Genomic epidemiology reveals geographically structured co-circulation of AFR10 and AFR13 Vibrio cholerae lineages driving cholera outbreaks in Tanzania (2022-2024).

Cholera remains a persistent public health challenge in Tanzania, with recurrent outbreaks across multiple regions overwhelming the health systems. Despite the ongoing surveillance to control the transmission, limited genomic characterization of circulating Vibrio cholerae strains has constrained traceability of outbreak sources and transmission dynamics including virulence determinants and antimicrobial resistance profiles to guide clinical and public health interventions. We conducted a genome analysis of 144 archived isolates from 2022 to 2024 cholera outbreaks from 16 regions across Tanzania. Sequencing was performed using Oxford Nanopore platforms. Comprehensive bioinformatics analysis was applied including genome assembly, multi-locus sequence typing, resistance gene detection, virulence profiling and phylogenetic reconstruction. Of 144 isolates, sequencing confirmed 119 to be V. cholerae, of which 109 (91.6%) belonged to sequence type ST69. Phylogenetic analysis identified two distinct transmission lineages: AFR10 (formerly T10) and AFR13 (formerly T13), demonstrating marked geographic stratification. AFR10 isolates clustered predominantly around Lake Tanganyika basin, bordering the Democratic Republic of the Congo and Zambia, whereas AFR13 isolates were widely distributed across different zones of Tanzania. All isolates harboured key virulence genes (ctxA, ctxB, tcpA) and multiple resistance genes conferring resistance to co-trimoxazole and macrolides. The catB gene was found to be silenced in these isolates. Notably, tetracycline resistance genes (tetA/B/C) were absent. The qacEdelta gene was only detected in AFR13 isolates and has been linked to resistance to quaternary ammonium compounds (antiseptics and disinfectants) in Gram-negative bacteria from other studies. These findings demonstrate the co-circulation of two sublineages (AFR10 and AFR13) of the seventh pandemic El Tor lineage with distinct geographic distribution, suggesting both localized persistence and regional circulation. The observed genotypic and phenotypic susceptibility to tetracycline supports the continued use of doxycycline as a first-line therapy in adults. However, the detected macrolide resistance (an alternative treatment for children) and resistance genes associated with reduced susceptibility to antiseptics underscore the need for extensive monitoring. Strengthened national and cross-border genomic surveillance is essential for effective cholera control.