Coli id agar plates

An aliquot of 50 μl of each sample was spread onto Coli-ID agar plates (bioMérieux, Marcy l’Etoile, France) and incubated at 37°C. Colony-forming units (CFUs) were counted at 24 and 48 h. Five colonies of each sample were further sub-cultured on Modified Scholtens’ Agar (MSA) plates and incubated 24 h at 37°C. The isolates were stocked in 20% (wt/vol) glycerol (Sigma-Aldrich, St. Louis, MO, United States) at −80°C.

Isolates from E. coli were quantified on Coli-ID agar plates (bio-Mérieux, Marcy l’Etoile, France) and S. aureus on Aureus Agar Base HiCrome (Sigma-Aldrich, MO, USA) by plating 50 µL of the rinsing solution. For the detection of the presumed resistant bacteria, five colonies were collected from each environmental sample and incubated for 24 h at 37 °C on Mueller-Hinton agar (Oxoid Ltd., Basingstoke, UK) containing appropriate antimicrobials. E. coli were tested for resistance towards ampicillin at 35 µg/mL, tetracycline at 20 µg/mL, kanamycin at 30 µg/mL, and chloramphenicol at 35 µg/mL, while S. aureus were tested for penicillin-G at 0.5 µg/mL, erythromycin at 4 µg/mL, clindamycin at 1 µg/mL, and tetracycline at 20 µg/mL (Sigma-Aldrich, St. Louis, MO, USA). The choice of antibiotics was based on their widespread use in veterinary and human medicine [32 (link)]. Disc diffusion was performed using the above-mentioned antimicrobials, according to the hospital’s laboratory routine following guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [33 ] (see below). Resistant bacteria were re-cultured on AB-plates with appropriate antibiotics. The isolated bacterial colonies were stored in Modified Scholtens’ Broth (MSB) or 2× Yeast Tryptone (YT) media with the addition of 20% (w/v) glycerol (Sigma-Aldrich, St. Louis, MO, USA) at −80 °C.

An aliquot of 100 µL of urine and 1:10 dilutions thereof were spread on Coli-ID agar plates (bioMérieux, Marcy l’Etoile, France) and incubated under aerobic conditions at 37 °C for 24 h. A total number of 57 single colonies were randomly isolated and sub-cultured on plate count agar for further analyses and for each isolate a separate stock in Luria broth (Merck, Darmstadt, Germany) with 20% glycerin (Sigma-Aldrich, St. Louis, MO, USA) was prepared and stored at −80 °C for further use. All isolates were typed biochemically as E. coli using the API 20E test system (bioMérieux, Marcy l’Etoile, France).
For bacteriophage isolation, samples (urine and liquids extracted from sponges and swabs) were filtered through a 0.22 µm filter. Coliphages were isolated according ISO 107006-2:2000 as previously described [4 (link),27 ]. Single phage plaques (n = 60) were isolated and cultured as previously reported [28 (link)]. Lysates were filtered through a 0.22 µm filter to remove bacterial cell debris and stored at 4 °C for further analysis.