Figure 2.

(a–c) In vitro planktonic MSSA susceptibility to free antibiotics assessment through broth microdilution method followed by turbidity measurement (OD_{570 nm}), after 24 h of incubation. The negative control corresponds to planktonic MSSA in MHB (CTR). Antibiotic concentrations of (a) RFB (0.002–0.200 µg/mL), (b) VCM (0.195–6.250 µg/mL) and (c) LEV (0.004–1.000 µg/mL); (d–i) MSSA biofilm susceptibility to free antibiotics was evaluated through the broth microdilution method, performed in mature biofilms (24 h old), followed by determination of (d–f) viable bacterial cells (MTT assay) and by (g–i) biofilm biomass quantification (CV method), after 24 h of incubation. Bacterial cell viability (%) in biofilm structure after incubation (d) with RFB (0.001–10.000 µg/mL), (e) with VCM (6.250–200.000 µg/mL) and (f) with LEV (0.098–100.000 µg/mL). Biofilm biomass (%) present after incubation (g) with RFB (0.001–10.000 µg/mL), (h) with VCM (6.250–200.000 µg/mL) and (i) with LEV (0.098–100.000 µg/mL). (j,k) Viable bacteria recovered from mature biofilm structure after 24 h of incubation with the selected antibiotics, RFB, VCM and LEV, were determined by CFU counts. Statistical comparisons were assayed by one-way ANOVA (Tukey’s multiple comparisons test) analysis of variance compared with control group (CTR, biofilm in TSB 0.25%) and between antibiotic groups. (j) Biofilm incubated with 0.2 µg/mL of each antibiotic. (k) Biofilm incubated with 0.8 µg/mL of each antibiotic. Results are expressed as mean ± SD of at least three independent experiments.