*2.3. Bacteriological Analyses and E. coli Isolation*

The bacteriological investigations were carried out as previously described [25]. In brief: Cloacal swab samples were directly streaked on Gassner agar plates, following an incubation overnight at 37 ◦C. For manure samples 50 mL of peptone water (Oxoid, Wesel, Germany) as well as the manure sample itself (25 g each) were put into a sterile Whirl-Pak® Bag (Nasco, Fort Atkinson, WI, USA). Bags were mixed for three minutes with a Bag Mixer® 400 VW (Interscience, Saint Nom, France). Using a sterile loop, 10 μL of each mixed-sample was streaked on Gassner agar (Oxoid, Wesel, Germany) and incubated at 37 ◦C for 18–24 h.

One single blue color colony from each plate was selected and spread onto Columbia blood agar (Oxoid, Wesel, Germany) and Tryptone Bile X-glucuronide (TBX) agar (Oxoid, Wesel, Germany). Incubation was done overnight at 37 ◦C. Bluegreen colonies on TBX agar detected glucuronidase activity. The positive indole test with Kovac's indole reagent (Merck, Darmstadt, Germany) was used to confirm the diagnosis.

#### *2.4. Antibacterial Susceptibility Testing*

The guidelines of the Clinical and Laboratory Standards Institute (CLSI) and the manufacturer's recommendations were the basis for testing the resistance of *E. coli* isolates using the broth microdilution technique. Micronaut plates (Merlin, Bornheim-Hersel, Germany) with Mueller–Hinton Broth (Merlin, Bornheim-Hersel, Germany) were used to determine minimal inhibitory concentrations (MICs) of enrofloxacin (ENR) and ampicillin (AMP). Dried antibacterial agents in serial dilutions of enrofloxacin and ampicillin were placed in wells of these plates, as previously described by Chuppava et al. [25]. MIC values were determined by visually reading and interpreting the results. As the reference strain, *E. coli* ATCC 25922 was tested concurrently on each testing day.
