Reemergence of Cholera in Haiti

Cholera was absent from Haiti until an inadvertent introduction by United Nations security forces in October 2010. The ensuing epidemic sickened 820,000 and caused 9,792 reported deaths1. The last cholera case in Haiti was recorded in January 2019, and in February 2022, Haiti was declared to have eliminated cholera2. In late September of 2022, a new outbreak began in Port-au-Prince and rapidly expanded to 964 suspected cases by mid-October of which 115 were confirmed by culture.3 Here, we present genomic and phenotypic analysis of the Vibrio cholerae isolated from a stool sample collected on September 30th, 2022 of an index case – a 10-year-old girl who presented with watery diarrhea and severe dehydration – to address the origins of the epidemic.


Strains and Growth Conditions
Strain H22 was imported from Haiti with CDC PHS Permit No. 20221004-3616A. Clinical samples of Vibrio cholerae (VC) were streaked overnight on lysogeny broth (LB) agar (Difco) plates. Individual colonies were then subcultured into LB (Difco) overnight. For induction of cholera toxin, strains were grown in AKI medium (1.5% Bacto peptone, 0.5% sodium chloride, 0.4% yeast extract, 0.3% sodium bicarbonate) with four hours stationary followed by four hours of shaking 1 . All growth was performed at 37°C. Clinical isolates presented in this study are presented in Supplementary Table 1.

Cholera toxin analysis and serotyping
Immunoblot analysis was performed as previously described 2 . Proteins were separated by SDS-PAGE using 4 to 12% NuPAGE Bis-Tris precast gels (Life Technologies) and transferred to nitrocellulose using an iBlot gel transfer device (Life Technologies). The prestained protein marker, SeeBlue (Invitrogen) was used as a molecular mass standard. Rabbit anti-CT polyclonal antibody (Abcam; catalog no. ab123129) and horseradish peroxidase (HRP)-linked anti-rabbit IgG were used as primary and secondary antibodies, respectively. Blots were developed with the SuperSignal West Pico Plus chemiluminescence substrate (Thermo Fisher) for five minutes and exposed in a ChemiDoc system (Bio-Rad Laboratories). This experiment was performed with three independent replicates. Serotyping was performed using slide agglutination with Ogawaand Inaba-specific antisera (BD Difco).

Minimum inhibitory concentration (MIC)-assays
For MIC assays, overnight cultures grown in LB were shifted into fresh LB at a final OD600 of 0.02. Three-fold serial dilutions of antimicrobial agents were tested across the following range of concentrations: 100µg/ml to 0.000564503µg/ml. After 18 h incubation of growth at 37°C, OD600 was measured. The MIC was defined as the minimum antimicrobial agent concentration which inhibited bacterial growth. Growth was defined by an at least four-fold increase of the OD600 compared to a sterile control. All MICs were performed with four independent replicates.

Whole-genome Sequencing and Phylogenetic Analysis
Genomic DNA (gDNA) was first isolated from V. cholerae strains using the GeneJet genomic DNA purification kit (Thermo Fisher). DNA was fragmented using a sonicator (Peak power 50, duty factor 10, cycles per burst 200, duration 90 seconds) (Covaris M220). Fragments were then prepared for short read sequencing via the NEBNext Ultra II DNA Library Prep Kit (NEB). Libraries were then sequenced on a NextSeq 550 (Illumina).
Contigs were assembled using SPAdes v3.15.5 27 (filtering contigs based on length > 500 bp and coverage > 4) and annotated using Geneious v2022.2 (Biomatters) from the H1 genome (aka KW3, Genbank accession no. GCA_001318185.1, 95% similarity). Alleles of interest were identified through either direct mapping of reads to the H1 or N16961 genomes via Geneious using default settings or through BLAST 28 of the N16961 allele on assembled genomes.

IBC Approval
The Waldor lab has IBC approval (2011B000082) to work with pathogenic V. cholerae and a CDC permit (20221004-3616A) to import V. cholerae from Haiti.

Data Availability
Sequencing from this study is available at GenBank BioProject PRJNA903489.