scholarly journals Longitudinal assessment of sputum microbiome by sequencing of the 16S rRNA gene in non-CF bronchiectasis patients

2016 ◽  
Author(s):  
Michael J Cox ◽  
Elena M Turek ◽  
Catherine Hennessy ◽  
Ghazala K Mirza ◽  
Phillip L James ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Antibiotic Therapy ◽  
Bacterial Overgrowth ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Microbial Culture ◽  
Study Objective ◽  
Bronchial Infection

AbstractBackgroundBronchiectasis is accompanied by chronic bronchial infection that may drive disease progression. However, the evidence base for antibiotic therapy is limited. DNA based methods offer better identification and quantification of microbial constituents of sputum than standard clinical culture and may help inform patient management strategies. Our study objective was to determine the longitudinal variability of the non-CF bronchiectasis microbiome in sputum with respect to clinical variables.Eighty-five patients with non-cystic fibrosis (CF) bronchiectasis and daily sputum production were recruited from outpatient clinics and followed for six months. Monthly sputum samples and clinical measurements were taken, together with additional samples during exacerbations. 16S rRNA gene sequencing of the sputum microbiota was successful for 381 samples from 76 patients and analysed in conjunction with clinical data.ResultsMicrobial communities were highly individual in composition and stability, usually with limited diversity and often containing multiple pathogens. When compared to DNA sequencing, microbial culture had restricted sensitivity in identifying common pathogens. With some exceptions, community characteristics showed poor correlations with clinical features including underlying disease, antibiotic use and exacerbations, with the subject showing the strongest association with community structure. When present, certain pathogens may also shape the structure of the rest of the microbial community.ConclusionsThe use of microbial community analysis of sputum added to information from microbial culture. A simple model of exacerbations driven by bacterial overgrowth was not supported, suggesting a need for revision of principles for antibiotic therapy. In individual patients, the management of chronic bronchial infection may be improved by therapy specific to their microbiome, taking into account pathogen load, community stability, and acute and chronic community responses to antibiotics.

scholarly journals Microbial community dynamics of surface water in British Columbia, Canada

2019 ◽  
Author(s):  
Miguel I. Uyaguari-Diaz ◽  
Matthew A. Croxen ◽  
Kirby Cronin ◽  
Zhiyao Luo ◽  
Judith Isaac-Renton ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Dynamics ◽  
Fecal Contamination ◽  
Rrna Gene ◽  
Microbial Culture ◽  
Sample Collection ◽  
Fecal Indicator ◽  
E Coli

AbstractTraditional methods for monitoring the microbiological quality of water focus on the detection of fecal indicator bacteria such as Escherichia coli, often tested as a weekly grab sample. To understand the stability of E.coli concentrations over time, we evaluated three approaches to measuring E. coli levels in water: microbial culture using Colilert, quantitative PCR for uidA and next-generation sequencing of the 16S rRNA gene. Two watersheds, one impacted by agricultural and the other by urban activities, were repeatedly sampled over a simultaneous ten-hour period during each of the four seasons. Based on 16S rRNA gene deep sequencing, each watershed showed different microbial community profiles. The bacterial microbiomes varied with season, but less so within each 10-hour sampling period. Enterobacteriaceae comprised only a small fraction (<1%) of the total community. The qPCR assay detected significantly higher quantities of E. coli compared to the Colilert assay and there was also variability in the Colilert measurements compared to Health Canada’s recommendations for recreational water quality. From the 16S data, other bacteria such as Prevotella and Bacteroides showed promise as alternative indicators of fecal contamination. A better understanding of temporal changes in watershed microbiomes will be important in assessing the utility of current biomarkers of fecal contamination, determining the best timing for sample collection, as well as searching for additional microbial indicators of the health of a watershed.

scholarly journals Population Abundance of Potentially Pathogenic Organisms in Intestinal Microbiome of Jungle Crow (Corvus macrorhynchos) Shown with 16S rRNA Gene-Based Microbial Community Analysis

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Isamu Maeda ◽  
Mohammad Shohel Rana Siddiki ◽  
Tsutomu Nozawa-Takeda ◽  
Naoki Tsukahara ◽  
Yuri Tani ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pathogenic Bacteria ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Intestinal Microbiome ◽  
Rrna Gene ◽  
Jungle Crow ◽  
Corvus Macrorhynchos

