Background: Chlamydia genital infections continue to be a serious health concern globally. Previous studies have reported that Chlamydia trachomatis infection alters the vaginal microbiota of infected women. This study aimed to investigate alterations in the vaginal microbiome of pregnant women with and without C. trachomatis infection.

Methods: This was a cross-sectional study among 385 pregnant women, recruited from the antenatal clinic (ANC) at the King Edward VIII Hospital in Durban, South Africa.C. trachomatis was detected using the Applied Biosystems™ TaqMan^® Assays. A total of 40 samples, 20C. trachomatis positive and 20 C. trachomatis negative were selected for bacterial vaginosis (BV) screening using the Allplex™ Bacterial Vaginosis plus Assay. Sequencing of the microbiomes were performed using the PacBio platform. Statistical analysis was performed on IBM SPSS version 26 (IBM Corporation, Armonk, NY).

Results: The prevalence of BV and C. trachimatisco-infection was 65.00%. At the genus level, Gardnerella was more abundant in the C. trachomatis infected group (32.14%) compared to the uninfected group (24.02%). Similarly, at species level, thespecies annotated as Gardnerella were more abundant in the C. trachomatis infected group (31.97%) compared to the uninfected group (24.03%).In addition, L. iners was also more abundant in the C. trachomatis infected women (28.30%) compared to the uninfected women. However, pairwise comparisons and the Holm-Sidak method showed no significant differences between women with C. trachomatis infection and the control group. The discriminant analysis showed that the class Alpha-Proteobacteria(p=0.029); order Bacillales(p=0.018); family Enterococcaceae(p=0.002); genus Enhydrobacter(p=0.044),Enterococcus(p=0.004) and Parabacteroides(p=0.020); and species Enterococcus spp. (p=0.005) and Pseudomonasstutzeri(p=0.009) were significantly associated with C. trachomatis infection.

Conclusion: The organisms that were significantly associated with C. trachomatis in this study cohort have not been previously observed in association with C. trachomatis infection or the vaginal microbiota. Future studies in larger cohorts that will investigate the role of these microorganisms in C. trachomatis infection and in the vaginal microbiota are required.

Keywords: Chlamydia trachomatis, Vaginal microbiota, Pregnant women, Bacterial vaginosis