Drug-Resistance Mechanisms in Vibrio cholerae O1 Outbreak Strain, Haiti, 2010

To increase understanding of drug-resistant Vibrio cholerae, we studied selected molecular mechanisms of antimicrobial drug resistance in the 2010 Haiti V. cholerae outbreak strain. Most resistance resulted from acquired genes located on an integrating conjugative element showing high homology to an integrating conjugative element identified in a V. cholerae isolate from India.

To increase understanding of drug-resistant Vibrio cholerae, we studied selected molecular mechanisms of antimicrobial drug resistance in the 2010 Haiti V. cholerae outbreak strain. Most resistance resulted from acquired genes located on an integrating conjugative element showing high homology to an integrating conjugative element identifi ed in a V. cholerae isolate from India. V ibrio cholerae is the bacterium that causes cholera, a disease characterized by acute watery diarrhea, vomiting, muscle cramps, and severe dehydration (1). The bacterium has many serogroups, but only toxin-producing serogroups O1 and O139 cause epidemic cholera. The primary treatment for cholera is rehydration with oral or intravenous fl uids (2). For severe cases, antimicrobial agents may reduce the volume and duration of diarrhea (1,2). Tetracyclines (e.g., doxycycline), fl uoroquinolones (e.g., ciprofl oxacin), macrolides (e.g., erythromycin), and trimethoprim/sulfamethoxazole have commonly been used to treat cholera (2).
Antimicrobial drug resistance can undermine the success of antimicrobial therapy. Several reports have documented tetracycline-and fl uoroquinolone-resistant V. cholerae, and multidrug resistance is increasing (3). Antimicrobial drug resistance in Vibrio spp. can develop through mutation or through acquisition of resistance genes on mobile genetic elements, such as plasmids, transposons, integrons, and integrating conjugative elements (ICEs). ICEs integrate and replicate with the host chromosome and can excise themselves and transfer between bacteria by conjugation (4). ICEs commonly carry several antimicrobial drug resistance genes and play a major role in the spread of antimicrobial drug resistance in V. cholerae (5). The fi rst V. cholerae ICE described was in an O139 isolate in Madras, India, in 1992 and was named SXT after the resistance phenotype it conferred (trimethoprim/sulfamethoxazole) (6). Many O139 and O1 isolates have since acquired SXT or a closely related ICE (4,5).
We describe antimicrobial drug resistance mechanisms in the 2010 Haiti V. cholerae O1 outbreak strain. Most of the resistance is caused by acquired genes located on an ICE with high similarity to an ICE identifi ed in a V. cholerae O1 isolated in India.

