Nisin penetrates Staphylococcus aureus biofilms but shows differences in killing effects against sessile and planktonic cells.

Abstract

Biofilms may restrict antimicrobial penetration and contribute to the recalcitrance of bacterial infections. In this work, we investigated the penetration of nisin into S. aureus biofilms and compared the susceptibility of S. aureus planktonic and sessile cells to this lantibiotic. Biofilms were grown under continuous flow in CDC reactors and calcein fluorescence was used to monitor the effect of nisin on the cytoplasmic membrane of S. aureus cells. Confocal scanning laser microscopy (CLSM) showed that calcein was lost within approximately 20 min in CDC biofilms, demonstrating that nisin penetrated to the bottom of the biofilm and caused membrane permeabilization. Viability analysis using PI staining showed that nisin was bactericidal against S. aureus sessile cells. Time-kill assays were performed against S. aureus in the following conditions: homogenized exponential planktonic (HEP), homogenized stationary planktonic (HSP), homogenized CDC biofilm (HB) and intact CDC biofilm (IB). The mean viability reduction of HEP and HSP were 6.71 and 1.64 log CFU.ml-1, respectively, confirming that stationary S. aureus cells were much less susceptible than exponential cells. The HB and IB treatments showed mean viability reductions of 1.25 and 0.50 log CFU.ml-1, respectively. Nisin activity against S. aureus was not limited by its ability to penetrate the bacterial biofilm, but the killing efficacy of the antimicrobial peptide was reduced by the physiological status of the biofilm-grown cells.\n\nImportanceBiofilms represent a major problem to control microorganisms in industrial environments and medical devices. We developed a direct real-time microscopic visualization technique to demonstrate experimentally that the antimicrobial peptide nisin is able to penetrate S. aureus biofilms. Our results confirmed that nisin caused membrane permeabilization of sessile bacteria and revealed qualitative agreement between viability loss and membrane integrity loss. This approach could improve the evaluation of antibacterial susceptibility breakpoints when testing the efficacy of standard and novel antimicrobials.