Oxacillin

During the intervention periods (period two and period four), the nasal swab was plated on blood agar plates, which were incubated at 35°C with 5% CO₂ in ambient air, and were checked for the presence of S. aureus after 16 to 18 hours of incubation. To shorten the time interval from culture to reporting MRSA, suspected S. aureus isolates were tested for the presence of coagulase using BD BBL Coagulase Plasma Rabbit with EDTA (BD, Franklin Lakes, NJ, USA) (35°C overnight), and plated on oxacillin screen agar plates containing 6 μg/ml oxacillin and 4% NaCl (BD, Franklin Lakes, NJ, USA) at 35°C for 24 hours to test for oxacillin resistance.

Antimicrobial susceptibility of the SAR Bari strain was determined by a BD PHOENIX^™ 100 instrument (Becton Dickinson, Franklyn Lake, NJ). Data were elaborated by the BD Epicenter Expert System according to EUCAST rules (http://www.eucast.org). The PMIC/ID-88 (BD) panel was used to test susceptibility to ampicillin, cefoxitin, ceftaroline, ciprofloxacin, clindamycin, daptomycin, erythro-mycin, fosfomycin, fusidic acid, gentamicin, imipenem, linezolid, moxifloxacin, mupirocin, nitro-furantoin, oxacillin, penicillin, rifampin, teicoplanin, tetracyclin, tigecycline, trimethoprim/ sulfamethoxazole, and vancomycin. The Epsilometer Test (ETest) was used for testing resistance to ciprofloxacin, daptomycin, erythromycin, gentamicin, moxifloxacin, tetracyclin, tigecycline, trimethoprim/sulfamethoxazole, and vancomycin (bioMérieux, Marcy-L’Étolie, France and Liofilchem, Roseto degli Abruzzi, Italy). All tests were repeated on four independent technical replicates. MIC interpretative breakpoints were defined according to EUCAST recommendations. Staphylococcus aureus ATCC 29213 was used as a control strain.

Microbial identification was confirmed by Bruker Biotyper MALDI-TOF MS (Bruker, Billerica, MA). For MRSA designation, a PBP2a SA culture colony test (Alere) was performed according to the manufacturer’s instructions using 18- to 24-h subculture growth. The presence of mecA and the homolog mecC were determined by in-house PCRs (22 (link), 23 (link)). Susceptibility testing included that for cefoxitin by disk diffusion and for oxacillin by disk diffusion and gradient diffusion. Methods followed the procedural guidelines outlined by the Clinical and Laboratory Standards Institute (documents M02 and M23) (55 , 56 ). Disk diffusion testing of cefoxitin (Hardy Diagnostics) and oxacillin (BD) was performed on conventional Mueller-Hinton agar (MHA) (Hardy Diagnostics). Gradient diffusion testing of oxacillin (bioMérieux) was performed on MHA with 2% NaCl agar (Remel). Detection of beta-lactamase production was assessed by the disk diffusion penicillin zone edge test (Hardy Diagnostics) and nitrocefin-based Cefinase disk test (Hardy Diagnostics). Beta-lactamase inhibitor rescue phenotype was determined by assessing the fold change in MIC from amoxicillin (bioMérieux) and amoxicillin-clavulanic acid (bioMérieux) gradient diffusion testing.