scholarly journals 219 Relationship between host-genetics and the vaginal microbiome in commercial gilts

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 43-44
Author(s):  
Leticia Sanglard ◽  
Stephan Schmitz-Esser ◽  
Kent Gray ◽  
Daniel C L Linhares ◽  
Carl J Yeoman ◽  
...  
Keyword(s):  
16S Rrna Gene Sequencing ◽  
Vaginal Swab ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Live Virus ◽  
Abundant Otus ◽  
Host Genetic ◽  
Heritability Estimates ◽  
Rrna Gene Sequencing ◽  
Genetic Contributions

Abstract The objective of this study was to investigate host-genetic contributions to the vaginal microbiome of commercial gilts vaccinated for Porcine Reproductive and Respiratory Syndrome (PRRS). Vaginal swab samples (n = 576) from 308 F1 gilts (183±12 days old) were collected on day 4 (D4) and 52 (D52) post-vaccination with a commercial modified live virus PRRS vaccine. Samples were used to profile the vaginal microbiome by 16S rRNA gene sequencing, with sequences clustered into operational taxonomic units (OTUs) and taxonomically classified. All animals were genotyped for 45,536 SNPs. Arcsine of the square root-transformed OTUs abundance data were analyzed using a linear mixed animal model with age at vaccination as a covariate and animal (random) for estimation of genetic parameters. The same model was used for GWAS for the 100 most abundant OTUs but with addition of genotype of SNPs as a covariate, one at a time. For D4, heritability estimates ranged from < 0.001±0.01(13 OTUs) to 0.60±0.13 (Fusobacterium), with OTUs corresponding to the genera Fusobacterium, Pasteurellaceae, Clostridiales, Prevotellaceae, and Lactobacillus having high estimates (0.41±0.13 to 0.60±0.13). For D52, heritability estimates ranged from < 0.001±0.01 (10 OTUs) to 0.63±0.12 (Terrisporobacter), with OTUs corresponding to Clostridium, Terrisporobacter, Romboutsia, Turicibacter, Phascolarctobacterium, Muribaculaceae, and Ruminococcaceae having high estimates (0.42±0.14 to 0.63±0.12). Forty-six QTLs were significantly (P < 0.00001) associated with OTU across days. Among these, one main QTL on chromosome 12 (20–23Mb), a gene-rich region with previously identified QTL for immune-related traits, was identified for 5 and 6 OTUs on D4 and D52, respectively. These OTUs were mainly of the phyla Proteobacteria and Firmicutes on D4 and D52, respectively. In conclusion, there is evidence of substantial host genetic variation for vaginal microbiome in commercial PRRS-vaccinated gilts, including the identification of many QTLs. Additional research is needed to investigate the genetic relationship between vaginal microbiome, health, and production in pigs.

scholarly journals The profiling of microbiota in vaginal swab samples using 16S rRNA gene sequencing and IS-pro analysis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
M. Singer ◽  
R. Koedooder ◽  
M. P. Bos ◽  
L. Poort ◽  
S. Schoenmakers ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Diagnostic Techniques ◽  
Vaginal Swab ◽  
Reproductive Age ◽  
Rrna Gene ◽  
Rrna Gene Sequencing ◽  
High Level

Abstract Background 16S rRNA gene sequencing is currently the most common way of determining the composition of microbiota. This technique has enabled many new discoveries to be made regarding the relevance of microbiota to the health and disease of the host. However, compared to other diagnostic techniques, 16S rRNA gene sequencing is fairly costly and labor intensive, leaving room for other techniques to improve on these aspects. Results The current study aimed to compare the output of 16S rRNA gene sequencing to the output of the quick IS-pro analysis, using vaginal swab samples from 297 women of reproductive age. 16S rRNA gene sequencing and IS-pro analyses yielded very similar vaginal microbiome profiles, with a median Pearson’s R2 of 0.97, indicating a high level of similarity between both techniques. Conclusions We conclude that the results of 16S rRNA gene sequencing and IS-pro are highly comparable and that both can be used to accurately determine the vaginal microbiota composition, with the IS-pro analysis having the benefit of rapidity.

scholarly journals Optimal protocols for sequence-based characterization of the human vaginal microbiome

