

192

Illumina sequencing of the V4 hypervariable region 16S rRNA gene reveals extensive changes in bacterial communities in the cecum following carbohydrate oral infusion and development of early-stage acute laminitis in the horse.  

PubMed

In the equine carbohydrate overload model of acute laminitis, disease progression is associated with changes in bacteria found in the cecum. To date, research has focused on changes in specific Gram-positive bacteria in this portion of the intestinal tract. Metagenomic methods are now available making it possible to interrogate microbial communities using animal protocols that sufficiently power a study. In this study, the microbiota in cecal fluid collected from control, non-laminitic horses (n=8) and from horses with early-stage acute laminitis induced with either oligofructan (n=6) or cornstarch (n=6) were profiled. The microbiota were identified based on sequencing the V4 hypervariable region of the 16S rRNA gene. The results of the study show that the relative abundance of Lactobacillus sp. and Streptococcus sp. increased significantly (p<0.05) following OF and CS infusion. Other significant changes included an increase (p<0.05) in relative abundance of Veillonella sp. and Serratia sp., two potentially pathogenic, Gram-negative bacteria. Significant decreases in the relative abundance of presumptive normal flora were detected as well. Although changes in cecal microbiota described in this communication are from a pilot study, it is hypothesized that an overgrowth of pathogenic Gram-negative bacteria develops and contributes to enterocolitis, pyrexia and lameness in the carbohydrate overload model of acute laminitis. PMID:24355533

Moreau, Michael M; Eades, Susan C; Reinemeyer, Craig R; Fugaro, Michael N; Onishi, Janet C

2014-01-31

193

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

194

Novel Approach to Quantitative Detection of Specific rRNA in a Microbial Community, Using Catalytic DNA  

PubMed Central

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities. PMID:16085888

Suenaga, Hikaru; Liu, Rui; Shiramasa, Yuko; Kanagawa, Takahiro

2005-01-01

195

Novel approach to quantitative detection of specific rRNA in a microbial community, using catalytic DNA.  

PubMed

We developed a novel method for the quantitative detection of the 16S rRNA of a specific bacterial species in the microbial community by using deoxyribozyme (DNAzyme), which possesses the catalytic function to cleave RNA in a sequence-specific manner. A mixture of heterogeneous 16S rRNA containing the target 16S rRNA was incubated with a species-specific DNAzyme. The cleaved target 16S rRNA was separated from the intact 16S rRNA by electrophoresis, and then their amounts were compared for the quantitative detection of target 16S rRNA. This method was used to determine the abundance of the 16S rRNA of a filamentous bacterium, Sphaerotilus natans, in activated sludge, which is a microbial mixture used in wastewater treatment systems. The result indicated that this DNAzyme-based approach would be applicable to actual microbial communities. PMID:16085888

Suenaga, Hikaru; Liu, Rui; Shiramasa, Yuko; Kanagawa, Takahiro

2005-08-01

196

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

197

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

198

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

199

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

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

200

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

PubMed Central

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

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

1996-01-01

201

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

PubMed Central

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

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

2011-01-01

202

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

PubMed Central

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

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

2001-01-01

203

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

204

Effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population.  

PubMed

This study characterizes the effects of concentrated pomegranate-peel extract (CPE) addition to the TMR at levels of 1, 2, or 4% on voluntary intake, in vivo digestibility, milk yield and composition, and profile of rumen bacterial and archaeal populations in lactating Holstein cows. Supplementation of CPE significantly affected the abundance of methanogenic archaea and specific ruminal bacterial species related to cellulolytic activities and soluble sugar and lactic acid fermentation, as revealed by real-time PCR quantification. Furthermore, CPE supplementation had a significant dose-dependent effect on the whole ruminal bacterial community, as determined by automated ribosomal intergenic spacer analysis. These changes were accompanied by a significant increase in digestibility of dry matter, crude protein, and neutral detergent fiber, as well as milk and energy-corrected milk yields in cows fed the 4% CPE supplement. These results suggest that CPE supplementation significantly affects the rumen bacterial communities, which in turn may be related to a beneficial effect on dairy cow performance. PMID:22863105

Jami, E; Shabtay, A; Nikbachat, M; Yosef, E; Miron, J; Mizrahi, I

2012-10-01

205

Analyses of bacterial communities in meju, a Korean traditional fermented soybean bricks, by cultivation-based and pyrosequencing methods.  

PubMed

Despite the importance of meju as a raw material used to make Korean soy sauce (ganjang) and soybean paste (doenjang), little is known about the bacterial diversity of Korean meju. In this study, the bacterial communities in meju were examined using both culture-dependent and independent methods in order to evaluate the diversity of the bacterial population. Analyses of the 16S rRNA gene sequences of the bacterial strains isolated from meju samples showed that the dominant species were related to members of the genera Bacillus, Enterococcus, and Pediococcus. The community DNAs extracted from nine different meju samples were analyzed by barcoded pyrosequencing method targeting of the V1 to V3 hypervariable regions of the 16S rRNA gene. In total, 132,374 sequences, with an average read length of 468 bp, were assigned to several phyla, with Firmicutes (93.6%) representing the predominant phylum, followed by Proteobacteria (4.5%) and Bacteroidetes (0.8%). Other phyla accounted for less than 1% of the total bacterial sequences. Most of the Firmicutes were Bacillus and lactic acid bacteria, mainly represented by members of the genera Enterococcus, Lactococcus, and Leuconostoc, whose ratio varied among different samples. In conclusion, this study indicated that the bacterial communities in meju were very diverse and a complex microbial consortium containing various microorganisms got involved in meju fermentation than we expected before. PMID:21717316

Kim, Yi-Seul; Kim, Min-Cheol; Kwon, Soon-Wo; Kim, Soo-Jin; Park, In-Cheol; Ka, Jong-Ok; Weon, Hang-Yeon

2011-06-01

206

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

207

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

PubMed Central

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

2014-01-01

208

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

PubMed

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

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

2012-02-01

209

Diversity of Bacterial Communities in Container Habitats of Mosquitoes  

PubMed Central

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

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

2010-01-01

210

A broad-host-range, generalized transducing phage (SN-T) acquires 16S rRNA genes from different genera of bacteria.  

PubMed

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 x 10(-9) transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria. PMID:16332816

Beumer, Amy; Robinson, Jayne B

2005-12-01

211

A Broad-Host-Range, Generalized Transducing Phage (SN-T) Acquires 16S rRNA Genes from Different Genera of Bacteria  

PubMed Central

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire 16S rRNA genes and gene sequences. Using PCR and primers specific to conserved regions of the 16S rRNA gene, we have found that generalized transducing phages (D3112, UT1, and SN-T), but not specialized transducing phages (D3), acquired entire bacterial 16S rRNA genes. Furthermore, we show that the broad-host-range, generalized transducing phage SN-T is capable of acquiring the 16S rRNA gene from two different genera: Sphaerotilus natans, the host from which SN-T was originally isolated, and Pseudomonas aeruginosa. In sequential infections, SN-T harbored only 16S rRNA gene sequences of the final host as determined by restriction fragment length polymorphism analysis. The frequency of 16S rRNA gene sequences in SN-T populations was determined to be 1 × 10?9 transductants/PFU. Our findings further implicate transduction in the horizontal transfer of 16S rRNA genes between different species or genera of bacteria. PMID:16332816

Beumer, Amy; Robinson, Jayne B.

2005-01-01

212

Effect of dietary prebiotic (mannan oligosaccharide) supplementation on the caecal bacterial community structure of turkeys.  

PubMed

The identification of specific bacterial species influenced by mannan oligosaccharide (MOS) supplementation may assist in the formulation of new and improved diets that promote intestinal health and improve bird performance, offering suitable alternatives to antimicrobials in feed for sustainable poultry production. This study has been conducted to evaluate the use of a MOS compound derived from the yeast cell wall of Saccharomyces cerevisiae on turkey performance, bacterial community structure and their phylogenetic associations. A 42-day turkey trial was carried out on birds fed control and MOS-supplemented diets. Bird performance data (weight gains, feed consumption and feed efficiency ratios) were collected, and caecal contents were extracted from randomly caught poults on days 28, 35 and 42 posthatch. Bird performance data showed no improvements as a result of dietary supplementation. Automated ribosomal intergenic spacer analysis (ARISA) revealed the bacterial community structure to be significantly altered on days 28 and 35 posthatch but not day 42 as a result of dietary supplementation. This technique was coupled with 16S rRNA gene sequence analysis to elucidate phylogenetic identities of bacteria. The dominant bacteria of the caecum on all days in both treatment groups were members of phylum Firmicutes, followed by the Bacteroidetes and Proteobacteria phyla, respectively. Statistical analysis of the 16S rRNA gene libraries showed that the composition of the MOS clone library differed significantly to the control on day 35 posthatch. It can be concluded that MOS alters the bacterial community structure in the turkey caecum. PMID:22538976

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

2012-10-01

213

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...

214

A Broad-Host-Range, Generalized Transducing Phage (SNT) Acquires 16S rRNA Genes from Different Genera of Bacteria  

Microsoft Academic Search

Genomic analysis has revealed heterogeneity among bacterial 16S rRNA gene sequences within a single species; yet the cause(s) remains uncertain. Generalized transducing bacteriophages have recently gained recognition for their abundance as well as their ability to affect lateral gene transfer and to harbor bacterial 16S rRNA gene sequences. Here, we demonstrate the ability of broad-host-range, generalized transducing phages to acquire

Amy Beumer; Jayne B. Robinson

2005-01-01

215

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

Microsoft Academic Search

The development of fast and easy on-site molecular detection and quantification methods for hazardous microbes on solid surfaces\\u000a is desirable for several applications where specialised laboratory facilities are absent. The quantification of bacterial\\u000a contamination necessitates the assessment of the efficiency of the used methodology as a whole, including the preceding steps\\u000a of sampling and sample processing. We used quantitative real-time

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

216

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

217

PCR-Enzyme-Linked Immunosorbent Assay and Partial rRNA Gene Sequencing: a Rational Approach to Identifying Mycobacteria  

Microsoft Academic Search

A PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid identification of myco- bacterial DNA coding for 16S rRNA was developed. The PCR selectively targeted and amplified part of the 16S rRNA gene from all mycobacteria while simultaneously labelling one strand of the amplified product with a 5* fluorescein-labelled primer. The identity of the labelled strand was subsequently determined by hybridization

SUSHIL PATEL; MALCOLM YATES; NICHOLAS A. SAUNDERS

1997-01-01

218

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

219

rRNA Operon Copy Number Reflects Ecological Strategies of Bacteria  

PubMed Central

Although natural selection appears to favor the elimination of gene redundancy in prokaryotes, multiple copies of each rRNA-encoding gene are common on bacterial chromosomes. Despite this conspicuous deviation from single-copy genes, no phenotype has been consistently associated with rRNA gene copy number. We found that the number of rRNA genes correlates with the rate at which phylogenetically diverse bacteria respond to resource availability. Soil bacteria that formed colonies rapidly upon exposure to a nutritionally complex medium contained an average of 5.5 copies of the small subunit rRNA gene, whereas bacteria that responded slowly contained an average of 1.4 copies. In soil microcosms pulsed with the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), indigenous populations of 2,4-D-degrading bacteria with multiple rRNA genes (x? = 5.4) became dominant, whereas populations with fewer rRNA genes (x? = 2.7) were favored in unamended controls. These findings demonstrate phenotypic effects associated with rRNA gene copy number that are indicative of ecological strategies influencing the structure of natural microbial communities. PMID:10742207

Klappenbach, Joel A.; Dunbar, John M.; Schmidt, Thomas M.

2000-01-01

220

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

PubMed Central

Background The nutrition and health of horses is closely tied to their gastrointestinal microflora. Gut bacteria break down plant structural carbohydrates and produce volatile fatty acids, which are a major source of energy for horses. Bacterial communities are also essential for maintaining gut homeostasis and have been hypothesized to contribute to various diseases including laminitis. We performed pyrosequencing of 16S rRNA bacterial genes isolated from fecal material to characterize hindgut bacterial communities in healthy horses and those with chronic laminitis. Results Fecal samples were collected from 10 normal horses and 8 horses with chronic laminitis. Genomic DNA was extracted and the V4-V5 segment of the 16S rRNA gene was PCR amplified and sequenced on the 454 platform generating a mean of 2,425 reads per sample after quality trimming. The bacterial communities were dominated by Firmicutes (69.21% control, 56.72% laminitis) and Verrucomicrobia (18.13% control, 27.63% laminitis), followed by Bacteroidetes, Proteobacteria, and Spirochaetes. We observed more OTUs per individual in the laminitis group than the control group (419.6 and 355.2, respectively, P = 0.019) along with a difference in the abundance of two unassigned Clostridiales genera (P = 0.03 and P = 0.01). The most abundant bacteria were Streptococcus spp., Clostridium spp., and Treponema spp.; along with unassigned genera from Subdivision 5 of Verrucomicrobia, Ruminococcaceae, and Clostridiaceae, which together constituted ~ 80% of all OTUs. There was a high level of individual variation across all taxonomic ranks. Conclusions Our exploration of the equine fecal microflora revealed higher bacterial diversity in horses with chronic laminitis and identification of two Clostridiales genera that differed in abundance from control horses. There was large individual variation in bacterial communities that was not explained in our study. The core hindgut microflora was dominated by Streptococcus spp., several cellulytic genera, and a large proportion of uncharacterized OTUs that warrant further investigation regarding their function. Our data provide a foundation for future investigations of hindgut bacterial factors that may influence the development and progression of chronic laminitis. PMID:23186268

2012-01-01

221

A new rapid method of glycolate test by diethyl ether extraction, which is applicable to a small amount of bacterial cells of less than one milligram.  