Jungle Crows (Corvus macrorhynchos) prefer human habitats because of their versatility in feeding accompanied with human food consumption. Therefore, it is important from a public health viewpoint to characterize their intestinal microbiota. However, no studies have been involved in molecular characterization of the microbiota based on huge and reliable number of data acquisition. In this study, 16S rRNA gene-based microbial community analysis coupled with the next-generation DNA sequencing techniques was applied to the taxonomic classification of intestinal microbiome for three jungle crows. Clustering of the reads into 130 operational taxonomic units showed that at least 70% of analyzed sequences for each crow were highly homologous toEimeriasp., which belongs to the protozoan phylumApicomplexa. The microbiotas of three crows also contained potentially pathogenic bacteria with significant percentages, such as the generaCampylobacterandBrachyspira. Thus, the profiling of a large number of 16S rRNA gene sequences in crow intestinal microbiomes revealed the high-frequency existence or vestige of potentially pathogenic microorganisms.

scholarly journals Comparison of 16S rRNA Gene Based Microbial Profiling Using Five Next-Generation Sequencers and Various Primers

2021 ◽  
Vol 12 ◽  
Author(s):  
Changwoo Park ◽  
Seung Bum Kim ◽  
Sang Ho Choi ◽  
Seil Kim
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Lactococcus Lactis ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Next Generation ◽  
Universal Primer ◽  
Mock Communities

Microbial community analysis based on the 16S rRNA-gene is used to investigate both beneficial and harmful microorganisms in various fields and environments. Recently, the next-generation sequencing (NGS) technology has enabled rapid and accurate microbial community analysis. Despite these advantages of NGS based metagenomics study, sample transport, storage conditions, amplification, library preparation kits, sequencing, and bioinformatics procedures can bias microbial community analysis results. In this study, eight mock communities were pooled from genomic DNA of Lactobacillus acidophilus KCTC 3164T, Limosilactobacillus fermentum KCTC 3112T, Lactobacillus gasseri KCTC 3163T, Lacticaseibacillus paracasei subsp. paracasei KCTC 3510T, Limosilactobacillus reuteri KCTC 3594T, Lactococcus lactis subsp. lactis KCTC 3769T, Bifidobacterium animalis subsp. lactis KCTC 5854T, and Bifidobacterium breve KCTC 3220T. The genomic DNAs were quantified by droplet digital PCR (ddPCR) and were mixed as mock communities. The mock communities were amplified with various 16S rRNA gene universal primer pairs and sequenced by MiSeq, IonTorrent, MGIseq-2000, Sequel II, and MinION NGS platforms. In a comparison of primer-dependent bias, the microbial profiles of V1-V2 and V3 regions were similar to the original ratio of the mock communities, while the microbial profiles of the V1-V3 region were relatively biased. In a comparison of platform-dependent bias, the sequence read from short-read platforms (MiSeq, IonTorrent, and MGIseq-2000) showed lower bias than that of long-read platforms (Sequel II and MinION). Meanwhile, the sequences read from Sequel II and MinION platforms were relatively biased in some mock communities. In the data of all NGS platforms and regions, L. acidophilus was greatly underrepresented while Lactococcus lactis subsp. lactis was generally overrepresented. In all samples of this study, the bias index (BI) was calculated and PCA was performed for comparison. The samples with biased relative abundance showed high BI values and were separated in the PCA results. In particular, analysis of regions rich in AT and GC poses problems for genome assembly, which can lead to sequencing bias. According to this comparative analysis, the development of reference material (RM) material has been proposed to calibrate the bias in microbiome analysis.

scholarly journals A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis

1998 ◽  
Vol 64 (12) ◽  
pp. 4870-4876 ◽  
Author(s):  
Frank Schwieger ◽  
Christoph C. Tebbe
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pure Culture ◽  
Community Analysis ◽  
Single Strand Conformation Polymorphism ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Pcr Products ◽  
Conformation Polymorphism

ABSTRACT Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.

scholarly journals rpoB-Based Microbial Community Analysis Avoids Limitations Inherent in 16S rRNA Gene Intraspecies Heterogeneity

2000 ◽  
Vol 66 (8) ◽  
pp. 3376-3380 ◽  
Author(s):  
Ingela Dahllöf ◽  
Harriet Baillie ◽  
Staffan Kjelleberg
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rdna ◽  
Pattern Analysis ◽  
Community Analysis ◽  
Microbial Community Analysis ◽  
Rrna Gene ◽  
Community Pattern ◽  
16S Rdna Pcr

