ABSTRACT
Vibrio cholerae inhabits aquatic environments worldwide and has over 200 recognized serogroups classified by O-polysaccharide specificity. Here, we report that V. cholerae selects either of two genetic traits during their evolution. Sequencing of the specific gene locus MS6_A0927 revealed that 339 of 341 strains of V. cholerae and closely related Vibrio species originating from 34 countries over a century carried either metY (M) (~1,269 bp) or luxR-hchA (LH) (~1,600 bp) genes, and consequently those vibrios were separated into two clusters, M (45.4%) and LH (54.6%). Only two strains contained both M and LH in the same locus. Moreover, extensive polymorphisms in those genes were detected in M and LH with 79 and 46 sequence variations, respectively. V. cholerae O1 strains isolated from cholera outbreaks worldwide, and some non-O1 strains evolving from O1 via exchange of genes encoding cell surface polysaccharides possessed LH alleles. Analysis of polymorphisms in the gene locus implicated a high degree of genetic diversity and identical subpopulations among the V. cholerae species.
Subject(s)
Evolution, Molecular , Genes, Bacterial , Genetic Variation , Genotype , Vibrio cholerae/classification , Vibrio cholerae/genetics , Cholera/microbiology , Cluster Analysis , Global Health , Humans , Sequence Analysis, DNAABSTRACT
BACKGROUND: The cholera outbreaks in Thailand during 2007-2010 were exclusively caused by the Vibrio cholerae O1 El Tor variant carrying the cholera toxin gene of the classical biotype. We previously isolated a V. cholerae O1 El Tor strain from a patient with diarrhea and designated it MS6. Multilocus sequence-typing analysis revealed that MS6 is most closely related to the U. S. Gulf Coast clone with the exception of two novel housekeeping genes. METHODOLOGY/PRINCIPAL FINDINGS: The nucleotide sequence of the genome of MS6 was determined and compared with those of 26 V. cholerae strains isolated from clinical and environmental sources worldwide. We show here that the MS6 isolate is distantly related to the ongoing seventh pandemic V. cholerae O1 El Tor strains. These strains differ with respect to polymorphisms in housekeeping genes, seventh pandemic group-specific markers, CTX phages, two genes encoding predicted transmembrane proteins, the presence of metY (MS6_A0927) or hchA/luxR in a highly conserved region of the V. cholerae O1 serogroup, and a superintegron (SI). We found that V. cholerae species carry either hchA/luxR or metY and that the V. cholerae O1 clade commonly possesses hchA/luxR, except for MS6 and U. S. Gulf Coast strains. These findings illuminate the evolutionary relationships among V. cholerae O1 strains. Moreover, the MS6 SI carries a quinolone-resistance gene cassette, which was closely related with those present in plasmid-borne integrons of other gram-negative bacteria. CONCLUSIONS/SIGNIFICANCE: Phylogenetic analysis reveals that MS6 is most closely related to a U. S. Gulf Coast clone, indicating their divergence before that of the El Tor biotype strains from a common V. cholerae O1 ancestor. We propose that MS6 serves as an environmental aquatic reservoir of V. cholerae O1.
Subject(s)
Phylogeny , Vibrio cholerae O1/genetics , Vibrio cholerae O1/isolation & purification , Chromosomes/genetics , Clone Cells , Conserved Sequence/genetics , Evolution, Molecular , Genome, Viral/genetics , Humans , Likelihood Functions , Molecular Sequence Data , Open Reading Frames/genetics , Reference Standards , Synteny/genetics , Thailand , United StatesABSTRACT
A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was used to generate MAb-secreting hybridomas denoted 2-4F, 7-3G, and 11-11A. An indirect competitive ELISA (icELISA) was applied to measure the sensitivity and specificity of each MAb in terms of its 50% inhibitory concentration (IC50) and percentage of cross-reactivity, respectively. MAb 2-4F exhibited the highest sensitivity, with an IC50 of 7.01 ng/ml. This MAb showed strong cross-reactivity to rolitetracycline, but no cross-reactivity to other unrelated antibiotics. When MAb 2-4F was used to detect OTC from shrimp samples, the recoveries were in the range of 82%-118% for an intra-assay and 96%-113% for an inter-assay. The coefficients of variation of the assays were 3.9%-13.9% and 5.5%-14.9%, respectively.
