Lactobacillus species are common inhabitants of the ‘healthy’ female urinary and vaginal communities, often associated with a lack of symptoms in both anatomical sites. Given identification by prior studies of similar bacterial species in both communities, it has been hypothesized that the two microbiotas are in fact connected. Here, we carried out whole-genome sequencing of 49 Lactobacillus strains, including 16 paired urogenital samples from the same participant. These strains represent five different Lactobacillus species: L. crispatus, L. gasseri, L. iners, L. jensenii, and L. paragasseri. Average nucleotide identity (ANI), alignment, single-nucleotide polymorphism (SNP), and CRISPR comparisons between strains from the same participant were performed. We conducted simulations of genome assemblies and ANI comparisons and present a statistical method to distinguish between unrelated, related, and identical strains. We found that 50 % of the paired samples have identical strains, evidence that the urinary and vaginal communities are connected. Additionally, we found evidence of strains sharing a common ancestor. These results establish that microbial sharing between the urinary tract and vagina is not limited to uropathogens. Knowledge that these two anatomical sites can share lactobacilli in females can inform future clinical approaches.

The recognition of the Aerococcus urinae complex (AUC) as an emerging uropathogen has led to growing concerns due to a limited understanding of its disease spectrum and antibiotic resistance profiles. Here, we investigated the prevalence of macrolide resistance within urinary AUC isolates, shedding light on potential genetic mechanisms. Phenotypic testing revealed a high rate of macrolide resistance: 45%, among a total of 189 urinary AUC isolates. Genomic analysis identified integrative and conjugative elements (ICEs) as carriers of the macrolide resistance gene ermA, suggesting horizontal gene transfer as a mechanism of resistance. Furthermore, comparison with publicly available genomes of related pathogens revealed high ICE sequence homogeneity, highlighting the potential for cross-species dissemination of resistance determinants. Understanding mechanisms of resistance is crucial for developing effective surveillance strategies and improving antibiotic use. Furthermore, the findings underscore the importance of considering the broader ecological context of resistance dissemination, emphasizing the need for community-level surveillance to combat the spread of antibiotic resistance within the urinary microbiome.

While Escherichia coli can be found in the bladders of females without lower urinary tract symptoms, its presence is often associated with urinary tract infections (UTIs). The genomic plasticity of E. coli, including urogenital strains, is largely shaped by the integration of prophages. Although genomic and metagenomic analyses of urinary E. coli and the urinary microbiome suggest that prophages are abundant, many represent uncharacterized species. Sequence analysis suggests that these prophages represent temperate phages. This study aimed to fill this gap, isolating and characterizing temperate phages from urinary E. coli strains. We assessed phage host range across a panel of urinary isolates, providing a critical first step for future work investigating their putative role in shaping E. coli populations within the urinary community. In total, 20 temperate urinary phages were evaluated. Phage morphology and genic content of these phages were determined via transmission electron microscopy (TEM) and whole-genome sequencing, respectively. Together, these analyses provide insight into the diversity, infectivity, and genomic composition of temperate coliphages from the female urinary tract.