2408 Genital microbiomes of women with recurrent bacterial vaginosis and their regular male sexual partner

OBJECTIVES/SPECIFIC AIMS: Epidemiologic data suggest that BV is sexually transmitted with male partners colonized or infected with the responsible organism(s). The objective of this study was to compare the genital microbiota of women with recurrent BV and their regular male sexual partner using 16S rRNA gene sequencing and quantitative PCR targeting BV-candidate bacteria (Gardnerella vaginalis, Atopobium vaginae, BVAB1-3, Sneathia, Leptotrichia, and Megasphaera type I). METHODS/STUDY POPULATION: Women with recurrent BV (≥3 prior episodes, including a current episode) and their regular male partner participating in a BV treatment trial and providing genital specimens (women: vaginal; men: urethral, coronal sulcus, urine) at enrollment were included. Male specimens for each participant were pooled. 250 bp 16S rRNA V4 region PCR amplicons were sequenced and analyzed using the QIIME pipeline. Taxonomy was assigned using the RDP Classifier against a modified Greengenes database with additional vaginal taxonomies added. An average relative abundance cutoff of 0.5% was used for analysis. qPCR was also performed for specific BV-candidate bacteria. Spearman correlation coefficients were used to investigate associations between all genital bacteria in addition to BV-candidate bacteria between partnerships. To determine positive associations between partnerships, the Wilcoxon signed-rank test was used. RESULTS/ANTICIPATED RESULTS: In total, 45 partnerships were included. Mean partnership age was 31.3 (SD=7.9), 91.1% partnerships were African-American. The majority of partnerships (70.0%) reported condomless sex during the past 3 months. Regarding 16S data, 37 genital bacteria had an average relative abundance of ≥0.5%. The average Spearman correlation across all 45 partnerships was 0.28 (SD=0.27) (median=0.27, minimum=−0.21, maximum=0.84). Overall, a positive association of all genital bacteria existed across the partnerships (p<0.0001). However, regarding specific BV-candidate bacteria, Spearman correlation tests for G. vaginalis, A. vaginae, Prevotella bivia, Megasphaera type I, BVAB1, and BVAB2 were nonsignificant. In contrast, Sneathia spp. were positively correlated between partnerships (r=0.37, p=0.01). With regards to qPCR results, RNA Cq analyses provided significant evidence for a linear association between male and females for only A. vaginae (r=0.52, p=0.006). DISCUSSION/SIGNIFICANCE OF IMPACT: In monogamous heterosexual couples in which the female has BV, the vaginal microbiota of women and the penile/urine microbiota of men were significantly correlated, particularly with regards to Sneathia spp. and A. vaginae, supporting the hypothesis that BV-associated bacteria are exchanged during sex.

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Soil Depth Determines the Composition and Diversity of Bacterial and Archaeal Communities in a Poplar Plantation

Forests ◽

10.3390/f10070550 ◽

2019 ◽

Vol 10 (7) ◽

pp. 550 ◽

Cited By ~ 6

Author(s):

Huili Feng ◽

Jiahuan Guo ◽

Weifeng Wang ◽

Xinzhang Song ◽

Shuiqiang Yu

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Relative Abundance ◽

Rrna Gene ◽

Poplar Plantation ◽

Plantation Forest ◽

Bacterial And Archaeal Communities ◽

Different Depths ◽

Archaeal Communities

Understanding the composition and diversity of soil microorganisms that typically mediate the soil biogeochemical cycle is crucial for estimating greenhouse gas flux and mitigating global changes in plantation forests. Therefore, the objectives of this study were to investigate changes in diversity and relative abundance of bacteria and archaea with soil profiles and the potential factors influencing the vertical differentiation of microbial communities in a poplar plantation. We investigated soil bacterial and archaeal community compositions and diversities by 16S rRNA gene Illumina MiSeq sequencing at different depths of a poplar plantation forest in Chenwei forest farm, Sihong County, Jiangsu, China. More than 882,422 quality-filtered 16S rRNA gene sequences were obtained from 15 samples, corresponding to 34 classified phyla and 68 known classes. Ten major bacterial phyla and two archaeal phyla were found. The diversity of bacterial and archaeal communities decreased with depth of the plantation soil. Analysis of variance (ANOVA) of relative abundance of microbial communities exhibited that Nitrospirae, Verrucomicrobia, Latescibacteria, GAL15, SBR1093, and Euryarchaeota had significant differences at different depths. The transition zone of the community composition between the surface and subsurface occurred at 10–20 cm. Overall, our findings highlighted the importance of depth with regard to the complexity and diversity of microbial community composition in plantation forest soils.

