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Comparison of storage conditions for human vaginal microbiome studies.

ABSTRACT: The effect of storage conditions on the microbiome and metabolite composition of human biological samples has not been thoroughly investigated as a potential source of bias. We evaluated the effect of two common storage conditions used in clinical trials on the bacterial and metabolite composition of the vaginal microbiota using pyrosequencing of barcoded 16S rRNA gene sequencing and (1)H-NMR analyses.Eight women were enrolled and four mid-vaginal swabs were collected by a physician from each woman. The samples were either processed immediately, stored at -80°C for 4 weeks or at -20°C for 1 week followed by transfer to -80°C for another 4 weeks prior to analysis. Statistical methods, including Kolmogorovo-Smirnov and Wilcoxon tests, were performed to evaluate the differences in vaginal bacterial community composition and metabolites between samples stored under different conditions. The results showed that there were no significant differences between samples processed immediately after collection or stored for varying durations. (1)H-NMR analysis of the small molecule metabolites in vaginal secretions indicated that high levels of lactic acid were associated with Lactobacillus-dominated communities. Relative abundance of lactic acid did not appear to correlate with relative abundance of individual Lactobacillus sp. in this limited sample, although lower levels of lactic acid were observed when L. gasseri was dominant, indicating differences in metabolic output of seemingly similar communities.These findings benefit large-scale, field-based microbiome and metabolomic studies of the vaginal microbiota.

SUBMITTER: Bai G

PROVIDER: S-EPMC3360033 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Comparison of storage conditions for human vaginal microbiome studies.

Bai Guoyun G Gajer Pawel P Nandy Melissa M Ma Bing B Yang Hongqiu H Sakamoto Joyce J Blanchard May H MH Ravel Jacques J Brotman Rebecca M RM

PloS one 20120524 5

<h4>Background</h4>The effect of storage conditions on the microbiome and metabolite composition of human biological samples has not been thoroughly investigated as a potential source of bias. We evaluated the effect of two common storage conditions used in clinical trials on the bacterial and metabolite composition of the vaginal microbiota using pyrosequencing of barcoded 16S rRNA gene sequencing and (1)H-NMR analyses.<h4>Methodology/principal findings</h4>Eight women were enrolled and four mi ...[more]

PMID: 22655031

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Project description:BackgroundMultiple factors can influence stool sample integrity upon sample collection. Preservation of faecal samples for microbiome studies is therefore an important step, particularly in tropical regions where resources are limited and high temperatures may significantly influence microbiota profiles. Freezing is the accepted standard to preserve faecal samples however, cold chain methods are often unfeasible in fieldwork scenarios particularly in low and middle-income countries and alternatives are required. This study therefore aimed to address the impact of different preservative methods, time-to-freezing at ambient tropical temperatures, and stool heterogeneity on stool microbiome diversity and composition under real-life physical environments found in resource-limited fieldwork conditions.MethodsInner and outer stool samples collected from one specimen obtained from three children were stored using different storage preservation methods (raw, ethanol and RNAlater) in a Ugandan field setting. Mixed stool was also stored using these techniques and frozen at different time-to-freezing intervals post-collection from 0-32 h. Metataxonomic profiling was used to profile samples, targeting the V1-V2 regions of 16S rRNA with samples run on a MiSeq platform. Reads were trimmed, combined and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology and R software.ResultsChild donor was the greatest predictor of microbiome variation between the stool samples, with all samples remaining identifiable to their child of origin despite the stool being stored under a variety of conditions. However, significant differences were observed in composition and diversity between preservation techniques, but intra-preservation technique variation was minimal for all preservation methods, and across the time-to-freezing range (0-32 h) used. Stool heterogeneity yielded no apparent microbiome differences.ConclusionsStool collected in a fieldwork setting for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout.

