*2.2. Strain Isolation and Identification*

Sludge samples were homogenized by vortexing for two minutes. For qualitative analysis, an amount of 1 mL from the homogenized sludge sample was suspended in 9 mL sterile saline solution (0.9% NaCl). In order to reduce the bacterial concentration, a decimal dilution series with saline solution was prepared.

ESBL isolation: 0.1 mL of each homogenized sludge sample was plated on chromID™ ESBL Agar (bioMérieux, Marcy-l'Etoile, France) and incubated for 24 h at 37 ◦C. Following incubation, ESBL positive colonies were determined based on the colour reaction of the ESBL-media (according to the manufacturer's protocol). Additionally 0.1 mL of the sludge samples was incubated (24 h, 37 ◦C) in thioglycolate nutrient broth for enrichment, then 10 μL of the material was inoculated on ESBL-media and incubated for 24 h at 37 ◦C [21].

MRSA isolation: 0.1 mL of the homogenized solutions were plated on oxacillin agar (OXOID Ltd., Basingstoke, UK) and incubated for 48 h at 37 ◦C. Following incubation, MRSA positive colonies were determined based on the colour reaction of the OXA-media. Blue colonies were presumed to be MRSA.

VRE isolation: For selective enrichment of VRE, an amount of 1 mL from the homogenized sludge sample was inoculated in 9 mL BBL™ Enterococcosel™ broth (BD, Sparks, MD, USA) containing 6 mg/L Vancomycin. Enterococci growing in the media turn the colour of the media from light brown to dark brown or black. In order to reduce the bacterial concentration, a decimal dilution series with saline solution was prepared. Subsequently, 0.1 mL from each of the homogenized solutions were plated on chromID™ VRE Agar (bioMérieux, Marcy-l'Etoile, France) and incubated for 24 h at 37 ◦C. VRE positive colonies were determined based on the colour reaction of the VRE-media (according to the manufacturer's protocol).

To obtain pure cultures, colonies growing on selective-media were transferred to blood agar (24 h, 37 ◦C). Identification was done using the Vitek® MS (bioMérieux, Marcy-l'Etoile, France), an automated microbial identification system using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and the biochemical-based VITEK®2 system (bioMérieux, Marcy-l'Etoile, France).
