Interaction of Pseudomonas aeruginosa and Staphylococcus aureus with Listeria innocua in dual species biofilms and inactivation following disinfectant treatments

Abstract

Biofilms pose a serious hygienic concern in the food processing environment due to the strong antimicrobial tolerance and thus higher risk of food contamination. The aim of this study was to investigate dual species biofilm formation of P. aeruginosa ATCC 27853 or S. aureus FI 2 with L. innocua FI 2 and biofilm inactivation using QAC-, tertiary alkyl amine-, and chlorine-based disinfectants; the effect of biofilm maturity and comparison with single species biofilms are reported. We used epifluorescence microscopy (EFM) coupled with fluorescence in situ hybridization (FISH) and LIVE/DEAD staining to evaluate species contribution and cell inactivation; cells were prepared using the LabTek Chamber Slide System. In dual species biofilms during 72-h incubation time, P. aeruginosa produced a higher and S. aureus slightly lower cell numbers than the companion species of L. innocua. In dual species biofilm inactivation experiments, P. aeruginosa was more resistant to disinfectant treatment of 24-h compared to 72-h; however, S. aureus was more resistant at 72-h compared to 24-h. In addition, P. aeruginosa was more resistant to tertiary alkyl amine- and chlorine-based disinfectants and S. aureus was more resistant to all three disinfectants in dual species biofilms compared to single species biofilms. In the LabTek™ experiment, when nutrients were repeatedly provided, FISH showed almost equal contribution of P. aeruginosa and L. innocua into the biofilm as well as a predominance of S. aureus when grown with L. innocua. Regardless of biofilm composition, the LIVE/DEAD staining revealed that the majority of cells had damaged membranes after the treatment with the QAC-based disinfectant, while approximately half of the cells remained intact following the treatment with the tertiary alkyl amine-based disinfectant. As for chlorine, cells in P. aeruginosa-L. innocua biofilm were mostly dead whereas cells in S. aureus-L. innocua biofilm were largely viable, and thus were protected under the applied conditions. This study shows that dual species biofilms with foodborne pathogens are more resistant to disinfectant treatment than their single species counterparts and that biofilm maturity as well as environmental conditions, i.e. access to nutrients, play an important role in their response to the treatments tested.