title: Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1.
abstract: BACKGROUND
  Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal.
  
  
  METHODS
  V. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE).
  
  
  RESULTS
  All V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498-511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.
  
  
  CONCLUSIONS
  In 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.
authors.forename: Sameer M
authors.initials: SM
authors.lastname: Dixit
authors.forename: Fatema-Tuz
authors.initials: FT
authors.lastname: Johura
authors.forename: Sulochana
authors.initials: S
authors.lastname: Manandhar
authors.forename: Abdus
authors.initials: A
authors.lastname: Sadique
authors.forename: Rajesh M
authors.initials: RM
authors.lastname: Rajbhandari
authors.forename: Shahnewaj B
authors.initials: SB
authors.lastname: Mannan
authors.forename: Mahamud-Ur
authors.initials: MU
authors.lastname: Rashid
authors.forename: Saiful
authors.initials: S
authors.lastname: Islam
authors.forename: Dibesh
authors.initials: D
authors.lastname: Karmacharya
authors.forename: Haruo
authors.initials: H
authors.lastname: Watanabe
authors.forename: R Bradley
authors.initials: RB
authors.lastname: Sack
authors.forename: Alejandro
authors.initials: A
authors.lastname: Cravioto
authors.affiliation: International Centre for Diarrheal Disease Research, GPO Box 128, 1000 Dhaka, Bangladesh. munirul@icddrb.org.
authors.forename: Munirul
authors.initials: M
authors.lastname: Alam
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002771:Cholera / Q000453:epidemiology*; D016172:DNA Fingerprinting; D003967:Diarrhea / Q000453:epidemiology; D004196:Disease Outbreaks; D024901:Drug Resistance, Multiple, Bacterial; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D009390:Nepal / Q000453:epidemiology; D016366:Open Reading Frames; D010641:Phenotype; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D041241:Vibrio cholerae O1 / Q000187:drug effects* / Q000472:pathogenicity; D014774:Virulence
chemical_list: D000900:Anti-Bacterial Agents