ABSTRACT Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (rpoB), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga Delisea pulchra and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of rpoB, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and rpoB PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from D. pulchra. Eight out of 14 isolates from D. pulchra, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when rpoB was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from D. pulchra proved to be more accurately described using rpoB, compared to the 16S rDNA-based PCR-DGGE.

scholarly journals The diversity of active microbial groups in an activated sludge process treating painting process wastewater

2020 ◽  
Vol 148 ◽  
pp. 01002
Author(s):  
Herto Dwi Ariesyady ◽  
Mentari Rizki Mayanda ◽  
Tsukasa Ito
Keyword(s):  
Wastewater Treatment ◽  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Activated Sludge ◽  
Biological Treatment ◽  
Rrna Gene ◽  
Activated Sludge Process ◽  
Painting Process ◽  
Process Wastewater

Activated sludge process is one of the wastewater treatment method that is applied for many wastewater types including painting process wastewater of automotive industry. This wastewater is well-known to have high heavy metals concentration which could deteriorate water environment if appropriate performance of the wastewater treatment could not be achieved. In this study, we monitored microbial community diversity in a Painting Biological Treatment (PBT) system. We applied a combination of cultivation and genotypic biological methods based on 16S rRNA gene sequence analysis to identify the diversity of active microbial community. The results showed that active microbes that could grow in this activated sludge system were dominated by Gram-negative bacteria. Based on 16S rRNA gene sequencing analysis, it was revealed that their microbial diversity has close association with Bacterium strain E286, Isosphaera pallida, Lycinibacillus fusiformis, Microbacterium sp., Orchobactrum sp., Pseudomonas guariconensis, Pseudomonas sp. strain MR84, Pseudomonas sp. MC 54, Serpens sp., Stenotrophomonas acidaminiphila, and Xylella fastidiosa with similarity of 86 – 99%. This findings reflects that microbial community in a Painting Biological Treatment (PBT) system using activated sludge process could adapt with xenobiotics in the wastewater and has a wide range of diversity indicating a complex metabolism mechanism in the treatment process.

scholarly journals Microbial community in persistent apical periodontitis: a 16S rRNA gene clone library analysis

2014 ◽  
Vol 48 (8) ◽  
pp. 717-728 ◽  
Author(s):  
M. N. Zakaria ◽  
T. Takeshita ◽  
Y. Shibata ◽  
H. Maeda ◽  
N. Wada ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Clone Library ◽  
Apical Periodontitis ◽  
Gene Clone ◽  
Rrna Gene ◽  
Clone Library Analysis

scholarly journals Arsenic mobilization in a high arsenic groundwater revealed by metagenomic and Geochip analyses

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zhou Jiang ◽  
Ping Li ◽  
Yanhong Wang ◽  
Han Liu ◽  
Dazhun Wei ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Arsenic Mobilization ◽  
Rrna Gene Sequencing ◽  
High Arsenic ◽  
High Arsenic Groundwater

Abstract Microbial metabolisms of arsenic, iron, sulfur, nitrogen and organic matter play important roles in arsenic mobilization in aquifer. In this study, microbial community composition and functional potentials in a high arsenic groundwater were investigated using integrated techniques of RNA- and DNA-based 16S rRNA gene sequencing, metagenomic sequencing and functional gene arrays. 16S rRNA gene sequencing showed the sample was dominated by members of Proteobacteria (62.3–75.2%), such as genera of Simplicispira (5.7–6.7%), Pseudomonas (3.3–5.7%), Ferribacterium (1.6–4.4%), Solimonas (1.8–3.2%), Geobacter (0.8–2.2%) and Sediminibacterium (0.6–2.4%). Functional potential analyses indicated that organics degradation, assimilatory sulfate reduction, As-resistant pathway, iron reduction, ammonification, nitrogen fixation, denitrification and dissimilatory nitrate reduction to ammonia were prevalent. The composition and function of microbial community and reconstructed genome bins suggest that high level of arsenite in the groundwater may be attributed to arsenate release from iron oxides reductive dissolution by the iron-reducing bacteria, and subsequent arsenate reduction by ammonia-producing bacteria featuring ars operon. This study highlights the relationship between biogeochemical cycling of arsenic and nitrogen in groundwater, which potentially occur in other aquifers with high levels of ammonia and arsenic.

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