PubMed

The glycolate test is a method to discriminate N-acyl groups of muramyl residue in peptidoglycan of bacterial cell walls by color reaction without purification of the cell walls. The glycolyl residue presents red purple color by heating with 0.02% 2,7-dihydroxynaphthalene (DON) dissolved in concentrated H(2)SO(4). Instead of the previous column methods for quantitative analysis, a qualitative method by solvent works was developed to simplify and to miniaturize the analysis. In this method, solvents played two roles, removal of interfering materials and extraction of glycolic acid from the cell hydrolysates. Of several solvent systems tested, diethyl ether was studied in detail on such properties as the efficiency of glycolic acid extraction under several conditions, the ability of removing various interfering compounds, and the advantage on evaporation procedure of the solvent from extracts. DON reaction of the second diethyl ether extract from cell hydrolysate of "Micromonospora nigra" JCM 3328 showed a clear red purple color of a strong absorbance at 530 nm, which is the same as that of authentic glycolic acid. The solvent method was applied to 20 strains of typical actinomycete species whose acyl types have already been known (Uchida and Seino, 1997). All glycolate test positive strains showed the clear red purple color mentioned above, whereas acetyl type strains revealed no apparent color by the same procedures. Additional experiments indicated that the glycolate test could be determined with less than 1 mg of actinomycete cells by using a smaller amount of DON reagent and ordinary polypropylene tubes. The new method was discussed for advantages in the identification of actinomycetes and for possible applications to other fields. PMID:12501387

Uchida, Kinya; Kudo, Takuji; Suzuki, Ken-Ichiro; Nakase, Takashi

1999-04-01

222

Biocidal activity of partially purified fractions from methanolic extract of Garcinia kola (Heckel) seeds on bacterial isolates.  

PubMed

The in vitro antibacterial activity of crude methanolic extract of the seeds of Garcinia kola was investigated. The extracts exhibited antibacterial activities with zones of inhibition ranging from 10 mm to 25 mm. The minimum inhibitory concentration of the diethyl ether fraction was between 0.313 and 5.0 mg/ml, while that of butanol fraction varied from 0.157 to 5.0 mg/ml. The butanol fraction killed about 77% of Bacillus anthracis and 79% of Escherichia coli cells within 120 min at a concentration of 5.0 mg/ml. Protein leakage from the B. anthracis and E. coli cells when exposed to the butanol and diethyl ether fractions was observed. We conclude that Garcinia kola seed extract has a broad spectrum antibacterial activity, with the butanol and diethyl ether fractions being bactericidal as exemplified by the killing rate and protein leakage regimes, which suggest cell membrane disruption as a mechanism of action of the extract. PMID:19399341

Akinpelu, D A; Adegboye, M F; Adeloye, O A; Okoh, A I

2008-01-01

223

Biological and antibacterial activities of the natural herb Houttuynia cordata water extract against the intracellular bacterial pathogen salmonella within the RAW 264.7 macrophage.  

PubMed

Salmonellosis is a major bacterial zoonosis that causes a variety of disease syndromes, from self-limiting enteritis to fatal infection in animals and food-borne infection and typhoid fever in humans. Recently, the emergence of multidrug-resistant strains of Salmonella sp. has caused more serious problems in public health. The present study investigated the antibacterial effects of Houttuynia cordata water extract (HCWE) against murine salmonellosis. In RAW 264.7 cells, there was no detectable cytotoxic effect of HCWE at any concentration between 25 and 100 microg/ml after 8-h incubation. The antibacterial activity of HCWE was then examined in a Salmonella enterica serovar (Salmonella typhimurium), and was found to increase in a dose-dependent manner at concentrations from 25 to 100 microg/ml during 8-h incubation. HCWE also affected RAW 264.7 cells including morphologic change and bacterial uptake, but there was no significant difference in bacterial replication in RAW 264.7 cells. With HCWE alone, nitric oxide (NO) production by RAW 264.7 cells did not increase, but when RAW 264.7 cells were infected by S. typhimurium, with or without HCWE, NO production with HCWE was 2-fold higher than that without HCWE. Treatment with HCWE did not affect inducible NO synthase (iNOS) mRNA expression by RAW 264.7 cells, but when RAW 264.7 cells with HCWE were infected by S. typhimurium, iNOS mRNA expression was increased during 8-h incubation. Furthermore, HCWE showed virulence reduction effects in S. typhimurium-infected BALB/c mice. After a lethal dose of S. typhimurium, the mortality rate in the HCWE untreated group was 100% at 7 d, but the HCWE 25, 50, and 100 microg/ml groups survived until 11, 17, and 23 d, respectively. These data suggest that HCWE is stable and beneficial in the treatment of bacterial infection including intracellularly replicating pathogens and may solve antimicrobial misuse and overuse. PMID:18981565

Kim, Gon Sup; Kim, Dong Hyeok; Lim, Jeong Ju; Lee, Jin Ju; Han, Dae Yong; Lee, Whi Min; Jung, Won Chul; Min, Won Gi; Won, Chung Gil; Rhee, Man Hee; Lee, Hu Jang; Kim, Suk

2008-11-01

224

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

225

Metatranscriptomics reveals overall active bacterial composition in caries lesions  

PubMed Central

Background Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms. Objective To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay. Design Non-cavitated enamel caries lesions (n=15) and dentin caries lesions samples (n=12) were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition. Results A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.02–0.73% of the total bacterial community. Conclusions The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci detected confirms that they are a minority and questions its importance as the main etiological agent of tooth decay. Future experimental work should be performed to confirm the cariogenicity of the identified bacteria. PMID:25626770

Simón-Soro, Aurea; Guillen-Navarro, Miriam; Mira, Alex

2014-01-01

226

Bacterial protein signals are associated with Crohn’s disease  

PubMed Central

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

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

2014-01-01

227

Technical challenges in metatranscriptomic studies applied to the bacterial communities of freshwater ecosystems.  

PubMed

Metatranscriptome analysis relates to the transcriptome of microbial communities directly sampled in the environment. Accessing the mRNA pool in natural bacterial communities presents some technical challenges such as the RNA extraction, rRNA depletion, and the choice of the high-throughput sequencing technique. The lack of technical details in scientific articles is a major problem to correctly obtained mRNA from a microbial community and thus the corresponding sequencing data. In our study, we present the methodological procedure that was developed in order to access to the metatranscriptome of the microbial communities during two cyanobacterial blooms successively occurring in a freshwater eutrophic lake. Each procedure step was detailed and discussed with regard to the choices and difficulties encountered and to the recent literature. Finally, the two major limits for metatranscriptomic approaches targeting bacterial communities from natural environments were (i) the removal of rRNA in order to increase the putative mRNA reads number after sequencing, and (ii) for most of the bacterial communities living in natural environments, the lack of reference genomes in databases that leads to the non-assignation of numerous reads. Once these challenges overcome, we managed to access putative mRNA of dominant species, i.e. cyanobacteria (from 6 to 72 % of mRNA assigned), and of the surrounding bacteria (from 1 to 5 % of mRNA assigned). PMID:25216965

Pascault, Noémie; Loux, Valentin; Derozier, Sandra; Martin, Véronique; Debroas, Didier; Maloufi, Selma; Humbert, Jean-François; Leloup, Julie

2015-04-01

228

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

229

Feature extraction by statistical contact potentials and wavelet transform for predicting subcellular localizations in gram negative bacterial proteins.  

PubMed

Predicting the localization of a protein has become a useful practice for inferring its function. Most of the reported methods to predict subcellular localizations in Gram-negative bacterial proteins make use of standard protein representations that generally do not take into account the distribution of the amino acids and the structural information of the proteins. Here, we propose a protein representation based on the structural information contained in the pairwise statistical contact potentials. The wavelet transform decodes the information contained in the primary structure of the proteins, allowing the identification of patterns along the proteins, which are used to characterize the subcellular localizations. Then, a support vector machine classifier is trained to categorize them. Cellular compartments like periplasm and extracellular medium are difficult to predict, having a high false negative rate. The wavelet-based method achieves an overall high performance while maintaining a low false negative rate, particularly, on "periplasm" and "extracellular medium". Our results suggest the proposed protein characterization is a useful alternative to representing and predicting protein sequences over the classical and cutting edge protein depictions. PMID:25219623

Arango-Argoty, G A; Jaramillo-Garzón, J A; Castellanos-Domínguez, G

2015-01-01

230

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...

231

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

232

Culture-independent analysis of bacterial diversity in a child-care facility  

PubMed Central

Background Child-care facilities appear to provide daily opportunities for exposure and transmission of bacteria and viruses. However, almost nothing is known about the diversity of microbial contamination in daycare facilities or its public health implications. Recent culture-independent molecular studies of bacterial diversity in indoor environments have revealed an astonishing diversity of microorganisms, including opportunistic pathogens and many uncultured bacteria. In this study, we used culture and culture-independent methods to determine the viability and diversity of bacteria in a child-care center over a six-month period. Results We sampled surface contamination on toys and furniture using sterile cotton swabs in four daycare classrooms. Bacteria were isolated on nutrient and blood agar plates, and 16S rRNA gene sequences were obtained from unique (one of a kind) colony morphologies for species identification. We also extracted DNA directly from nine representative swab samples taken over the course of the study from both toy and furniture surfaces, and used "universal" 16S rRNA gene bacterial primers to create PCR-based clone libraries. The rRNA gene clones were sequenced, and the sequences were compared with related sequences in GenBank and subjected to phylogenetic analyses to determine their evolutionary relationships. Culturing methods identified viable bacteria on all toys and furniture surfaces sampled in the study. Bacillus spp. were the most commonly cultured bacteria, followed by Staphylococcus spp., and Microbacterium spp. Culture-independent methods based on 16S rRNA gene sequencing, on the other hand, revealed an entirely new dimension of microbial diversity, including an estimated 190 bacterial species from 15 bacterial divisions. Sequence comparisons and phylogenetic analyses determined that the clone libraries were dominated by a diverse set of sequences related to Pseudomonas spp., as well as uncultured bacteria originally identified on human vaginal epithelium. Other sequences were related to uncultured bacteria from wastewater sludge, and many human-associated bacteria including a number of pathogens and opportunistic pathogens. Our results suggest that the child-care facility provided an excellent habitat for slime-producing Pseudomonads, and that diaper changing contributed significantly to the bacterial contamination. Conclusion The combination of culture and culture-independent methods provided powerful means for determining both viability and diversity of bacteria in child-care facilities. Our results provided insight into the source of contamination and suggested ways in which sanitation might be improved. Although our study identified a remarkable array of microbial diversity present in a single daycare, it also revealed just how little we comprehend the true extent of microbial diversity in daycare centers or other indoor environments. PMID:17411442

2007-01-01

233

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

234

16S rRNA methyltransferase RmtC in Salmonella enterica serovar Virchow.  

PubMed

We screened Salmonella and Escherichia coli isolates, collected 2004-2008 in the United Kingdom, for 16S rRNA methyltransferases. rmtC was identified in S. enterica serovar Virchow isolates from clinical samples and food. All isolates were clonally related and bore the rmtC gene on the bacterial chromosome. Surveillance for and research on these resistance determinants are essential. PMID:20350396

Hopkins, Katie L; Escudero, Jose A; Hidalgo, Laura; Gonzalez-Zorn, Bruno

2010-04-01

235

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

PubMed Central

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

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

2008-01-01

236

Intragenomic heterogeneity in the 16S rRNA genes of Flavobacterium columnare and relevance to genomovar assignment  

Technology Transfer Automated Retrieval System (TEKTRAN)

Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the USA and may be emerging as an important pathogen in the rainbow trout industry. The 16S rRNA gene is a housekeeping gene commonly used for bacterial taxonomy and genotyping...

237

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

238

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

239

Molecular Analysis of Oral and Respiratory Bacterial Species Associated with Ventilator-Associated Pneumonia?  

PubMed Central

Trauma intensive care unit (TICU) patients requiring mechanical respiratory support frequently develop ventilator-associated pneumonia (VAP). Oral and oropharyngeal bacteria are believed to be responsible for many cases of VAP, but definitive evidence of this relationship is lacking. Earlier studies used conventional culture-based methods for identification of bacterial pathogens, but these methods are insufficient, as some bacteria may be uncultivable or difficult to grow. The purpose of this study was to use a culture-independent molecular approach to analyze and compare the bacterial species colonizing the oral cavity and the lungs of TICU patients who developed VAP. Bacterial samples were acquired from the dorsal tongue and bronchoalveolar lavage fluid of 16 patients. Bacterial DNA was extracted, and the 16S rRNA genes were PCR amplified, cloned into Escherichia coli, and sequenced. The sequencing data revealed the following: (i) a wide diversity of bacterial species in both the oral and pulmonary sites, some of them novel; (ii) known and putative respiratory pathogens colonizing both the oral cavity and lungs of 14 patients; and (iii) a number of bacterial pathogens (e.g., Dialister pneumosintes, Haemophilus segnis, Gemella morbillorum, and Pseudomonas fluorescens) in lung samples that had not been reported previously at this site when culture-based methods were used. Our data indicate that the dorsal surface of the tongue serves as a potential reservoir for bacterial species involved in VAP. Furthermore, it is clear that the diversity of bacterial pathogens for VAP is far more complex than the current literature suggests. PMID:17301280

Bahrani-Mougeot, Farah K.; Paster, Bruce J.; Coleman, Shirley; Barbuto, Sara; Brennan, Michael T.; Noll, Jenene; Kennedy, Thomas; Fox, Philip C.; Lockhart, Peter B.

