Background: Human papillomavirus (HPV) infection is the primary risk factor for cervical cancer. Recently, the cervical bacterial community has been considered an additional risk factor for HPV infection and carcinogenesis. A decrease in Lactobacillus proportions, along with an increase in bacterial diversity, known as dysbiosis, has been associated with HPV persistence. In tumors, bacterial profiles have been linked to intratumoral immune responses and the efficacy of treatment across cancer types. Understanding the taxonomic and functional composition of the cervical cancer microbiome is essential to understand each microorganism’s role and its contribution to cancer development and prognosis. Objectives: To characterize the microbiota in cervical cancer tumor tissue using shotgun sequencing. Methods: DNA was extracted from tumor samples of 29 patients followed at Instituto Nacional de Câncer (INCA) and sequenced on the MGI Tech DNBSEQ-T7 platform. Sequences were quality-filtered and taxonomically assigned using the Kraken2 program with the RefSeq Standard-16 database. Relative abundances were normalized in R (vegan v.2.6-10). Taxonomic diversity was assessed and compared between samples of different histological types, tumor stages, and HPV genotypes using statistical analyses. Results: A total of 917 species and 359 genera of bacteria were identified. The most abundant species were Fusobacterium vincentii, Bacteroides fragilis, Porphyromonas somerae and Campylobacter ureolyticus. Clinical stage II samples showed higher alpha diversity (Shannon Index), compared to stage I and stage III samples. In the multivariate analysis, a lower abundance of the Mycolicibacterium genus was observed in squamous cell carcinoma samples and a higher abundance of the Kocuria genus in samples from non-smoking women, when compared to adenocarcinomas and smoking samples, respectively. A correlation analysis was performed using Spearman’s method (prevalence ≥20%; r > 0.85), identifying 15 strong positive associations between bacterial taxa. Notably, Murdochiella vaginalis – Anaerococcus obesiensis (r = 0.98), Cruoricaptor ignavus – Anaerococcus mediterraneensis (r = 0.93), Campylobacter hominis – Filifactor alocis (r = 0.90), and correlated abundance patterns among Prevotella species were observed, indicating anaerobic consortia associated with cervical dysbiosis in cervical cancer. Conclusions: The characterization of the intratumoral microbiota in cervical cancer revealed a diverse microbial community potentially linked to disease development and progression, with specific associations observed between certain microorganisms and clinical features, as well as the identification of strong positive correlations between bacterial taxa. These findings highlight the need to evaluate the microbiome e and its role in the tumor microenvironment to develop more effective therapeutic and screening strategies.

Funding: National Council for Scientific and Technological Development (CNPq), Ministério da Saúde do Brasil, SwissBridge Foundation.