Elaboration of a Toxigenic Vibrio cholerae Typing Scheme Based on Bioinformatiсs Analysis Data

Introduction: The current stage of the seventh cholera pandemic is characterized by the emergence of novel Vibrio cholerae gene variants, gradually replacing their predecessors and occupying a dominant position in the etiology of the disease. Determining their epidemic potential by identifying the number of genetic markers is unsuitable for operational analysis. Thus, the development of a method for differentiating pathogens based on PCR detection of a limited number of markers seems relevant. Objective: To create a database of whole genome V. cholerae sequences containing different alleles of cheA3 (VCA1095) and rtxA genes based on bioinformatics analysis data and to elaborate a simple and informative toxigenic vibrio typing scheme. Materials and methods: The NCBI database-extracted results of whole genome sequencing of 3,309 strains of Vibrio cholerae isolated in 1962–2021 were used for the analysis. The software was developed in Java. Results: The bioinformatics analysis of the database of whole genome V. cholerae sequences, including 3,309 genomes of third wave strains, enabled us to divide them into three groups: “pre-Haitian”, “Haitian”, and “post-Haitian”. All of them contained alleles of the genes of toxin-co-regulated tcpACIRS101 pili and the MARTX rtxA4 cytotoxin with a null mutation that caused a premature stop codon. However, in the “pre-Haitian” strains, the gene of the cholera toxin subunit B of the classical ctxB1 type and the prototype gene of histidine kinase cheA3 (VCA1095) were always detected, which in PCR formed a 95 bp long amplicon and was designated as VCA1095-95. In the “Haitian” strains, a deletion of 8 bp occurred in this gene, and the PCR amplicon was shortened to 87 bp (VCA1095-87). Its mandatory combination with the ctxB7 allele was revealed. The “post-Haitian” strains contained an even shorter rtxA4a allele due to the deletion of 60 bp in the proximal part. Conclusion: Since the analysis of a large number of genomes revealed strict correlations between certain alleles in each group, we consider it possible to use only two markers for operational analysis, i.e. alleles of the cheA3 and rtxA genes. The typing scheme based on their PCR detection can be used to facilitate determination of the epidemic potential of newly isolated cultures.