2020 ◽  
Author(s):  
Luisa W. Hugerth ◽  
Marcela Pereira ◽  
Yinghua Zha ◽  
Maike Seifert ◽  
Vilde Kaldhusdal ◽  
...  
Keyword(s):  
16S Rrna ◽  
Best Practices ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Wide Range ◽  
Rrna Gene Sequencing ◽  
Good Agreement

AbstractThe vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment, but also to the use of conflicting methodologies to study its microbial composition. Here we systematically assess best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region has the best theoretical properties, but limitations of current sequencing technologies mean that the V3-V4 region performs equally well. Both of these approaches present very good agreement with qPCR quantification of key taxa, provided an appropriate bioinformatic pipeline is used. Shotgun metagenomic sequencing presents an interesting alternative to 16S amplification and sequencing, but it is not without its challenges. We have assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and – use, reference database of vaginal taxa. This strategy performed as well as the best performing previously published strategies. Despite the many advantages of shotgun sequencing none of the shotgun approaches assessed here had as good agreement with the qPCR data as 16S rRNA gene sequencing.ImportanceThe vaginal microbiome has been connected to a wide range of health outcomes, from susceptibility to sexually transmitted infections to gynecological cancers and pregnancy outcomes. This has led to a thriving research environment, but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail for them to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.

scholarly journals Vaginal microbiome and serum metabolite differences in late gestation commercial sows at risk for pelvic organ prolapse

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zoë E. Kiefer ◽  
Lucas R. Koester ◽  
Lucas Showman ◽  
Jamie M. Studer ◽  
Amanda L. Chipman ◽  
...  
Keyword(s):  
Pelvic Organ Prolapse ◽  
16S Rrna Gene Sequencing ◽  
Pelvic Organ ◽  
Late Gestation ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Swine Industry ◽  
Vaginal Microflora ◽  
Vaginal Swabs ◽  
Rrna Gene Sequencing

AbstractSow mortality attributable to pelvic organ prolapse (POP) has increased in the U.S. swine industry and continues to worsen. Two main objectives of this study were, (1) to develop a perineal scoring system that can be correlated with POP risk, and (2) identify POP risk-associated biological factors. To assess POP risk during late gestation, sows (n = 213) were scored using a newly developed perineal scoring (PS) system. Sows scored as PS1 (low), PS2 (moderate), or PS3 (high) based on POP risk. Subsequently, 1.5, 0.8, and 23.1% of sows scored PS1, PS2, or PS3, respectively, experienced POP. To identify biomarkers, serum and vaginal swabs were collected from late gestation sows differing in PS. Using GC–MS, 82 serum metabolite differences between PS1 and PS3 animals (P < 0.05) were identified. Vaginal swabs were utilized for 16S rRNA gene sequencing and differences in vaginal microbiomes between PS1 and PS3 animals were detected on a community level (P < 0.01) along with differences in abundances of 89 operational taxonomic units (P < 0.05). Collectively, these data demonstrate that sows with greater POP risk have differential serum metabolites and vaginal microflora. Additionally, an initial and novel characterization of the sow vaginal microbiome was determined.

scholarly journals Assessment of In Vitro and In Silico Protocols for Sequence-Based Characterization of the Human Vaginal Microbiome

mSphere ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Luisa W. Hugerth ◽  
Marcela Pereira ◽  
Yinghua Zha ◽  
Maike Seifert ◽  
Vilde Kaldhusdal ◽  
...  
Keyword(s):  
16S Rrna ◽  
Best Practices ◽  
16S Rrna Gene ◽  
In Silico ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Rrna Gene Sequencing

ABSTRACT The vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment but also to the use of conflicting methodologies to study its microbial composition. Here, we systematically assessed best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region performed best in silico, but limitations of current sequencing technologies meant that the V3-V4 region performed equally well. Both approaches presented very good agreement with qPCR quantification of key taxa, provided that an appropriate bioinformatic pipeline was used. Shotgun metagenomic sequencing presents an interesting alternative to 16S rRNA gene amplification and sequencing but requires deeper sequencing and more bioinformatic expertise and infrastructure. We assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and -use reference database of vaginal taxa. This curated database performed as well as the best-performing previously published strategies. Despite the many advantages of shotgun sequencing, none of the shotgun approaches assessed here agreed with the qPCR data as well as the 16S rRNA gene sequencing. IMPORTANCE The vaginal microbiome has been connected to various aspects of host health, including susceptibility to sexually transmitted infections as well as gynecological cancers and pregnancy outcomes. This has led to a thriving research environment but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.