Project description:The human vaginal microbiome is dominated by bacteria from the genus Lactobacillus, which create an acidic environment thought to protect women against sexually transmitted pathogens and opportunistic infections. Strikingly, lactobacilli dominance appears to be unique to humans; while the relative abundance of lactobacilli in the human vagina is typically >70%, in other mammals lactobacilli rarely comprise more than 1% of vaginal microbiota. Several hypotheses have been proposed to explain humans' unique vaginal microbiota, including humans' distinct reproductive physiology, high risk of STDs, and high risk of microbial complications linked to pregnancy and birth. Here, we test these hypotheses using comparative data on vaginal pH and the relative abundance of lactobacilli in 26 mammalian species and 50 studies (N = 21 mammals for pH and 14 mammals for lactobacilli relative abundance). We found that non-human mammals, like humans, exhibit the lowest vaginal pH during the period of highest estrogen. However, the vaginal pH of non-human mammals is never as low as is typical for humans (median vaginal pH in humans = 4.5; range of pH across all 21 non-human mammals = 5.4-7.8). Contrary to disease and obstetric risk hypotheses, we found no significant relationship between vaginal pH or lactobacilli relative abundance and multiple metrics of STD or birth injury risk (P-values ranged from 0.13 to 0.99). Given the lack of evidence for these hypotheses, we discuss two alternative explanations: the common function hypothesis and a novel hypothesis related to the diet of agricultural humans. Specifically, with regard to diet we propose that high levels of starch in human diets have led to increased levels of glycogen in the vaginal tract, which, in turn, promotes the proliferation of lactobacilli. If true, human diet may have paved the way for a novel, protective microbiome in human vaginal tracts. Overall, our results highlight the need for continuing research on non-human vaginal microbial communities and the importance of investigating both the physiological mechanisms and the broad evolutionary processes underlying human lactobacilli dominance.

Project description:The vaginal microbiota plays an important role in women's reproductive and urogenital health. It is now well accepted that a "healthy" vaginal microbiome is dominated by Lactobacillus species. Disturbances in this microbial community can lead to several adverse outcomes, including pelvic inflammatory disease and bacterial vaginosis (BV), as well as increased susceptibility to sexually transmitted infections, miscarriage, and preterm births. However, vaginal communities, especially those of women in the developing world, can be comprised of a diverse set of microorganisms in the absence of overt clinical symptoms. The implications of these diverse vaginal microbiomes for women's health remain poorly understood. Rhesus macaques are an excellent translational animal model to address these questions due to significant physiological and genetic homology with humans. In this study, we performed a longitudinal analysis of clinical and microbiome data from 16 reproductive-age female rhesus macaques. At both the taxonomic and functional levels, the rhesus macaque vaginal microbiome was most similar to that of women who harbor a diverse vaginal community associated with asymptomatic/symptomatic bacterial vaginosis. Specifically, rhesus macaque vaginal microbiomes harbored a diverse set of anaerobic Gram-negative bacteria, including Sneathia, Prevotella, Porphyromonas, and Mobiluncus Interestingly, some animals were transiently colonized by Lactobacillus and some with Gardnerella Our in-depth and comprehensive analysis highlights the importance of the model to understand the health implications of a diverse vaginal microbiome and test interventions for manipulating this community.IMPORTANCE It is widely accepted that the "healthy" vaginal microbiome of women in the developed world is dominated by Lactobacillus species. However, in the developing world, many asymptomatic women harbor diverse vaginal microbial communities that are typically associated with bacterial vaginosis. Many questions remain about the drivers and health implications of a diverse vaginal microbial community. Rhesus macaques provide an excellent translational model to address these questions due to significant physiological and genetic homology with humans. In this study, we performed a longitudinal analysis of clinical and microbiome data from a large cohort of reproductive-age rhesus macaques. At the taxonomic, genomic, and functional levels, the rhesus macaque vaginal microbiome was most similar to that of humans, who harbor a diverse vaginal community associated with asymptomatic/symptomatic bacterial vaginosis. Our in-depth and comprehensive analysis highlights the utility of macaques as a model to study diverse vaginal community state types and test interventions for manipulating the vaginal microbiome.

Dataset Information

Comparison of storage conditions for human vaginal microbiome studies.

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Comparison of storage conditions for human vaginal microbiome studies.

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