3%). The remaining were from colonization (C; 13.3%), pneumonia (P; 6.7%), skin/soft tissue infections (SSTI; 5%), urinary tract infections (UTI; 3.3%) and prosthesis fragment (PF; 1.7%). The infection sites had not been reported for 4 isolates. The agr-knockout MNY474 (Δagr::tetM) and the rnaIII-trans-complemented mutant CMNY474 (Δagr::tetM, pbla-rnaIII) were previously constructed from the clinical S. aureus isolate NY474 [27].
BMB9393 (ST239-SCCmecIII) was used as positive control for biofilm and gene expression experiments [27]. The S. aureus RN4220 and RN6390B, a gift from Richard Novick (New York University), were used for hemolytic activity and gene expression analyses; respectively. This study was approved (#1055/09) by the Human Research Ethics Committee from Federal University of Rio de Janeiro, RJ, Brazil. Minimal inhibitory Etomoxir concentration (MIC) Oxacillin MIC was determined using Müller Hinton plates and performed in accordance with the Clinical Laboratories Standards Institutes (CLSI) guidelines [50]. In vitro biofilm assay For all 60 isolates, biofilm was tested using 96-well inert polystyrene microtiter plates buy Batimastat (Nunclon; Nunc A/S, Roskilde, Denmark) as previously described [28]. The biofilm unit (BU) was defined as indicated by Amaral et al. [14] and the isolates were classified as non-producers (BU≤0.230), weak (BU>0.230
and ≤0.460), moderate (BU>0.460 and ≤0.920) or strong producers (BU>0.920), as suggested [14]. For 19 isolates, biofilm assays were also carried out on surfaces covered with human fibronectin Aspartate (Merck; Darmstadt, Germany) as previously described [28]. In some experiments, before treatment with crystal violet, the biofilm was treated with sodium metaperiodate (10mM/well; Sigma; St. Louis, MO, USA) or proteinase K (6U/well, Invitrogen; Carlsbad, California, EUA) [27]. Confocal
laser scanning microscopy (CLSM) was SBI-0206965 datasheet employed to record and contrast structural images of the biofilm as described [28]. eDNA was quantified in biofilm supernatants using Qubit® 2.0 Fluorometer (Invitrogen; Eugene, Oregon, USA), after ethanol precipitation. For some experiments, biofilms were formed in the presence of DNase I (28U/well or 56U/well Invitrogen; Carlsbad, California, EUA). Animal model A pair of isolates showing differential agr expression (08–008, agr-dysfunctional, obtained from BSI and 96/05, agr-functional, from CT) was used. The mouse subcutaneous catheter implant model was described in detail by Ferreira et al. [28]. Briefly, two intravenous polyurethane catheter segments (C-UDLM-953J model; Cook Medical, Bloominaton, USA) were implanted in the back of each anesthetized young-adult BALB/c male mice. Infection was induced 24 h after the implantation procedure by injecting a mid-exponential growth phase culture (106 CFU/10 µL) into the lumen of the implanted catheter segment.