scholarly journals Preterm birth is associated with xenobiotics and predicted by the vaginal metabolome

2021 ◽  
Author(s):  
William F Kindschuh ◽  
Federico Baldini ◽  
Martin C Liu ◽  
Kristen D Gerson ◽  
Jingqui Liao ◽  
...  
Keyword(s):  
Preterm Birth ◽  
Black Women ◽  
Neonatal Morbidity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Spontaneous Preterm Birth ◽  
Potential Risk Factors ◽  
Rrna Gene Sequencing ◽  
Singleton Pregnancies

Spontaneous preterm birth (sPTB) is a leading cause of maternal and neonatal morbidity and mortality, yet both its prevention and early risk stratification are limited. The vaginal microbiome has been associated with PTB risk, possibly via metabolic or other interactions with its host. Here, we performed untargeted metabolomics on 232 vaginal samples, in which we have previously profiled the microbiota using 16S rRNA gene sequencing. Samples were collected at 20-24 weeks of gestation from women with singleton pregnancies, of which 80 delivered spontaneously before 37 weeks of gestation. We find that the vaginal metabolome correlates with the microbiome and separates into six clusters, three of which are associated with spontaneous preterm birth (sPTB) in Black women. Furthermore, while we identify five metabolites that associate with sPTB, another five associate with sPTB only when stratifying by race. We identify multiple microbial correlations with metabolites associated with sPTB, including intriguing correlations between vaginal bacteria that are considered sub-optimal and metabolites that were enriched in women who delivered at term. We propose that several sPTB-associated metabolites may be exogenous, and investigate another using metabolic models. Notably, we use machine learning models to predict sPTB risk using metabolite levels, weeks to months in advance, with high accuracy. We show that these predictions are more accurate than microbiome-based and maternal covariates-based models. Altogether, our results demonstrate the potential of vaginal metabolites as early biomarkers of sPTB and highlight exogenous exposures as potential risk factors for prematurity.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

2019 ◽  
Vol 13 (1) ◽  
pp. 90-101
Author(s):  
Sanju Kumari ◽  
Utkarshini Sharma ◽  
Rohit Krishna ◽  
Kanak Sinha ◽  
Santosh Kumar
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Characterization ◽  
Biochemical Characterization ◽  
Evolutionary Relationship ◽  
Gene Sequencing ◽  
Cellulase Activity ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Background: Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels. Objective: The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions. Methods: Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers. Results: Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 &#181;mol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively. Conclusion: Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

scholarly journals Diversity, Composition and Functional Inference of Gut Microbiota in Indian Cabbage white Pieris canidia (Lepidoptera: Pieridae)

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 254
Author(s):  
Ying Wang ◽  
Jianqing Zhu ◽  
Jie Fang ◽  
Li Shen ◽  
Shuojia Ma ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
16S Rrna Gene Sequencing ◽  
Adult Survival ◽  
Rrna Gene ◽  
Life Stages ◽  
Microbial Composition ◽  
Significant Difference ◽  
Functional Inference ◽  
Rrna Gene Sequencing ◽  
Insect Fitness

We characterized the gut microbial composition and relative abundance of gut bacteria in the larvae and adults of Pieris canidia by 16S rRNA gene sequencing. The gut microbiota structure was similar across the life stages and sexes. The comparative functional analysis on P. canidia bacterial communities with PICRUSt showed the enrichment of several pathways including those for energy metabolism, immune system, digestive system, xenobiotics biodegradation, transport, cell growth and death. The parameters often used as a proxy of insect fitness (development time, pupation rate, emergence rate, adult survival rate and weight of 5th instars larvae) showed a significant difference between treatment group and untreated group and point to potential fitness advantages with the gut microbiomes in P. canidia. These data provide an overall view of the bacterial community across the life stages and sexes in P. canidia.

scholarly journals Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Janis R. Bedarf ◽  
Naiara Beraza ◽  
Hassan Khazneh ◽  
Ezgi Özkurt ◽  
David Baker ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
False Positive ◽  
Gene Sequencing ◽  
Bacterial Biomass ◽  
16S Rrna Gene Sequencing ◽  
Amplicon Sequencing ◽  
Rrna Gene ◽  
Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

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