Antimicrobial susceptibility for β-lactam resistance of the isolates was performed by disk susceptibility testing using Mueller-Hinton agar (Oxoid) and cefoxitin disk (30 mg Oxoid). The results were interpreted using the Clinical and Laboratory Standards Institute guidelines M100 to identify methicillin resistance [26 ]. Staphylococcus aureus ATTC®25923 was used as a control MSSA strain and S. aureus ATCC®43300 was used as MRSA control strain for antimicrobial drug susceptibility testing.
Cefoxitin disk
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Cefoxitin disk is a laboratory equipment used for antimicrobial susceptibility testing. It is a standardized paper disk impregnated with a fixed concentration of the antibiotic cefoxitin. The disk is placed on a culture medium inoculated with a bacterial sample, and the size of the resulting inhibition zone around the disk is measured to determine the susceptibility of the bacteria to the antibiotic.
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6 protocols using cefoxitin disk
1
Methicillin Resistance Screening Protocol
2
Molecular Characterization of lukSF-PV+ MRSA
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Fifty-one consecutive, nonduplicate lukSF-PV+ MRSA isolates from various clinical specimens of individual patients were obtained from Zhejiang Xiaoshan Hospital (a tertiary hospital), Hangzhou City, China. The isolates were collected between January 2010 and December 2011 and identified using VITEK 32 instrument (bioMérieux, Marcy l′ Etoile, France). Methicillin resistance was determined using the Kirby–Bauer method with cefoxitin disk (30 µg, Oxoid, Basingstoke, UK) and PCR for mecA33 ,34 (link). All isolates had been previously described by PVL isoform detection and PFGE-SCCmec-MLST typing (including CC) (Table 1 )35 (link). The strains ATCC 49775 and MRSA N315 were used as positive and negative controls for the PVL production, respectively.
3
Detecting Methicillin Resistance in S. aureus
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S. aureus isolates were tested for methicillin resistance using the cefoxitin and oxacillin disk diffusion methods outlined by the Clinical and Laboratory Standards Institute [32] . Cefoxitin disk (30 µg, Oxoid) and oxacillin disk (1 µg, Oxoid) were used in this study. The zones of inhibition were measured after 18–20 hours incubation at 35°C. Isolates with zone sizes less than 21 mm for cefoxitin and 10 mm for oxacillin were considered methicillin resistant according to the criteria of CLSI [32] .
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S. aureus isolates were tested for methicillin resistance using the cefoxitin and oxacillin disk diffusion methods outlined by the Clinical and Laboratory Standards Institute [32] . Cefoxitin disk (30 µg, Oxoid) and oxacillin disk (1 µg, Oxoid) were used in this study. The zones of inhibition were measured after 18–20 hours incubation at 35°C. Isolates with zone sizes less than 21 mm for cefoxitin and 10 mm for oxacillin were considered methicillin resistant according to the criteria of CLSI [32] .
4
Methicillin Sensitivity Screening Protocol
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Methicillin sensitivity was screened by the Kirby–Bauer disk diffusion method using a cefoxitin disk (Fox) (30 µg) (Oxoid, Altrincham, UK) and analyzed in consonance with Clinical and Laboratory Standard Institute (CLSI) breakpoints [11 ]. The diameter of the inhibitory zone was estimated in millimeters. Susceptible isolates have a zone diameter equal to or less than the susceptible breakpoint. It was inhibited by the antimicrobial agent at the achievable concentrations when the recommended dosage for treatment at the site of infection was utilized, indicating potential clinical efficacy; otherwise, the isolate is regarded as resistant [11 ].
5
Antibiotic Susceptibility Testing of MRSA
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Susceptibility testing was carried out according to EUCAST recommendations [14 (link)]. MRSA isolates were identified using cefoxitin disks (30 μg) (Oxoid Ltd., Cambridge, UK), which was further confirmed by the detection of mecA gene in the DNA microarray analysis. The inducible macrolide-lincosamide-streptogramin B (MLSB) resistance was detected by disk diffusion method with use of clindamycin disks (2 μg) and erythromycin disks (15 μg) set 15–26 mm apart on Mueller-Hinton agar inoculated with S. aureus bacterial suspension (turbidity adjusted to 0.5 McFarland standard). The inducible phenotype causes a D-shaped inhibition zone around the clindamycin disk, blunted from the side of erythromycin disk. In the case of efflux mechanism of resistance, the entire zone of inhibition surrounding clindamycin disk is round. Other antibiotics tested via disc-diffusion method included: gentamicin (10 μg), linezolid (30 μg) or netilmicin (30 μg). Teicoplanin and vancomycin susceptibilities were determined by using E-test strips (bioMérieux, Marcy l'Etoile, France) according to the manufacturer’s instructions.
6
AmpC and ESBL Screening Protocol
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The strains included in the study were screened for AmpC-blactamase-production by cefoxitin disks (30 mg; Oxoid, Basingstoke, UK) [27] . Phenotypic verification of AmpC-blactamase and ESBL-production was carried out using AmpC-ESBL Detection Set (MAST Diagnostica, Reinfeld, Germany) and VITEK 2 Compact ID/AST (bioM erieux, Marcy-l' Etoile, France), according to the manufacturer's instructions.
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