Laboratories (n = 276) participating in the POLMICRO scheme sent five consecutive clinical isolates of S. aureus (one isolate/patient) (n = 1376) during a 3-month period in 1999–2000 to the CQCM. Data on susceptibility to antimicrobial agents (obtained by disk diffusion tests) and additional information (e.g., hospital or ambulatory care, type of ward, site of infection) were collected.
Centre of Quality Control in Microbiology
Most (n = 1005; 73.7%) of the S. aureus isolates were from hospital-acquired infections, including skin and soft tissue infections (n = 685; 50.2%), deep abscesses (n = 121; 8.9%), blood (n = 103; 7.6%), and bone and joint infections (n = 90; 6.6%). Isolates were collected from surgical (n = 388), internal medicine (n = 96), intensive care (n = 53), obstetric and gynaecology (n = 56), paediatric (n = 38), dermatology (n = 37), neonatology (n = 32) and other unspecified wards. Most community-derived isolates were recovered from skin and soft tissue infections. The isolates were re-identified by standard procedures , based on free coagulase production and clumping factor (rabbit plasma; Biomed, Warsaw, Poland) and DNase production (DNase agar; Mast Diagnostics, Bootle, UK).
Methicillin resistance was determined by disk diffusion with a 1-µg oxacillin disk, an oxacillin agar screening test (performed when discrepancies between local laboratory and CQCM results were encountered) [12,13] and detection of the mecA gene by PCR (performed on all MRSA isolates identified by oxacillin disk diffusion) . S. aureus ATCC 29213 (methicillin-susceptible, MSSA) and S. aureus ATCC 43300 (methicillin-resistant) were included as reference strains.
Antimicrobial susceptibility testing was performed by both disk diffusion and agar dilution methods according to NCCLS guidelines [12,13]. The following antimicrobial agents were used for disk diffusion tests by local laboratories: penicillin, oxacillin, erythromycin, clindamycin, lincomycin, gentamicin, tetracycline, doxycycline, ciprofloxacin, ofloxacin, trimethoprim–sulphamethoxazole, teicoplanin, vancomycin, rifampicin, chloramphenicol, nitrofurantoin, fusidic acid (Becton Dickinson, Franklin Lakes, NJ, USA) and mupirocin (Oxoid, Basingstoke, UK). S. aureus ATCC 25923 (methicillin-susceptible) was used as the control strain for disk diffusion testing.
MICs were determined for 19 antibiotics (MRSA isolates) and 24 antibiotics (MSSA isolates) by agar dilution on Mueller–Hinton-II Agar (Becton Dickinson). An inoculum of 104 CFU was applied to antibiotic-containing plates with a multipoint inoculator (West Sussex Instruments Ltd, Denley, UK). The following antimicrobial agents were tested: penicillin (Sigma, Munich, Germany), cloxacillin (Polfa Tarchomin, Warsaw, Poland), amoxycillin–clavulanic acid (SmithKline Beecham, Philadelphia, PA, USA), cefazolin (Fluka, Buchs, Switzerland), cefuroxime (Sigma), erythromycin (Fluka), clindamycin (Pharmacia Upjohn, Kalamazoo, MI, USA), gentamicin (Polfa Tarchomin), tetracycline (Sigma), doxycycline (Sigma), minocycline (Wyeth Ayerst, St Davids, PA, USA), ciprofloxacin (KRKA, Novo Mesto, Slovenia), moxifloxacin (Bayer, Wuppertal, Germany), trimethoprim–sulphamethoxazole (Roche, Basel, Switzerland), trimethoprim (Roche), teicoplanin (Marion Merrell, Denham, UK), vancomycin (Lilly, Indianapolis, IN, USA), rifampicin (Lepetit, Lainate, Italy), chloramphenicol (Sigma), linezolid (Pharmacia Upjohn), quinupristin–dalfopristin (Aventis Pharma, Romainville, France), nitrofurantoin (Terpol, Sieradz, Poland), fusidic acid (Aldrich, Taufkirchen, Germany) and mupirocin (SmithKline Beecham). Breakpoints for fusidic acid, mupirocin, cloxacillin and doxycycline were those recommended by Comité de l'Antibiogramme de la Societé Francaise de Microbiologie guidelines .
S. aureus ATCC 29213 (MSSA), Escherichia coli ATCC 35218 (for β-lactam/β-lactamase inhibitor combinations) and Pseudomonas aeruginosa ATCC 27853 (for Mueller–Hinton agar) were used for quality control of the susceptibility tests . The inducible MLSB (iMLSB) phenotype was detected by the double erythromycin–clindamycin disk test.