Cefoxitin

The susceptibility of S. aureus strains to 30 µg cefoxitin (Becton Dickinson, Franklin Lakes, NJ, USA) and 1 µg oxacillin (Becton Dickinson, Franklin Lakes, NJ, USA) was determined by the disc diffusion test, according to the CLSI guidelines [29 ]. The suspensions 0.5 McFarland standard, equivalent to 1.5 × 10⁸ colony-forming units/mL (CFU/mL), were spread with a sterile swab on Mueller-Hinton agar (MHA) plates (Oxoid, Milan, Italy) and the disks were applied to the surface. After incubation for 24 h at 35 °C, the diameter inhibition zone was determined. S. aureus ATCC 6538 was used as a reference strain (negative control).

Antibiotic susceptibility pattern of isolates was performed using the disc diffusion method on Mueller Hinton Agar (Condalab, Spain) according to the Clinical and Laboratory Standards Institute (CLSI) guideline for the following antibiotics: penicillin (10 μg), cefepime (30 μg), cefoxitin (30 μg), sulfamethoxazole (23/75 μg), tazobactam (10 μg) + piperacillin (100 μg), cefotaxime (30 μg), ceftriaxone (30 μg), meropenem (10 µg), clindamycin (2 μg), clarithromycin (15 μg), levofloxacin (5 μg), azithromycin (15 μg) (BD-BBL Company, USA). The Minimum Inhibitory Concentration (MIC) of colistin and vancomycin was determined using the broth microdilution method. Bacterial isolates resistant to three or more different antimicrobial classes were identified as MDR isolates. S. aureus (ATCC 25923) and E. coli (ATCC 25922) were used as control strains.

Antimicrobial susceptibility testing was done by the Kirby-Bauer standard disk diffusion method [11] according to CLSI guidelines [12] for different antimicrobial agents like: ceftazidime (30 µg), cefotaxime (30 µg), cefpodoxime (10 µg), ceftriaxone (30 µg), cefepime (30 µg), aztreonam (30 µg), ampicillin (10 µg), piperacillin (100 µg), cefoxitin (30 µg), gentamicin (120 µg), amikacin (30 µg), ciprofloxacin (5 µg), tetracycline (30 µg), minocycline (30 µg), chloramphenicol (30 µg), trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg), colistin (10 µg), ertapenem (10 µg) and meropenem (10 µg) (BD Diagnostics, Franklin Lakes, NJ, USA).
The MIC values (mg/L) of cefotaxime, ertapenem, meropenem, amikacin, gentamicin and tigecycline were determined using Etest method (AB Biodisk, Solna, Sweden) and were interpreted according to CLSI guidelines as modified in 2013. The clinical breakpoints for meropenem were as follows: susceptible (S) ≤1.0 mg/L, intermediate (I) 2.0–3.0 mg/L, and resistant (R) ≥4.0 mg/L. The same for ertapenem were as follows: S ≤0.5 mg/L, I: 1.0 mg/L, R ≥2 mg/L. MIC50 and MIC90 of meropenem were calculated as the MIC at which 50% and 90% of the isolates were inhibited.

Fecal samples were collected from patients previously identified as colonized or infected with C. difficile and plated anaerobically onto brain heart infusion (BHI) agar plates supplemented with yeast extract, cysteine, and the antibiotics cycloserine and cefoxitin (BHI and yeast extract were from BD Biosciences, and the other components were from Sigma-Aldrich). Individual colonies that were able to grow in the presence of these antibiotics and that had the characteristic phenotype of C. difficile were selected, isolated, and then typed using MLST (24 (link)).