2007-01-01

240

Extremely Acidophilic Protists from Acid Mine Drainage Host Rickettsiales-Lineage Endosymbionts That Have Intervening Sequences in Their 16S rRNA Genes  

Microsoft Academic Search

During a molecular phylogenetic survey of extremely acidic (pH < 1), metal-rich acid mine drainage habitats in the Richmond Mine at Iron Mountain, Calif., we detected 16S rRNA gene sequences of a novel bacterial group belonging to the order Rickettsiales in the Alphaproteobacteria. The closest known relatives of this group (92% 16S rRNA gene sequence identity) are endosymbionts of the

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

2003-01-01

241

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

242

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

243

Changes in Vaginal Bacterial Concentrations with Intravaginal Metronidazole Therapy for Bacterial Vaginosis as Assessed by Quantitative PCR  

Microsoft Academic Search

Several fastidious bacteria have been associated with bacterial vaginosis (BV) using broad-range bacterial PCR methods such as consensus sequence 16S rRNA gene PCR, but their role in BV remains poorly defined. We describe changes in vaginal bacterial concentrations following metronidazole therapy for BV. Vaginal swabs were collected from women with BV diagnosed using Amsel clinical criteria, and vaginal fluid was

David N. Fredricks; Tina L. Fiedler; Katherine K. Thomas; Caroline M. Mitchell; Jeanne M. Marrazzo

244

Genotoxicity evaluation of Guibi-Tang extract using an in vitro bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus test  

PubMed Central

Background Guibi-Tang is a traditional herbal prescription made from 12 different herbs that is used in the treatment of amnesia and poor memory. Methods In the present study, we evaluated the acute oral toxicity and genotoxic potential of Guibi-Tang water extract (GBT) at doses up to 2000 ?g/plate an using a bacterial reverse mutation test (Ames test) with Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537, and Escherichia coli strain WP2uvrA. Acute toxicity and genotoxic potential were measured in the presence and absence of an exogenous source of metabolic activation, in an in vitro chromosome aberration assay with Chinese hamster lung (CHL) cells, and in an in vivo micronucleus test using ICR mice bone marrow as recommended by the Korean Food and Drug Administration. An acute oral toxicity test of GBT was performed in Sprague Dawley rats. The Ames test showed that GBT did not induce gene mutations in S. typhimurium or in E. coli in the presence or absence of S9 activation. Results GBT did not significantly increase the number of structural aberrations in CHL cells with or without S9 activation. The oral administration of GBT at a dose of up to 2000 mg/kg caused no significant increase in the number of micronucleated polychromatic erythrocytes or in the mean ratio of polychromatic to total erythrocytes. Conclusions However, as we did not identify the components of GBT responsible for these effects, other assays are needed to confirm its genotoxicity. PMID:24985139

2014-01-01

245

Novel Bacterial Diversity in an Anchialine Blue Hole on Abaco Island, Bahamas  

E-print Network

within the platform. Dense microbial assemblages which occurred as mats on the cave walls below the halocline were investigated through construction of 16S rRNA clone libraries, finding representatives across several bacterial lineages including...

Gonzalez, Brett Christopher

2012-02-14

246

Babesia bovis: transcriptional analysis of rRNA gene unit expression.  

PubMed

The complex life cycle of Babesia bovis includes erythrocytic stages in the bovine host and other stages occurring inside its common tick vector Rhipicephalus microplus. In related apicomplexa, changing environmental conditions affect the expression of ribosomal RNA, but it remained unknown whether the polymorphic A, B, and C rRNA coding units of B. bovis are differentially expressed. Northern blot analysis confirmed that polymorphic regions in the B. bovis 18s and ITS-2 rRNA coding units are transcribed. Then, rRNA transcript expression profiles were compared by analyzing cDNA libraries generated from total RNA extracted from in vitro cultured parasites, B. bovis infected cattle, R. microplus larvae and egg sources. The 18s and ITS-2 expression profiles indicate that rRNA unit B is almost exclusively expressed in cultured parasites while units A, B, and C are co-transcribed in the in vivo total RNA sources. Collectively, the data indicate that differential transcription of rRNA occurs in B.bovis, depending on the life stage of the parasite and on the environment. PMID:19508867

Laughery, Jacob M; Lau, Audrey O T; White, Stephen N; Howell, Jeanne M; Suarez, Carlos E

2009-09-01

247

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

248

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

PubMed

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

249

Partial Bacterial Proteome Extraction Kit  

E-print Network

to their very specific characteristics, some proteins may be well denatured and solubilized by a given detergent is to find mixtures of detergents and chaotropes able to cope with the diversity of proteins encountered-positive bacteria in the presence of different detergents and chaotropes. P-PEK is designed for a serial sample

Lebendiker, Mario

250

The rRNA enhancer regulates rRNA transcription in Saccharomyces cerevisiae.  

PubMed Central

In Saccharomyces cerevisiae, the rRNA genes are organized as a tandem array of head-to-tail repeats. An enhancer of rRNA transcription is present just at the end of each transcription unit, 2 kb away from the next one. This enhancer is unusual for S. cerevisiae in that it acts both upstream and downstream of, and even across, genes. The role of the enhancer in the nutritional regulation of rRNA transcription was studied by introducing a centromere plasmid carrying two rRNA minigenes in tandem, flanking a single enhancer, into cells. Analysis of the transcripts from the two minigenes showed that the enhancer was absolutely required for the stimulation of transcription of rRNA that occurs when cells are shifted from a poor carbon source to a good carbon source. While full enhancer function is provided by a 45-bp region at the 3' end of the 190-bp enhancer, some activity was also conferred by other elements, including both a T-rich stretch and a region containing the binding sites for the proteins Reb1p and Abf1p. We conclude that the enhancer is composed of redundant elements and that it is a major element in the regulation of rRNA transcription. Images PMID:8423793

Morrow, B E; Johnson, S P; Warner, J R

1993-01-01

251

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

252

Analysis of the intestinal microbiota using SOLiD 16S rRNA gene sequencing and SOLiD shotgun sequencing  

PubMed Central

Background Metagenomics seeks to understand microbial communities and assemblages by DNA sequencing. Technological advances in next generation sequencing technologies are fuelling a rapid growth in the number and scope of projects aiming to analyze complex microbial environments such as marine, soil or the gut. Recent improvements in longer read lengths and paired-sequencing allow better resolution in profiling microbial communities. While both 454 sequencing and Illumina sequencing have been used in numerous metagenomic studies, SOLiD sequencing is not commonly used in this area, as it is believed to be more suitable in the context of reference-guided projects. Results To investigate the performance of SOLiD sequencing in a metagenomic context, we compared taxonomic profiles of SOLiD mate-pair sequencing reads with Sanger paired reads and 454 single reads. All sequences were obtained from the bacterial 16S rRNA gene, which was amplified from microbial DNA extracted from a human fecal sample. Additionally, from the same fecal sample, complete genomic microbial DNA was extracted and shotgun sequenced using SOLiD sequencing to study the composition of the intestinal microbiota and the existing microbial metabolism. We found that the microbiota composition of 16S rRNA gene sequences obtained using Sanger, 454 and SOLiD sequencing provide results comparable to the result based on shotgun sequencing. Moreover, with SOLiD sequences we obtained more resolution down to the species level. In addition, the shotgun data allowed us to determine a functional profile using the databases SEED and KEGG. Conclusions This study shows that SOLiD mate-pair sequencing is a viable and cost-efficient option for analyzing a complex microbiome. To the best of our knowledge, this is the first time that SOLiD sequencing has been used in a human sample. PMID:24564472

2013-01-01

253

Variable rRNA gene copies in extreme halobacteria  

SciTech Connect

Using PFG electrophoresis techniques, the authors have examined the organization of rRNA gene in halobacterium species. The results show that the organization of rRNA genes among closely related halobacteria is quite heterogeneous. This contrasts with the high degree of conservation of rRNA sequence. The possible mechanism of such rRNA gene amplification and its evolutionary implications are discussed.

Sanz, J.L.; Marin, I.; Ramirez, L.; Amils, R. (Centro de Biologia Molecular, Madrid (Spain)); Abad, J.P.; Smith, C.L. (Columbia Univ., New York, NY (USA))

1988-08-25

254

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

255

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

256

Effects of pH on chemical stability and de-esterification of fenoxaprop-ethyl by purified enzymes, bacterial extracts, and soils.  

PubMed

De-esterification is an initial step in the metabolism of certain herbicides, for example, fenoxaprop-ethyl [(+/-)-ethyl 2-[4-[(6-chloro-2-benzoxaolyl)oxy]phenoxy]propanoate] (FE). The ethyl-ester bond cleavage of FE to fenoxaprop acid (FA) by purified enzymes, crude bacterial enzyme preparations, and soils was investigated. In similar experiments fluorescein diacetate (FDA) was used as an alternative substrate. FE stability was pH sensitive in acidic buffered solutions; that is, below pH 4.6, rapid nonenzymatic hydrolysis of the benzoxazolyl-oxy-phenoxy ether linkage occurred, forming 6-chloro-2,3-dihydro-benzoxazol-2-one (CDHB) and ethyl 4-hydroxyphenoxypropanoate or 4-hydroxyphenoxypropanoate. With porcine esterase and cell-free Pseudomonas fluorescens extracts, activity on FE and FDA was most rapid at pH 7.6-8.6 but decreased 80-90% at pH 5.6. Yeast (Candida cylindrica) lipase-mediated de-esterification of FE and FDA was not as sensitive to pH; that is, activity at pH 4.6 was 70% of that at pH 7.6. Short-term incubations (20 h) were conducted in eight soils (pH 4.5-6.9) treated with (14)C-chlorophenyl ring-labeled FE (2 mg kg(-)(1)). In the most acidic soils (pH 4.4-4.5) 25% of the (14)C was recovered as FA, versus 30-40% in moderately acid soils (pH 5.0-5.6) and 55% in neutral soils (pH 6.8-6.9). There was a similar correlation between soil pH and FDA de-esterification. CDHB was formed in all acidic soils with levels 4-fold greater in pH 4.4-4.5 soils than in pH 5. 0-5.6 soils. CDHB was not formed in neutral soils. Results demonstrate some chemical hydrolysis (benzoxazolyl-oxy-phenoxy ether linkage) of FE in acid soils, the sensitivity of enzymatic de-esterification of FE to pH, and the potential of FDA as a colorimetric indicator for esterase hydrolysis of FE. PMID:11052722

Zablotowicz, R M; Hoagland, R E; Staddon, W J; Locke, M A

2000-10-01

257

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

Microsoft Academic Search

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

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

2010-01-01

258

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

259

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

260

Optimized microbial DNA extraction from diarrheic stools  

PubMed Central

Background The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction. Findings A total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4%) non-lyophilised (Ct value 28.78?±?9.1) versus 39/41 (95.1%) lyophilised aliquots (Ct value 22.04?±?5.5); bacterial 16S rRNA was detected in 33/41 (80.5%) non-lyophilised (Ct value 28.11?±?5.9) versus 40/41 (97.6%) lyophilised aliquots (Ct value 24.94?±?6.6); and S. enterica was detected in 6/6 (100%) non-lyophilized and lyophilized aliquots (Ct value 26.98?±?4.55 and 26.16?±?4.97, respectively). S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p?=?0.00043) and Bacteria (p?=?0.015). Conclusion Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea. PMID:23273000

2012-01-01

261

Importance of Micromonospora spp. as Colonizers of Cellulose in Freshwater Lakes as Demonstrated by Quantitative Reverse Transcriptase PCR of 16S rRNA  

PubMed Central

The relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, water columns, and sediments of two freshwater lakes was determined by quantitative PCR (qPCR) of reverse-transcribed 16S rRNA. Micromonospora spp. were shown to be significant members of the active bacterial population colonizing cellulosic substrates in the lake sediment, and their increased prevalence with greater depth was confirmed by enumeration of CFU. PMID:22389367

de Menezes, Alexandre B.; McDonald, James E.; Allison, Heather E.

2012-01-01

262

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

PubMed Central

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

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

2012-01-01

263

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

264

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

PubMed

In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life. PMID:23316189

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

2012-01-01

265

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

266

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

267

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

268

Non-Watson-Crick Basepairing and Hydration in RNA Motifs: Molecular Dynamics of 5S rRNA Loop E  

Microsoft Academic Search

Explicit solvent and counterion molecular dynamics simulations have been carried out for a total of >80ns on the bacterial and spinach chloroplast 5S rRNA Loop E motifs. The Loop E sequences form unique duplex architectures composed of seven consecutive non-Watson-Crick basepairs. The starting structure of spinach chloroplast Loop E was modeled using isostericity principles, and the simulations refined the geometries

Kamila Réblová; Nad’a Špa?ková; Richard Štefl; Kristina Csaszar; Jaroslav Ko?a; Neocles B. Leontis; Ji?í Šponer

2003-01-01

269

Detection and Quantification of Dehalogenimonas and “Dehalococcoides” Populations via PCR-Based Protocols Targeting 16S rRNA Genes? †  

PubMed Central

Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to “Dehalococcoides” but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site. PMID:19820163

Yan, Jun; Rash, Brian A.; Rainey, Fred A.; Moe, William M.

