Genomic characterization of Vibrio cholerae Isolated from Clinical and Environmental sources during the 2022-2023 cholera outbreak in Kenya

Lydia M Mageto^1,2*Gabriel Oluga Aboge¹Zelalem Mekuria³Peter Gathura¹John Juma⁴Kelvin Kimutai Kering⁵Collins kipkorir Kebenei⁵Beatrice Atieno Ongadi⁵Michael Mugo⁵Cecilia Kathure Mbae⁵Samuel Kariuki⁵

• ¹University of Nairobi, Nairobi, Kenya
• ²WSU Global Health-Kenya, Paul G. Allen School for Global Health, College of Veterinary Medicine, Washington State University, Nairobi, Kenya
• ³Department of Veterinary Preventive Medicine, College of Veterinary Medicine, Ohio State University, Columbus, Ohio, United States
• ⁴International Livestock Research Institute (ILRI), Nairobi, Kenya
• ⁵Centre for Microbiology Research, Kenya Medical Research Institute (KEMRI), Nairobi, Kenya

Background: Cholera remains a public health challenge in Kenya. To better understand its dynamics, we analysed Vibrio cholerae genomes from clinical and environmental samples collected during the 2022-2023 outbreak. These strains were compared with historical genomes from Kenya, Uganda, Tanzania and Haiti to inform strategies for cholera prevention, control and elimination in Kenya.Methods: Clinical (stool) and environmental (wastewater, drinking water and household effluent) samples were collected from Nairobi County.Samples were analysed for Vibrio cholerae using culture and real time PCR. The environmental (n=17) and clinical (n=70) isolates were then subjected to phenotypic antimicrobial susceptibility testing using the Kirby-Bauer disk diffusion method. Whole genome sequencing was employed to characterize the genome, detect antimicrobial resistance genes, virulence factors and mobile genetic elements. Phylogenetic analysis was performed to assess the genetic relationship and diversity of isolates from 2022-2023 outbreak, comparing them with isolates from historical outbreaks.Results: Clinical isolates carried key virulence genes (ctxA, ctxB7, zot, hlyA) and were 100% resistant to multiple antibiotics, including ampicillin, cefotaxime, ceftriaxone, and cefpodoxime, but remained susceptible to gentamicin and chloramphenicol. In contrast, environmental isolates lacked ctxB gene but harbored toxR, als and hlyA, showing variable antibiotic resistance (59% to ampicillin, 41% to trimethoprim-sulfamethoxazole, and 47% to nalidixic acid). All clinical isolates from 2022-2023 outbreak harbored IncA/C2 plasmids and several antimicrobial resistance genes including blaPER-7. Phylogenetic analysis revealed high genetic diversity in environmental strains, clustering outside the 7 th Pandemic El Tor lineage, while clinical isolates were highly clonal. Genomes from 2022-2023 outbreak were closely related to Kenyan cholera outbreak genomes from 2016 (15 Single Nucleotide Polymorphisms, T13 lineage).The 2022-2023 outbreak likely resulted from re-emergence of previously circulating strains rather than a new introduction. While the role of environmental reservoirs as a source of human infection remains unclear in our study, environmental isolates possess virulent and antimicrobial resistance genes that may spread via horizontal gene transfer. This highlights the need for continuous genomic surveillance to monitor V. cholerae evolution, track transmission patterns, and mitigate the spread of antimicrobial resistance.