Intra-individual genetic diversity of vaginal Lactobacillus crispatus revealed through citizen science-driven isolation and pangenome analysis

Aims A vaginal microbiome dominated by Lactobacillus crispatus is associated with positive reproductive and sexual health outcomes, yet intra-individual genetic diversity within this species remains largely unexplored. This study characterised inter- and intra-individual genomic variation in L. crispatus strains isolated through a citizen science initiative and assessed implications for multi-strain probiotic development. Methods and Results Fifty-three women participated in this citizen science project. Self-sampling resulted in 48 shotgun metagenomes. Twenty-two participants isolated their own L. crispatus strains using selective enrichment and LAMP-based species confirmation, resulting in 53 whole-genome-sequenced isolates. L. crispatus dominated 20 of 48 metagenomes (50.1–99.6% relative abundance). Pangenome analysis revealed 3 456 gene families, of which 43.7% were core and 56.3% accessory. A 14-kb plasmid harbouring a Fic-domain toxin-antitoxin protein, but devoid of antimicrobial resistance genes, was present in 44 of 53 strains. Strains from the same individual clustered closely together yet harboured 1–123 gene differences. Intra-individual variation was observed in the pullulanase type I gene required for glycogen degradation: 40 strains were predicted to grow on glycogen, six showed genetic disruptions with unknown consequences, and seven were predicted to lack this ability entirely. Variation within individuals was also found for bacteriocin classes and CRISPR-Cas genes. Conclusions Substantial functional diversity exists within L. crispatus, even among strains from the same individual, supporting the rationale for multi-strain vaginal probiotics. This citizen science approach enabled discovery of host-specific adaptations while ensuring participant ownership of their strains.