Enterococcosel agar

Serial dilutions of all samples were performed with sterile saline solution and aliquots of 30µl of each sample were seeded in two different culture medias: Enterococcosel agar (Becton, Dickinson and Company Sparks, MD, USA) for
E. faecalis; and MacConkey agar (Himedia Laboratories, Mumbai, India) for
E. coli. The plates were kept at 37°C for 24h and then CFU/mL were counted.

Enterococcal strains used in the study were obtained from the Biodefense and Emerging Infections Research Resources Repository (BEI Resources) (Manassas, VA, USA), and the American Type Culture Collection (ATCC) (Manassas, VA, USA). Media and reagents were purchased from commercial vendors: tryptic soy broth (TSB), tryptic soy agar (TSA), enterococcosel agar (Becton, Dickinson and Company, Cockeysville, MD, USA), and phosphate-buffered saline (PBS) (Corning, Manassas, VA, USA). Drugs used in the study were purchased commercially: dorzolamide (TCI America, Portland, OR, USA), linezolid and vancomycin (Chem-Impex International, Wood Dale, IL, USA), and ampicillin (IBI Scientific, Peosta, IA, USA).

Tank Collected from Hospitals, Washed Market Fluid, Market Leachate, Washed Fluid from Garbage Truck, and Stabilized Leachate from Landfill Facility
An undiluted sample of 0.1 mL was inoculated on MacConkey agar supplemented with ceftriaxone 2 µg/mL, on Enterococcosel agar (BBL, Becton Dickinson, USA) supplemented with vancomycin 6 µg/mL, and on mannitol salt agar supplemented with oxacillin 6 µg/mL. The inoculated agar plates were incubated at 35 °C for 18–24 h.
A diluted sample with buffered peptone water supplemented with ceftriaxone 2 µg/mL at 1:10 was prepared. A diluted sample of 0.1 mL was also inoculated on all of the aforementioned agars and they were incubated at 35 °C for 18–24 h.
Colony count of grown bacteria was performed on the plate inoculated with either undiluted or diluted sample. Each type of grown bacteria was inoculated on a blood agar plate and incubated at 35 °C for 18–24 h. The bacteria grown on blood agar plate were identified up to species level and were used for antibiotic susceptibility testing as described in Section 2.2.4.

For VRE, MRSA, and C. difficile cultures, media included Enterococcosel agar (Becton Dickinson, Cockeysville, MD) containing 20 µg/mL of vancomycin, CHROMagar (CHROMagar, Paris, France) containing 6 µg/mL of cefoxitin, and cycloserine-cefoxitin-brucella agar containing 0.1% taurocholic acid and lysozyme 5 mg/L (CDBA), respectively [19] (link). Plates containing MRSA or VRE were incubated aerobically at 37°C for 48 hours. C. difficile plates were incubated in a Whitley Workstation MG1000 anaerobic chamber (Microbiology International, Frederick, MD) at 37°C for 48 hours.

Collected samples were inoculated onto Enterococcosel agar (Becton–Dickinson Japan, Tokyo, Japan) containing 32 µg/mL of vancomycin and incubated aerobically at 37 °C for 48 h. Positive cultures were subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI Biotyper; Bruker Daltonik GmbH, Bremen, Germany) to identify the species. Susceptibility of VRE isolates to vancomycin and teicoplanin was tested using E-test (bioMèrieux, Durham, NC, USA) and was interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) document M100 ED29. Types of van genes were confirmed through multiplex PCR, as previously described^{38 (link)}.