Amplification of the V5 – V8 region of the 16S rRNA gene effectively speciates medically important genital tract Lactobacillus species in the upper female genital tract

Background The endometrial cavity is an upper genital tract site largely heralded as sterile, however, advances in culture-independent, next generation sequencing technology have revealed that this site harbours a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most common non-pathogenic genital tract commensals. Next-generation sequencing of the female lower genital tract has revealed significant variation amongst microbial community composition with respect to Lactobacillus sp. in samples collected from healthy and diseased women. The aim of this study was to evaluate the ability of the 16S rRNA gene to characterize genital tract lactobacilli to species-level taxonomy. Methods Samples were interrogated for the presence of microbial DNA using two-step next generation sequencing technology to exploit the V5–V8 regions of the 16S rRNA gene and compared to standard speciation using qPCR. Results The V5-V8 region of the 16S rRNA gene has sufficient sequence variation within frequently encountered genital tract lactobacilli to allow accurate determination of relative abundance within the community, and speciation for several key community members without completing additional experimentation. Conclusions Next-generation sequencing of clinical genital tract isolates is an effective method for high throughput identification to species-level of key Lactobacillus sp. IMPORTANCE Human microbiome experiments, including the low biomass organs such as the upper genital tract, require the development of consensus protocols to ensure accurate comparison between such studies and our data forms an important foundation for future protocols. This paper provides evidence to support the selection of the V5-V8 regions of the 16S rRNA gene improved Lactobacillus speciation using next generation sequencing technology. The choice of variable region for broad-range amplification in microbiome studies is important due to preferential primer binding associated with some genera based on nucleotide sequence patterns. By utilising the V5-V8 region, multiple species of Lactobacillus can be characterised with relative confidence. * ### List of abbreviations ATCC : American type culture collection DGC : dysmenorrhea progestin effect endocervix DGE : dysmenorrhea progestin effect endometrium DNA : deoxyribonucleic acid DPC : dysmenorrhea proliferative endocervix DPE : dysmenorrhea proliferative endometrium DSC : dysmenorrhea secretory endocervix DSE : dysmenorrhea secretory endometrium HRM : high resolution melt MGC : menorrhagia progestin effect endocervix MGE : menorrhagia progestin effect endometrium MPC : menorrhagia proliferative endocervix MPE : menorrhagia proliferative endometrium MSC : menorrhagia secretory endocervix MSE : menorrhagia secretory endometriumx OTU : operational taxonomic unit rRNA : ribosomal ribonucleic acid VIC : virgo intacta