We characterized the strains in this study by using basic phenotypic markers and genotypic tests that in other studies have been shown to be useful for characterizing V. cholerae
outbreak strains. Epidemic cholera strains cause human illness by expression of specific genes that enable V. cholerae
to exist in the environment long enough to be ingested, overcome host immunity, colonize the intestinal tract, and produce cholera toxin in the host (13,23–25
). The horizontal acquisition of genes and expression of the transcriptional co-regulated pilus (located on Vibrio
pathogenicity island 1) and CTXAB (carried on CTXΦ) determine primary virulence (9,23,24
). CTXΦ has 2 regions, a core that includes ctxAB
and the RS2 region that carries phage replication genes, such as rstR
. Allelic variations in ctxB
, and rstR
can be useful markers to characterize CTXФ types and track cholera strains. Whereas nucleotide sequence analysis of the acquired virulence genes ctxAB
, and tcpA
differentiates among V. cholerae
O1, the VC2346 gene is part of the nontransferable genomic backbone that identifies seventh pandemic V. cholerae
O1 El Tor strains currently in circulation.
The adaptability of V. cholerae
as a pathogen is facilitated by extensive genetic diversity driven by acquisition and recombination of various genetic elements. Community defense mechanisms, mediated by cellular signaling, such as quorum sensing in biofilms, gives the species additional resiliency against changing environmental conditions (26
). All V. cholerae
have these attributes, but only O1 and O139 serogroups currently have pandemic potential. The El Tor biotype was first identified 100 years ago, and 50 years ago it emerged from Indonesia to begin the ongoing seventh pandemic, displacing the classical biotype of the fifth and sixth pandemics because of its superior ability to survive in the environment, increased frequency of asymptomatic carriers, and more efficient transmissibility (27
Genes encoding hemolysis, such as hlyA
, may be a virulence factor in some Vibrio
spp., and in the past, hemolysis patterns were used to distinguish biotypes (28
). Classical biotype strains carry deletion mutations in the hlyA
locus, and many El Tor strains produce no detectable hemolysis on blood agar plates. Our findings confirm observations that hemolysis is not a reliable marker for strain discrimination. Of interest, hemolytic colonies were often observed within a streak predominated by nonhemolytic colonies.
The predominant KZGS12.0088/KZGN11.0092 SfiI/NotI PFGE pattern in the strains from Haiti is a relatively new subtype that was first seen in 2005 in the PulseNet USA database in isolates from travelers returning from India. Although the isolates from Haiti show diversity in their PFGE subtyping patterns, the constancy of the main pattern coupled with virulence genotyping results indicate a high clonality of the outbreak strains, which is consistent with a point-source introduction. Such PFGE diversity, similar to what we observed, has been noticed during outbreaks in the Bengal region (T. Ramamurthy, pers. comm.). This primary KZGS12.0088/KZGN11.0092 pattern and its close variant, KZGS12.0089/KZGN11.0092, were found in strains from Afghanistan, Cameroon, India, Nepal, Oman, Pakistan, and South Africa. Among the strains from Haiti, we identified 4 new NotI restriction patterns, 5 new SfiI patterns, and 9 new SfiI/NotI combinations, a finding suggestive of continuing evolution of the outbreak strain. The PFGE pattern combination KZGS12.0019/KZGN.0092 was commonly seen in serotype Inaba strains originating from East Africa and the Middle East and was recently seen in isolates from Togo; in this study, the pattern was associated with the ctxB-1 allele.
Although the strains from Haiti are genetically an El Tor biotype, they contain the classical ctxB-
7 allele. This allele was first identified in 2007 in strains from an outbreak in Orissa, India (20
). The appearance of a classical ctxB
gene in El Tor strains is not unprecedented. In the early 2000s, hybrid El Tor strains emerged carrying CTXΦClassical
with the ctxB
gene of the classical biotype, which is thought to cause more severe clinical disease (29
). These hybrid strains most likely arose through horizontal transmission of CTXΦClassical
, and the resulting genotypes with the classical ctxB-
1 allele have spread to Asia and Africa. In this study, we show that V. cholerae
carrying the ctxB-
7 allele are also disseminating globally.
