V. cholerae isolates and travel histories from cholera case-patients in the United States were referred to CDC. A strain from an outbreak in Cameroon in 2010, isolated from a specimen received at CDC, and an isolate from South Africa likely linked to an outbreak in Zimbabwe in 2009 were also included in this study (10). Isolates C6706 and 3569–08 were acquired during the outbreak in Latin America in 1991 and from the US Gulf Coast in 2008, respectively. All strains were characterized as V. cholerae O1 on the basis of standard biochemical, cholera toxin, and serologic testing performed as described (11,12). PFGE was performed according to the PulseNet standardized protocol with restriction enzymes SfiI and NotI; PFGE patterns were designated by using BioNumerics version 5.10 (Applied Maths Inc., Sint-Martins-Latem, Belgium) and compared by unweighted pair group method with arithmetic mean analysis (DICE coefficient 1.5% tolerance and optimization). Strain designations and other information are shown in Table 1.

Single-end pyrosequencing reads (GS FLX-Titanium; Roche Diagnostics, Indianapolis, IN, USA) and single-end 36-bp or 76-bp Illumina reads (GAIIe sequencer; Illumina, San Diego, CA, USA) were acquired and yielded >99% genome coverage and 32× and 240× average coverage depths, respectively (Table 2). Pyrosequencing reads were first assembled de novo by using Newbler version 2.5.3 (Roche Diagnostics). To correct potential base-calling errors attributed to homopolymers, Illumina GAIIe reads (average 14 million reads/genome) were mapped to the Newbler contigs by using CLC Genomics Workbench version 4.5 (www.clcbio.com/index.php?id=1042) and yielded an average combined coverage depth of 270× per genome.

Both chromosomes of Haiti outbreak isolate 2010EL-1786 were sequenced to full closure by using PCR and Sanger sequence-based bridging of contigs and a fosmid library of templates. Optical mapping also supported the contig ordering derived for 2010EL-1786. For all remaining isolates, Illumina-supplemented, homopolymer-corrected, Newbler-assembled contigs were prepared as pseudogenomes by first linking contigs with a linker sequence containing stop codons in all 6 translation reading frames. These high-coverage pseudogenomes were used for downstream analyses. Identification of coding sequences was achieved by using Glimmer3 (14). Genome annotation was achieved by using an automated, in-house, modified version of GenDB version 2.2 (15) and manual curation for regions of interest.

Nine V. cholerae isolates directly associated with the outbreak on Hispaniola were examined, 7 of which had indistinguishable SfiI and NotI PFGE patterns designated PulseNet USA patterns KZGS12.0088 and KZGN11.0092, respectively (Table 1). Also sequenced were a hemolytic variant and a nonhemolytic variant that harbored a minor variation of the main Haiti outbreak PFGE pattern and were derived from an isolate from 1 patient in Haiti (Table 1). Twelve contemporary V. cholerae isolates from global sources with matched PFGE fingerprints were also sequenced. Infections for these 12 contemporary isolates originated (by documented patient travel) from regions of Pakistan, India, or Nepal. Two additional isolates were from patients in outbreaks in Cameroon and South Africa likely connected to the cholera outbreak in Zimbabwe in 2009 (21). Although all sequenced clinical isolates were serogroup O1, Inaba and Ogawa serotypes were observed among PFGE pattern-matched isolates (Table 1). All strains were biotype El Tor and all produced cholera toxin.

Haiti outbreak isolates and 12 global PFGE pattern-matched V. cholerae isolates belong to phylogroup 1 of the seventh pandemic clade. The phylogenetic tree based on whole-genome sequencing showed clustering of the 9 Hispaniola isolates (8 from Haiti and 1 related isolate from the Dominican Republic) with 12 other PFGE pattern-matched isolates. All 21 isolates were in 1 cluster relative to non-PFGE–pattern-matched outliers (Figure 1). When compared with historical reference genomes, the closest ancestors for Haiti genome sequences (2010–2011; derived herein) were isolates CIRS101 from Dhaka, Bangladesh (2002) and MJ-1236 from Matlab, Bangladesh (1994). These data confirm the genetic relatedness also inferred by PFGE subtyping and further support inclusion of the Haiti outbreak isolates in phylogroup 1 of the seventh pandemic clade (Figure 1). The whole-genome sequencing dataset showed that additional underlying genetic diversity was present across PFGE pattern-matched isolates (including 9 isolated from Hispaniola) not observed by PFGE subtyping.

