Public health investigators use PFGE, the current standard technique for subtyping most bacterial enteric pathogens, to link patients infected with a particular pathogen to a specific infection source(s) by fingerprint matching to pathogens isolated from environmental samples. Whole-genome sequencing has recently emerged as an enhanced laboratory tool for high-resolution analysis of microbial diversity and has been successfully used to investigate bacterial disease outbreaks (24
). Because whole-genome sequencing can provide pathogen genetic fingerprints at single-nucleotide resolution, it should revolutionize the diagnosis, surveillance, and control of microbial diseases.
For molecular epidemiologic investigations using whole-genome sequencing, an expansive number of isolates from an outbreak would ideally be selected to ensure broad coverage for possible genotype variants within that population that might otherwise be masked with lower-resolution typing methods. In addition, outlier isolates from different locations that are indistinguishable or related by several diverse subtyping methods should also be subjected to whole-genome sequencing to contextualize the diversity seen within the outbreak population and to find other clonal relationships In this study, a temporal and geographic distribution of outbreak isolates was selected to confirm clonality of the outbreak strain and to gain insight into the microevolution of V. cholerae during an outbreak. Additionally, minor PFGE and nonhemolytic variants observed among outbreak isolates were also sequenced to confirm their clonal relationships with isolates exhibiting the main outbreak pattern and phenotype.
The PulseNet USA database substantially contributed to this work by identifying genetically related (using PFGE typing) and epidemiologically relevant isolates for whole-genome sequencing analyses. Notably, one 2008 isolate from a traveler from the United States to Nepal was identified and included in this study, although we acknowledge that the evolutionary relationship of the Haiti strain to strain(s) circulating in Nepal during 2010 may not be ideally represented by this 2008 isolate. Microbial evolution will have occurred during 2008–2010, and global travel may have introduced additional strains into Nepal in the interim, such that the 2008 isolate from Nepal may differ substantially from a strain circulating in Nepal in 2010, the suggested progenitor of the outbreak strain. Unfortunately, 2010 isolates from Nepal were not available for analysis.
Also identified in the PulseNet USA database was 1 PFGE pattern-matched isolate from western Africa. The close genetic relationship of this isolate from Cameroon to the Haiti strain suggests that a potential link between western Africa and the Haiti outbreak cannot be ignored. Further studies on additional isolates from western Africa are required to confirm or refute this possibility. Similarity of whole-genome sequences for Haiti isolates, PFGE pattern-matched isolates, and other seventh pandemic strains confirmed the clonal nature of the 2010–2011 cholera outbreak strain and the close genetic relationships for the studied strains initially suggested by PFGE subtyping (). Previous V. cholerae
studies have reported that seventh pandemic strains are clonal, sharing near identical gene content on a highly related genome backbone but containing variable mobile genetic elements or gene cassettes (27
). Despite dynamic horizontal gene transfer (22
), we identified only a few nucleotide differences among mobile sequences of the 9 sequenced 2010–2011 outbreak-related Hispaniola isolates and the 12 recent PFGE pattern-matched clinical isolates ().
Extensive recombination in V. cholerae
genomes may confound evolutionary relationship analyses as strains and lineages undergo reassortment (1
). However, base substitutions acquired horizontally as recombination segments generally occur with localized density (28
). Although we cannot guarantee that recombinant segments were absent from the core genome phylogeny (Technical Appendix 2
Figure 2), the even spatial and genome-wide distribution of core genome hqSNPs suggests that they were vertically inherited. We have derived a useful phylogenetic approximation of isolate relatedness on the basis of hqSNPs, which supports shared ancestry for the Haiti outbreak isolates and 12 recent clinical isolates sharing PFGE patterns (Technical Appendix 2
Figure 2). Sequenced isolates from India and Cameroon (2009–2010) were shown to be the closest genetic relatives among the non-Hispaniola isolates (isolated in 1991–2010; this study) and 4 other available reference V. cholerae
genomes (isolated in 1937–2002). The ctxB
allele variant (ctxB
-7) of the Haiti strain (and its genetic relatives) was first observed among isolates from a cholera outbreak in Orissa, India, in 2007 (29
), but the ctxB
-7 allele has since also been observed in isolates from southern Asia and more recently from western Africa (8
The genetic makeup of the Haiti outbreak strain will likely have substantial public health implications for Haiti and other susceptible locations. Our reasoning is that the atypical O1 El Tor V. cholerae
strains (CIRS101 and CIRS101-like variants) have already emerged as the predominant clone causing cholera in Asia and Africa and have displaced prototypical O1 El Tor strains (3
). Unfortunately, atypical O1 El Tor V. cholerae
strains appear to have retained the relative environmental fitness of their prototypical O1 El Tor ancestors while acquiring enhanced virulence traits, such as classical or hybrid CTX prophage and SXT-ICE (4
). Thus, with higher relative fitness and virulent and antimicrobial drug–resistant phenotypes, the Haiti outbreak strain harbors infectivity and ecologic persistence advantages over other seventh pandemic strains. Consequently, the Haiti outbreak strain (or its genetic ancestor) may easily replace current El Tor V. cholerae
strains circulating in the Western Hemisphere to become endemic (like other atypical El Tor strains) and will likely cause future outbreaks. Such dire predictions warrant enhanced epidemiologic surveillance and renewed priorities aimed at cholera prevention.
Absence of cholera in Haiti over the past century; the clonal nature of the outbreak strain; and a massive influx of international travelers, aid workers, and supplies after the 2010 earthquake suggest an outside infection source for the 2010–2011 outbreak. Our core genome phylogeny (Technical Appendix 2
Figure 2) suggests that the Haiti outbreak strain most likely derived from an ancestor related to isolates from within or near the Indian subcontinent. However, concurrent identification of a 2010 isolate from Cameroon as a close genetic relative of the Haiti outbreak strain illustrates that whole-genome sequencing on such a relatively small number (n = 27) of V. cholerae
isolates is insufficient to exclude other plausible ancestral geographic locations.
Our study results are consistent with recent findings of Chin et al. (9
), who concluded that two 2010 Haiti outbreak isolates shared ancestry with variant O1 El Tor strains isolated in Bangladesh in 2002 and 2008 and a more distant relationship with an isolate from an outbreak in Latin American in 1991. The vertical inheritance pattern of hqSNPs in our study provide unequivocal genetic evidence for introduction of the outbreak strain into Haiti from an external source as opposed to local aquatic emergence. However, the specific geographic source and mode of entry of the outbreak strain into Haiti cannot be proven by microbiological investigations. Only large-scale epidemiologic studies and microbiological data can provide conclusive evidence of how cholera was introduced into Haiti. This whole-genome sequencing study provides expanded evidence that variant O1 El Tor V. cholerae
appeared in Haiti by importation and has generated a whole-genome sequencing dataset for future study.