Anaerobes in the microbiomeQuantification, isolation and characterization of Bifidobacterium from the vaginal microbiomes of reproductive aged women
Introduction
Bifidobacteria were first described by Tissier in 1899, who isolated a bacterium from breast-fed infant feces and named it Bacillus bifidus [1]. In 1924, Orla-Jensen proposed the genus Bifidobacterium as a separate taxon for these organisms [2], which currently includes more than 30 species [3]. Bifidobacteria are Gram-positive, anaerobic, non-motile, non-spore forming rod-shaped bacteria, with varied branching. They belong to the Bifidobacteriaceae family and have high genomic G + C content (55–67 mol%) [3]. Bifidobacteria are known to colonize the human vagina, oral cavity and, more abundantly, the gastrointestinal tract (GIT) [4]. Several studies have shown their influence on human physiology and nutrition [5], [6], [7], [8], [9]. In newborns, bifidobacteria play an important role as one of the primary colonizers of the GIT, representing 60–91% of fecal bacteria in breast-fed infants [10], [11]. This proportion decreases with age and it may represent less than 10% of the adult fecal microbiota [12], [13]. Bifidobacteria provide protection from pathogens in the GIT through the production of bacteriocins [7], inhibition of pathogen adhesion [5], and modulation of the immune system [14], [15]. Due to these health-promoting effects, bifidobacteria have been extensively studied as probiotics [8], [16], [17], [18].
Early microbial colonization is an essential process in the maturation of the immune system [19]. This initial colonization may be affected by many factors, such as the mode of delivery (vaginal or caesarean section), feeding type (breast-fed or formula-fed), exposure to antibiotics and hygiene [20]. However, the relative contributions of maternal microbiota (gut, breast milk, vaginal) and environmental sources to the bifidobacteria population of the neonatal gut remain unresolved.
While Bifidobacterium spp. present in the gut are well described, vaginal bifidobacteria remain relatively poorly characterized, and it is not known if vaginal adaptation has resulted in distinct phenotypic features that distinguish them from gut populations. Although a healthy vaginal microbiota is defined as Lactobacillus-dominated, several studies have identified vaginal Bifidobacterium-dominated profiles in 5–10% of healthy, reproductive aged women [21], [22], [23], [24]. Furthermore, vaginal bifidobacteria are reported to produce lactic acid and hydrogen peroxide; attributes of vaginal lactobacilli credited with maintaining homeostasis in the vaginal microbiome [25].
Culture-independent techniques are useful tools in microbiome characterization, but methods based on amplification and sequencing of 16S rRNA genes, have been reported to underrepresent Bifidobacterium in microbial communities [26]. The abundance of Bifidobacterium in the vaginal microbiota may also be underestimated due to the similarity of their 16S rRNA sequences to those of Gardnerella vaginalis. G. vaginalis is also a member of the Bifidobacteriaceae family and is a commonly detected microorganism associated with bacterial vaginosis (BV) [27]. The use of the cpn60 “universal target” (UT) region as a barcode for microbiome profiling results in better resolution of closely related species, including those within Bifidobacteriaceae [28], and cpn60 based human fecal microbiome profiles have been shown to more accurately represent Bifidobacterium content than a 16S rRNA based approach [26]. Previous studies of the vaginal microbiome [21] or synthetic mixtures of vaginal organisms [29] have demonstrated a strong correlation between cpn60 sequence read abundance and organism abundance determined by quantitative PCR. However, regardless of the target used, relative abundance of specific organisms within complex communities may not be represented accurately by methods that rely on polymerase chain reaction (PCR) amplification and its inherent biases.
Considering the lack of information about Bifidobacterium spp. of vaginal origin, their importance as a potential source for the neonatal gut microbiome, and their potential health-promoting effects in the vagina, a better understanding of the properties of vaginal bifidobacteria is needed. In this study, our main objectives were: 1) to apply species-specific quantitative PCR to confirm the relative abundance of Bifidobacterium in the vaginal microbiomes of reproductive aged women previously determined based on cpn60 barcode sequencing, and 2) to characterize vaginal Bifidobacterium isolates based on carbohydrate fermentation patterns, hydrogen peroxide production, lactic acid production, resistance to low pH and lactic acid, and susceptibility to antibiotics.
Section snippets
Samples and microbiome profiles
Vaginal microbiome profiles from 492 healthy women were previously published by our research group [30], [31]. Profiles were created by PCR amplification and deep sequencing of the cpn60 UT region. Total bacterial load in each sample was also estimated as part of these studies using a SYBR Green assay based on the amplification of the V3 region of the 16S rRNA gene. The remaining vaginal swabs and DNA extracts from these studies, archived at −80 °C, were available for use in the current study.
Bifidobacterium quantitative PCR assays
Confirmation of Bifidobacterium-dominated vaginal microbial profiles
We identified 21/492 (4.2%) of the previously published vaginal microbiome profiles that were dominated (>50% of sequence reads) by Bifidobacterium-like sequences, of which eight were dominated by B. breve, five by B. longum, three by B. dentium and five by A. omnicolens. An additional 6% (29/492) of microbiome profiles had intermediate (1–50%) levels of Bifidobacterium-like sequences and 59.5% (293/492) of profiles had low (<1%) levels. Bifidobacterium-like sequences were undetected in 30.3%
Discussion
As a result of microbiome characterization by culture-independent, DNA sequence based methods there is a growing appreciation of “atypical” vaginal microbiomes in healthy women, such as the Bifidobacterium-dominated profiles. However, it is known that DNA extraction methods and PCR amplification biases affect the sequencing outcome, resulting in a view of the abundances of species within the community that is inevitably distorted to some degree. Thus, for a careful investigation of the
Conclusion
In this study we confirmed that a subset of healthy, reproductive aged women have vaginal microbiomes dominated by Bifidobacterium spp. We also demonstrated that vaginal bifidobacteria have the potential to be as protective as lactobacilli according to the current understanding of a “healthy” vaginal microbiome. These results have significant implications for women's health diagnostics since current protocols based on Gram staining and Nugent score would likely result in a diagnosis of
Conflict of interest
The authors declared that they have no conflict of interests.
Acknowledgements
The authors would like to thank John Schellenberg for providing some of the bifidobacteria isolates and Champika Fernando for insightful discussions during the execution of this study. The authors are grateful to the Vaginal Microbiome Group Initiative (VOGUE) team members Deborah Money, Alan Bocking, Sean Hemmingsen, Janet Hill, Gregor Reid, Tim Dumonceaux, Gregory Gloor, Matthew Links, Kieran O'Doherty, Patrick Tang, Julianne Van Schalkwyk and Mark Yudin for access to samples, and helpful
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