Rifampin

To measure sensitivity to β-lactams, Sensi-Discs were prepared on the day of the assay by adding a 20-µl drop containing 10 µg amdinocillin, 10 µg ampicillin, or 30 µg cephalexin dissolved in water on 6-mm paper discs (Becton Dickinson). To measure susceptibility to other antibiotics (see Fig. S1E in the supplemental material), we used 6-mm paper discs preloaded with 30 µg vancomycin, 5 µg novobiocin, 10 µg bacitracin, 15 µg erythromycin, or 25 µg rifampin (Becton Dickinson). Cells at an OD₆₀₀ of 0.5 (100 µl or 1 ml) were mixed with LB top agar (3 or 10 ml), containing spectinomycin when required for the maintenance of pAM238 vectors, and poured on top of an LB agar plate. Sensi-Discs were placed on the solidified mixture at a minimum 3-cm distance from each other and from the plate border. The assay was performed in triplicate for each strain. The diameter of growth inhibition around each disc was measured after overnight incubation at 37°C. When indicated, the relative growth inhibition was calculated as follows: each diameter value was normalized by the average diameter obtained from three replicates of the control strain, and the resulting triplicates of normalized values were then averaged for each tested strain. Bar graphs were prepared using Prism 6 (GraphPad Software, Inc.).

S. aureus isolates were identified by MALDI-TOF mass spectrometry (Bruker Daltonic GmBH, Bremen, Germany), and MRSA isolates were detected by antibiotic susceptibility testing using the agar disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [18 ]. The antibiotics tested were penicillin, cefoxitin, gentamicin, erythromycin, clindamycin, tetracycline, ciprofloxacin, trimethoprim-sulfamethoxazole, rifampin, linezolid, and mupirocin (BD, Sparks, USA). The minimal inhibitory concentration (MIC) of vancomycin and teicoplanin was determined using the E-test (bioMerieux, Marcy-l’Etoile, France).

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.

Staphylococci were identified according to standard microbiological procedures, whereby isolates that were Gram-positive cocci (grape-like clusters), which produced catalase and positive to both slide and tube coagulase tests with human plasma, were considered as S. aureus [41 ]. Antibiotic susceptibility was determined using standard Clinical and Laboratory Standards Institute methods for antimicrobial disk diffusion using the following antibiotics: penicillin (10 IU), cefoxitin (30 µg), gentamicin (10 µg), erythromycin (15 µg), tetracycline (30 µg), doxycycline (30 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), clindamycin (2 µg), trimethoprim/sulfamethoxazole (1.25/23.75 mcg), rifampin (5 µg), and linezolid (30 µg) (BD, Sparks, MD). Antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds (qacA/B, smr), as well as mupirocin (both mupA and mupB) and methicillin resistance (mecA), were screened with a multiplex polymerase chain reaction (PCR) assay, as described [42 (link)]. DNA was extracted by rapid boiling method, as previously described [43 (link)].