2009-01-01

270

Bacterial vaginosis  

Microsoft Academic Search

Bacterial vaginosis, the most prevalent cause of vaginal discharge in the United States, is characterized microbiologically\\u000a by a shift in the vagina away from a lactobacillus-predominant flora and toward a predominantly anaerobic milieu. The cause\\u000a of bacterial vaginosis is unknown, but the epidemiology of the syndrome suggests that it is sexually associated. Bacterial\\u000a vaginosis has been associated with various complications,

Jane R. Schwebke

2000-01-01

271

16S rRNA gene-based identification of bacteria in postoperative endophthalmitis by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprinting  

PubMed Central

Conventional microbiological culture techniques are frequently insufficient to confirm endophthalmitis clinical cases which could require urgent medical attention because it could lead to permanent vision loss. We are proposing PCR-DGGE and 16S rRNA gene libraries as an alternative to improve the detection and identification rate of bacterial species from endophthalmitis cases. PMID:24031830

Navarro-Noya, Yendi; Hernández-Rodríguez, César; Zenteno, Juan C.; Buentello-Volante, Beatriz; Cancino-Díaz, Mario E.; Jan-Roblero, Janet; Cancino-Díaz, Juan C.

2012-01-01

272

RmtC introduces G1405 methylation in 16S rRNA and confers high-level aminoglycoside resistance on Gram-positive microorganisms.  

PubMed

Seven plasmid-mediated 16S rRNA methyltransferases (MTases), RmtA, RmtB, RmtC, RmtD, RmtE, ArmA, and NpmA, conferring aminoglycoside resistance have so far been found in Gram-negative pathogenic microorganisms. In the present study, by performing an RNase protection assay, primer extension, and HPLC, we confirmed that RmtC indeed methylates at the N7 position of nucleotide G1405 in 16S rRNA as found in ArmA and RmtB. RmtC has an MTase activity specific for the bacterial 30S ribosomal subunit consisting of 16S rRNA and several ribosomal proteins, but not for the naked 16S rRNA, as seen in ArmA, RmtB, and NpmA. All seven 16S rRNA MTases have been found exclusively in Gram-negative bacilli to date, and no plasmid-mediated 16S rRNA MTase has been reported in Gram-positive pathogenic microorganisms. Thus, we checked whether or not the RmtC could function in Gram-positive bacilli, and found that RmtC could indeed confer high-level resistance to gentamicin and kanamycin in Bacillus subtilis and Staphylococcus aureus. 16S rRNA MTases seemed to be functional to some extent in any bacterial species, regardless of the provenance of the 16S rRNA MTase gene responsible for aminoglycoside resistance. PMID:20722735

Wachino, Jun-Ichi; Shibayama, Keigo; Kimura, Kouji; Yamane, Kunikazu; Suzuki, Satowa; Arakawa, Yoshichika

2010-10-01

273

Microbial community of salt crystals processed from Mediterranean seawater based on 16S rRNA analysis.  

PubMed

Phylogenetic analysis of 16S rRNA was used to investigate for the first time the structure of the microbial community that inhabits salt crystals retrieved from the bottom of a solar saltern, located in the coastal area of the Mediterranean Sea (Sfax, Tunisia). This community lives in an extremely salty environment of 250-310 g/L total dissolved salt. A total of 78 bacterial 16S rRNA clone sequences making up to 21 operational taxonomic units (OTUs), determined by the DOTUR program to 97% sequence similarity, was analyzed. These OTUs were affiliated to Bacteroidetes (71.4% of OTUs), and gamma-Proteobacteria and alpha-Proteobacteria (equally represented by 14.2% of the OTUs observed). The archaeal community composition appeared more diverse with 68 clones, resulting in 44 OTUs, all affiliated with the Euryarchaeota phylum. Of the bacterial and archaeal clones showing <97% 16S rRNA sequence identity with sequences in public databases, 47.6% and 84.1% respectively were novel clones. Both rarefaction curves and diversity measurements (Simpson, Shannon-Weaver, Chao) showed a more diverse archaeal than bacterial community at the Tunisian solar saltern pond. The analysis of an increasing clone's number may reveal additional local diversity. PMID:20130693

Baati, Houda; Guermazi, Sonda; Gharsallah, Neji; Sghir, Abdelghani; Ammar, Emna

2010-01-01

274

Inter-laboratory evaluation of the ISO standard 11063 "Soil quality - Method to directly extract DNA from soil samples".  

PubMed

Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring. PMID:21256879

Petric, I; Philippot, L; Abbate, C; Bispo, A; Chesnot, T; Hallin, S; Laval, K; Lebeau, T; Lemanceau, P; Leyval, C; Lindström, K; Pandard, P; Romero, E; Sarr, A; Schloter, M; Simonet, P; Smalla, K; Wilke, B-M; Martin-Laurent, F

2011-03-01

275

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

276

Antibacterial activity of aqueous extract of pomegranate peel against Pseudomonas stutzeri isolated from poultry meat.  

PubMed

In this study antibacterial activity of pomegranate peel (PPE) was evaluated against bacteria isolated from poultry meat. The bacteria were identified using 16S rRNA gene and DNA sequencing. Results of molecular characterization showed that the bacteria isolated were having 100% homology with the Pseudomonas stutzeri strain CTSP36 and further analysis showed that sample sequence clustered with the P. stutzeri strain CTSP36. Antibacterial activity of PPE was demonstrated by clear zone of inhibition in plates inoculated with extract. The diameter of inhibition zones were significantly (p?bacterial growth in test plates. Further, a decrease in the growth of bacterial cells and a gradual decline in protein content of bacterial cells were also observed when bacterial culture was grown with different concentration of PPE along with a control. These results showed the potential application of pomegranate peel extract as antibacterial agent against P. stutzeri. PMID:24425952

Devatkal, Suresh K; Jaiswal, Parnita; Jha, Shyam N; Bharadwaj, Rishi; Viswas, K N

2013-06-01

277

High-throughput 16S rRNA gene sequencing reveals alterations of intestinal microbiota in myalgic encephalomyelitis/chronic fatigue syndrome patients.  

PubMed

Human intestinal microbiota plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. Intestinal dysfunction is a frequent complaint in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients, and previous reports suggest that dysbiosis, i.e. the overgrowth of abnormal populations of bacteria in the gut, is linked to the pathogenesis of the disease. We used high-throughput 16S rRNA gene sequencing to investigate the presence of specific alterations in the gut microbiota of ME/CFS patients from Belgium and Norway. 43 ME/CFS patients and 36 healthy controls were included in the study. Bacterial DNA was extracted from stool samples, PCR amplification was performed on 16S rRNA gene regions, and PCR amplicons were sequenced using Roche FLX 454 sequencer. The composition of the gut microbiota was found to differ between Belgian controls and Norwegian controls: Norwegians showed higher percentages of specific Firmicutes populations (Roseburia, Holdemania) and lower proportions of most Bacteroidetes genera. A highly significant separation could be achieved between Norwegian controls and Norwegian patients: patients presented increased proportions of Lactonifactor and Alistipes, as well as a decrease in several Firmicutes populations. In Belgian subjects the patient/control separation was less pronounced, however some abnormalities observed in Norwegian patients were also found in Belgian patients. These results show that intestinal microbiota is altered in ME/CFS. High-throughput sequencing is a useful tool to diagnose dysbiosis in patients and could help designing treatments based on gut microbiota modulation (antibiotics, pre and probiotics supplementation). PMID:23791918

Frémont, Marc; Coomans, Danny; Massart, Sebastien; De Meirleir, Kenny

2013-08-01

278

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

279

Bacterial vaginosis  

Microsoft Academic Search

Bacterial vaginosis is the most common cause of vaginitis, affecting over 3 million women in the United States annually. Depopulation of lactobacilli from the normal vaginal flora and overgrowth of Gardnerella vaginalis and other anaerobic species are the presumed etiology. To date, no scientific evidence shows that bacterial vaginosis is a sexually transmitted disease. Malodorous vaginal discharge is the most

Jeff Wang

2000-01-01

280

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

281

Specified Species in Gingival Crevicular Fluid Predict Bacterial Diversity  

PubMed Central

Background Analysis of gingival crevicular fluid (GCF) samples may give information of unattached (planktonic) subgingival bacteria. Our study represents the first one targeting the identity of bacteria in GCF. Methodology/Principal Findings We determined bacterial species diversity in GCF samples of a group of periodontitis patients and delineated contributing bacterial and host-associated factors. Subgingival paper point (PP) samples from the same sites were taken for comparison. After DNA extraction, 16S rRNA genes were PCR amplified and DNA-DNA hybridization was performed using a microarray for over 300 bacterial species or groups. Altogether 133 species from 41 genera and 8 phyla were detected with 9 to 62 and 18 to 64 species in GCF and PP samples, respectively, per patient. Projection to latent structures by means of partial least squares (PLS) was applied to the multivariate data analysis. PLS regression analysis showed that species of genera including Campylobacter, Selenomonas, Porphyromonas, Catonella, Tannerella, Dialister, Peptostreptococcus, Streptococcus and Eubacterium had significant positive correlations and the number of teeth with low-grade attachment loss a significant negative correlation to species diversity in GCF samples. OPLS/O2PLS discriminant analysis revealed significant positive correlations to GCF sample group membership for species of genera Campylobacter, Leptotrichia, Prevotella, Dialister, Tannerella, Haemophilus, Fusobacterium, Eubacterium, and Actinomyces. Conclusions/Significance Among a variety of detected species those traditionally classified as Gram-negative anaerobes growing in mature subgingival biofilms were the main predictors for species diversity in GCF samples as well as responsible for distinguishing GCF samples from PP samples. GCF bacteria may provide new prospects for studying dynamic properties of subgingival biofilms. PMID:21049043

Asikainen, Sirkka; Do?an, Ba?ak; Turgut, Zeynep; Paster, Bruce J.; Bodur, Aysen; Oscarsson, Jan

2010-01-01

282

Effects of Functionalized and Raw Multi-Walled Carbon Nanotubes on Soil Bacterial Community Composition  

PubMed Central

Carbon nanotubes (CNTs) are widely used in industry, but their environmental impacts on soil microbial communities are poorly known. In this paper, we compare the effect of both raw and acid treated or functionalized (fCNTs) multi-walled carbon nanotubes (MWCNTs) on soil bacterial communities, applying different concentrations of MWCNTs (0 µg/g, 50 µg/g, 500 µg/g and 5000 µg/g) to a soil microcosm system. Soil DNA was extracted at 0, 2 and 8 weeks and the V3 region of the 16S rRNA gene was PCR-amplified and sequenced using paired-end Illumina bar-coded sequencing. The results show that bacterial diversity was not affected by either type of MWCNT. However, overall soil bacterial community composition, as illustrated by NMDS, was affected only by fMWCNT at high concentrations. This effect, detectable at 2 weeks, remained equally strong by 8 weeks. In the case of fMWCNTs, overall changes in relative abundance of the dominant phyla were also found. The stronger effect of fMWCNTs could be explained by their intrinsically acidic nature, as the soil pH was lower at higher concentrations of fMWCNTs. Overall, this study suggests that fMWCNTs may at least temporarily alter microbial community composition on the timescale of at least weeks to months. It appears, by contrast, that raw MWCNTs do not affect soil microbial community composition. PMID:25825905

Kerfahi, Dorsaf; Tripathi, Binu M.; Singh, Dharmesh; Kim, Hyoki; Lee, Sujin; Lee, Junghoon; Adams, Jonathan M.

2015-01-01

283

Phylogenetic Diversity and Spatial Distribution of the Microbial Community Associated with the Caribbean Deep-water Sponge Polymastia cf. corticata by 16S rRNA, aprA , and amoA Gene Analysis  

Microsoft Academic Search

Denaturing gradient gel electrophoresis (DGGE)-based analyses of 16S rRNA, aprA, and amoA genes demonstrated that a phylogenetically diverse and complex microbial community was associated with the Caribbean deep-water\\u000a sponge Polymastia cf. corticata Ridley and Dendy, 1887. From the 38 archaeal and bacterial 16S rRNA phylotypes identified, 53% branched into the sponge-specific,\\u000a monophyletic sequence clusters determined by previous studies (considering predominantly

Birte Meyer; Jan Kuever

2008-01-01

284

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

285

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

286

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

287

Different bacterial populations associated with the roots and rhizosphere of rice incorporate plant-derived carbon.  

