New Approaches for Genotyping.

Easy-to-perform, fast, and inexpensive methods of differentiation of strains beyond the species level are highly required. Herein two new, original tools for genotyping of E. coli isolates are proposed. The first of the developed method, a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) test uses a highly variable gene, encoding the H antigen as a molecular target. The designing of universal pair of primers and selection of the optimal restriction enzyme I was preceded by in silico comparative analysis of the sequences of the genes coding for 53 different serotypes of H-antigen ( flagellin). The target fragments of genomes for MLST method were selected on the basis of bioinformatics analysis of complete sequences of 16 genomes of . Initially, seven molecular targets were proposed (seven pairs of primers) and five of them were found useful for effective genotyping of strains. Both developed methods revealed high differentiation power, and a high genetic diversity of the strains tested was observed. Within the group of 71 strains tested, 29 and 47 clusters were revealed with RFLP-PCR and MLST methods, respectively. Differentiation of the strains with the reference BOX-PCR method revealed 31 different genotypes. The in silico analysis revealed that the discriminatory power of the new MLST method is comparable to the Pasteur and Achtman schemes and is higher than the discriminatory power of the method developed by Clermont. From the epidemiology point of view, the outcomes of our investigation revealed that in most cases, the patients were infected with unique strains, probably from environmental sources. However, some strains isolated from different patients of the wards of pediatrics, internal medicine, and neurology were classified to the same genotype when the results of all three methods were taken into account. It could suggest that they were transferred between the patients.