Biofilm Sanitizer Tolerance of Vermont Dairy Listeria monocytogenes

Listeria monocytogenes is a foodborne pathogen found in biofilms on surfaces and equipment in the food processing environment. Sodium hypochlorite (SH) and quaternary ammonium compounds (QAC) are readily available and commonly used sanitizers. However, due to the structure and additional organic material produced in a biofilm, killing bacteria within the biofilm may be a challenge for one or both of these sanitizers. The objective of this work was to determine if immature and mature biofilms from L. monocytogenes isolated from Vermont artisan dairy environments were more tolerant to QAC and SH compared to planktonic cultures’ tolerance. To determine sanitizer minimum inhibitory concentration (MIC), cultures were incubated statically in 1x or 1/20x Brain Heart Infusion broth (BHI) in polystyrene microtiter plates for 24 hours at room temperature (22°C) with serial dilutions of sanitizer. Sanitizer efficacy on biofilms was determined by growing isolates on one cm stainless steel coupons in 1x or 1/20x BHI to simulate nutrient rich and poor conditions on a surface commonly seen in food processing environments. Coupons were incubated statically for 1, 3, or 10 days. Media was replaced every 48 hours to prevent nutrient depletion. After incubation, coupons were rinsed 3 times with phosphate buffered saline and placed into 0, 50, 100, or 200ppm SH or QAC for the manufacturer recommended contact time. Sanitizer was neutralized and adherent cells were removed by vortexing with beads. Cell suspensions were diluted and plated, then counted via spot plating on BHI agar. Significant differences for biofilm survival were assessed using Analysis of Variance in R (v.4.0.4). MICs for isolates grown in nutrient poor (1/20X BHI) conditions were lower than nutrient rich conditions (1X BHI) for both sanitizers. All isolates’ biofilms reached ~6-8 log10 CFU/coupon on stainless steel. Reductions from different QAC concentrations differed (padj \u3c0.05) in 1/20x BHI but were not significantly different in 1x BHI. In both biofilm growth conditions SH was more effective at 200 ppm than 50 ppm (padj \u3c 0.05). Biofilms from both persistent and transient L. monocytogenes environmental isolates from Vermont dairies are resistant to working concentrations of QAC sanitizer, but sodium hypochlorite bleach more adequately reduces L. monocytogenes biofilm on stainless steel