Each strain was plated on the selective and non-selective LB agar plates and incubated at 37°C. Rifampicin selecting concentrations were 2 and 20 mg/L for the reference strain, and 20 mg/L for the RIF-R MRSA strains. In these experimental Ku-0059436 manufacturer conditions OD620 = 0.125 corresponded

to 5 × 107 cfu/ml. The equivalent to 107, 108 and 109 cfu were spread on selective plates, and appropriated diluted samples were plated on non-selective plates. After 24 h to 36 h, colonies that grew on selective and non-selective plates were counted and mutation frequencies were calculated. Three independent experiments were performed to ensure reproducibility. Molecular typing Pulsed Field Gel Electrophoresis (PFGE) was performed after SmaI restriction of chromosomal DNA according to Chung et al. [20]. Pulses run from 5 s to 15 s for 10 h for block 1, and from 15 s to 60 s for 13 h for block 2 [21]. Isolates with PFGE patterns differing in four or less restriction fragments were considered to be subtypes of a Staurosporine purchase single genotype. Isolates with differences in more than four fragments were ascribed to distinct genotypes [22]. SCCmec typing Molecular typing based on the amplification

of the mobile region mec was performed according to previously described procedures [23, 24]. Control strains for SCCmec typing were: ATCCBAA44 (SCCmec type I) [18, 19], ATCCBAA-41 (SCCmec type II) [19], ATCCBAA-39 (SCCmec type III) [19] and HGSA60 (SCCmec type IV-A) [24]. Multilocus sequence typing (MLST). Analysis of the seven Adenosine triphosphate housekeeping gene sequences was performed according to previously described procedures http://​saureus.​mlst.​net/​[25]. spa typing The polymorphic region of protein A was studied according to previously described procedures at http://​spa.​ridom.​de/​[26]. The interest region was amplified with primers spa-1113f (5′-TAA AGA CGA TCC TTC GGT GAG C-3′) and spa-1514r (5′-CAG CAG TAG TGC CGT TTG CTT-3′). Results Rifampicin resistance levels and associated rpoB mutations The majority (n = 104, 96%) of the 108 RIF-R MRSA isolates, showed rifampicin MICs between 2 and

4 mg/L. Two isolates had rifampicin MICs of 128 mg/L and the remaining two had MICs ≥ 256 mg/L. Corresponding E-test and disk diffusion results are shown in table 1. On the basis of these results and following other authors’ categorisation [13, 17, 27] the strains were classified into categories of rifampicin susceptible (MICs, ≤ 0.5 mg/L), low-level rifampicin resistance (MICs, 1 to 4 mg/L), and high-level rifampicin resistance (MICs, ≥ 8 mg/L). Interestingly, 20 strains with rifampicin MICs of 2 mg/L showed inhibition zones between 20 and 23 mm, borderline to the susceptible CLSI breakpoint (inhibition zones ≥ 20 mm). The five RIF-S MRSA isolates, with the same multi-resistance pattern, had rifampicin MICs of 0.012 mg/L and inhibition zones > 30 mm.