**2.3 Bacterial isolates**

Isolation of *E. coli* was carried out onto eosin methylene blue agar (Liofilchem, Italy), and the plates were incubated at +37°C for 18–24 h. After this stage, the suspected *E. coli* colonies were selected and streaked onto Mueller-Hinton agar plate (Liofilchem, Italy). Confirmation was carried out by a biochemical microbiology method based on negative urease (Bio-Rad, France), negative citrate (Liofilchem, Italy), positive indole (Bio-Rad, France), positive

lactose (Liofilchem, Italy), and positive orthonitrophenyl-β-D-galactopyranoside (ONPG) (bioMerieux, France). *E. coli* strains isolated were confirmed by API 20E (bioMérieux, France).

The five main pathogroups of *E. coli* (Enteroaggregative *E. coli:* EAEC, Enteropathogenic *E. coli*: EPEC, Enteroinvasive *E. coli*: EIEC, Enterohemorrhagic *E. coli*: EHEC, and Enterotoxigenic *E. coli*: ETEC) were characterized by the 16-plex polymerase chain reaction (PCR) as described by Antikainen et al. [20].
