Marisa Di Pietro1, Simone Filardo1, Maria Grazia Porpora2, Nadia Recine2, Maria Agnese Latino3,. Rosa Sessa1* .... beads (Beckman Coulter, USA). The final ...
Full Paper HPV/Chlamydia trachomatis co-infection: metagenomic analysis of cervical microbiota in asymptomatic women Marisa Di Pietro1 , Simone Filardo1 , Maria Grazia Porpora2 , Nadia Recine2 , Maria Agnese Latino 3 , Rosa Sessa1 * 1 Section
of Microbiology, Department of Public Health and Infectious Diseases, University of Rome
"Sapienza", Rome, Italy; 2 Department 3 Unit
of Gynecology, Obstetrics and Urology, University of Rome "Sapienza", Rome, Italy;
of Bacteriology, STIs Diagnostic Centre, Sant’Anna Hospital, Turin, Italy
M. Di Pietro and S. Filardo contributed equally to the manuscript Running title: HPV/C. trachomatis co-infection and cervical microbiome SUMMARY HPV and Chlamydia trachomatis are the most common causes of sexually transmitted diseases worldwide. Most infections are asymptomatic and left untreated lead to severe reproductive tract sequelae such as cervical cancer and infertility. Interestingly, C. trachomatis may also increase the susceptibility to HPV infection as well as contribute to viral persistence. Recently, a growing body of evidence has suggested that the composition of the cervico-vagina l microbiota plays a key role in the susceptibility and outcome of genital infections caused by several pathogens, including HPV and C. trachomatis. The aim of our study was to undertake a metagenomic analysis of sequenced 16s rRNA gene amplicons to characterize the cervical microbiota from asymptomatic women with HPV/C. trachomatis co-infection. The composition of the cervical microbiota from HPV-positive or C. trachomatis-positive women was also analysed. The main finding of our study showed that the cervical microbiota in HPV/C. trachomatis co-infected women had a higher microbial diversity than the cervical microbiota in healthy controls (p2 have been included in the analysis. Statistical analysis Nonparametric T-test based on Monte Carlo permutations was used for alpha diversity comparisons, Kruskal-Wallis test for taxa level comparisons, and Adonis for category comparisons of distance matrixes, all calculated in QIIME. Bonferroni correction was used to correct for multiple hypothes is testing when necessary. All remaining statistical calculations were performed in Excel (Microsoft, USA) and R 3.1.2 (R development core team). Chi-squared test was used for assessment of association of frequencies among groups (Fisher’s exact test was used when any cell had expected values < 5). Mann-Whitney U test for non-parametric data was used for comparison of means. The single or multiple inference significance level was set to 5%. RESULTS Study subject characteristics Thirty- five women of reproductive age were enrolled in this study. Ten women were positive to C. trachomatis, 10 women were positive to HPV, 5 women had a HPV/C. trachomatis co-infection and 10 were negative to any genital pathogen (healthy controls). No women had any specific genita l symptoms related to HPV and/or chlamydial infection. All 35 samples underwent 16s rRNA amplicon-based microbiome analysis and 10 samples were excluded from downstream analysis due to a number of reads 4), a recently developed algorithm for high dimensional biomarker discovery in metagenomic data. Aerococcus christensenii in cervical microbiota might represent, thus, a potential biomarker of C. trachomatis infection, even though this needs to be further evaluated. However, to date, the role of A. christensenii in the cervical environment is still not known, although this microorganism is usually found more frequently in women with bacterial vaginosis, the most common dysbiosis condition (Ling et al., 2010). Lastly, unlike HPV/C. trachomatis coinfection and C. trachomatis infection, the cervical microbiota from HPV-positive women did not show a higher species diversity as compared to cervical microbiota from healthy controls. Indeed, the cervical microbiota from HPV-positive women was similar to the cervical microbiota from healthy controls, since both were characterized by the predominance of Lactobacillus species. The similarity between HPV-positive women and healthy controls was further confirmed by the weighted UniFrac analysis. Specifically, we found that the cervical microbiota from HPV-infected women and healthy controls significantly defined a well separated cluster compared to the cervical microbiota from C. trachomatis-positive and co-infected women (p
