Evaluation of the efficacy of antimicrobial agents to prevent the transfer of Listeria monocytogenes from biofilms to produce or processing surfaces

Field: Immunology and Microbiology (Life Sciences)

Bacteria that are part of a biofilm community are considerably less susceptible to antimicrobial agents than their planktonic counterparts. It is therefore very difficult to eradicate biofilm-associated bacteria from surfaces of fruits and vegetables or harvesting and processing surfaces. Biofilm-associated bacteria surviving treatments with antimicrobials can be released from the biofilm and colonize other surfaces. This research focused on biofilms consisting of either a mixture of six Listeria monocytogenes (Lm) isolates or a mixture of the Lm isolates and four stains isolated from surfaces at a produce processing plant (PPPI). The isolates were identified by 16S rRNA sequencing as belonging to the genera Acinetobacter, Pedobacter, Chryseobacterium and Pantoea. The efficacy of sodium hypochlorite (NaOCl) and peroxyacetic acid (PAA) in preventing Lm released from biofilms to take refuge on fresh produce (spinach) or processing surfaces was studied. Pure Lm and multi-species biofilms were grown on flat bottom 96-well microtiter plates or stainless coupon surfaces and the effectiveness of the antimicrobials dissolved in tap water in preventing the transfer of Lm to polystyrene pegs (sterile and non-sterile surfaces) or fresh produce (spinach) was determined. Complete inactivation of Lm in a mono-species biofilm on Calgary Biofilm Device (CBD) pegs required a 30-min exposure to 50 ppm or a 7.5-min exposure to 100 ppm NaOCl in tap water. Lm in mixed species biofilms were inactivated after a 30 min exposure to 25 ppm or 7.5 min exposure to 250 ppm of NaOCl in tap water. PAA achieved complete inactivation of pure Lm species biofilm at a concentration of 120 ppm and 15 ppm for 7.5 and 30 min, respectively. When present in multispecies biofilms, Lm was inactivated when exposed to 240 ppm for 7.5 mins or 120 ppm for 30 mins. Lm in mixed-species biofilms enjoyed a higher level of protection from antimicrobials than Lm in pure biofilms. Also, side-by-side comparison of the effects of different concentrations of PAA or NaOCl and exposure times indicated that Lm added to existing biofilms was about as sensitive to the anti-microbial as Lm that was part of the biofilm from the start. Transfer of Lm from existing biofilms was prevented by NaOCl at concentrations between 25 and 50 ppm and 50 and 100 ppm for sterile and non-sterile surfaces, respectively. The concentration of PAA that achieved the same effect was between 7.5 and 120 ppm and 60 and 240 ppm for sterile surfaces and non-sterile surfaces, respectively. Prevention of transfer of Lm from a biofilm to spinach was achieved at concentrations of NaOCl lower than 20 ppm; however, even the highest concentration of NaOCl (250 ppm) applied was not sufficient to completely inactivate the residual Lm on the stainless coupons after a 20-min exposure time. The information gathered from this study highlights the need of produce processors to make every effort to prevent the development of Lm-containing biofilms and to carefully monitor the concentration of the antimicrobials used throughout the wash phase.