PubMed

Microorganisms associated with the roots of plants have an important function in plant growth and in soil carbon sequestration. Rice cultivation is the second largest anthropogenic source of atmospheric CH4, which is a significant greenhouse gas. Up to 60% of fixed carbon formed by photosynthesis in plants is transported below ground, much of it as root exudates that are consumed by microorganisms. A stable isotope probing (SIP) approach was used to identify microorganisms using plant carbon in association with the roots and rhizosphere of rice plants. Rice plants grown in Italian paddy soil were labeled with (13)CO2 for 10 days. RNA was extracted from root material and rhizosphere soil and subjected to cesium gradient centrifugation followed by 16S rRNA amplicon pyrosequencing to identify microorganisms enriched with (13)C. Thirty operational taxonomic units (OTUs) were labeled and mostly corresponded to Proteobacteria (13 OTUs) and Verrucomicrobia (8 OTUs). These OTUs were affiliated with the Alphaproteobacteria, Betaproteobacteria, and Deltaproteobacteria classes of Proteobacteria and the "Spartobacteria" and Opitutae classes of Verrucomicrobia. In general, different bacterial groups were labeled in the root and rhizosphere, reflecting different physicochemical characteristics of these locations. The labeled OTUs in the root compartment corresponded to a greater proportion of the 16S rRNA sequences (?20%) than did those in the rhizosphere (?4%), indicating that a proportion of the active microbial community on the roots greater than that in the rhizosphere incorporated plant-derived carbon within the time frame of the experiment. PMID:25616793

Hernández, Marcela; Dumont, Marc G; Yuan, Quan; Conrad, Ralf

2015-03-15

288

Bacterial Vaginosis  

MedlinePLUS

... Archive STDs Home Page Bacterial Vaginosis (BV) Chlamydia Gonorrhea Genital Herpes HIV/AIDS & STDs Human Papillomavirus (HPV) ... of getting other STDs, such as chlamydia and gonorrhea . These bacteria can sometimes cause pelvic inflammatory disease ( ...

289

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

290

Influence of Commonly Used Primer Systems on Automated Ribosomal Intergenic Spacer Analysis of Bacterial Communities in Environmental Samples  

PubMed Central

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected. PMID:25749323

Francioli, Davide; Reitz, Thomas; Buscot, François; Schloter, Michael; Krüger, Dirk

2015-01-01

291

Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples.  

PubMed

Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected. PMID:25749323

Purahong, Witoon; Stempfhuber, Barbara; Lentendu, Guillaume; Francioli, Davide; Reitz, Thomas; Buscot, François; Schloter, Michael; Krüger, Dirk

2015-01-01

292

A randomized, double-blind, placebo-controlled trial to assess the bacterial anti-adhesion effects of cranberry extract beverages.  

PubMed

In this study, we examined the ex vivo urinary anti-adhesion activity of low-calorie cranberry extract beverages in both a pilot study (n = 10) and a randomized, double-blind, placebo controlled clinical trial (n = 59). In the pilot study, subjects consumed a cranberry extract beverage (CEB) or a cranberry extract and juice beverage (CEJB), compared to placebo. Both cranberry beverages utilized a standardized cranberry extract powder at a level equivalent to low-calorie cranberry juice cocktail (LCJC) on a PAC content basis. Clean-catch urine samples collected at baseline and post intervention were tested for anti-adhesion activity utilizing a mannose-resistant human red blood cell hemagglutination assay specific for P-fimbriated E. coli. Results from the pilot study indicated that ex vivo anti-adhesion activity for both cranberry treatments were higher (p < 0.05) than placebo. In the clinical trial, we compared CEJB to LCJC and a placebo beverage. Post-consumption urine from both cranberry treatment groups showed significantly higher (p < 0.05) anti-adhesion activity compared to placebo. There were no differences observed in anti-adhesion activity between CJEB and LCJC, indicating similar bioactivity. Therefore, acute beverage consumption of cranberry extract and/or juice provides ex vivo anti-adhesion activity, which may help to improve urinary tract health. PMID:25723356

Kaspar, Kerrie L; Howell, Amy B; Khoo, Christina

2015-04-01

293

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

PubMed Central

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

1996-01-01

294

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

PubMed Central

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

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

2002-01-01

295

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

PubMed Central

The high throughput and cost-effectiveness afforded by short-read sequencing technologies, in principle, enable researchers to perform 16S rRNA profiling of complex microbial communities at unprecedented depth and resolution. Existing Illumina sequencing protocols are, however, limited by the fraction of the 16S rRNA gene that is interrogated and therefore limit the resolution and quality of the profiling. To address this, we present the design of a novel protocol for shotgun Illumina sequencing of the bacterial 16S rRNA gene, optimized to amplify more than 90% of sequences in the Greengenes database and with the ability to distinguish nearly twice as many species-level OTUs compared to existing protocols. Using several in silico and experimental datasets, we demonstrate that despite the presence of multiple variable and conserved regions, the resulting shotgun sequences can be used to accurately quantify the constituents of complex microbial communities. The reconstruction of a significant fraction of the 16S rRNA gene also enabled high precision (>90%) in species-level identification thereby opening up potential application of this approach for clinical microbial characterization. PMID:23579286

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

2013-01-01

296

In-Vitro, Anti-Bacterial Activities of Aqueous Extracts of Acacia catechu (L.F.)Willd, Castanea sativa, Ephedra sinica stapf and shilajita mumiyo Against Gram Positive and Gram Negative Bacteria  

PubMed Central

Objective: Evaluations of the in-vitro anti-bacterial activities of aqueous extracts of Acacia catechu (L.F.)Willd, Castanea sativa, Ephedra sinica stapf and Shilajita mumiyo against gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumonia) and gram-negative bacteria (Escherichia coli, klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa) are reasonable since these ethnomedicinal plants have been used in Persian folk medicine for treating skin diseases, venereal diseases, respiratory problems and nervous disorders for ages. Methods: The well diffusion method (KB testing) with a concentration of 250 ?g/disc was used for evaluating the minimal inhibitory concentrations (MIC). Maximum synergistic effects of different combinations of components were also observed. Results: A particular combination of Acacia catechu (L.F.) Willd, Castanea sativa, Ephedra sinica stapf and shilajita mumiyo extracts possesses an outstanding anti-bacterial activity. It's inhibiting effect on microorganisms is significant when compared to the control group (P< 0.05). Staphylococcus aureus was the most sensitive microorganism. The highest antibacterial activity against gram-positive bacteria (Staphylococcus aureus, Streptococcus pneumonia) or gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, Proteus mirabilis and Pseudomonas aeruginosa) was exerted by formula number 2 (Table1). Conclusion: The results reveal the presence of antibacterial activities of Acacia catechu, Castanea sativa husk, Ephedra sp. and Mumiyo against gram-positive and gram-negative bacteria. Synergistic effects in a combined formula, especially in formula number 2 (ASLAN?) can lead to potential sources of new antiseptic agents for treatment of acute or chronic skin ulcers. These results considering the significant antibacterial effect of the present formulation, support ethno-pharmacological uses against diarrheal and venereal diseases and demonstrate use of these plants to treat infectious diseases.

Dashtdar, Mehrab; Dashtdar, Mohammad Reza; Dashtdar, Babak; shirazi, Mohammad khabaz; Khan, Saeed Ahmad

2013-01-01

297

Changes in Bacterioplankton Community Structure and Activity with Depth in a Eutrophic Lake as Revealed by 5S rRNA Analysis  

PubMed Central

The community structure of bacterioplankton was studied at different depths (0 to 25 m) of a temperate eutrophic lake (Lake Plußsee in northern Germany) by using comparative 5S rRNA analysis. The relative amounts of taxonomic groups were estimated from 5S rRNA bands separated by high-resolution electrophoresis. Comparison of partial 5S rRNA sequences enabled detection of changes in single taxa over space and during seasons. Overall, the bacterioplankton community was dominated by 3 to 14 abundant (>4% of the total 5S rRNA) taxa. In general, the number of 5S rRNA bands (i.e., the number of bacterial taxa) decreased with depth. In the fall, when thermal stratification and chemical stratification were much more pronounced than they were in the spring, the correlation between the depth layers and the community structure was more pronounced. Therefore, in the fall each layer had its own community structure; i.e., there were different community structures in the epilimnion, the metalimnion, and the hypolimnion. Only three 5S rRNA bands were detected in the hypolimnion during the fall, and one band accounted for about 70% of the total 5S rRNA. The sequences of individual 5S rRNA bands from the spring and fall were different for all size classes analyzed except two bands, one of which was identified as Comamonas acidivorans. In the overall analysis of the depth profiles, the diversity in the epilimnion contrasted with the reduced diversity of the bacterioplankton communities in the hypolimnion, and large differences occurred in the composition of the epilimnion at different seasons except for generalists like C. acidivorans. PMID:12089049

Dominik, Katja; Höfle, Manfred G.

2002-01-01

298

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

299

Extract from Rumex acetosa L. for Prophylaxis of Periodontitis: Inhibition of Bacterial In Vitro Adhesion and of Gingipains of Porphyromonas gingivalis by Epicatechin-3-O-(4??8)-Epicatechin-3-O-Gallate (Procyanidin-B2-Di-Gallate)  

PubMed Central

Background The aerial parts of Rumex acetosa L. have been used in traditional European medicine for inflammatory diseases of the mouth epithelial tissue. The following study aimed to investigate the influence of a proanthocyanidin-enriched extract from R. acetosa extract against the adhesion of Porphyromonas gingivalis (P. gingivalis), a pathogen strongly involved in chronic and aggressive periodontitis. A further goal was to define the bioactive lead structures responsible for a potential antiadhesive activity and to characterize the underlying molecular mechanisms of the antiadhesive effects. Methodology An extract of R. acetosa (RA1) with a defined mixture of flavan-3-ols, oligomeric proanthocyanidins and flavonoids, was used. Its impact on P. gingivalis adhesion to KB cells was studied by flow cytometry, confocal laser scanning microscopy and in situ adhesion assay using murine buccal tissue. RA1 and its compounds 1 to 15 were further investigated for additional effects on gingipain activity, hemagglutination and gene expression by RT-PCR. Principal Findings RA1 (5 to 15 ?g/mL) reduced P. gingivalis adhesion in a dose-dependent manner to about 90%. Galloylated proanthocyanidins were confirmed to be responsible for this antiadhesive effect with epicatechin-3-O-gallate-(4?,8)-epicatechin-3’-O-gallate (syn. procyanidin B2-di-gallate) being the lead compound. Ungalloylated flavan-3-ols and oligomeric proanthocyanidins were inactive. RA1 and the galloylated proanthocyanidins strongly interact with the bacterial virulence factor Arg-gingipain, while the corresponding Lys-gingipain was hardly influenced. RA1 inhibited also hemagglutination. In silico docking studies indicated that epicatechin-3-O-gallate-(4?,8)-epicatechin-3’-O-gallate interacts with the active side of Arg-gingipain and hemaglutinin from P. gingivalis; the galloylation of the molecule seems to be responsible for fixation of the ligand to the protein. In conclusion, the proanthocyanidin-enriched extract RA1 and its main active constituent procyanidin B2-di-gallate protect cells from P. gingivalis infection by inhibiting bacterial adhesion to the host cell. RA1 and procyanidin B2-di-gallate appear to be promising candidates for future cytoprotective preparations for oral mouth care products. PMID:25803708

Schmuch, Jana; Beckert, Sabine; Brandt, Simone; Löhr, Gesine; Hermann, Fabian; Schmidt, Thomas J.; Beikler, Thomas; Hensel, Andreas

2015-01-01

300

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

301

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

E-print Network

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

302

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

PubMed Central

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

Dohrmann, Anja B.; Tebbe, Christoph C.

2005-01-01

303

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

304

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

Microsoft Academic Search

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

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

1993-01-01

305

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

306

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

PubMed Central

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

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

1991-01-01

307

Dominant obligate anaerobes revealed in lower respiratory tract infection in horses by 16S rRNA gene sequencing.  

PubMed

Obligate anaerobes are important etiological agents in pneumonia or pleuropneumonia in horses, because they are isolated more commonly from ill horses that have died or been euthanized than from those that survive. We performed bacterial identification and antimicrobial susceptibility testing for obligate anaerobes to establish effective antimicrobial therapy. We used 16S rRNA gene sequencing to identify 58 obligate anaerobes and compared the results with those from a phenotypic identification kit. The identification results of 16S rRNA gene sequencing were more reliable than those of the commercial kit. We concluded that genera Bacteroides and Prevotella-especially B. fragilis and P. heparinolytica-are dominant anaerobes in lower respiratory tract infection in horses; these organisms were susceptible to metronidazole, imipenem and clindamycin. PMID:24366152

Kinoshita, Yuta; Niwa, Hidekazu; Katayama, Yoshinari; Hariu, Kazuhisa

2014-04-01

308

Up to Species-level Community Analysis of Human Gut Microbiota by 16S rRNA Amplicon Pyrosequencing  

PubMed Central

Pyrosequencing-based 16S rRNA profiling has become a common powerful tool to obtain the community structure of gastrointestinal tract microbiota, but it is still hard to process the massive amount of sequence data into microbial composition data, especially at the species level. Here we propose a new approach in combining the quantitative insights into microbial ecology (QIIME), Mothur and ribosomal database project (RDP) programs to efficiently process 454 pyrosequence data to bacterial composition data up to the species level. It was demonstrated to precisely convert batch sequence data of 16S rRNA V6-V8 amplicons obtained from adult Singaporean fecal samples to taxonomically annotated biota data. PMID:24936364

NAKAYAMA, Jiro; JIANG, Jiahui; WATANABE, Koichi; CHEN, Kangting; NINXIN, Huang; MATSUDA, Kazunori; KURAKAWA, Takashi; TSUJI, Hirokazu; SONOMOTO, Kenji; LEE, Yuan-Kun

2013-01-01

309

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

310

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods  

Microsoft Academic Search

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly\\u000a used blood culture materials, i.e. BACTEC and BacT\\/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation\\u000a for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex\\u000a assay was

A. J. M. Loonen; A. R. Jansz; H. Kreeftenberg; C. A. Bruggeman; P. F. G. Wolffs

2011-01-01

311

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in the genetic diversity of bacterial populations  

Microsoft Academic Search

A mesocosm experiment was performed to study the influence of nutrients on activity and diversity of bacterial assemblages from the Mediterranean Sea. Changes in the diversity of the predominant bacterial populations were monitored by DGGE fingerprinting of PCR products derived from 16S rRNA encoding genes. Fluctuations in the diversity of the most active populations was inferred by performing the DGGE

Hendrik Schäfer; Laetitia Bernard; Claude Courties; Philippe Lebaron; Pierre Servais; Rüdiger Pukall; Erko Stackebrandt; Marc Troussellier; Teresa Guindulain; Josep Vives-Rego; Gerard Muyzer

2001-01-01

312

Terrestrial Runoff Controls the Bacterial Community Composition of Biofilms along a Water Quality Gradient in the Great Barrier Reef  

PubMed Central

16S rRNA gene molecular analysis elucidated the spatiotemporal distribution of bacterial biofilm communities along a water quality gradient. Multivariate statistics indicated that terrestrial runoff, in particular dissolved organic carbon and chlorophyll a concentrations, induced shifts of specific bacterial communities between locations and seasons, suggesting microbial biofilms could be suitable bioindicators for water quality. PMID:22904059

Wild, Christian; Uthicke, Sven

2012-01-01

313

Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing.  

