The frequency of SCVs

is defined as the number of SCVs per total CFU counts on antibiotic-free TSA. The pinpoint colonies detected by this gentamicin-plate method were confirmed to be SCVs by streaking several of them on TSA plates (See Additional file 3). We have also evaluated the auxotrophism (as described below) of several HQNO-induced SCVs generated from strains CF1A-L and CF07-L in order to further validate the ability of this technique to detect typical SCVs (see Additional file 4). Antibiotic susceptibility The minimal inhibitory concentrations (MICs) of gentamicin for all strains were determined by a broth microdilution technique, following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) guidelines Ganetespib manufacturer [66], except that the incubation period was extended to 48 h and that the medium used was BHI in order to allow SCVs to reach maximal growth. Auxotrophism of SCVs In the context of SCVs, auxotrophism is defined as the requirement of specific compounds in order to regain a normal growth phenotype [41]. An agar diffusion method was used to characterize the auxotrophism of SCVs using hemin or menadione (10 μg each/well) on an inoculated Mueller-Hinton agar (MHA) plate. Thymidine at 1.5 μg/well was also tested as previously described [67]. Auxotrophy for specific supplements was detected

by a zone of normal growth surrounding the well after 18 h of incubation at 35°C. The photography of the Additional file 5 shows the normal growth of NewbouldhemB SHP099 in proximity of a well loaded with

hemin as an example of a positive auxotrophism result. Preparation of supernatants from P. aeruginosa and E. coli strains Overnight cultures were used to inoculate TSB at a dilution of 1:100. Cultures were then incubated 20 h at 35°C/225 RPM before collecting the culture supernatants by Momelotinib in vitro centrifugation. Similar culture conditions were previously Phospholipase D1 shown to allow maximal production of HQNO by P. aeruginosa PAO1 [68]. The supernatants were then filter-sterilized using 0.22 μ pore size (Millipore, MA, USA) and used immediately. The sterility of the supernatants was confirmed by plating samples on TSA plate. Biofilm formation For studying the effect of HQNO on biofilm production by S. aureus, three colonies grown on blood agar plates were used to inoculate BHI broths containing 0.25% glucose with or without 10 μg/ml of HQNO and cultures were incubated for 18 h. These cultures were used to adjust an appropriate volume of BHI-0.25% glucose to 0.5 Mcfarland for transfer into wells of a flat-bottom polystyrene microtiter plate containing half volume of the same medium with or without HQNO (final concentration 10 μg/ml). For experiments evaluating the effect of culture supernatants from P. aeruginosa and E. coli on S. aureus biofilm production, a S. aureus 0.5 Mcfarland suspension was prepared in BHI-0.