Study of the CRISPR-cas System of Vibrio cholerae O1 Serogroup Strains

The aim of this study was to analyze the CRISPR-Cas system of V. cholerae serogroup O1 strains of the classical biovar and nontoxigenic isolates of the El Tor biovar and determine their resistance to cholera phages. Materials and methods. Fifty-six strains of V. cholerae serogroup O1 were analyzed. Phage assays were performed using the two-layer method proposed by A. Gratia. Rostov 7 cholera phage was used as a model phage. Sequencing was performed on the MGI DNBSEQ-G50 platform (MGI, China). CRISPRCas typer ( https://github.com/Russel88/CRISPRCasTyper ) and CRISPRCas Finder ( https://github. com/dcouvin/CRISPRCasFinder ) were used to analyze the CRISPR-Cas system. Results and discussion. Classic biovar strains contain a class I CRISPR-cas system of type I-E, which is also prevalent in the studied nontoxigenic ctxAB–tcpA– strains of V. cholerae O1 El Tor. The latter strains also exhibit a canonical variant of the I-F system, as well as I-C, III-B, and a reduced I-Fmini type. The CRISPR array of classical vibrios contains spacers homologous to the protospacer sequences of cholera phages only, while El Tor vibrios possess spacers homologous to both cholera phages and phages of the Vibrio genus, unrelated bacteria, as well as plasmids and a transposon of V. cholerae. The obtained data on the presence of spacers to various cholera phages in the CRISPR array of a number of studied classical strains, as well as nontoxigenic isolates of V. cholerae O1 El Tor, demonstrate its activity. It was established that 82% of V. cholerae O1 classical biovar strains and 50% of El Tor isolates containing spacers to the Rostov 7 phage are resistant to it. The presence of phage-resistant isolates that lack spacers to this phage serves as evidence of the presence of alternative defense systems. The detection of spacers homologous to the genetic material of the ICP1 phage circulating in endemic areas in a number of V. cholerae O1 El Tor strains may indicate their imported nature. The sensitivity of all V. cholerae O1 El Tor strains containing only the reduced I-Fmini system to the Rostov 7 phage may indicate the involvement of this system in other cellular processes unrelated to adaptive immunity to cholera phages. Conclusions. V. cholerae O1 serogroup strains of the classical biovar contain exclusively the class I CRISPR-cas system of the canonical type I-E. In nontoxigenic V. cholerae O1 El Tor strains, in addition to type I-E, the presence of types I‑F, I-C, III-B, and the reduced I-Fmini was detected. The presence of spacers to various cholera phages in the CRISPR array of the studied strains indicates its active state. The resistance of V. cholerae O1 strains of the classical biovar to the Rostov 7 phage is due to the CRISPR-cas system, while the resistance of El Tor vibrios to this phage, along with the indicated system, is ensured by the presence of alternative defense systems.