PubMed

This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1-V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs. PMID:25656184

Pajarillo, Edward Alain B; Chae, Jong Pyo; Balolong, Marilen P; Kim, Hyeun Bum; Seo, Kang-Seok; Kang, Dae-Kyung

2015-04-01

314

Characterization of the Fecal Microbial Communities of Duroc Pigs Using 16S rRNA Gene Pyrosequencing  

PubMed Central

This study characterized the fecal bacterial community structure and inter-individual variation in 30-week-old Duroc pigs, which are known for their excellent meat quality. Pyrosequencing of the V1–V3 hypervariable regions of the 16S rRNA genes generated 108,254 valid reads and 508 operational taxonomic units at a 95% identity cut-off (genus level). Bacterial diversity and species richness as measured by the Shannon diversity index were significantly greater than those reported previously using denaturation gradient gel electrophoresis; thus, this study provides substantial information related to both known bacteria and the untapped portion of unclassified bacteria in the population. The bacterial composition of Duroc pig fecal samples was investigated at the phylum, class, family, and genus levels. Firmicutes and Bacteroidetes predominated at the phylum level, while Clostridia and Bacteroidia were most abundant at the class level. This study also detected prominent inter-individual variation starting at the family level. Among the core microbiome, which was observed at the genus level, Prevotella was consistently dominant, as well as a bacterial phylotype related to Oscillibacter valericigenes, a valerate producer. This study found high bacterial diversity and compositional variation among individuals of the same breed line, as well as high abundance of unclassified bacterial phylotypes that may have important functions in the growth performance of Duroc pigs. PMID:25656184

Pajarillo, Edward Alain B.; Chae, Jong Pyo; Balolong, Marilen P.; Kim, Hyeun Bum; Seo, Kang-Seok; Kang, Dae-Kyung

2015-01-01

315

Phylogenetic mapping of bacterial morphology  

NASA Technical Reports Server (NTRS)

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

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

1998-01-01

316

Bacterial Wilt  

Microsoft Academic Search

Bacterial wilt is caused by the bacterium Curtobacterium flaccumfaciens subsp. flaccumfaciens. This pathogen grows throughout the water conducting tissues of the plant and impedes water movement, resulting in a wilt. Symptom development is favored by temperatures greater than 90°F. Infection is often caused by the planting of infected seed, but the pathogen may also survive in infested crop debris. Wilt

Howard F. Schwartz; David H. Gent; Gary D. Franc; Robert M. Harveson

317

Bacterial vaginosis  

Microsoft Academic Search

Bacterial vaginosis is the commonest cause of abnormal vaginal discharge in women of childbearing age, with a prevalence as high as 50% in some communities. The symptoms of discharge and offensive smell can cause considerable distress, although 50% of women are asymptomatic when diagnosed. Microbiologically the usually dominant lactobacillus flora is overwhelmed by an overgrowth of predominantly anaerobic organisms, accompanied

Phillip Hay

2010-01-01

318

Bacterial vaginosis  

Microsoft Academic Search

Bacterial vaginosis is a common cause of abnormal discharge in women of child-bearing age. It is present in 10–20% women in the UK, and may recur or regress spontaneously. It is not regarded as an STI because it can occur in virgin women, but it is more common in sexually active women. Other associations include smoking, partner change, having a

Phillip Hay

2005-01-01

319

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

320

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

321

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

PubMed

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

Schulz, C; Faisal, M

2010-06-01

322

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

PubMed Central

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

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

2012-01-01

323

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

324

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

325

Biodegradation of Leonardite by an alkali-producing bacterial community and characterization of the degraded products.  

PubMed

In this study, three bacterial communities were obtained from 12 Leonardite samples with the aim of identifying a clean, effective, and economic technique for the dissolution of Leonardite, a type of low-grade coal, in the production of humic acid (HA). The biodegradation ability and characteristics of the degraded products of the most effective bacterial community (MCSL-2), which degraded 50% of the Leonardite within 21 days, were further investigated. Analyses of elemental composition, (13)C NMR, and Fourier transform infrared revealed that the contents of C, O, and aliphatic carbon were similar in biodegraded humic acid (bHA) and chemically (alkali) extracted humic acid (cHA). However, the N and carboxyl carbon contents of bHA was higher than that of cHA. Furthermore, a positive correlation was identified between the degradation efficiency and the increasing pH of the culture medium, while increases of manganese peroxidase and esterase activities were also observed. These data demonstrated that both alkali production and enzyme reactions were involved in Leonardite solubilization by MCSL-2, although the former mechanism predominated. No fungus was observed by microscopy. Only four bacterial phylotypes were recognized, and Bacillus licheniformis-related bacteria were identified as the main group in MCSL-2 by analysis of amplified 16S rRNA genes, thus demonstrating that Leonardite degradation ability has a limited distribution in bacteria. Hormone-like bioactivities of bHA were also detected. In this study, a bacterial community capable of Leonardite degradation was identified and the products characterized. These data implicate the use of such bacteria for the exploitation of Leonardite as a biofertilizer. PMID:22075634

Gao, Tong-Guo; Jiang, Feng; Yang, Jin-Shui; Li, Bao-Zhen; Yuan, Hong-Li

2012-03-01

326

Atmospheric cloud water contains a diverse bacterial community  

NASA Astrophysics Data System (ADS)

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 17-21 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, Peter S.; Hill, Kimberly A.; Shepson, Paul B.; Konopka, Allan

2011-09-01

327

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

328

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

329

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

330

Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker†  

PubMed Central

For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions. PMID:11375206

Cubero, J.; Graham, J. H.; Gottwald, T. R.

2001-01-01

331

Comparison of two approaches for the classification of 16S rRNA gene sequences.  

PubMed

The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80?% of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5?%. Up to 1.4?% of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods. PMID:25062942

Chatellier, Sonia; Mugnier, Nathalie; Allard, Françoise; Bonnaud, Bertrand; Collin, Valérie; van Belkum, Alex; Veyrieras, Jean-Baptiste; Emler, Stefan

2014-10-01

332

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

333

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

PubMed Central

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

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

2014-01-01

334

Wide Distribution and Diversity of Members of the Bacterial Kingdom Acidobacterium in the Environment  

PubMed Central

To assess the distribution and diversity of members of the recently identified bacterial kingdom Acidobacterium, members of this kingdom present in 43 environmental samples were surveyed by PCR amplification. A primer designed to amplify rRNA gene sequences (ribosomal DNAs [rDNAs]) from most known members of the kingdom was used to interrogate bulk DNA extracted from the samples. Positive PCR results were obtained with all temperate soil and sediment samples tested, as well as some hot spring samples, indicating that members of this kingdom are very widespread in terrestrial environments. PCR primers specific for four phylogenetic subgroups within the kingdom were used in similar surveys. All four subgroups were detected in most neutral soils and some sediments, while only two of the groups were seen in most low-pH environments. The combined use of these primers allowed identification of a novel lineage within the kingdom in a hot spring environment. Phylogenetic analysis of rDNA sequences from our survey and the literature outlines at least six major subgroups within the kingdom. Taken together, these data suggest that members of the Acidobacterium kingdom are as genetically and metabolically diverse, environmentally widespread and perhaps as ecologically important as the well-known Proteobacteria and gram-positive bacterial kingdoms. PMID:10103274

Barns, Susan M.; Takala, Shannon L.; Kuske, Cheryl R.

1999-01-01

335

Assessment of fecal bacterial diversity among healthy piglets during the weaning transition.  

PubMed

The high level of genetic diversity in the microflora of the gastrointestinal tract has the potential to provide numerous beneficial functions to the host. Thus it is now acknowledged that the complexity in animal functioning is linked to the interacting microbiome in the gut. Despite the importance of gut microbiome, there is a lack of information concerning the microbial communities in the pig gut during the weaning transition. This study describes the fecal microbial shifts of healthy piglets during the weaning transition using barcoded pyrosequencing of the prokaryotic 16S rRNA gene. Fecal samples were obtained from 15 piglets during the pre-weaning period (fourth week after birth) and post-weaning (sixth week after birth) and were subjected to community genomic DNA extraction for pyrosequencing analysis. As the piglets underwent the weaning transition a trend toward increased bacterial diversity was observed, based on species abundance as measured by the Shannon-Weaver index. Firmicutes (54.0%) and Bacteroidetes (59.6%) were the most dominant phyla during pre-weaning and post-weaning, respectively. During the weaning transition a distinct shift from Bacteroides to Prevotella as the most abundant genus was observed. Additionally, we detected a number of abundant gut bacterial species that have not been reported previously. Clostridium rectum, C. clostridioforme, C. lactatifermentans and Butyricimonas virosa were uniquely detected prior to weaning while Roseburia cecicola and Blautia wexlerae were detected during the post-weaning period only. PMID:25273987

Alain B Pajarillo, Edward; Chae, Jong-Pyo; P Balolong, Marilen; Bum Kim, Hyeun; Kang, Dae-Kyung

2014-01-01

336

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

PubMed

Oceanic oxygen minimum zones (OMZs) have a central role in biogeochemical cycles and are expanding as a consequence of climate change, yet how deoxygenation will affect the microbial communities that control these cycles is unclear. Here we sample across dissolved oxygen gradients in the oceans' largest OMZ and show that bacterial richness displays a unimodal pattern with decreasing dissolved oxygen, reaching maximum values on the edge of the OMZ and decreasing within it. Rare groups on the OMZ margin are abundant at lower dissolved oxygen concentrations, including sulphur-cycling Chromatiales, for which 16S rRNA was amplified from extracted RNA. Microbial species distribution models accurately replicate community patterns based on multivariate environmental data, demonstrate likely changes in distributions and diversity in the eastern tropical North Pacific Ocean, and highlight the sensitivity of key bacterial groups to deoxygenation. Through these mechanisms, OMZ expansion may alter microbial composition, competition, diversity and function, all of which have implications for biogeochemical cycling in OMZs. PMID:24162368

Beman, J Michael; Carolan, Molly T

2013-01-01

337

Analysis of stomach bacterial communities in Australian feral horses.  

PubMed

We investigated the community structure of bacteria that populate the stomach of the Brumby, a breed of feral horses from the Australian outback. Using a 16S rRNA gene clone library, we identified 155 clones that were assigned to 26 OTUs based on a 99.0 % sequence identity cutoff. Two OTUs represented 73.5 % of clones, while 18 OTUs were each assigned only a single clone. Four major bacterial types were identified in the Brumby stomach: Lactobacillaceae, Streptococcaceae, Veillonellaceae and Pasteurellaceae. The first three groups, which represented 98.1 % of the Brumby stomach library clones, belonged to the bacterial phylum Firmicutes. We found that 49.7 % of clones were related to bacterial species previously identified in the equine hindgut, and that 44.5 % of clones were related to symbiotic bacterial species identified in the mouth or throat of either horses or other mammals. Our results indicated that the composition of mutualistic bacterial communities of feral horses was consistent with other studies on domestic horses. In addition to bacterial sequences, we also identified four plastid 16S rRNA gene sequences, which may help in further characterizing the type of vegetation consumed by Brumby horses in their natural environment. PMID:23065252

St-Pierre, Benoit; de la Fuente, Gabriel; O'Neill, Sean; Wright, André-Denis G; Al Jassim, Rafat

2013-01-01

338

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

339

Hot-alkaline DNA extraction method for deep-subseafloor archaeal communities.  

PubMed

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

Morono, Yuki; Terada, Takeshi; Hoshino, Tatsuhiko; Inagaki, Fumio

2014-03-01

340

16S rRNA gene sequence analysis of halophilic and halotolerant bacteria isolated from a hypersaline pond in Sichuan, China  

Microsoft Academic Search

One hundred and twenty bacterial isolates were obtained from a hypersaline pond (c. 22% salinity) in Sichuan, China. Bacteria\\u000a were isolated from hypersaline water, sediment and soil samples using three culture media and an incubation temperature of\\u000a 37°C. Of these isolates, 47 were selected and examined by phylogenetic analysis of 16S rRNA gene sequences and by tests of\\u000a salt tolerance.