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Impact of Biochar Application to Soil on the Root-Associated Bacterial Community Structure of Fully Developed Greenhouse Pepper Plants

Applied and Environmental Microbiology ◽

10.1128/aem.00148-11 ◽

2011 ◽

Vol 77 (14) ◽

pp. 4924-4930 ◽

Cited By ~ 195

Author(s):

Max Kolton ◽

Yael Meller Harel ◽

Zohar Pasternak ◽

Ellen R. Graber ◽

Yigal Elad ◽

...

Keyword(s):

Bacterial Community ◽

16S Rrna ◽

Relative Abundance ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Community Composition ◽

Systemic Resistance ◽

Rrna Gene ◽

Molecular Fingerprinting ◽

Content Type ◽

Biochar Amendment

ABSTRACTAdding biochar to soil has environmental and agricultural potential due to its long-term carbon sequestration capacity and its ability to improve crop productivity. Recent studies have demonstrated that soil-applied biochar promotes the systemic resistance of plants to several prominent foliar pathogens. One potential mechanism for this phenomenon is root-associated microbial elicitors whose presence is somehow augmented in the biochar-amended soils. The objective of this study was to assess the effect of biochar amendment on the root-associated bacterial community composition of mature sweet pepper (Capsicum annuumL.) plants. Molecular fingerprinting (denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism) of 16S rRNA gene fragments showed a clear differentiation between the root-associated bacterial community structures of biochar-amended and control plants. The pyrosequencing of 16S rRNA amplicons from the rhizoplane of both treatments generated a total of 20,142 sequences, 92 to 95% of which were affiliated with theProteobacteria,Bacteroidetes,Actinobacteria, andFirmicutesphyla. The relative abundance of members of theBacteroidetesphylum increased from 12 to 30% as a result of biochar amendment, while that of theProteobacteriadecreased from 71 to 47%. TheBacteroidetes-affiliatedFlavobacteriumwas the strongest biochar-induced genus. The relative abundance of this group increased from 4.2% of total root-associated operational taxonomic units (OTUs) in control samples to 19.6% in biochar-amended samples. Additional biochar-induced genera included chitin and cellulose degraders (ChitinophagaandCellvibrio, respectively) and aromatic compound degraders (HydrogenophagaandDechloromonas). We hypothesize that these biochar-augmented genera may be at least partially responsible for the beneficial effect of biochar amendment on plant growth and viability.

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Impact of a probiotic, inulin, or their combination on the piglets’ microbiota at different intestinal locations

Beneficial Microbes ◽

10.3920/bm2014.0030 ◽

2015 ◽

Vol 6 (4) ◽

pp. 473-483 ◽

Cited By ~ 10

Author(s):

V.A. Sattler ◽

K. Bayer ◽

G. Schatzmayr ◽

A.G. Haslberger ◽

V. Klose

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

16S Rrna Gene ◽

Real Time ◽

Relative Abundance ◽

Real Time Pcr ◽

Denaturing Gradient Gel Electrophoresis ◽

Feed Additives ◽

Rrna Gene ◽

16S Rrna Gene Pyrosequencing

Natural feed additives are used to maintain health and to promote performance of pigs without antibiotics. Effects of a probiotic, inulin, and their combination (synbiotic), on the microbial diversity and composition at different intestinal locations were analysed using denaturing gradient gel electrophoresis (DGGE), real-time PCR, and 16S rRNA gene pyrosequencing. Bacterial diversity assessed by DGGE and/or pyrosequencing was increased by inulin in all three gut locations and by the synbiotic in the caecum and colon. In contrast, the probiotic did only affect the microbiota diversity in the ileum. Shifts in the DGGE microbiota profiles of the caecum and colon were detected for the pro- and synbiotic fed animals, whereas inulin profiles were more similar to the ones of the control. 16S rRNA gene pyrosequencing revealed that all three additives could reduce Escherichia species in each gut location, indicating a potential beneficial effect on the gut microbiota. An increase of relative abundance of Clostridiaceae in the large intestine was found in the inulin group and of Enterococcaceae in the ileum of probiotic fed pigs. Furthermore, real-time PCR results showed that the probiotic and synbiotic increased bifidobacterial numbers in the ileum, which was supported by sequencing results. The probiotic and inulin, to different extents, changed the diversity, relative abundance of phylotypes, and community profiles of the porcine microbiota. However, alterations of the bacterial community were not uniformly between gut locations, demonstrating that functionality of feed additives is site specific. Therefore, gut sampling from various locations is crucial when investigations aim to identify the composition of a healthy gut microbiota after its manipulation through feed additives.

