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).