Jie Tang; Ai-ping Zheng; Eden S. P. Bromfield; Jun Zhu; Shuang-cheng Li; Shi-quan Wang; Qi-ming Deng; Ping Li

2011-01-01

341

Dominant bacterioplankton populations in the meromictic Lake Suigetsu as determined by denaturing gradient gel electrophoresis of 16S rRNA gene fragments  

Microsoft Academic Search

Lake Suigetsu is a typical meromictic lake in Japan characterized by a permanent chemocline at a depth of between 3 and 8 m\\u000a separating the oxic freshwater mixolimnion from anoxic saline sulfidogenic monimolimnion. Dominant bacterioplankton populations\\u000a in Lake Suigetsu were investigated using PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The\\u000a bacterial population was vertically stratified, and temporal shifts

Ryuji Kondo; Akie Nakagawa; Lisa Mochizuki; Kyoko Osawa; Yukiyasu Fujioka; Junki Butani

2009-01-01

342

Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments  

Microsoft Academic Search

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was\\u000a used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and ?-Proteobacteria. Sequencing\\u000a of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying\\u000a the new primer sets, the bacterial diversity in the chemoclines of a eutrophic

Jörg Overmann; Marco J. L. Coolen; Christian Tuschak

1999-01-01

343

Multiplex PCR with 16S rRNA Gene-Targeted Primers of Bifidobacterium spp. To Identify Sources of Fecal Pollution  

Microsoft Academic Search

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B.

X. Bonjoch; E. Balleste; A. R. Blanch

2004-01-01

344

Ecological Significance of Microdiversity: Identical 16S rRNA Gene Sequences Can Be Found in Bacteria with Highly Divergent Genomes and Ecophysiologies  

Microsoft Academic Search

A combination of cultivation-based methods with a molecular biological approach was used to investigate whether planktonic bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of 11 strains of Brevundimonas alba were isolated from a bacterial freshwater community by conventional plating or by using a liquid most-probable-number (MPN) dilution series. These strains had identical

Elke Jaspers; Jorg Overmann

2004-01-01

345

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

Microsoft Academic Search

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

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

2005-01-01

346

Oxazolidinone Resistance Mutations in 23S rRNA of Escherichia coli Reveal the Central Region of Domain V as the Primary Site of Drug Action  

Microsoft Academic Search

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A,

LIQUN XIONG; PATRICIA KLOSS; STEPHEN DOUTHWAITE; NIELS MØLLER ANDERSEN; STEVEN SWANEY; DEAN L. SHINABARGER; ALEXANDER S. MANKIN

2000-01-01

347

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

348

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

349

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

PubMed

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained. PMID:16391020

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

2006-01-01

350

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

351

In silico approach to study adaptive divergence in nucleotide composition of the 16S rRNA gene among bacteria thriving under different temperature regimes.  

PubMed

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

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

2014-10-01

352

Jellyfish Modulate Bacterial Dynamic and Community Structure  

PubMed Central

Jellyfish blooms have increased in coastal areas around the world and the outbreaks have become longer and more frequent over the past few decades. The Mediterranean Sea is among the heavily affected regions and the common bloom - forming taxa are scyphozoans Aurelia aurita s.l., Pelagia noctiluca, and Rhizostoma pulmo. Jellyfish have few natural predators, therefore their carcasses at the termination of a bloom represent an organic-rich substrate that supports rapid bacterial growth, and may have a large impact on the surrounding environment. The focus of this study was to explore whether jellyfish substrate have an impact on bacterial community phylotype selection. We conducted in situ jellyfish - enrichment experiment with three different jellyfish species. Bacterial dynamic together with nutrients were monitored to assess decaying jellyfish-bacteria dynamics. Our results show that jellyfish biomass is characterized by protein rich organic matter, which is highly bioavailable to ‘jellyfish - associated’ and ‘free - living’ bacteria, and triggers rapid shifts in bacterial population dynamics and composition. Based on 16S rRNA clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis, we observed a rapid shift in community composition from unculturable Alphaproteobacteria to culturable species of Gammaproteobacteria and Flavobacteria. The results of sequence analyses of bacterial isolates and of total bacterial community determined by culture independent genetic analysis showed the dominance of the Pseudoalteromonadaceae and the Vibrionaceae families. Elevated levels of dissolved proteins, dissolved organic and inorganic nutrient release, bacterial abundance and carbon production as well as ammonium concentrations characterized the degradation process. The biochemical composition of jellyfish species may influence changes in the amount of accumulated dissolved organic and inorganic nutrients. Our results can contribute insights into possible changes in bacterial population dynamics and nutrient pathways following jellyfish blooms which have important implications for ecology of coastal waters. PMID:22745726

Tinta, Tinkara; Kogovšek, Tjaša; Malej, Alenka; Turk, Valentina

2012-01-01

353

Nonradioactive method to study genetic profiles of natural bacterial communities by PCR-single-strand-conformation polymorphism.  

PubMed Central

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 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems. PMID:8795197

Lee, D H; Zo, Y G; Kim, S J

1996-01-01

354

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

PubMed

Silver nanoparticles (AgNPs) were synthesised using Kalopanax septemlobus plant leaf extracts. UV-visible spectrophotometric, Fourier-transform infrared, electron dispersive X-ray spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses confirmed synthesis of AgNPs. TEM micrographs revealed presence of well-dispersed AgNPs predominantly of small size and different shapes with an average particle size of 30.8 nm. Antimicrobial susceptibility tests of AgNP treatments revealed variability in sensitivity of bacteria Bacillus cereus and Saccharophagus degradans under study. Minimum inhibitory concentration (MIC) values of the AgNPs for B. cereus and S. degradans were found to be 30 and 10 ?g/mL, respectively. The mixed culture of B. cereus and S. degradans treated with AgNPs at 10 ?g/mL showed increase in growth with time, suggesting survival of bacteria in liquid media. The plating of mixed culture before AgNP treatment showed presence of both bacteria, but 24-h-old mixed culture treated with AgNPs at the concentration of 10 ?g/mL showed presence of B. cereus colonies. SEM micrographs revealed damage to S. degradans cells but no effect on B. cereus cells after AgNP treatment. Confocal microscopic observations of AgNP-treated mixed cultures by Nile blue A staining indicated intact polyhydroxyalkanoates producing flourescent cells of B. cereus but damage and deformities in S. degradans cells. This study suggests that AgNPs can selectively inhibit growth of S. degradans and retain B. cereus at MIC of S. degradans. This report is a case study for selective inhibition of one bacteria and growth of the other in a culture using plant-synthesized silver nanoparticles. PMID:25085530

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

2014-09-01

355

The B chromosomes of the African cichlid fish Haplochromis obliquidens harbour 18S rRNA gene copies  

PubMed Central

Background Diverse plant and animal species have B chromosomes, also known as accessory, extra or supernumerary chromosomes. Despite being widely distributed among different taxa, the genomic nature and genetic behavior of B chromosomes are still poorly understood. Results In this study we describe the occurrence of B chromosomes in the African cichlid fish Haplochromis obliquidens. One or two large B chromosome(s) occurring in 39.6% of the analyzed individuals (both male and female) were identified. To better characterize the karyotype and assess the nature of the B chromosomes, fluorescence in situ hybridization (FISH) was performed using probes for telomeric DNA repeats, 18S and 5S rRNA genes, SATA centromeric satellites, and bacterial artificial chromosomes (BACs) enriched in repeated DNA sequences. The B chromosomes are enriched in repeated DNAs, especially non-active 18S rRNA gene-like sequences. Conclusion Our results suggest that the B chromosome could have originated from rDNA bearing subtelo/acrocentric A chromosomes through formation of an isochromosome, or by accumulation of repeated DNAs and rRNA gene-like sequences in a small proto-B chromosome derived from the A complement. PMID:20051104

2010-01-01

356

Structure of ERA in complex with the 3? end of 16S rRNA: Implications for ribosome biogenesis  

SciTech Connect

ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is essential for bacterial cell viability. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the {sub 1531}AUCACCUCCUUA{sub 1542} sequence at the 3' end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in noneukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a 'twist' for noneukaryotic ERA proteins by also recognizing the CCUCC.

Tu, Chao; Zhou, Xiaomei; Tropea, Joseph E.; Austin, Brian P.; Waugh, David S.; Court, Donald L.; Ji, Xinhua; (NCI)

2009-10-09

357

Usefulness of the MicroSeq 500 16S Ribosomal DNA-Based Bacterial Identification System for Identification of Clinically Significant Bacterial Isolates with Ambiguous Biochemical Profiles  

PubMed Central

Due to the inadequate automation in the amplification and sequencing procedures, the use of 16S rRNA gene sequence-based methods in clinical microbiology laboratories is largely limited to identification of strains that are difficult to identify by phenotypic methods. In this study, using conventional full-sequence 16S rRNA gene sequencing as the “gold standard,” we evaluated the usefulness of the MicroSeq 500 16S ribosomal DNA (rDNA)-based bacterial identification system, which involves amplification and sequencing of the first 527-bp fragment of the 16S rRNA genes of bacterial strains and analysis of the sequences using the database of the system, for identification of clinically significant bacterial isolates with ambiguous biochemical profiles. Among 37 clinically significant bacterial strains that showed ambiguous biochemical profiles, representing 37 nonduplicating aerobic gram-positive and gram-negative, anaerobic, and Mycobacterium species, the MicroSeq 500 16S rDNA-based bacterial identification system was successful in identifying 30 (81.1%) of them. Five (13.5%) isolates were misidentified at the genus level (Granulicatella adiacens was misidentified as Abiotrophia defectiva, Helcococcus kunzii was misidentified as Clostridium hastiforme, Olsenella uli was misidentified as Atopobium rimae, Leptotrichia buccalis was misidentified as Fusobacterium mortiferum, and Bergeyella zoohelcum was misidentified as Rimerella anatipestifer), and two (5.4%) were misidentified at the species level (Actinomyces odontolyticus was misidentified as Actinomyces meyeri and Arcobacter cryaerophilus was misidentified as Arcobacter butzleri). When the same 527-bp DNA sequences of these seven isolates were compared to the known 16S rRNA gene sequences in the GenBank, five yielded the correct identity, with good discrimination between the best and second best match sequences, meaning that the reason for misidentification in these five isolates was due to a lack of the 16S rRNA gene sequences of these bacteria in the database of the MicroSeq 500 16S rDNA-based bacterial identification system. In conclusion, the MicroSeq 500 16S rDNA-based bacterial identification system is useful for identification of most clinically important bacterial strains with ambiguous biochemical profiles, but the database of the MicroSeq 500 16S rDNA-based bacterial identification system has to be expanded in order to encompass the rarely encountered bacterial species and achieve better accuracy in bacterial identification. PMID:12734240

Woo, Patrick C. Y.; Ng, Kenneth H. L.; Lau, Susanna K. P.; Yip, Kam-tong; Fung, Ami M. Y.; Leung, Kit-wah; Tam, Dorothy M. W.; Que, Tak-lun; Yuen, Kwok-yung

2003-01-01

358

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

359

Empirical and Theoretical Bacterial Diversity in Four Arizona Soils  

Microsoft Academic Search

Understanding patterns of biodiversity in microbial communities is severely constrained by the difficulty of adequately sampling these complex systems. We illustrate the problem with empirical data from small surveys (200-member 16S rRNA gene clone libraries) of four bacterial soil communities from two locations in Arizona. Among the four surveys, nearly 500 species-level groups (Dunbar et al., Appl. Environ. Microbiol. 65:662-1669,

John Dunbar; Susan M. Barns; Lawrence O. Ticknor; Cheryl R. Kuske

2002-01-01

360

[Atopobium vaginae: characterization and association with pathogenesis of bacterial vaginosis].  

PubMed

Atopobium vaginae was described in 1999 by Rodriguez et al. It is a Gram-positive bacterium producing organic acids (lactic acid, acetic acid, formic acid) as a results of glucose fermentation. It was first found in vaginal swab taken from a healthy woman using 16S rRNA analysis. A. vaginae is associated with bacterial vaginosis and its consequences in upper part of women reproductive organs. PMID:16958231

Romanik, Ma?gorzata; Friedek, Daniela; Wojciechowska-Wieja, Anna; Martirosian, Gayane

2006-05-01

361

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

362

Partitioning of Bacterial Communities between Seawater and Healthy, Black Band Diseased, and Dead Coral Surfaces  

PubMed Central

Distinct partitioning has been observed in the composition and diversity of bacterial communities inhabiting the surface and overlying seawater of three coral species infected with black band disease (BBD) on the southern Caribbean island of Curaçao, Netherlands Antilles. PCR amplification and sequencing of bacterial 16S rRNA genes (rDNA) with universally conserved primers have identified over 524 unique bacterial sequences affiliated with 12 bacterial divisions. The molecular sequences exhibited less than 5% similarity in bacterial community composition between seawater and the healthy, black band diseased, and dead coral surfaces. The BBD bacterial mat rapidly migrates across and kills the coral tissue. Clone libraries constructed from the BBD mat were comprised of eight bacterial divisions and 13% unknowns. Several sequences representing bacteria previously found in other marine and terrestrial organisms (including humans) were isolated from the infected coral surfaces, including Clostridium spp., Arcobacter spp., Campylobacter spp., Cytophaga fermentans, Cytophaga columnaris, and Trichodesmium tenue. PMID:11976091

Frias-Lopez, Jorge; Zerkle, Aubrey L.; Bonheyo, George T.; Fouke, Bruce W.

