Integrated multi-omics analysis uncovers cervicovaginal ecological networks and their association with <i>Chlamydia trachomatis</i> load

ABSTRACT Chlamydia trachomatis (Ct) is a causal agent of upper reproductive tract pathology. There is a broad spectrum of cervical Ct load in infected women, and upper tract infection is associated with higher cervical Ct load. Recent studies indicate that bacterial vaginosis (BV) can modulate host-Ct outcomes. To identify features associated with BV status and Ct load, we performed an integrated multi-omics analysis of the cervicovaginal microbiome, tryptophan metabolome, and cytokines. Samples were analyzed using 16S rRNA gene sequencing, targeted UPLC-MS/MS quantification of tryptophan metabolites, and multiplex cytokine profiling. Ordination analyses showed that BV status was separated by the microbiome, metabolome, and cytokines, whereas Ct load was separated only by cytokines. K-means clustering of tryptophan metabolites defined three metabolome state types (MSTs). MST I, associated primarily with Lactobacillus crispatus -dominated community state type (CST) I, exhibited high tryptophan availability, indole-3-lactic acid, and complete kynurenine-pathway activity. Both MST II and MST III were associated with BV-associated CST IV and showed marked tryptophan depletion. MST II was broadly depleted of most tryptophan metabolites, while MST III was enriched in downstream microbially derived indole pathway metabolites and kynurenic acid. Hierarchical all-against-all association testing revealed coordinated relationships linking clusters of bacterial taxa, metabolites, and cytokines. Importantly, multi-omics network analyses identified integrated microbial-metabolic-immune modules that predicted high versus low Ct load, highlighting CXCL9, CXCL10, IL-17, BV-associated taxa, and indole pathway metabolites as key discriminative features. Results demonstrate that cervical Ct load reflects coordinated microbial-metabolic-immune ecological states rather than microbiome composition alone and refine current models of Ct-BV interactions.