Assessment of tolerance development in L. monocytogenes - E. coli dual-species biofilms to pronase and benzalkonium chloride treatments

Tolerance against disinfectant compounds is a common phenomenon that occurs among sessile bacterial communities present in industrial settings. As a consequence, the development of any new and efficient disinfection strategy should be evaluated respecting to its capacity to develop resistance for a target before application. In the present study, the development of tolerance in a Listeria monocytogenes – Escherichia coli mixed-species biofilm grown on stainless steel coupons was assessed following the exposure to sublethal concentrations of a pronase (PRN) solution followed by a benzalkonium chloride (BAC) solution. Three different approaches were followed: (a) two sequential exposures at 24-hour intervals with growing PRN-BAC concentrations, (b) same exposure interval using same PRN-BAC concentrations and (c) two 24-hour exposures with growing concentrations of PRN-BAC with 24-hour incubation intervals with fresh medium in between. Samples were subsequently treated with a 700 UI/l PRN solution followed by a 50 ppm BAC solution and remaining cells were determined by means of plate count and LIVE/DEAD staining followed by fluorescence microscopy image acquisition and analysis using Metamorph MM AF. No tolerance development was observed among biofilm samples tested presenting a different behaviour depending on the strain when facing the final treatment. In all cases L. monocytogenes was more affected to the treatment used whereas E. coli did not present any significant difference in the amount of the remaining attached cells recovered after treatments. Fluorescence microscopy image analysis demonstrated a severe alteration of the biofilm architecture not detected in plate count assay and dependent of the PRN-BAC treatment applied. In the light of these results, the combination of enzymes and chemical compounds in order to remove attached communities on stainless steel while preventing the development of tolerance to those treatments seems to be an attractive alternative to be considered to apply in industrial settings related with food product manufacturing.