2002-01-01

363

Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats  

PubMed Central

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied. PMID:9925563

Nübel, Ulrich; Garcia-Pichel, Ferran; Kühl, Michael; Muyzer, Gerard

1999-01-01

364

Characterization of a Bacterial Community in an Abandoned Semiarid Lead-Zinc Mine Tailing Site  

Microsoft Academic Search

Bacterial diversity in mine tailing microbial communities has not been thoroughly investigated despite the correlations that have been observed between the relative microbial diversity and the success of revegetation efforts at tailing sites. This study employed phylogenetic analyses of 16S rRNA genes to compare the bacterial communities present in highly disturbed, extremely (pH 2.7) and moderately (pH 5.7) acidic lead-zinc

Monica O. Mendez; Julia W. Neilson; Raina M. Maier

2008-01-01

365

Investigation of postpartum dairy cows' uterine microbial diversity using metagenomic pyrosequencing of the 16S rRNA gene.  

PubMed

The objective of this study was the use of metagenomic pyrosequencing of the 16S rRNA gene for the investigation of postpartum dairy cows' uterine bacterial diversity. The effect of subcutaneous supplementation of a trace mineral supplement containing Zn, Mn, Se, and Cu (Multimin North America, Inc., Fort Collins, CO) at 230 days of gestation and 260 days of gestation on dairy cows' uterine microbiota was also evaluated. Uterine lavage samples were collected at 35 DIM and were visually scored for the presence of purulent or mucopurulent secretion. The same samples were also used for the acquisition of bacterial DNA. The 16S rRNA genes were individually amplified from each sample. Pyrosequencing of the samples was carried at the Cornell University Life Sciences Core Laboratories Center using Roche 454 GS-FLX System Titanium Chemistry. The Ribosomal Database Project online tools were used for the analysis of the obtained sequences library. Bacteroides spp., Ureaplasma spp., Fusobacterium spp., Peptostreptococcus spp., Sneathia spp., Prevotella spp. and Arcanobacterium spp. prevalence was significantly (P<0.05) higher in samples derived from cows that had a higher uterine lavage sample score. Bacteroides spp., Ureaplasma spp., Fusobacterium spp., and Arcanobacterium spp. prevalence was significantly (P<0.05) higher in samples derived from cows that were not pregnant by 200 DIM. Anaerococcus spp., Peptostreptococcus spp., Parabacteroides spp., and Propionibacterium spp. prevalence was significantly (P<0.05) lower in samples derived from cows that were trace mineral supplemented. PMID:22595139

Machado, V S; Oikonomou, G; Bicalho, M L S; Knauer, W A; Gilbert, R; Bicalho, R C

2012-10-12

366

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

367

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

368

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

369

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

PubMed

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

2014-05-01

370

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

371

Metagenomic evaluation of bacterial and archaeal diversity in the geothermal hot springs of manikaran, India.  

PubMed

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. PMID:25700403

Bhatia, Sonu; Batra, Navneet; Pathak, Ashish; Green, Stefan J; Joshi, Amit; Chauhan, Ashvini

2015-01-01

372

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions  

PubMed Central

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; Bælum, Jacob; Feld, Louise; Holben, William E.

2014-01-01

373

Ten Cases of Actinobaculum schaalii Infection: Clinical Relevance, Bacterial Identification, and Antibiotic Susceptibility  

PubMed Central

Nine of 10 strains of Actinobaculum schaalii caused urinary tract infections in predisposed individuals. Identification included 16S rRNA gene sequence analysis and use of the API Coryne and Rapid ID32A test systems. A. schaalii is easily overlooked due to its slow growth in ambient air and its resemblance to the normal bacterial flora on skin and mucosa. PMID:16208004

Reinhard, Mark; Prag, Jørgen; Kemp, Michael; Andresen, Keld; Klemmensen, Belinda; Højlyng, Niels; Sørensen, Susan Hildebrand; Christensen, Jens Jørgen

2005-01-01

374

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

375

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. PMID:25700403

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

2015-01-01

376

Enterobacter morus sp. nov., a novel Enterobacter species associated with bacterial wilt on mulberry  

Technology Transfer Automated Retrieval System (TEKTRAN)

A mulberry pathogenetic bacterial strain R18-2T isolated from the diseased mulberry root was analyzed by a polyphasic taxonomic study. Comparative 16S rRNA gene sequence analysis combined with rpoB gene sequence analysis allocated the strain R18-2T to the genus Enterobacter. The strain was Gram nega...

377

New Treatments for Bacterial Keratitis  

PubMed Central

Purpose. To review the newer treatments for bacterial keratitis. Data Sources. PubMed literature search up to April 2012. Study Selection. Key words used for literature search: “infectious keratitis”, “microbial keratitis”, “infective keratitis”, “new treatments for infectious keratitis”, “fourth generation fluoroquinolones”, “moxifloxacin”, “gatifloxacin”, “collagen cross-linking”, and “photodynamic therapy”. Data Extraction. Over 2400 articles were retrieved. Large scale studies or publications at more recent dates were selected. Data Synthesis. Broad spectrum antibiotics have been the main stay of treatment for bacterial keratitis but with the emergence of bacterial resistance; there is a need for newer antimicrobial agents and treatment methods. Fourth-generation fluoroquinolones and corneal collagen cross-linking are amongst the new treatments. In vitro studies and prospective clinical trials have shown that fourth-generation fluoroquinolones are better than the older generation fluoroquinolones and are as potent as combined fortified antibiotics against common pathogens that cause bacterial keratitis. Collagen cross-linking was shown to improve healing of infectious corneal ulcer in treatment-resistant cases or as an adjunct to antibiotics treatment. Conclusion. Fourth-generation fluoroquinolones are good alternatives to standard treatment of bacterial keratitis using combined fortified topical antibiotics. Collagen cross-linking may be considered in treatment-resistant infectious keratitis or as an adjunct to antibiotics therapy. PMID:22991650

Wong, Raymond L. M.; Gangwani, R. A.; Yu, Lester W. H.; Lai, Jimmy S. M.

2012-01-01

378

Expression of CD64 on neutrophils can be used to predict the severity of bloodstream infection before broad range 16S rRNA PCR.  

PubMed

The aging population and increased incidence of severe bacterial infection can lead to sepsis. Interest to early identification of endangered patients and identification of pathogen do not always confirm the infection. To use biomarkers can help in early identification of infection and opportunity to start therapy timeously. All biomarkers were defined in 33 out of 96 patients. Thirty-two (97 %) patients had bacterial infection and 1 (3 %) patient had systemic inflammatory response syndrome (SIRS) without infection. PCR confirmed the infection in 27 cases and blood cultures in 8. Area under curve (AUC) for CD64 was 1.00, meanwhile other biomarkers showed 2-fold smaller AUC for positive infection. CD64 index was associated with bacterial infection (p<0.001) and could be used to confirm assessment of SIRS severity (p=0.037). As regards to our results, limited to only 33 patients, CD64 index served as a good parameter to predict bacterial infection and determine severity. The use of broad range 16S ribosomal RNA (rRNA) PCR proved to be an excellent tool to confirm bloodstream infection. The CD64 index had the highest AUC, which exceeded all the others, and could be used to predict the outcome of broad range 16S rRNA PCR from whole blood. However, C-reactive protein (CRP), procalcitonin (PCT) and sCD14 are much easier and faster to measure, but the values could be elevated in other clinical assessments. PMID:25253263

Stubljar, David; Skvarc, Miha

2015-03-01

379

Quantification of Hyphomicrobium Populations in Activated Sludge from an Industrial Wastewater Treatment System as Determined by 16S rRNA Analysis  

PubMed Central

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869T in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge. PMID:10698787

Layton, A. C.; Karanth, P. N.; Lajoie, C. A.; Meyers, A. J.; Gregory, I. R.; Stapleton, R. D.; Taylor, D. E.; Sayler, G. S.

2000-01-01

380

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

PubMed

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

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

2012-04-01

381

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

NASA Astrophysics Data System (ADS)

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

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

2006-12-01

382

Shift in prokaryotic diversity in Arctic sediment along a continuum Glacier -River - Fjord using massive 16S rRNA gene tag sequencing  

NASA Astrophysics Data System (ADS)

In Arctic environment, one of indirect consequences of the global climate warming is the significant amplification of the amount of inland water during the spring thaw resulting from the snow cover and permafrost melting. These freshwater transfers to the coast cause sedimentary transfers. The Arctic fjords that represent deep glacial valleys of the sea are particularly vulnerable systems. Although the previous studies have highlighted potentially the high bacterial diversity in Arctic environment by the pyrosequencing, a new-generation sequencing and high throughput method, does not escape the same bias as the one of classical molecular biology techniques involved at different stages of the analysis. In this context, our objective was to characterize the prokaryotic diversity associated to the sediment transfer along a gradient from the head of the glacier to mud patch sediment in the Goule river streaming in Kongsfjorden (Svalbard) during an active thaw. The prokaryotic diversity in sediment was characterized by combining a massive of 16S rRNA gene tag sequencing with a specific and original approach in order to overcome the bias associated to the sampling and extraction. The sediment was extracted by three different methods. One method was done in duplicate. Negative controls performed at extraction and PCR stages were also sequenced. The phylogenetic analysis of the environmental samples below phylum level revealed significantly changes in the diversity and the function of the prokaryotic community along the gradient. The subglacial Goule river sediment is characterized by bacteria with specific functions methylotroph bacteria, aerobic chemoautolithotrophic bacteria (Alphaproteobacteria with Methylobacteriaceae) whereas the mouth of the river Goule and the freshwater part of the Goule River was dominated by sulphate-reducing-bacteria, anaerobic chemooorganotroph (Deltaprotobacteria with the Desulfobulbaceae and Desulfuromonadaceae) and by chemoheteroorganotrophic aerobic bacteria (Bacteroidetes with the Flavobactericeae and Verrucomicrobia with Verrucomicrobiaceae). It is interesting to note that the Cyanobacteria, phototroph organisms, most of which are specialized in the nitrogen fixation (Nostocaceae), are mainly present in the Goule river sediment. The study of the prokaryotic diversity with our approach was very relevant and important in the context of the representativeness of diversity explored in cold sediment. It allowed to reveal rigorously changes in prokaryotic diversity and specific functions associated to sediment transfer along the Goule River.

Laghdass, M.; Deloffre, J.; Lafite, R.; Hänni, C.; Gillet, B.; Cecillon, S.; Simonet, P.; Petit, F.

2012-04-01

383

Time between collection and storage significantly influences bacterial sequence composition in sputum samples from cystic fibrosis respiratory infections.  

PubMed

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

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

2014-08-01

384

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

385

Bacterial Enteritides of Poultry  

Microsoft Academic Search

Enteric bacterial infections in poultry pose a threat to intestinal health and can contribute to poor feed efficiency and livability of a flock. A variety of enteric bacterial diseases are recognized in poultry. Three of these bacterial diseases, necrotic enteritis, ulcerative enteritis, and spirochetosis, primarily infect the intestine, whereas other bacterial diseases, such as salmonellosis, colibacillosis, mycobacteriosis, erysipelas, and fowl

ROBERT E. PORTER

386

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

387

Ice formation and growth shape bacterial community structure in Baltic Sea drift ice.  

PubMed

Drift ice, open water and under-ice water bacterial communities covering several developmental stages from open water to thick ice were studied in the northern Baltic Sea. The bacterial communities were assessed with 16S rRNA gene terminal-restriction fragment length polymorphism and cloning, together with bacterial abundance and production measurements. In the early stages, open water and pancake ice were dominated by Alphaproteobacteria and Actinobacteria, which are common bacterial groups in Baltic Sea wintertime surface waters. The pancake ice bacterial communities were similar to the open-water communities, suggesting that the parent water determines the sea-ice bacterial community in the early stages of sea-ice formation. In consolidated young and thick ice, the bacterial communities were significantly different from water bacterial communities as well as from each other, indicating community development in Baltic Sea drift ice along with ice-type changes. The thick ice was dominated by typical sea-ice genera from classes Flavobacteria and Gammaproteobacteria, similar to those in polar sea-ice bacterial communities. Since the thick ice bacterial community was remarkably different from that of the parent seawater, results indicate that thick ice bacterial communities were recruited from the rarer members of the seawater bacterial community. PMID:25764550

Eronen-Rasimus, Eeva; Lyra, Christina; Rintala, Janne-Markus; Jürgens, Klaus; Ikonen, Vilma; Kaartokallio, Hermanni

2015-02-01

388

Cell Size Distributions of Soil Bacterial and Archaeal Taxa  

PubMed Central

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

Portillo, Maria C.; Leff, Jonathan W.; Lauber, Christian L.

2013-01-01

389

Different bacterial communities in ectomycorrhizae and surrounding soil  

PubMed Central

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

Vik, Unni; Logares, Ramiro; Blaalid, Rakel; Halvorsen, Rune; Carlsen, Tor; Bakke, Ingrid; Kolstø, Anne-Brit; Økstad, Ole Andreas; Kauserud, Håvard

2013-01-01

390

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

391

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

392

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

PubMed Central

Background Bacterial vaginosis (BV) is a common condition that is associated with numerous adverse health outcomes and is characterized by poorly understood changes in the vaginal microbiota. We sought to describe the composition and diversity of the vaginal bacterial biota in women with BV using deep sequencing of the 16S rRNA gene coupled with species-level taxonomic identification. We