Development of an INDEL typing system for ctx+ strains of Vibrio cholerae from the seventh pandemic

Abstract

BACKGROUND: The seventh cholera pandemic is accompanied by the formation of Vibrio cholerae clones with new genetic properties, including those with the ability to spread pandemically and cause diseases with a more severe clinical course. The widespread distribution of such genetic variants of Vibrio cholerae and the possibility of their introduction into the territory of the Russian Federation necessitate constant comprehensive monitoring using modern molecular genetic technologies.

AIM: To improve INDEL typing of ctx+ strains of V. cholerae of the seventh pandemic by using additional INDEL loci.

Materials and methods: A bioinformatic analysis of 2105 full-genome sequences of toxigenic ctxAB+tcpA+ strains of Vibrio cholerae O1 El Tor from open databases was carried out in order to search for INDEL loci for molecular typing. Based on the convenience criterion for allele size identification, eight INDEL loci were selected. Three loci have been described previously, and five were identified as a result of this work. The designed primers formed amplicons ranging in size from 67 to 390 base pairs, which made it possible to confidently identify them during gel electrophoresis.

Results: The distribution of alleles formed 11 unique INDEL clusters, which we designated A-K. Based on the number of strains within the clusters, three types of clusters were identified: major (A, B and C) made up 89% of the total number of sequences studied, intermediate (D, E, F, G and H) 10.5% of the genomes. Three minor clusters (I, J and K) were represented by single strains. Four clusters united strains isolated in the 20^th century (A — in 1941, F — in 1957, G — in 1993, E — in 1999), and seven clusters — in the 21^st century in the period from 2003 to 2016. In the period from 2019 to 2023, representatives of INDEL clusters were active: A, B, D and E.

ConclusionS: The study of the timing of circulation suggested that representatives of different clusters have different epidemic potential, which was manifested in the absence of isolation of strains of some clusters in recent years. A comparative study of INDEL typing with SNP typing in the in silico analysis of 378 genomes of strains isolated on the African continent indicates that the proposed INDEL typing method is not inferior to SNP typing in terms of resolution.

Keywords

Vibrio cholerae / molecular typing / INDEL loci / INDEL genotyping

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