Identification of cholix toxin gene in Vibrio cholerae non-O1/non-O139 isolated from diarrhea patients in Bushehr, Iran

Background and Objectives: Cholixin (cholix toxin) is a novel exotoxin in Vibrio cholerae identified as an elongation factor II specific ADP-ribosyltransferase which inhibits protein synthesis in the eukaryotic cell. Previous researches have suggested that cholixin probably is an important virulence factor in non-O1/non-O139 V. cholerae (NAG) serotypes that could be related to extra-intestinal rather than intestinal infections. This study was aimed to investigate the frequency and genetic diversity of colixin gene (chxA) in clinical V. cholerae NAG isolates. Materials and Methods: The presence of chxA gene in 44 clinical V. cholerae NAG isolates were screened using PCR through specific primers designed for the receptor-binding domain (RBD) of chxA gene. The five PCR products of chxA gene were sequenced. Results: This study showed that chxA gene presented in 19 V. cholerae NAG isolates. The sequences analysis of 5 out of 19 the partial chxA genes amplicon showed that 4 of them belonged to chxA I and the other one belonged to chxA II subtypes. Two distinct clusters were revealed for these isolates by phylogenic analysis, too. Conclusion: The chxA gene contained high frequency among V. cholerae NAG isolates in Bushehr, Iran. The polymorphism study on RBD of cholixin gene is suggested as an appropriate method for phylogenic characterization of the various chxA gene subtypes.

Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae

Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

Prevalence of Vibrio cholerae O1 El Tor variant in a cholera-endemic zone of Kenya

Since 2007, Kenya has experienced an increase in cholera outbreaks characterized by a high fatality rate. In this study, we characterized 81 Vibrio cholerae isolates from diarrhoeal stool samples in Nyanza, a cholera-endemic lake region of Kenya, for virulence properties, clonality and antibiotic susceptibility. Eighty of these isolates were V. cholerae O1 El Tor variants carrying the classical ctxB gene sequence, while one isolate was V. cholerae non-O1/O139. All of the El Tor variants were of clonal origin, as revealed by PFGE, and were susceptible to ampicillin, tetracycline, ciprofloxacin, fosfomycin, kanamycin and norfloxacin. However, the isolates showed resistance to sulfamethoxazole/trimethoprim and streptomycin, and intermediate resistance to nalidixic acid, chloramphenicol and imipenem. The non-O1/O139 isolate carried the cholix toxin II gene (chxA II) and was susceptible to all antimicrobials tested except ampicillin. We propose that an El Tor variant clone caused the Nyanza cholera outbreak of 2007–2008.

Novel Cholix Toxin Variants, ADP-Ribosylating Toxins in Vibrio cholerae Non-O1/Non-O139 Strains, and Their Pathogenicity

ABSTRACTCholix toxin (ChxA) is a recently discovered exotoxin inVibrio choleraewhich has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A ofPseudomonas aeruginosaand diphtheria toxin ofCorynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence ofchxAand its genetic variations and pathogenic mechanisms. In this study, we screened thechxAgene in a large number (n= 765) ofV. choleraestrains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n= 485) or O139 (n= 84). Sequencing of these 53chxAgenes generated 29 subtypes which were grouped into three clusters designatedchxAI,chxAII, andchxAIII.chxAI belongs to the prototype, whilechxAII andchxAIII are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.