During October 2010–January 2011, a total of 122 clinical isolates of laboratory-confirmed 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 modifications: Mueller-Hinton broth without blood was used on the CAMPY plate, and for both plates, a final inoculum concentration of 5 × 104
to 5 × 105
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 specific 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
). 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 ciprofloxacin (MIC 0.25–1.0 mg/L), and resistance to furazolidone, nalidixic acid, sulfisoxazole, 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
, and dfrA12
). In addition, the gyrA
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, sulfisoxazole/sulfamethoxazole, and trimethoprim, respectively. Nalidixic acid resistance and decreased susceptibility to ciprofloxacin were attributed to mutations in gyrA (Ser83Ile) and parC (Ser85Leu). The mechanism responsible for furazolidone resistance was not identified. 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.
Location of the resistance genes was analyzed by whole-genome sequencing of 2010EL-1786. Single-end 454 pyrosequencing (GS FLX-Titanium; Roche Diagnostics, Indianapolis, IN, USA) reads and single-end 36-bp Illumina Solexa (GAIIe; Illumina, San Diego, CA, USA) reads were assembled de novo by using Newbler (Roche Diagnostics) and CLC Genomics Workbench (CLC bio, Cambridge, MA, USA) software. Sequence finishing was performed by using Sanger sequencing of fosmid clones (11
Whole-genome sequencing identified an ICE inserted in the prfC gene. This ICE, designated ICEVchHai1, was 97.9 kb and contained 95 open reading frames (). All resistance genes identified were located on ICEVchHai1. In addition, floR, a chloramphenicol resistance gene, was detected. The strA, strB, sul2, and floR 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.
Figure 1 Genetic organization of the 2010 Haiti Vibrio cholerae O1 integrating conjugative element (ICE), ICEVchHai1. The ICE contained 97,915 bp and 95 open reading frames. Coding sequences were identified and manually annotated by using an in-house modified (more ...)
The genetic relatedness of ICEVch
Hai1 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). ICEVch
Hai1 showed highest homology to ICEVch
Ind5, an ICE derived from a V. cholerae
isolate from India (). These ICEs differed by only 5 single-nucleotide polymorphisms.
Figure 2 Phylogenetic tree illustrating the genetic relatedness between the Haiti integrating conjugative element (ICE) ICEVchHai1 and other ICEs described in Vibrio cholerae (ICEVchBan1, ICEVchBan9, ICEVchInd1, ICEVchInd4, ICEVchInd5, ICEVchMex1, and SXT). Each (more ...)