The Study
During October 2010-January 2011, a total of 122 clinical isolates of laboratory-confi rmed V. cholerae O1 were recovered by the National Public Health Laboratory in Haiti and submitted to the Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA) for characterization. Disk-diffusion antimicrobial drug susceptibility testing was performed at the National Public Health Laboratory and CDC. MICs were determined by broth microdilution at CDC by using Sensititer plates (CAMPY and CMV1AGNF; Trek Diagnostics, Cleveland, OH, USA) according to the manufacturer's instructions with the following modifi cations: Mueller-Hinton broth without blood was used on the CAMPY plate, and for both plates, a fi nal inoculum concentration of 5 × 10 4 to 5 × 10 5 CFU/mL was targeted. Escherichia coli American Type Culture Collection (ATCC; Manassas, VA, USA) 25922, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Pseudomonas aeruginosa ATCC 27853 were used for quality control testing. Where available, Clinical and Laboratory Standards Institute criteria specifi c for V. cholerae were used (7). For drugs lacking such criteria, manufacturers' criteria, Clinical and Laboratory Standards Institute criteria for Enterobacteriaceae, or consensus breakpoints used by the National Antimicrobial Resistance Monitoring System were applied (8,9). Furazolidone was tested only by disk diffusion, and azithromycin was tested only by broth microdilution.
Results for all 122 outbreak isolates were similar. They showed susceptibility to azithromycin and tetracycline, reduced susceptibility to ciprofl oxacin (MIC 0.25-1.0 mg/L), and resistance to furazolidone, nalidixic acid, sulfi soxazole, streptomycin, and trimethoprim/ sulfamethoxazole. With a common susceptibility pattern among all outbreak isolates, 1 isolate, 2010EL-1786 (deposited under ATCC BAA-2163), was chosen for molecular characterization. PCR was used to screen the isolate for the following resistance genes: strA, strB, sul1, sul2, dfrA1, dfrA10, and dfrA12 (10). In addition, the gyrA and parC genes were sequenced to identify quinolone resistance-determining region mutations. PCR was performed according to standard protocols by using the HotStarTaq PCR Master Mix (QIAGEN, Valencia, CA, USA). DNA sequencing was performed by using a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
The isolate 2010EL-1786 contained strA/B, sul2, and dfrA1, which mediate resistance to streptomycin, sulfi soxazole/sulfamethoxazole, and trimethoprim, respectively. Nalidixic acid resistance and decreased susceptibility to ciprofl oxacin were attributed to mutations in gyrA (Ser83Ile) and parC (Ser85Leu). The mechanism responsible for furazolidone resistance was not identifi ed. Mutations in the nfsA and nfsB genes are associated with furazolidone resistance in E. coli, but inspection of the 2010EL-1786 sequence failed to identify these genes.
Whole-genome sequencing identifi ed an ICE inserted in the prfC gene. This ICE, designated ICEVchHai1, was 97.9 kb and contained 95 open reading frames ( Figure 1). All resistance genes identifi ed were located on ICEVchHai1. In addition, fl oR, a chloramphenicol resistance gene, was detected. The strA, strB, sul2, and fl oR genes were part of an ≈17-kb fragment inserted into the rumB gene, whereas the dfrA1 gene was located ≈70 kb further downstream. Whole-genome sequencing also indicated a chloramphenicol acetyltransferase gene, catB9, that was not part of the ICE.
The genetic relatedness of ICEVchHai1 was assessed by comparison with 7 other ICE sequences (4). Sequence alignments were performed by using Progressive Mauve (http://asap.ahabs.wisc.edu/mauve/download.php) and visualized with PHYLIP version 3.69 (distributed by J. Felsenstein, Department of Genome Sciences, University of Washington, Seattle, WA, USA). ICEVchHai1 showed highest homology to ICEVchInd5, an ICE derived from a V. cholerae isolate from India ( Figure 2). These ICEs differed by only 5 single-nucleotide polymorphisms.

Conclusions
In October 2010, an epidemic caused by toxigenic V. cholerae O1, serotype Ogawa, biotype El Tor strain, was reported from Haiti. We confi rmed that the outbreak strain was multidrug resistant and displayed resistance to furazolidone, nalidixic acid, sulfi soxazole, streptomycin, and trimethoprim/sulfamethoxazole and decreased susceptibility to ciprofl oxacin. Genetic mechanisms responsible for resistance to 5 of these drugs were identifi ed. Sequencing also detected fl oR, a gene commonly associated with chloramphenicol resistance in Enterobacteriaceae (MICs >32 mg/L) (12). However, in this study, fl oR was not associated with resistance; isolates from Haiti displayed chloramphenicol MICs of 4-16 mg/L (7  (13). ICEVchHai1 showed high homology to ICEVchInd5, an ICE fi rst identifi ed in a V. cholerae isolate from Sevagram, India, in 1994. Since then, ICEVchInd5 has persisted among clinical isolates in India; a recent study of O1 strains isolated in India during 1994-2005 confi rmed that ICEVchInd5 was the only ICE that persisted during the study period (14).
Drug-resistant V. cholerae is a global health concern because resulting infections can be more severe and diffi cult to treat. Infections with drug-resistant V. cholerae can result in higher case-fatality rates, prolonged hospitalizations, more secondary infections, and increased health care costs. During an outbreak in Guinea-Bissau, case-fatality rates increased from 1% to 5.3% after the outbreak strain acquired multidrug resistance (15). To limit development and spread of antimicrobial drug resistance among V. cholerae, treatment with antimicrobial agents should be restricted to patients with severe dehydration or other conditions that truly warrant their use. Surveillance should continue for antimicrobial drug resistance among V. cholerae isolates from Haiti.