Macconkey agar plate

All samples were inoculated into Trypticase soy broth tubes and were incubated at 37 °C for 24 h. A loop-ful from the previously incubated tubes were streaked onto MacConkey agar plates (Oxoid Ltd., Cairo, Egypt) and Eosin methylene blue agar (EMB) (Oxoid) and were incubated aerobically at 37 °C for 24 h. Suspected colonies were purified through subculture on MacConkey agar plates and were examined for morphology and phenotype traits according to Collee et al. [23 ].

For each selected broiler farm, cloacal swabs were collected using sterile cotton swabs directly from cloaca of 10 randomly selected birds. The cloacal swab samples in each farm were pooled in Falcon tubes containing 25 ml Luria-Bertani (LB) broth (Merck, Darmstadt, Germany). A paired boot swab samples were obtained by walking along the whole length of the broiler house. Boot swab samples were placed in a 500 ml beaker containing 250 ml of LB broth for enrichment. A total of 156 samples (78 pooled cloacal swabs and 78 boot swabs) from 78 broiler farms were processed and subjected to microbiological analysis. Samples were incubated aerobically at 37 °C for 18–24 h. Thereafter, a loopful (10 μl) of each enriched sample was streaked onto MacConkey agar plate (Oxoid, United Kingdom) supplemented with 1 mg/L cefotaxime and incubated aerobically at 37 °C for 24 h. A replicate MacConkey agar plate without cefotaxime was also prepared for each sample. Subsequently, one bright pink colony, suggestive of lactose-fermenting bacteria and morphologically indicative of E. coli, was picked and streaked in a selective and differential medium, Eosin Methylene Blue agar plate (HiMedia, Mumbai, India) and incubated at 37 °C for 24 h. The bacteria isolated from all pooled fecal and swab samples were identified.

The tissue cylinders were lysed using Precellys Lysing Kit (Tissue homogenizing CKMix, Precellys Lysing Kit, Bertin, France), according to the manufacturer’s instruction and under consideration of hygiene standards as to avoid contamination. After dilution of the lysates at 1:5 and 1:100 in PBS, 50 μL were spread out on Columbia agar +5% sheep blood agar (COS), Columbia NaladixicAcid agar (CNA) (Biomerieux, Marcy l’Etoile, France), and Mac Conkey Agar plates (Thermo Fisher Scientific, Waltham, MA, USA) for 48 h at 37 °C, before the number of colony forming units was determined manually. Exemplary mass spectrometric analysis (matrix-assisted laser desorption/ionization (MALDI)) of each macroscopically distinctive unit was then carried out for bacterial identification.