The causative agents of colibacillosis in poultry: carriers of genes associated with extraintestinal and intestinal pathogenic Escherichia coli

The expansion and intensification of poultry farming increases the risk of spreading colibacillosis among poultry, so there is an urgent need to monitor avian pathogenic Escherichia coli (APEC), study their genetic diversity and identify strains that pose a threat to human health. Determination of virulence-associated genes and the degree of specific adhesion may be useful for a comprehensive assessment of the epidemic and epizootic significance of E. coli strains isolated from livestock. In this study, an extended molecular analysis of E. coli strains isolated from poultry during outbreaks of colibacillosis was performed with the objective to genotypically characterize the isolated E. coli strains and to evaluate the relationship between genes encoding adhesins and specific adhesion to erythrocytes. It was shown for the first time that the strains were characterized by a high potential for pathogenicity and could be carriers of genes for several pathotypes at once, while the genes of intestinal pathogenic E. coli (IPEC) were detected often than others. A positive adhesive profile for a number of genes correlated positively with the activity of strain adhesion to chicken ( Gallus gallus L.) and human erythrocytes. In the study 28 non-clonal E. coli strains, as determined by ERIC-PCR, isolated from various organs (except the intestine) of Ross 308 cross broilers ( Gallus gallus L.) with generalized colibacillosis in 2016-2018 were characterized. Polymerase chain reaction (PCR) was used to detect virulence-associated genes characteristic of four different E. coli pathotypes, the APEC, extraintestinal pathogenic (ExPEC), intestinal pathogenic E. coli (IPEC: Enteropathogenic E. coli EPEC, Enterotixigenic E. coli ETEC, Enterohemorrhagic E. coli EHEC, Enteroaggregative E. coli EaggEC), and uropathogenic E. coli (UPEC). Previously published protocols were used for all types of PCRs and amplifications were performed in the DNA Engine Dyad Thermal Cycler (Bio-Rad, USA). Band visualization and data documentation were performed using the Gel-Doc XR gel documentation system (Bio-Rad, USA). Formalinized human erythrocytes of the type 0(I) Rh(+) and avian erythrocytes were used as cell substrates for the determination of bacterial adhesion to erythrocytes. To evaluate the bacterial adhesion properties the adhesion index was calculated as the average number of bacteria bound to an erythrocyte in the adhesion assay. The obtained results showed that the characterized strains possessed a high pathogenic potential, as they carried genes associated with APEC, ExPEC as well as IPEC. The presence of APEC-specific marker genes identified most of the strains as APEC. However, potential for human pathogenicity was also found among the analyzed strains. As the IPEC-associated genes were found more frequently than ExPEC-associated genes, the E. coli strains studied were more similar to strains causing acute intestinal infections in humans, particularly due to the fact that they carried genes encoding toxins characteristic of IPEC (with the exception of genes for Shiga-like toxins and enterohemolysins). Based on cluster analysis of genetic profiles, the strains studied could be classified into three groups: (i) pathogenic to birds and humans, characterized by the presence of 2-6 genes associated with APEC and 2-6 genes associated with ExPEC or IPEC (24 strains), (ii) pathogenic to birds and nonpathogenic to humans, characterized by the presence of 2-6 genes associated with APEC and 0-1 gene associated with ExPEC or IPEC (2 strains), and (iii) nonpathogenic, characterized by the possession of none or one gene from each pathotype, APEC, ExPEC, IPEC (2 strains). It was found that 75 % of the first group, pathogenic to birds and humans, carried not only a high number of virulence-associated genes, but also pathogenicity island SHI-2, as well as genes for extended-spectrum beta-lactamases and class 1 integrons. Specific adhesion of E. coli strains was more pronounced on chicken erythrocytes than on human ones. Statistical analysis revealed several positive correlations between the chicken and human erythrocytes adhesion profiles and a number of genes encoding adhesins. The high adhesion activity of the bacteria, regardless of the type of erythrocyte, also correlated with longer survival in host blood serum (genotype iss +) and the possibility of erythrocyte lysis (genotype hlyF +). The obtained data on the molecular and adhesive properties of causative agents of colibacillosis in birds allow us to assess their zoonotic potential and epizootic significance and can also serve as the basis for improving the monitoring system for colibacillosis in poultry farms.