# Methicillin Resistance of Food-Borne Biofilm-Forming Staphylococcus-Crocei
## 2 Materials and Methods  
This study used 34 isolates of _Staphylococcus_ spp. previously recovered from RTE street food sold in Maputo [11]. This set of isolates included 10 _S. aureus_ (coagulase-positive staphylococci (CPS) and 24 coagulase-negative staphylococci (CNS): seven isolates of _S. warneri_ and _S. saprophyticus_, respectively, and five isolates of _S. pasteuri_ and _S. xylosus_, respectively (Table 1). The procedures for obtaining, identifying, and characterizing these isolates by biochemical and molecular tests were previously described in [PERSON] et al. [12]. Briefly, _Staphylococcus_ spp. presumptive colonies were obtained after incubation for 48 h at 37 \({}^{\circ}\)C onto Chromogenic Baird Parker Agar--CBPA (Biokar Diagnostics, Beauvaais, France) according to ISO 6888 [11; 13]. Biochemical tests were carried out to characterize the presumptive _Staphylococcus_ colonies from CBPA plates, namely: Gram staining, catalase, and oxidase tests [11]. Subsequently, coagulase tests were performed in Coagulase Rabbit Plasma (Biokar Diagnostics, Beauvaais, France) and in Baird Parker RPF Agar (Biokar Diagnostics, Beauvaais, France), according to ISO 6888-2/A1-2003 [14]. Molecular identification was performed targeting the 165 rRNA gene (1500 bp) with Bac27F forward primer (5-AGAGTTTGGATCMTGGCTCAG-3) and Univ1492R universal reverse primer (5-CGGTTACCTTGTTACGACT-3). The amplified products were sequenced (STAB VIDA, Caparica, Portugal) and the resulting sequences were submitted to Blast using the megablast algorithm against Reference RNA sequences database (RefSeq RNA) from the National Center of Biotechnology Information (NCBI) for isolate identification. The presence of seven virulence genes, namely encoding toxins (_hlb_ and _sak_) and staphylococcal enterotoxins (_sea_, _seb_, _sec_, _sed_, and _see_) were previously investigated by multiplex PCR [12]. Fourteen of the thirty-four (41.2%) isolates were also multidrug-resistant. These characteristics are presented in Table 1.  
The nine antibiotics tested were: Cefoxitin (FOX), Ampicillin (AMP), Methicillin (MET), Vancomycin (VAN), Penicillin G (PEN), Tetracycline (TET), Gentamicin (GEN), Trimethoprim/sulfamethoxazole (SXT), and Erythromycin (ERY). Isolates signed in bold and underlined are multidrug-resistant isolates. Multidrug resistance was considered as non-susceptibility to at least one agent in three or more antimicrobial categories and/or resistance to methicillin [15]. Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)  
The MIC was determined according to the standard broth dilution method (CLSI M07-A8) as indicated by the Clinical and Laboratory Standards Institute (CLSI) [15], evaluating the visible growth of microorganisms in Trypto-Casein-Soy Broth (TSB) (Biokar Diagnostics, Beauvais, France) at 37 \({}^{\circ}\)C for 18 \(\pm\) 2 h. Briefly, serial two-fold dilutions of methicillin at concentrations ranging from 64 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro}\)/mL to 1 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro}\)/mL were used in 96-well polystyrene microtiter plates (CellStar(r), Kremsmunster, Austria). Then, 100 \(\mathrm{\SIUnitSymbolMicro}\)/mL of inoculum was then added to obtain a final bacterial concentration of 10\({}^{8}\) CFU mL\({}^{-1}\) (0.5 [PERSON]'s standard) in each well, in a total volume of 200 \(\mathrm{\SIUnitSymbolMicro}\)/mL. Cells that were not subjected to the antibiotic at any time and TSB that was not inoculated at any time were controls for inoculum viability