Effect of Sub-Lethal Concentrations of Biocides on the Structural Parameters and Viability of the Biofilms Formed by Salmonella Typhimurium.

Abstract

This study aimed to investigate the effect of sub-minimum inhibitory concentrations (sub-MICs) of three food-grade biocides (benzalkonium chloride -BZK-, trisodium phosphate -TSP-, and sodium hypochlorite -SHY-) on Salmonella biofilms. The structural parameters and bacterial viability of the biofilms formed by a S. Typhimurium isolate from poultry was investigated by means of confocal laser scanning microscopy after staining with SYTO9 and propidium iodide. The MIC values for Salmonella cells before exposure to subinhibitory concentrations of biocides were 8.0 μg/mL (BZK), 18.0 mg/mL (TSP), and 6.0 mg/mL (SHY). The cultures exhibited a stable acquired tolerance to BZK and SHY. The maximum concentrations of biocides that allowed bacterial growth after several passages through gradually higher concentrations of such compounds were 30.4 μg/mL (BZK) and 10.1 mg/mL (SHY). The architecture and viability of S. Typhimurium biofilms varied in response to sub-MICs of different biocides. Previous adaptation to SHY enhanced (p < 0.001) biofilm formation (average biovolume in the observed field -14,161 μm2-: 139,856.15 ± 155,213.27 μm3) relative to unexposed cells (53,779.05 ± 55,535.62 μm3) and cells previously exposed to BZK (58,216.97 ± 58,644.45 μm3) or TSP (30,052.13 ± 28,290.56 μm3). This was particularly marked when biofilm was grown in the absence of biocides or in the presence of sub-MICs of SHY. The highest percentage of dead cells was shown by biofilms formed by cultures previously exposed to TSP relative to the other conditions tested (34.08% ± 13.74% vs. 23.70% ± 16.16%; p < 0.001). The importance of maintaining higher than MICs of SHY during sanitizing procedures to fight foodborne infections by Salmonella biofilms is highlighted.