Virulent drug resistant non-O1/non-O139 vibrio cholerae strains identified in wastewater in Tshwane district

ABSTRACT

¹Ramocha O,

¹Microbiological pathology, School of Medicine, Sefako Makgatho Health Sciences University

Background

Non-O1/ non-O139 Vibrio cholerae (NOVC) species are typically non-toxic and regarded as etiological agents of infrequent but mild to severe human gastroenteritis. NOVC strains have been shown to acquire virulence factors including outbreak associated toxin and toxin mediating genes ctxA and tcpA among others. In 2022-2023 there was a significant outbreak of cholera in Tshwane prompting this study to investigate NOVC isolates circulating in wastewater in Tshwane district between May 2023 and May 2024.

Method

Study isolates were obtained from 345 wastewater samples using TCBS selective media and confirmed using multiplex-PCR assays. REP-PCR was performed to ascertain genetic relatedness. Representative isolates were subjected to whole genome sequencing (WGS) using Oxford Nanopore Technologies MinION. The rapid barcoding kit was used for library preparation following the Nanopore-Only Microbial Isolate Sequencing Solution (NO-MISS) protocol. Sequence analysis was performed on the cloud-based EPI2ME software employing Medaka, Prokka, ResFinder and MLST programs. The genome mapping was constructed using Proksee.

Result

In total, 345 samples were processed from which 145 isolates were classified as NOVC. Rep-PCR clustered isolates into 12 groups. 12 representative isolates subjected to WGS were confirmed as NOVC. The isolates harboured pathogenicity related genes such hlyA, rtxA, ompW, ompU, trkH, tnaA and gyrA while toxigenic genes such as ctxA and tcpA were absent. The analysis revealed 30 genes mostly associated with resistance mechanisms such as efflux pump, enzymatic degradation and target replacement. Drug resistance was mostly observed in the first-line antibiotics, ciprofloxacin, chloramphenicol, trimethoprim sulfamethoxazole, tetracycline, ampicillin and erythromycin.

Conclusion

Although the isolates do not harbour toxigenic genes, they still possess other virulence factors responsible for pathogenicity, posing a potential risk for clinical infection. This highlights the critical need for ongoing surveillance to mitigate the risk of these strains emerging as significant public health concern.

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PRESENTING AUTHOR

Ms. Onalenna Ramocha, BSc in Biochemistry and Microbiology, Honors in Microbiology

Student, Sefako Makgatho Health Sciences University (SMU)

A dedicated and detail-oriented postgraduate student currently pursuing an MSc in Medicine with a specialization in Microbiology. Holds a BSc Honours in Microbiology, as well as a BSc in Biochemistry and Microbiology, reflecting a solid foundational knowledge in the biological sciences and a strong commitment to academic and scientific excellence.

The current research project focuses on the identification and characterization of virulent, drug-resistant non-O1/non-O139 Vibrio cholerae strains isolated from wastewater in the Tshwane District. This work contributes to the growing body of research on antimicrobial resistance and environmental microbiology, with implications for public health and wastewater management.

Possesses a strong background in microbiological techniques, molecular diagnostics, and data analysis, with a keen interest in infectious diseases, environmental pathogens, and microbial resistance mechanisms. Committed to advancing scientific understanding through rigorous research and contributing to solutions that address pressing global health challenges.

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