Methods to characterize the vaginal microbiome in a rhesus macaque model of simian human immunodeficiency virus (SHIV) transmission uncover epithelium-associated enrichment of Prevotella

The vaginal microbiome plays a crucial role in maintaining mucosal integrity and mitigating pathogen transmission, yet its comprehensive characterization remains challenging due to limited sampling and analysis methods. In this study, we aimed to characterize bacterial and fungal taxa diversities in the vaginal microbiomes of Simian Human Immunodeficiency (SHIV)-infected rhesus macaques, as well as their metabolic activities, using three sampling methods. The cervicovaginal lavage (CVL), vaginal swab, and vaginal mucosal tissue methods offer novel insights into microbial diversity and their potential impacts on HIV transmission. Using 16S rRNA and Internal Transcribed Spacer (ITS) sequencing, we assessed bacterial and fungal community composition and abundances, respectively, across all sampling methods. PICRUSt2 was used for functional predictions, and a modified glycosidase assay to further characterize glycan-degrading enzymatic activity in CVL samples. Our findings reveal that tissue samples were uniquely enriched for microbial taxa such as <i>Prevotella</i> spp. and <i>Helicobacter</i> spp., showing notable abundance differences compared to CVL and swab samples. Tissue samples exhibited higher alpha diversity and distinct metabolic prediction profiles, particularly elevated sialidase activity. While fewer differences were found in fungal microbiome composition and diversity, marked correlations were observed between bacterial and fungal taxa, emphasizing complex interkingdom interactions. These results highlight the significance of sampling methods in microbial ecology studies, which should be carefully considered due to their potential influence on pathogen transmission risk.