M enterococcus agar

To test microbiota, the samples of feces and appendix content (1 g) were treated using the standard microbiological diluton method (International Organization for Standardization (ISO)). The appropriate dilutions in Ringer solution (pH 7.0; Oxoid Ltd., Basingstoke, Hampshire, England) were plated onto following media: M-Enterococcus agar (NF-V04503, Difco Laboratories, Detroit, MI, USA) for enterococci, DeMann-Rogosa-Sharpe agar (ISO 15214, Merck, Germany) for lactic acid bacteria (LAB), mannitol salt agar for coagulase-negative staphylococci (CoNS, ISO 6888), Baird-Parker agar enriched with egg yolk tellurite supplement (ISO 21527-1, Difco) for coagulase-positive staphylococci and S. aureus (CoPS), Clostridium difficile agar with the supplement SR0096E 7% (v/v) defibrinated horse blood (SR0050, ISO 15883, Oxoid) for Clostridium species (anaerobic cultivation), MacConkey agar (ISO 7402, Oxoid) for coliforms, and CLED agar (Conda, Spain) for enterobacteria. Pseudomonads were isolated on Pseudomonas agar (Biomark, India). Cultivation was performed at 30 °C and/or 37 °C for 24–48 h depending on the bacterial genera. The bacterial counts were expressed in log 10 of colony forming units per gram (log 10 CFU/g ± SD). Randomly picked up representants of selected bacterial groups were confirmed by MALDI-TOF identfication system (Bruker Daltonics).

Feces (a total of 25 g) were diluted 1:10 in Brain Heart Infusion (BHI, Difco Laboratories, Detroit, MI, USA) broth supplemented with sterile NaCl 6.5% and incubated at 37 ± 1°C for 18 to 24 h. Subsequently, 20 µL of the incubated broth was subcultured onto m-Enterococcus Agar (Difco Laboratories) for the selective isolation of Enterococcus spp. Simultaneously, for the selective isolation of vancomycin-resistant enterococci (VRE), 10 µL of the initial broth was subcultured on BHI broth supplemented with 2 mg/L vancomycin (Sigma-Aldrich, Saint Louis, MO, USA). After incubation (37 ± 1°C for 18 to 24 h), 20 µL was subcultured onto m-Enterococcus plates with vancomycin (6 mg/mL) and incubated at 37 ± 1°C for 18 to 24 h. Ten colonies per plate were selected based on colony morphology and subcultured onto Blood Agar (Columbia agar + 5% sheep blood, bioMérieux, Marcy-l’Etoile, France) for further identification of Efs and Efm by the simultaneous real-time PCR amplification of the mltF gene of Efs and the ddlA gene present in Efm (RTi-PCR1), as described elsewhere (16 (link)).