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Methylothermus subterraneus sp. nov., a moderately thermophilic methanotroph isolated from a terrestrial subsurface hot aquifer

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.028092-0 ◽

2011 ◽

Vol 61 (11) ◽

pp. 2646-2653 ◽

Cited By ~ 52

Author(s):

Hisako Hirayama ◽

Yohey Suzuki ◽

Mariko Abe ◽

Masayuki Miyazaki ◽

Hiroko Makita ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Sequence ◽

Sequence Similarity ◽

16S Rrna Gene Sequence ◽

Rrna Gene ◽

Type I ◽

Rrna Gene Sequence ◽

Intracytoplasmic Membrane ◽

Moderately Thermophilic

A novel methane-oxidizing bacterium, strain HTM55T, was isolated from subsurface hot aquifer water from a Japanese gold mine. Strain HTM55T was a Gram-negative, aerobic, motile, coccoid bacterium with a single polar flagellum and the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain HTM55T was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 37–65 °C (optimum 55–60 °C). The isolate grew at pH 5.2–7.5 (optimum 5.8–6.3) and with 0–1 % NaCl (optimum 0–0.3 %). The ribulose monophosphate pathway was operative for carbon assimilation. The DNA G+C content was 54.4 mol% and the major fatty acids were C16 : 0 (52.0 %) and C18 : 1ω7c (34.8 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HTM55T was closely related to Methylothermus thermalis MYHTT (99.2 % 16S rRNA gene sequence similarity), which is within the class Gammaproteobacteria. However, DNA–DNA relatedness between strain HTM55T and Methylothermus thermalis MYHTT was ≤39 %. On the basis of distinct phylogenetic, chemotaxonomic and physiological characteristics, strain HTM55T represents a novel species of the genus Methylothermus, for which the name Methylothermus subterraneus sp. nov. is proposed. The type strain is HTM55T ( = JCM 13664T = DSM 19750T).

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Bacterial DNAemia is associated with serum zonulin levels in older subjects

10.1101/2020.04.10.035519 ◽

2020 ◽

Author(s):

Giorgio Gargari ◽

Valentina Taverniti ◽

Cristian Del Bo’ ◽

Stefano Bernardi ◽

Cristina Andres-Lacueva ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Serum Levels ◽

Gene Profiling ◽

Rrna Gene ◽

Bacterial Dna ◽

Blood Samples ◽

Gene Copies ◽

Older Subjects ◽

Pcr Targeting

AbstractThe increased presence of bacteria in blood is a plausible contributing factor in the development and progression of aging-associated diseases. In this context, we performed the quantification and the taxonomic profiling of the bacterial DNA in blood samples collected from a group of forty-three older subjects enrolled in a nursing home. Quantitative PCR targeting the 16S rRNA gene revealed that all the older volunteers contained detectable amounts of bacterial DNA in their blood. The total amount of 16S rRNA gene copies varied considerably between subjects. Correlation analyses revealed that the bacterial DNAemia (expressed as concentration of 16S rRNA gene copies in blood) significantly correlated with the serum levels of zonulin, an emerging marker of intestinal permeability. This result was confirmed by the analysis of a second set of blood samples collected after approximately four months from the same subjects. Analyses of 16S rRNA gene profiling revealed that most of the bacterial DNA detected in blood was ascribable to the phylum Proteobacteria with a predominance of Pseudomonadaceae and Enterobacteriaceae. Several control samples were also analyzed to assess the influence exerted by contaminant bacterial DNA potentially originating from reagents and materials. The date reported here suggest that para-cellular permeability of epithelial (and potentially also endothelial) cell layers may play an important role in bacterial migration into the bloodstream. Bacterial DNAemia is likely to impact on several aspects of host physiology and could underpin the development and prognosis of various diseases in older subjects.

