Emergence of Vibrio cholerae O1 Sequence Type 75, South Africa, 2018–2020

We describe the molecular epidemiology of cholera in South Africa during 2018–2020. Vibrio cholerae O1 sequence type (ST) 75 recently emerged and became more prevalent than the V. cholerae O1 biotype El Tor pandemic clone. ST75 isolates were found across large spatial and temporal distances, suggesting local ST75 spread.

T he seventh cholera pandemic, caused by Vibrio cholerae O1 biotype El Tor (7PET), arrived in Africa during 1970 and became endemic in many countries on the continent (1). Cholera was fi rst reported in South Africa in 1974 (2). However, South Africa is not considered a cholera-endemic area; outbreaks typically are associated with importation, particularly from neighboring countries. The last cholera outbreak in South Africa was triggered by imported cases from an outbreak in Zimbabwe during 2008; South Africa reported 12,706 cases during November 2008-April 2009 (3).
Globally, 7PET isolates are genetically homogeneous and linked to the Bay of Bengal in South Asia (4,5). Most 7PET isolates are multidrug-resistant sequence type (ST) 69 (6). Rarely, 7PET has a single-locus variant, ST515, in isolates from Africa belonging to lineage T10 (7). As of September 2021, all cholera isolates from South Africa have been characterized as 7PET ST69 by multilocus sequence typing (MLST).
South Africa actively surveils for cholera. Since the 2008-2009 outbreak, few cases have been identifi ed: Limpopo, and KwaZulu-Natal (3), but all were caused by imported cases from neighboring Zimbabwe and Mozambique. Therefore, given their experience, healthcare workers and laboratorians in these provinces typically will test for cholera in all cases of acute watery diarrhea.
In South Africa, the National Institute for Communicable Diseases (NICD) is notifi ed of suspected cholera cases. NICD's Centre for Enteric Diseases supports case investigations and receives all human and environmental V. cholerae isolates for further investigation. The case defi nition for confi rmed cholera is isolation of V. cholerae O1 or O139 from a person with diarrhea. We investigated the molecular epidemiology of V. cholerae in South Africa during 2018-2020.
Of 9 V. cholerae O1 isolates tested, we identifi ed 2 ST69 (7PET) and 7 ST75 isolates. The ST69 isolates were collected in October 2018 from 2 cholera patients in a family cluster. The index case-patient had traveled to Zimbabwe, where an outbreak was ongoing (8), within the 7-day cholera incubation period before symptom onset. We confi rmed these ST69 isolates belonged to the previously described highly antimicrobial-resistant Zimbabwe outbreak strain (8  We describe the molecular epidemiology of cholera in South Africa during 2018-2020. Vibrio cholerae O1 sequence type (ST) 75 recently emerged and became more prevalent than the V. cholerae O1 biotype El Tor pandemic clone. ST75 isolates were found across large spatial and temporal distances, suggesting local ST75 spread.
from patients with cholera, all adults 37-57 years of age; 2 isolates were from environmental samples collected during case investigations, 1 from sewage in Limpopo Province and 1 from river water in KwaZulu-Natal Province ( Table 1). The 3 KwaZulu-Natal cases occurred ≈200-600 km apart; the first occurred in February 2018 and the last in January 2020. The 2 Limpopo cases occurred ≈70 km apart in the same district during November 2018. The Limpopo cases were >900 km from the KwaZulu-Natal cases. Epidemiologic investigations involved interviewing case-patients by using a standard case investigation form; visiting case-patients' residences to inspect water and sanitation services and interview other household members; collecting stool samples from household members; and collecting environmental samples when indicated. Investigators found no evidence of importation from another country, epidemiologic links between cases, or secondary transmission.