The transcriptional co-regulated pilus serves a dual role as the major intestinal colonization factor and CTXΦ receptor. All tested isolates from Haiti had tcpA
sequences with an SNP at nt 266 (tcpETCIRS
), an allele previously reported in strain CIRS 101 from Bangladesh (22
), which is an El Tor biotype that produces a classical toxin yet carries CTXΦEl Tor
; this SNP produces a distinct allele that distinguishes it from classical and typical El Tor. In the present study, isolates from Haiti, Africa, and southern Asia carried tcpETCIRS
(). Our results show that the tcpETCIRS
allele is also spreading globally, although not in tandem with the ctxB-
7 allele because the tcpETCIRS
allele was also found in isolates with the ctxB-
1 allele from Afghanistan, India, Pakistan, South Africa, and Sri Lanka (). Our findings agree with those from a recent study suggesting a close relationship between 2 isolates from Haiti and isolates from Southeast Asia (30
). However, we also observed a relationship between isolates from Haiti and Africa.
The finding of these ctxB-
isolates in Cameroon, India, Nepal, and now Hispaniola is not surprising, given the ease of international travel; we are unable to identify the origin of the Haiti strains because of geographic and temporal limitations in our culture collection. In particular, our findings do not rule out the presence of ctxB-
isolates in countries not represented in our collection. Whole-genome sequencing results confirmed the genetic relationship of these isolates from Haiti, Cameroon, India, and Nepal (18
). More extensive whole-genome sequencing studies and other subtyping methods, such as multiple-locus variable-number tandem repeat analysis, hold promise for providing a better understanding of the relationships between isolates.
The strain from Haiti is distinct from the isolate from the US Gulf Coast. The isolate from the Gulf Coast was characterized as KZGS12.0055/KZGN11.0029, ctxB-1, tcpET, rstRClassical and negative for VC2346. The isolate from Haiti is also not related to the isolates from Peru from the 1991 Latin America outbreaks, which were characterized as KZGS12.0114/KZGN11.0033, ctxB-3, tcpET, and rstREl Tor.
The strain from Haiti has the core characteristics of the seventh pandemic El Tor clone. Our findings support the widespread observation that the typical El Tor strain, which started the seventh pandemic, is gradually being replaced by El Tor isolates with classical cholera toxin subunits.
The isolates from Haiti and those from other regions displayed a consistent resistance phenotype, with resistance to the clinically relevant antimicrobial drugs trimethoprim/sulfamethoxazole and sulfisoxazole but susceptibility to other primary antimicrobial drug options, including doxycycline and azithromycin. A discussion of integrating conjugative and other mobile genetic elements that can potentially mediate transfer of antimicrobial drug resistance is provided elsewhere in this issue (31
). Development of additional antimicrobial drug resistance, particularly to doxycycline and macrolides, remains a serious clinical threat, and Laboratoire National de Santé Publique and CDC continue to monitor for the emergence of such resistance.
The strains from Haiti are fully virulent and contain all the genes necessary for orchestrating the expression of Vibrio spp. virulence factors. These strain characteristics, coupled with the sudden and explosive course of the 2010 outbreak, are consistent with an introduction of this strain into a vulnerable population at a single point in time.
The adaptive immunity of the local population as well as climate variations will further drive bacterial evolution; for example, it will not be surprising to observe a gradual switch over time from the Ogawa to the Inaba serotype as population immunity to Ogawa rises, as has been reported during several outbreaks (23
). The primary PFGE patterns continue to diverge as the infections continue, likely reflecting interactions with the host immune system and between environmental and epidemic populations of bacteria networking in complex ways. Regardless, rapid diagnosis and continuing public health control of the current outbreak in Haiti as well as future outbreaks is paramount for limiting sickness and death, and intensive studies using a variety of basic science, diagnostic, and epidemiologic tools will remain useful for reducing the overall global impact of cholera.