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Molecular Detection of Borrelia Bacteria in Cerebrospinal Fluid-Optimisation of Pre-Analytical Sample Handling for Increased Analytical Sensitivity

Diagnostics ◽

10.3390/diagnostics11112088 ◽

2021 ◽

Vol 11 (11) ◽

pp. 2088

Author(s):

Malin Lager ◽

Peter Wilhelmsson ◽

Andreas Matussek ◽

Per-Eric Lindgren ◽

Anna J. Henningsson

Keyword(s):

Cerebrospinal Fluid ◽

16S Rrna ◽

Clinical Diagnostics ◽

Systematic Evaluation ◽

Clinical Samples ◽

Rrna Gene ◽

Analytical Sensitivity ◽

Gene 16S Rrna ◽

Pcr Targeting ◽

The 16S Rrna Gene

The main tools for clinical diagnostics of Lyme neuroborreliosis (LNB) are based on serology, i.e., detection of antibodies in cerebrospinal fluid (CSF). In some cases, PCR may be used as a supplement, e.g., on CSF from patients with early LNB. Standardisation of the molecular methods and systematic evaluation of the pre-analytical handling is lacking. To increase the analytical sensitivity for detection of Borrelia bacteria in CSF by PCR targeting the 16S rRNA gene, parameters were systematically evaluated on CSF samples spiked with a known amount of cultured Borrelia bacteria. The results showed that the parameters such as centrifugation time and speed, the use of complementary DNA as a template (in combination with primers and a probe aiming at target gene 16S rRNA), and the absence of inhibitors (e.g., erythrocytes) had the highest impact on the analytical sensitivity. Based on these results, a protocol for optimised handling of CSF samples before molecular analysis was proposed. However, no clinical evaluation of the proposed protocol has been done so far, and further investigations of the diagnostic sensitivity need to be performed on well-characterised clinical samples from patients with LNB.

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Identification and Removal of Potential Contaminants in 16S rRNA Gene Sequence Datasets from Low Microbial Biomass Samples: An Example from the Mosquito Gut

10.21203/rs.3.rs-45329/v1 ◽

2020 ◽

Author(s):

Sebastián Diaz ◽

Juan Sebastián Escobar ◽

Frank William Avila

Keyword(s):

Microbial Biomass ◽

16S Rrna ◽

Gut Microbiota ◽

Relative Abundance ◽

Vector Competence ◽

Negative Control ◽

Rrna Gene ◽

Physiological Processes ◽

Physiological Relevance ◽

The Impact

Abstract Background: The bacterial gut microbiota of the female mosquito influences numerous physiological processes, including vector competence. As a low-microbial-biomass ecosystem, mosquito gut tissue is prone to contamination from the laboratory environment and from reagents commonly used to dissect and/or isolate DNA from gut tissue. In this report, we analyze five 16S rRNA datasets, including new data obtained by us, to gain insight into the impact of potential contaminating sequences on the composition, diversity, and structure of the mosquito gut microbial community. Results: We present a clustering-free approach that, based on the relative abundance of amplicon sequence variants (ASVs) in gut and negative control samples , allowed for the identification of candidate contaminating sequences. Some of these sequences belong to bacterial taxa previously identified as common contaminants in metagenomic studies; they have also been identified as part of the mosquito core gut microbiota, with putative physiological relevance for the host. By using different relative abundance cutoffs, we show that contaminating sequences have a significant impact on gut microbiota diversity and structure.Conclusions: The approach presented here allows the identification and removal of purported contaminating sequences in datasets obtained from low-microbial biomass samples. While it was exemplified with the analysis of gut microbiota from mosquitos, it can easily extend to other datasets dealing with similar technical artifacts.

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Stability of Microbial Community Profiles Associated with Compacted Bentonite from the Grimsel Underground Research Laboratory

mSphere ◽

10.1128/msphere.00601-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 1

Author(s):

Katja Engel ◽

Sian E. Ford ◽

Sara Coyotzi ◽

Jennifer McKelvie ◽

Nikitas Diomidis ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Relative Abundance ◽