The only antimicrobial-resistance determinant found in all ST75 isolates was the qnrVC4 gene, located in the chromosomal superintegron. Various qnrVC alleles previously have been reported in the Vibrionaceae family and sometimes are associated with fluoroquinolone resistance (10,11). However, all ST75 isolates we analyzed showed fluoroquinolone susceptibility, MIC of ciprofloxacin 0.06 µg/mL, and susceptibility to all other tested antimicrobial drugs. This pansusceptibility sharply contrasts antimicrobial resistance trends observed in 7PET isolates from Africa, which reportedly became increasingly antimicrobial resistant over time; after the 2000s, none were susceptible to antimicrobial agents (5).  We further compared the ST75 isolates from South Africa with a larger global collection of 144 ST75, or closely related ST169, ST170, and ST182, genomes (Appendix 2), and constructed a maximum-likelihood phylogeny by using 49,540 SNPs (Figure). Our phylogenetic analysis showed that the 7 isolates from South Africa clustered in the L3b.1 clade, defined by H. Wang et al. (9), with a maximum pairwise distance of 22 SNPs. Isolates from Limpopo Province had a maximum pairwise distance of 1-6, but KwaZulu-Natal Province isolates had no SNP differences. Core-genome MLST showed Limpopo Province isolates differed from the KwaZulu-Natal Province isolates by 4-5 alleles (Appendix 1 Figure). The closest related isolates were collected in Russia from Rostov Oblast in 2005 and Republic of Kalmykia in 2011 and from Turkmenistan in Central Asia in 1965, but none of those isolates contained the CTX prophage. L3b.1 isolates from Taiwan containing the CTX prophage ctxB3 allele were more distant.
Emergence of ST75 L3b.1 clade in South Africa is cause for concern. Recent studies on V. cholerae O1 isolated in Taiwan (12) and China (13) reported emerging and potential toxigenic ST75. Genomic signatures of these ST75 isolates closely resembled the US Gulf Coast V. cholerae O1 clone that emerged in 1973 (14). In particular, an investigation of V. cholerae O1 isolated during 2002-2018 in Taiwan showed that ST75 emerged there in 2009 and now is more prevalent than the ST69 pandemic clone (12). Our findings from South Africa align with the findings from Taiwan, showing that ST75 isolates outnumber ST69 isolates.
One limitation of our study is that we used reference laboratory data and a review of published V. cholerae O1 data to conclude that all previous cholera isolates in South Africa characterized by MLST were V. cholerae O1 biotype El Tor ST69. However, we cannot exclude the possibility that V. cholerae O1 isolates not characterized by MLST, particularly those from environmental samples, could have been non-ST69.
Epidemic 7PET lineage cholera demands an aggressive public health response to prevent outbreaks. In contrast, sporadic V. cholerae O1 infections mediated by other lineages, including those carrying toxin co-regulated pilus and CTX genes, typically are not epidemic-prone; most are associated with sporadic cases that rarely lead to secondary transmission (15). Tailoring the public health response to the degree of epidemic risk would be invaluable, especially in resource-limited settings.
In countries that are not cholera-endemic but are at high risk for cholera introductions, conventional laboratory determination of V. cholerae O1, even complemented by identifying ctxA or ctxB genes, might be insufficient. Typing resolution of genomics, which distinguishes between 7PET and nonepidemic lineages, can elucidate the local and global epidemiology of cholera and inform public health decisions.

Conclusions
The emergence and dominance of nonepidemic, non-7PET, V. cholerae ST75 L3b.1 in South Africa requires close monitoring. The spatiotemporal pattern suggests local spread, possibly indicating a geographically widespread risk for sporadic disease from this strain. South Africa should strengthen its disease and environmental surveillance systems to identify nonpandemic ST75 strains, define local epidemiology, and inform an appropriate public health response.
This study was funded by the United Kingdom Department of Health and Social Care, managed by the Fleming Fund, and performed under the auspices of the SEQAFRICA project. The Fleming Fund is a £265 million aid program supporting <24 low-and middle-income countries to generate, share, and use data on antimicrobial resistance. The Fleming Fund works in partnership with Mott MacDonald, the Management Agent for the Country and Regional Grants and Fellowship Programme.

About the Author
Dr. Smith is a principal medical scientist at the Centre for Enteric Diseases, National Institute for Communicable Diseases, Johannesburg, South Africa. He also holds an extraordinary professor appointment at the University of Pretoria, Pretoria, South Africa. His research interests include surveillance and epidemiology of enteric bacterial pathogens in South Africa.