Stress response and survival of Salmonella Enteritidis in single and dual species biofilms with Pseudomonas fluorescens following repeated exposure to quaternary ammonium compounds

Abstract

Biofilms formed on food contact surfaces are frequently exposed to disinfectants at different concentrations. This study was designed to evaluate how S. Enteritidis in single species and dual species biofilms with P. fluorescens respond to quaternary ammonium compounds (QAC) residues on food contact surfaces. The 48 h-biofilms of S. Enteritidis and P. fluorescens in single/dual species were continuously exposed to 20 ppm QAC for 5 days, followed by QAC challenge at 200 ppm and 100 ppm for attached and detached cells, respectively. Biofilm structures were observed by confocal laser scanning microscopy (CLSM) and extracellular polymeric substances (EPS)-related gene expression was also evaluated. Results showed that QAC stress led to one log lower cell counts of S. Enteritidis and P. fluorescens single species biofilms. More cellulose observed by CLSM images and increased transcript levels of cellulose-related genes (csgD, bcsA and ardA) of S. Enteritidis were induced by QAC stress. Nevertheless, high percentage of membrane damaged cells in QAC pre-exposed biofilms might contribute to the increased sensitivity of S. Enteritidis in both attached and detached cells. Previous QAC exposure did not influence S. Enteritidis viable cell counts in dual specie biofilms, in which S. Enteritidis showed strong resistance to QAC with <2 log CFU/cm² reductions. Decreased transcript levels of cellulose-related genes were observed of S. Enteritidis in dual species biofilms, but EPS-related gene expression of P. fluorescens was not affected by single/duals species. The dual species biofilm matrix which has big microcolonies extruding from bottom layers with great amounts of polysaccharides mainly produced by P. fluorescens could possibly protect S. Enteritidis against disinfection. Enhanced survival of S. Enteritidis in dual species biofilms was also found when they were detached from the coupons. Overall, our findings highlight that although repeated exposures to low dose of QAC sensitized S. Enteritidis, the presence of P. fluorescens in dual species biofilms could enhance QAC resistance of S. Enteritidis, probably contributing to survival of S. Enteritidis in food processing plants.

Keywords: Biofilms; Pseudomonas fluorescens; Quaternary ammonium compounds (QAC); Salmonella Enteritidis.