Genomic Epidemiology of 3,076 Vibrio cholerae Isolates Reveals ST69 Clonal Expansion and Multidrug Resistance across Africa

Background Cholera remains a major public health threat across Africa, driven by complex interactions between environmental, socioeconomic, and microbial factors. However, the continental genomic epidemiology of Vibrio cholerae remains incompletely characterised. We aimed to conduct a comprehensive continental genomic analysis of V. cholerae to determine its spatiotemporal dynamics, virulence, and antimicrobial resistance (AMR) profiles. Methods We conducted a large-scale genomic analysis of 3,076 V. cholerae isolates collected from 29 African countries between 1949 and 2025. Genomic data were retrieved from publicly available databases and subjected to standardized quality control, assembly, and annotation pipelines. Phylogenomic reconstruction was performed using core genome single-nucleotide polymorphism (SNP) analysis with recombination filtering. We conducted multilocus sequence typing (MLST), virulence profiling, AMR gene detection, and mobile genetic element characterization using established bioinformatics tools to assess associations between lineage distribution, geographic regions, temporal trends, and resistance patterns. Results The population is highly clonal and dominated by sequence type 69 (ST69) (2,682 [87.2%] of 3,076 genomes). ST69 almost exclusively drove the 2022–2024 continent-wide outbreaks, representing 98.9% (463/468) of genomes in 2022, 99.3% (605/609) in 2023, and 97.3% (252/259) in 2024. Toxigenic O1 El Tor markers and ctxA/ctxB co-carriage exceeded 91% in clinical genomes but were significantly attenuated in environmental strains. We identified 101 distinct AMR genes, with 94.1% of isolates classified as multidrug-resistant. Ubiquitous chromosomal mutations, primarily gyrA_S83I (92.88%) and parC_S85L (84.82%), drove universal fluoroquinolone resistance. An SXT conjugative element conferring multidrug resistance was present in roughly 64% of the population. Clinical isolates exclusively harboured high-risk resistance determinants absent environmentally, including macrolide resistance genes (6.6%–6.8%) and extended-spectrum beta-lactamases such as blaPER-7 (6.4%). Conclusion Cholera transmission across Africa is persistently driven by the clonal expansion of the ST69 lineage. Universal fluoroquinolone resistance and clinically restricted emergence of macrolide and beta-lactamase resistance highlight a critical therapeutic challenge. Sustained genomic surveillance is essential to monitor this multidrug-resistant clone and inform regional outbreak control.