Isolation, identification, and selection of strains
Isolation and identification of resistant E. coli strains were carried out according to a procedure described earlier (Osińska et al., 2020). The material was inoculated on selective MacConkey agar media (Biomaxima, Lublin, Poland) supplemented with tetracycline (8 mg/L), chloramphenicol (16 mg/L), kanamycin (32 mg/L), and cefotaxime (2 mg/L) (Sigma Aldrich, Germany). Single isolates were selected as described in previous studies (Nowakiewicz et al., 2020; Osińska et al., 2020) using a two-step strain selection approach: the disc diffusion method (DDM) and Amplification of DNA Fragments Surrounding Rare Restriction Sites (ADSRRS).
Based on the analysis of susceptibility profiles consisting of six antimicrobials with DDM, the isolates were considered as different when the susceptibility of the strains differed for at least one drug.
In the second step, based on a similarity value below 90%, the ADSRRS-fingerprinting method was used for distinguishing separate genotypes among strains isolated from the same individual as described in a previous study (Osińska et al., 2020).
DNA was obtained using a ready-made isolation kit (Tissue and Bacterial DNA Purification Kit, Eurx, Gdańsk, Poland).