Mature Biofilms of Listeria Monocytogenes from VT Dairies Are Resistant to QACs

Listeria monocytogenes (Lm) is a foodborne pathogen that commonly persists in food processing environments. Under certain circumstances, Lm is resistant to quaternary ammonium compounds (QAC), sanitizers used in dairy operations. Our goal was to assess a set of Lm strains isolated from Vermont dairies and determine QAC tolerance in broth, biofilm forming capability, and the response of their mature biofilms to QACs in varying nutrient conditions. Sanitizer tolerance: Lm isolates were inoculated into 1x or 1/20x BHI in 96-well microtitre plates and incubated at 22°C for 24hr before measuring optical density (OD600) to determine QAC minimum inhibitory concentrations (MIC). Crystal violet assay: isolates were grown in 1x and 1/20x BHI at 22°C for 1, 3, and 5 days; they were then washed, stained, and OD600 measured to determine biofilm formation. Mature biofilm QAC MIC: 1cm stainless steel coupons were placed in Petri dishes containing BHI and inoculated; coupons incubated statically at 22°C for 10 days then were rinsed 3 times with PBS and placed in 0, 50, 100, or 200ppm QAC for 1 minute. Dey-Engley broth was used to neutralize QAC and remaining cells were removed for serial dilution and enumeration. Significant differences for MIC, attachment, and biofilm survival were assessed using Analysis of Variance in R (v. 3.6.1). QAC MIC for Lm isolates in broth was 3.25-25ppm. Biofilm formation in microtitre plates varied, but on coupons all isolates grew to ~5-8 log10 CFU/coupon by day 10. In nutrient poor conditions (1/20x BHI), log10 CFU/coupon decreased significantly between 50 and 200ppm, and 100 and 200ppm (p \u3c 0.05). In nutrient rich conditions (1x BHI), there was no significant decrease in log10 CFU/coupon across all isolates and QAC concentrations. Lm strains isolated from Vermont dairies are susceptible to QACs as planktonic cells, but form biofilms that are resistant to QAC sanitizers