Development of a multiplex assay technique of E. coli colicinotypes

Abstract

Intraspecific antagonistic relationships of E. coli by producing bacteriocins - colicins are the key factor determining the dynamics of microbial communities. Presumably, the risks of coli infection may depend on the diversity of E. coli colicinotypes in the animal body. A large number of E. coli strains in the intestinal microbiocenosis cannot be studied by conventional microbiology methods. The research goal was to develop a technique for analyzing the diversity of E. coli colicinotypes by using high-throughput sequencing. By using bioinformatics analysis, clustering of colicin genes was carried out, and conservative regions of colicin genes forming clusters were identified. The primers for PCR and high-throughput sequencing were designed with the NGS-PrimerPlex software. Based on the identified nucleotide sequences, a system of 17 primer pairs was developed to perform multiplex PCR reactions for the presence of a wide range of colicin genes, virulence markers FliC, PapF, FimH and the highly polymorphic locus of the GND genome providing an effective analysis of E. coli biodiversity using high-throughput sequencing technologies. The proposed primer system was tested for multilocus sequencing of individual E. coli strains. To test the technique, isolates of E. coli were taken from the heart and parenchymatous organs of birds and pigs (11 isolates). Some of the 11 E. coli strains isolated from poultry and pigs were successfully identified by increased representation of the corresponding colicin genes. In total, colicins M, Ib, B, V and 2 allelic variants of GND were identified. This methodological approach provides the opportunity to study E. coli microbiocenoses including their biodiversity, epizootic significance of genotypes and cooperative antagonism.