Seventh Pandemic Vibrio cholerae O1 Sublineages, Central African Republic

Four cholera outbreaks were reported in the Central African Republic during 1997–2016. We show that the outbreak isolates were Vibrio cholerae O1 serotype Inaba from 3 seventh pandemic El Tor sublineages originating from West Africa (sublineages T7 and T9) or the African Great Lakes Region (T10).

in southern CAR, along the Oubangui River, close to the border with the Democratic Republic of the Congo (DRC). In the second, 113 cases and 19 deaths were reported in northern CAR, close to the borders with Cameroon and Chad, after which cholera cases continued to be detected in southern CAR, along the Oubangui River. According to reports from the International Federation of Red Cross and Red Crescent Societies, 172 cholera cases and 16 deaths were reported in the region in 2011 (https://reliefweb.int/ sites/reliefweb.int/files/resources/MDRCF009finrep.pdf) and 265 cases and 20 deaths were reported in 2016 (https://reliefweb.int/report/central-africanrepublic/central-africa-republic-cholera-epidemicoutbreak-dref-operation).
The Institut Pasteur de Bangui in CAR performed microbial analyses to confirm the causal agent of these outbreaks and identified 30 V. cholerae O1 serotype Inaba isolates collected during 1997-2016 (Appendix 1 Table 1, https://wwwnc. cdc.gov/EID/article/27/1/20-0375-App1.x1sx). We used whole-genome sequencing to fully characterize all 30 V. cholerae O1 isolates in terms of virulence and antimicrobial resistance determinants. We also placed these genomes within a broader phylogenetic context to elucidate their origins and evolutionary history.

The Study
The 30 V. cholerae O1 isolates were received at the Institut Pasteur, Paris, France. We performed antimicrobial susceptibility testing, whole-genome sequencing, comparative genomics, and phylogenetic analyses by using methods previously described (3,(5)(6)(7)(8)(9)(10)(11)    Previous genomic studies described 12 introductions of the 7PET lineage from Southern Asia into Africa during 1970-2016 (3,6). The introduced sublineages were called T1 and T3-T13. The 2 cholera outbreaks in CAR in 1997 were caused by sublineage T7, which had been introduced into West Africa during the early 1980s, and T9, which was introduced in the late 1980s, according to Weill et al. (3). T9 isolates were identified in neighboring countries such as Chad and Cameroon (particularly northern Cameroon) before they were detected in northwest CAR, but T7 isolates were identified in Gabon and the western part of DRC, along the Congo River in Kinshasa, before being identified in CAR along the Oubangui River, a tributary of the Congo River (Figure 2, panel B; Appendix 2 Figures 1, 2).
The 2011 and 2016 outbreaks were caused by closely related bacterial populations from the same sublineage, T10, introduced into East Africa during the 1990s and later detected in the African Great Lakes Region (AGLR) (3) (Figure 2   Except during the outbreak in northwest CAR in 1997, all cholera cases were reported along or close to the Oubangui River, suggesting that 7PET strains probably moved from area to area along the river and with the displacement of human populations. The risk factors in these remote areas are unknown, but the prevailing conditions, such as poor hygiene and sanitary conditions, overcrowding, lack of latrines, and drinking water from the Oubangui River, likely would increase the risk for transmission via the fecal-oral route, as evidenced by the high attack rates observed at several sites when the 2011 outbreak began (15). Nevertheless, since the declaration of the first case in 1997, the small number (<1,000) of cholera cases in CAR contrasts with the much larger numbers in central Africa (2). The low population density of CAR, its poor transport infrastructure, and poor trading links are probably key factors limiting disease spread (15). Phylogenetic analyses showed no other isolates from Africa were derived from CAR isolates in the aftermath of the 4 outbreaks, which also suggests that the transmission of cholera is impeded in this country. Of note, all 4 outbreaks were caused by serotype Inaba 7PET strains. This serotype has a nonmethylated form of lipopolysaccharide caused by an alteration to the wbeT gene (3) ( Table 2). The implication of this serotype in all 4 outbreaks suggests that these 7PET sublineages circulated regionally for some time, long enough to acquire this alteration to the wbeT gene, before reaching CAR (Appendix 2 Figures 1-3).
All CAR isolates in this study displayed resistance to polymyxin B, consistent with the susceptibility pattern reported for the El Tor biotype until recently (6). All but 1 of the isolates collected along the Oubangui River in 1997 were susceptible to all other antimicrobial drugs tested; the outlying isolate contained the extended-spectrum β-lactamase bla SHV-2a gene on an IncA/C2 plasmid (Table 1; Appendix 1 Table 3). No susceptible isolates have been collected in CAR since. All the other isolates display mutations of the VC_0715 and VC_A0637 genes, conferring nitrofuran resistance, and carry an SXT/R391 genomic element called ICEVchInd5, encoding resistance to streptomycin (strAB), sulfonamides (sul2), trimethoprim, the O/129 vibriostatic agent (dfrA1), and trimethoprimsulfamethoxazole (sul2 and dfrA1). The 2011 and 2016 CAR isolates also recently acquired gyrA mutations (Table 1), resulting in resistance to nalidixic acid (Appendix 1 Table 3).

Conclusions
Strains from 3 7PET sublineages caused 4 cholera outbreaks identified in CAR during 1997-2016. The southern and southeastern parts of CAR are higher risk areas for cholera outbreaks, particularly when cases are reported in the western part of DRC. These findings highlight the need for an effective surveillance system, and for coordinated communication actions on cholera that target healthcare professionals and the populations living along the Oubangui River, to prevent and control cholera outbreaks in CAR.

About the Author
Dr. Breurec is a medical microbiologist on the faculty of medicine and the University Medical Center of Pointe-à-Pitre, Guadeloupe, and at the Institut Pasteur International Network. His research interests include genomic epidemiology, resistance, and the virulence determinants of bacterial pathogens.