Tiered Functional Screening Identifies an Autochthonous Vaginal Lactiplantibacillus plantarum Strain with Probiotic...

Persistent high-risk human papillomavirus (HPV) infection drives cervical cancer, a leading cause of cancer-related mortality among women in low- and middle-income countries; its clinical course is shaped by the cervicovaginal microbiome, in which Lactobacillus-dominated communities are associated with enhanced viral clearance. Despite this, vaginal probiotic interventions often demonstrate limited colonization efficiency, and autochthonous strain libraries from Central Asia remain absent. We applied a tiered functional screening workflow to a collection of 235 vaginal lactic acid bacterial isolates recovered from 400 women undergoing routine gynecological examination in Astana, Kazakhstan. The workflow sequentially filtered isolates on (i) antimicrobial activity against seven urogenital indicator pathogens using the deferred antagonism assay, (ii) surface adhesion by the Brilis erythrocyte assay, and (iii) biofilm-forming capacity by crystal violet retention and laser-capture-microdissection (LCM) microscopy. Species-level identification of the selected candidate was performed by whole-genome shotgun sequencing followed by Kraken2 taxonomic classification. From 235 isolates, three rounds of phenotypic filtering identified four broad-spectrum antimicrobial candidates (127-3, 127-4, 107-2, 107-4) with non-overlapping inhibitory profiles against seven urogenital indicator strains. Adhesion phenotyping segregated candidates into low- and moderate-adhesion groups, with none reaching the high-adhesion threshold. Among all four candidates, only strain 127-4 produced a reproducible biofilm-associated signal (crystal violet retention OD490 = 0.09 ± 0.07 at 24 h; 0.08 ± 0.03 at 48 h), consistent with early surface attachment under static conditions. Whole-genome shotgun sequencing assigned 97.81% of classified reads to Lactiplantibacillus plantarum, supporting preliminary identification of the selected isolate as L. plantarum strain 127-4. Composite ranking confirmed 127-4 as the only isolate combining broad antimicrobial activity (5/7 indicators), moderate adhesion (specific adhesion index, SPA = 2.95), and a detectable biofilm-associated phenotype. We report the first systematic functional screening of autochthonous cervicovaginal lactic acid bacteria from a Central Asian population and identify L. plantarum 127-4 as a probiotic candidate with an integrated trait profile rarely identified through single-criterion screening approaches. Beyond candidate identification, this work establishes a transferable workflow for assembling functionally annotated vaginal Lactobacillus collections from underrepresented populations, providing a foundation for future population-specific probiotic interventions targeting cervicovaginal health.