V. cholerae macrodiversity is commonly attributed to presence or absence of mobile genetic elements (22). The contiguous genome derived for Haiti isolate 2010EL-1786 was used as the outbreak type strain and harbored 2 circular chromosomes of 3.03 Mbp (chromosome I) and 1.05 Mbp (chromosome II), which encoded 2,920 and 1,051 predicted coding sequences, respectively. Pairwise comparisons of all coding sequences from each study genome with all coding sequences from reference isolate 2010EL-1786 (all vs. all comparison) showed congruent gene content and low overall diversity on larger chromosome I (Figure 2). One noteworthy exception was the absence of Vibrio pathogenicity island 1 in the 2005 isolate 3582–05 from Pakistan. This island contains essential cholera virulence factors, including the tcp gene cluster, which encodes toxin-coregulated pilus involved in V. cholerae colonization of the human intestine and necessary for horizontal transfer of the cholera toxin bacteriophage. This finding was the only macroscopic difference observed between isolate 3582–05 and PFGE matches. All Haiti outbreak and PFGE pattern-matched isolates contain an integrated conjugative element belonging to the SXT/R391 family (SXT-ICE) that carries genes conferring antimicrobial drug resistance. No macroscopic differences were observed in SXT-ICE among Haiti outbreak and PFGE pattern-matched isolates (Figure 2; Technical Appendix 2 Figure 1, panel A).

Smaller chromosome II was more content variable and divergent across study strains. These findings were largely attributable to the hypervariable superintegron region, an ≈120-kb gene capture system predominantly encoding hypothetical proteins (Figure 2; Technical Appendix 2 Figure 1, panel B) (13). Gene polymorphisms observed in the 9 sequenced isolates from Hispaniola also localized primarily within the superintegron region. Despite these observed differences, no major deletions in the superintegron were observed among PFGE pattern-matched isolates (Figure 2; Technical Appendix 2 Figure 1). Thus, phylogeny derived from V. cholerae whole-genome sequencing (Figure 1) showed genetic diversity within PFGE pattern-matched isolates. However, binary (present or absent) gene content assessment failed to pinpoint extensive contiguous diversity outside the superintegron region.

A core genome phylogeny was constructed on the basis of 4,376 hqSNPs found within 632 orthologous core genes (0.81 Mbp) that were universally present in all 27 study and reference genomes (Technical Appendix 1; Technical Appendix 2 Figure 2). Among 9 sequences from Hispaniola isolates, 0–2 SNPs were observed (Technical Appendix 2 Figure 2). Hispaniola isolates differed from PFGE pattern-matched genomes from other locations by 4–25 SNPs, and genomes with nonmatched PFGE patterns differed from the outbreak isolates by 13–3,361 SNPs. Notably, phylogeny based on hqSNPs showed clustering of the Haiti strain with 3 epidemiologically unrelated clinical isolates, which represented isolates from 2 travelers from the United States to India in 2009 and a patient in Cameroon in 2010. Isolates 2009V-1085 (India, 2009), 2009V-1096 (India, 2009), and 2010EL-1749 (Cameroon, 2010) were most related to the Haiti isolates. These 3 isolates had 4–7 core hqSNPs when compared with the outbreak strain, and the derived sequence for a 2008 clinical isolate from Nepal differed from outbreak isolates by 7–8 core hqSNPs (Technical Appendix 1; Technical Appendix 2 Figure 2).

Conversely, historical isolates (1970–2005) from Pakistan, Bangladesh, the US Gulf Coast, and South America, and recent clinical isolates (2009–2010) from cases linked to Pakistan or South Africa independently clustered away from Haiti outbreak isolates (Technical Appendix 2 Figure 2). Clade analysis of outbreak isolates and highly related isolates 2009V-1085, 2009V-1096, and 2010EL-1749, identified 25 hqSNPs in 24 conserved loci that distinguish members of this clade (Technical Appendix 2 Figure 3; Table A1). Resulting distances suggest that the outbreak isolates have a closer genetic relationship with 2009V-1085 and 2009V-1096 from India (7–10 hqSNPs) than with 2010EL-1749 from Cameroon (10–13 hqSNPs).

Across the 18 described hypervariable V. cholerae mobile genetic elements sequences (representing >300 kb of the total genome), no macroscopic differences were observed among the 9 Hispaniola isolate sequences (Figure 2; Technical Appendix 2 Figure 1), and as stated, only 2 hqSNPs were identified in the core genome. Pairwise alignment of the complete genome of study reference 2010EL-1786 with available genome data for 2 sequenced Haiti 2010 outbreak isolates, designated H1 and H2 (9), showed only 3 polymorphisms across the entire genome. However, because the available H1 and H2 consensus sequences contain ambiguous basecalls, these nucleotides were excluded from our comparative analyses. Nonetheless, these data confirm the clonal nature of the Haiti outbreak strain.

Structure and allelic profiles of the CTXϕ prophage have been used for V. cholerae lineage analysis (23). Chromosome I of Haiti isolate 2010EL-1786 harbors 1 hybrid CTXϕ characterized by a 1-nt variant of the classical ctxB allele (ctxB-7) and El Tor rstR flanked by a toxin-linked cryptic element and El Tor–type RS1 element with an intact rstC locus (Figure 3). The SNP at ctxB codon19 results in replacement of the classical cholera toxin B histidine residue with asparagine, and this ctxB-7 allele was observed among all Hispaniola isolates (Table 1). Five of the 12 PFGE pattern-matched isolates from other locations (2008–2010) also shared this variant ctxB allele. The remaining 7 PFGE pattern-matched isolates encoded classical ctxB alleles.