Biofilm formation assay Overnight cultures in TSB were corrected with fresh TSB to an OD550 of 1.00 (corresponding to about 1 × 109 CFU/ml). Two-hundred microliters of 1:100 diluted inoculum were dispensed to each well of a sterile flat-bottom polystyrene tissue culture 96-wells microtiter (Iwaki, Bibby srl; Milan, Italy) and incubated at 37°C for 24 h. Biofilm formation by ENV strains was also assessed at 25°C. Non-adherent cells were removed NU7026 order by being washed three times in sterile PBS (pH 7.3; Sigma-Aldrich Co; Milan, Italy), and biofilm biomass was then measured by crystal violet assay. JQ-EZ-05 Briefly, biofilm samples were fixed for

1 h at 60°C, stained for 5 min at RT with 200 μl Hucker-modified crystal violet, then rinsed in standing water and allowed to dry. Biofilm samples were estained with 250 μl of 33% glacial acetic acid for 15 min, and the optical density at 492 nm (OD492) was read. Considering a low cut-off (ODc) represented by 3×SD above the mean OD of control wells, strains were classified into the following categories: no biofilm producer (OD ≤ ODc), weak biofilm

producer (ODc < OD ≤ 2 × ODc), moderate biofilm producer (2 × ODc < OD ≤ 4 × ODc), and strong biofilm producer (4 × ODc < OD) [53]. Measurement of growth rate Two-hundred microliters of the 1:100 diluted standardized inoculum were dispensed in each well of a microtiter plate, and OD570 readings were taken every 15 min Luminespib price for a total time of 15 h by a microplate reader (SpectraMax 190; Molecular Devices

Inc.; Sunnyvale, CA, USA). Considering the exponential growth phase selected on a graph of ln OD570 versus time, mean generation Unoprostone time (MGT) was calculated as follows: MGT = ln2/μ, where μ (growth rate) = (lnOD t – lnODt0)/t. Swimming and twitching motilities Motility assays were performed according to the method described by Rashid et al. [54], with some modifications. i) Swimming assay: a single colony from an overnight MHA-growth was inoculated at the surface of swimming agar (10 g/liter tryptone, 5 g/liter NaCl, 3 g/liter agar); after inoculation, the plates were then wrapped to prevent dehydration and incubated at 37°C for 24 h, and results were expressed as diameter (mm) of growth zone. ii) Twitching motility: a single colony from an overnight MHA-growth was inoculated, by using an inoculation needle, to the bottom of the Petri dish plate containing twitching agar (1% TSB solidified with 1% agar); after incubation at 37°C for 72 h, agar was removed and the zone of motility at the agar/Petri dish interface was stained with crystal violet and measured in millimeters. Sensitivity to oxidative stress Assays were carried out by a disk assay adapted by Hassett et al. [55]. Briefly, 100-μl aliquots from TSB cultures in mid-log or stationary phases of growth were uniformly spread on TSA plates containing 2% agar. Sterile filter paper 7-mm diameter disks (Oxoid) were placed on TSA surface, and the disks were spotted, in triplicate on each plate, with 10 μl of 1.