Fig. 5.

Biofilm-associated cross-resistance of gentamicin (GM)-resistant strain to silver. A Inhibition of biofilm growth of P. aeruginosa GM-resistant strain by NAg and Ag⁺ (24 h exposure, 37 °C). B Eradication of established biofilm of GM-resistant strain by NAg and Ag⁺ (24 h exposure, 37 °C). Biofilm biomass is expressed as % relative to the cell-only control (no antimicrobial agent, 0 µg/mL). Error bars represent SEM (standard error of the mean) of three biological replicates (experiments with independent bacterial inocula from three isolates and different antimicrobial preparations, each with three technical replicates). *Indicates statistically significant inhibition and eradication effects with p > 0.05 (not significant, ns), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****), relative to the cell-only control. C Penetration of NAg particle (aggregates) in wild-type and GM-resistant P. aeruginosa biofilms. Biofilm biomass (green) were exposed to NAg at 20 × MIC dosage (3 h, 37 °C) and sectional z-stack depth images were acquired using multiphoton microscope. The second harmonic generation and hyper Rayleigh scattering signals of NAg particles (pink) were used to detect their presence in the biofilms. Scale bar = 20 µm. Refer to Additional file 1: Figure S4 for the NAg and Ag⁺ biofilm inhibition and eradication studies, as well as the multiphoton biofilm-nanoparticle imaging of the GM-resistant strain obtained from the second biological replicate of the passaging experiment