3) and 0.05 mL of a solution containing 2X the EtBr concentration previously selected and mTOR inhibitor 2X the EI concentration to be tested (final concentrations of

TZ: 12.5 mg/L, CPZ: 25 mg/L, VER: 200 mg/L, RES: 20 mg/L). All assays included control tubes containing only the isolate (0.05 mL of cellular suspension at OD600 nm of 0.6 plus 0.05 mL of 1X PBS) and only the EtBr concentration to be tested (0.05 mL of 2X EtBr stock solution plus 0.05 mL of 1X PBS). The assays were then run in a Rotor-Gene 3000™ at 37°C, and the fluorescence of EtBr was measured (530/585 nm) at the end of every cycle of 60 seconds, for a total period of 60 minutes. For the efflux assays, EtBr-loaded cells were prepared by incubating a cellular suspension with

an OD600 nm of 0.3 with either 0.25 or 1 mg/L EtBr for EtBrCW-negative or positive cultures, respectively and 200 mg/L VER at 25°C for 60 minutes. After EtBr accumulation, cells were collected by centrifugation and re-suspended in 1X PBS to an OD600 nm of 0.6. Several parallel assays were then run in 0.1 mL final volume corresponding to 0.05 mL of the EtBr loaded cells (final OD600 nm of 0.3) incubated with 0.05 mL of (1) PBS 1X only; (2) glucose 0.8% only (final concentration of 0.4%); (3) 2X VER only (final concentration of 200 mg/L); (4) glucose BIBW2992 0.8% (final concentration of 0.4%) plus 2X VER (final concentration of 200 mg/L). These efflux assays were conducted in the Rotor-Gene 3000™ at 37°C, and the fluorescence of EtBr was measured (530/585 nm) at the end of every cycle of 10 seconds, for a total period of 10 minutes. The raw data obtained was then normalized against data obtained from non-effluxing cells (cells from the control tube with only 200 mg/L VER), at each point, considering that these correspond to the maximum fluorescence values that can be obtained during the assay. The relative fluorescence thus corresponds to the ratio of fluorescence that remains per

unit of time, relatively to the EtBr-loaded cells. Macrorestriction analysis Isolates were typed by pulsed-field gel electrophoresis (PFGE) analysis, using well-established protocols. Briefly, agarose disks containing intact chromosomal DNA were prepared as previously described [29] and restricted with SmaI (New England Biolabs, Ipswich, Anacetrapib MA, USA), according to the manufacturer’s recommendations. Restriction fragments were then resolved by PFGE, which was carried out in a contour-clamped homogeneous electric field apparatus (CHEF-DRIII, Bio-Rad), as previously described [29]. Lambda ladder DNA (New England Biolabs) was used as molecular weight marker. PFGE types were defined according to the criteria of Tenover et al. [17]. Preparation of chromosomal DNA Genomic DNA was extracted with the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), with an additional step of 30 Rabusertib concentration minutes digestion with lysostaphin (Sigma) (200 mg/L) prior to extraction.