Cervicovaginal dysbiosis is an established co-factor of high-risk human papillomavirus (HPV) persistence and cervical neoplastic development, yet most studies address the bacterial compartment in isolation, leaving fungal communities and bacterial–fungal cross-kingdom interactions underexplored, particularly in Central Asian populations. We performed shotgun metagenomic sequencing (mNGS) of cervicovaginal samples from 311 Kazakhstani women undergoing routine cervical screening. HPV status was determined using combined PCR and mNGS methods, and cervical screening was completed using liquid-based cytology (NILM, ASC-US, LSIL, ASC-H). Bacterial, viral, and fungal taxa were profiled from a single shotgun dataset with Kraken2 pipeline. Bacterial community state types (CSTs) were determined based on dominant bacterial species, functional gene content was annotated against KEGG using eggNOG, and covariate-adjusted associations were estimated using MaAsLin3. Mycobiome β-diversity differed significantly by HPV status (p = 0.003). In particular, Candida positivity was significantly associated with HPV presence and with high-risk α-9 HPV in cytologically normal (NILM) samples (OR = 3.6, [1.6–9.6], p ≤ 0.001). Covariate-adjusted analysis was consistent with this positive association (q < 0.05). Concurrently, among CSTs, Lactobacillus iners-dominated CST III and dysbiotic Gardnerella vaginalis-dominated CST IV showed a 3-fold higher Candida albicans prevalence (p < 0.01). Further analysis demonstrated that, functionally, both of these CSTs had depleted capacity for lactate metabolism (ko00620, p < 0.0001) and, in particular, for the genetic capacity for pyruvate-dependent H₂O₂ generation (half that of the L. crispatus-dominated CST I). These findings support L. iners as a metabolically permissive rather than protective Lactobacillus and suggest cross-kingdom functional signatures as candidate biomarkers for HPV acquisition and persistence in Central Asia, a region previously absent from the cervicovaginal microbiome literature.

Introduction: The cervicovaginal microbiome is a key determinant of reproductive health. Its multi-kingdom structure and ecological interactions remain insufficiently characterized across diverse populations. This study aimed to define the composition and cross-kingdom dynamics of the cervical microbiome in women without HPV infection and with normal cytology in a Kazakhstani population. Methods: In this cross-sectional study, cervical samples from 92 reproductive-age women were analyzed using whole-genome metagenomic sequencing to characterize bacterial, viral, fungal, and archaeal communities, together with predicted functional pathways. Microbial communities were stratified into community state types based on dominant bacterial species. Results: Bacterial composition differed markedly across community states, with Lactobacillus -dominated profiles associated with low diversity and anaerobe-rich communities associated with higher diversity. In contrast, viral, fungal, and archaeal diversity remained relatively stable, although descriptive compositional shifts indicated variation in bacteriophages, methanogenic archaea, and opportunistic fungi in non- Lactobacillus communities. Functional analyses indicated CST-associated pathway differences, suggesting greater metabolic flexibility in dysbiotic states, and exploratory network analysis revealed CST-associated restructuring of bacterial and cross-kingdom co-variation patterns. Notably, more than half of participants exhibited non- Lactobacillus -dominated communities despite the absence of infection or cytological abnormalities, indicating population-specific microbiome configurations. Discussion: Study demonstrates that the cervical microbiome is accompanied by exploratory cross-kingdom compositional variation and ecological states traditionally considered dysbiotic may represent stable, population-specific configurations, highlighting the need for context-dependent definitions of microbial health.

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.

The cervicovaginal virome remains poorly characterized in relation to human papillomavirus (HPV) infection and bacterial community structure. We performed shotgun metagenomic sequencing of 311 cervicovaginal specimens from a Kazakhstani cohort spanning seven groups defined by HPV status and cervical cytology. Viral community composition differed according to HPV status, with increasing representation of the oncogenic Alphapapillomavirus 9 clade across more abnormal cytological categories. Predicted phage functional profiles also differed between NILM HPV-positive and NILM HPV-negative women. Integrase, excisionase, transcriptional repressor, anti-repressor Ant, and amidase annotations showed differential prevalence after false-discovery-rate correction. Auxiliary metabolic and host-interaction genes associated with nucleotide metabolism, DNA modification, anti-restriction functions, and toxin–antitoxin systems were also differentially represented. Stratification by bacterial community state type revealed contrasting predicted functional repertoires, with toxin–antitoxin-associated annotations enriched in Lactobacillus crispatus-dominated communities and anti-restriction-associated annotations enriched in Gardnerella vaginalis-dominated communities. These findings identify associations between HPV status, bacterial community structure, and cervicovaginal viral composition and predicted phage functions. Longitudinal and experimental studies are required to determine the directionality and biological activity of these associations.