Etest methodology

Antimicrobial susceptibility testing was performed by the disk diffusion technique on Mueller-Hinton agar and it included a panel of 26 antibiotic disks belonging to 15 classes (Supplementary Table S1). Additionally, carbapenems were tested by the E-test methodology (AB BIODISK, Solna, Sweden) to determine the minimal inhibitory concentrations (MICs) of ertapenem, imipenem, and meropenem. The obtained data was interpreted according to the CLSI guidelines (CLSI, 2017) and Galani et al.^{14 (link)}.

The minimal inhibiting concentrations (MICs) of antimicrobial agents, including erythromycin, penicillinG, co-trimoxazole, vancomycin, cefotaxime, ceftriaxone clindamycin, tetracycline, amoxicillin, chloromycetin and levofloxacin were measured using the E-test methodology (AB BioDisk, Switzerland). S.pneumoniae ATCC49619 was used as a quality control strain in antimicrobial susceptibility tests. The interpretations of MIC breakpoints and test results were made according to the recommended method of the Clinical and Laboratory Standards Institute (CLSI)criteria [20] . Isolates not susceptible to at least three antibiotic families were defined as multidrug-resistant (MDR) S.pneumoniae.

Toxin production of the isolates was tested using an immunochromatographic method, detecting toxin A and toxin B (C. difficile QuikChek Complete, Techlab, Blacksburg, VA, USA). Samples with a GDH-positive, but toxin A/B negative results were further tested with the TC (toxigenic culture) method [44 (link)]. Strain typing was performed by PCR ribotyping method as described by Stubbs et al. [53 (link)].
Metronidazole (MET), vancomycin (VAN), clindamycin (CLI) and rifampicin (RIF) susceptibility of the isolates were tested using the E-test methodology (AB BIODISK, Solna, Sweden). A Cd suspension (of 0.5 McFarland turbidity) of each strain was swabbed on Brucella agar (Oxoid, UK) supplemented with 5% defibrinated sheep blood, 1 mg/L hemin and 5 mg/L Vitamin K₁ [50 (link)]. E-test strips of MET, VAN, CLI and RIF were applied onto the agar surface and the plates were incubated in an anaerobic atmosphere for 24–48 h. MICs were read at the point at which the zone of complete inhibition intersected the MIC scale. For the evaluation of the susceptibility of the tested isolates, EUCAST breakpoints and epidemiological cut-off values (in case of RIF) were used (http://www.eucast.org). C. difficile ATCC 9689, C. perfringens ATCC 13,124, B. fragilis ATCC 25,285 and C. acnes ATCC 11,827 were used as control strains.