Corrosion Test ◽

Research Laboratory ◽

Bentonite Clay ◽

Rrna Gene ◽

Deep Geological Repository ◽

Geological Repository ◽

Underground Research Laboratory

ABSTRACT To assess the microbiology and corrosion potential of engineered components of a deep geological repository for long-term storage of high-level nuclear waste, the Materials Corrosion Test is being conducted at the Underground Research Laboratory in Grimsel, Switzerland. Modules containing metal coupons surrounded by highly compacted MX-80 bentonite, at two dry densities (1.25 and 1.50 g/cm3), were emplaced within 9-m-deep boreholes, and the first modules were retrieved after 13 months of exposure. Bentonite and associated module materials were sampled, and microbial communities and their distributions were assessed using 16S rRNA gene sequencing and phospholipid fatty acid (PLFA) analysis. Borehole fluid was dominated by amplicon sequence variants (ASVs) affiliated with Desulfosporosinus and Desulfovibrio, which are putatively involved in sulfate reduction. The relative abundance of these ASVs was lower for samples from inside the borehole module, and they were almost undetectable in samples of the inner bentonite layer. The dominant ASV in case and filter sample sequence data was affiliated with Pseudomonas stutzeri, yet its relative abundance decreased in the inner layer samples. Streptomyces sp. ASVs were relatively abundant in all bentonite core sample data both prior to emplacement and after 13 months of exposure, presumably as metabolically inactive spores or extracellular “relic” DNA. PLFA concentrations in outer and inner layer bentonite samples suggested cellular abundances of 1 × 106 to 3 × 106 cells/g, with similar PLFA distributions within all bentonite samples. Our results demonstrate consistent microbial communities inside the saturated borehole module, providing the first evidence for microbial stability under conditions that mimic a deep geological repository. IMPORTANCE The Materials Corrosion Test in Grimsel Underground Research Laboratory, Switzerland, enables an evaluation of microbiological implications of bentonite clay at densities relevant for a deep geological repository. Our research demonstrates that after 13 months of exposure within a granitic host rock, the microbial 16S rRNA gene signatures of saturated bentonite clay within the modules were consistent with the profiles in the original clay used to pack the modules. Such results provide evidence that densities chosen for this emplacement test are refractory to microbial activity, at least on the relatively short time frame leading to the first time point sampling event, which will help inform in situ engineered barrier system science. This study has important implications for the design of deep geological repository sites under consideration for the Canadian Shield.

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Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis

Applied and Environmental Microbiology ◽

10.1128/aem.65.11.4887-4897.1999 ◽

1999 ◽

Vol 65 (11) ◽

pp. 4887-4897 ◽

Cited By ~ 136

Author(s):

Mark G. Wise ◽

J Vaun McArthur ◽

Lawrence J. Shimkets

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Ribosomal Dna ◽

Rrna Gene ◽

Type I ◽

Type Ii ◽

Partial Sequencing ◽

16S Ribosomal Dna ◽

Culture Independent ◽

The 16S Rrna Gene

ABSTRACT The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobiumgroup, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly publishedMethylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described.

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Isolation and screening of Lactobacillus species from dogs for probiotic action

Indian Journal of Animal Research ◽

10.18805/ijar.b-3429 ◽

2017 ◽

Author(s):

N. Mohammad Sharif ◽

B. Sreedevi ◽

R. K. Chaitanya ◽

D. Sreenivasulu

Keyword(s):

Antibacterial Activity ◽

16S Rrna ◽

Cell Surface Hydrophobicity ◽

Lactobacillus Species ◽

Nucleotide Sequence Analysis ◽

Rrna Gene ◽

Acid And Bile Tolerance ◽

In Vitro Antibacterial Activity ◽

Pcr Targeting

The present study aimed at the detection of suitable Lactobacillus species from dogs for usage as probiotic. A total of 67 rectal swabs from healthy pups were analyzed and 49 (73.1%) Lactobacillus isolates were identified based on morphological, biochemical characteristics and confirmed by genus specific PCR targeting 16S rRNA gene. A total of 20 isolates that showed strong aggregation, high cell surface hydrophobicity, acid and bile tolerance were screened for in-vitro antibacterial activity by agar well diffusion assay, where prominent inhibitory zones were observed against majority of the test pathogens. Reduction in antibacterial activity was noticed after neutralization, proteinase K and heat treatment of supernatants. Partial 16S rRNA nucleotide sequence analysis of seven Lactobacillus isolates that showed prominent in-vitro antibacterial activity revealed maximum sequence homology with Lactobacillus fermentum (for six isolates) and Lactobacillus agilis (for one isolate). The in-vitro antibacterial activity results highlighted the need for in-vivo studies in India to establish probiotic potential of canine faecal Lactobacillus species in the near future.

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