Non-O1/non-O139 Vibrio cholerae (NOVC) isolates are non-toxigenic and regarded as etiological agents of infrequent but mild-to-severe human gastroenteritis. In response to the 2022–2023 cholera outbreak in Tshwane, this study investigated the occurrence of NOVC isolates in wastewater within the Tshwane district, South Africa. A total of 341 wastewater samples were screened using Thiosulfate citrate bile salts sucrose (TCBS) media, with subsequent confirmation of Vibrio cholerae (V. cholerae) isolates by multiplex-PCR assays. Rep-PCR was performed to determine the genetic relatedness of the isolates. Selected isolates were subjected to whole genome sequencing (WGS). Of the 341 samples, PCR confirmed 143 isolates as NOVC. Genetic fingerprinting grouped these isolates into 12 distinct clusters, from which 12 representative isolates were selected for WGS and 10 isolates were confirmed as NOVC. The isolates harboured pathogenicity-related genes, such as hlyA and rtxA, as well as other genes contributing to bacterial adaptation such as trkH and tnaA. Drug resistance was mostly observed to first-line antibiotics, ciprofloxacin, chloramphenicol, and trimethoprim–sulfamethoxazole. Plasmid analysis showed six isolates harboured plasmids (pA, p21L, and PVN84) bearing multiple resistance determinants. Phylogenetic analysis showed evidence of genetic diversity amongst isolates. Although the isolates lacked classical toxigenic genes, they carried other virulence determinants associated with pathogenicity, posing a potential risk for clinical infection, highlighting the need for sustained surveillance.

Non-O1/non-O139 Vibrio cholerae (NOVC) species are typically non-toxic and are regarded as etiological agents of infrequent, but mild to severe human gastroenteritis. In response to the 2022-2023 cholera outbreak in Tshwane, this study investigated the occurrence of NOVC isolates in wastewater within the Tshwane district, South Africa. A total of 341 wastewater samples were screened using Thiosulfate citrate bile salts sucrose (TCBS) media, with subsequent confirmation of Vibrio cholerae (V. cholerae) isolates by multiplex-PCR assays. Rep-PCR was performed to determine genetic relatedness of the isolates. Selected isolates were subjected to whole genome sequencing (WGS). Of the 341 samples; PCR confirmed 143 isolates as NOVC. Genetic fingerprinting grouped these isolates into 12 distinct clusters, from which 12 representative isolates were selected for WGS, all of which were confirmed as NOVC. The isolates harboured pathogenicity-related genes, such as hlyA, rtxA, trkH, tnaA, gyrB and gyrA. Drug resistance was mostly observed to first-line antibiotics, ciprofloxacin, chloramphenicol, and trimethoprim-sulfamethoxazole. Plasmid analysis showed seven isolates harboured plasmids (pA, p21L, PVN84) bearing multiple resistance determinants. Phylogenetic analysis showed evidence of genetic diversity amongst the isolates. Although the isolates lacked classical toxigenic genes, they carried other virulence determinants associated with pathogenicity, posing a potential risk for clinical infection, highlighting the need for sustained surveillance.