The persistence of certain strains of Listeria monocytogenes, even after the food processing environment has been cleaned and disinfected, suggests that this may be related to phenomena that reduce the concentration of the disinfectants to subinhibitory levels. This includes (i) the existence of environmental niches or reservoirs that are difficult for disinfectants to reach, (ii) microorganisms that form biofilms and create microenvironments in which adequate concentrations of disinfectants cannot be attained, and (iii) the acquisition of resistance mechanisms in L. monocytogenes, including those that lead to a reduction in the intracellular concentration of the disinfectants. The only available data with regard to the resistance of L. monocytogenes to disinfectants applied in food production environments refer to genotypic resistance to quaternary ammonium compounds (QACs). Although there are several well-characterized efflux pumps that confer resistance to QACs, it is a low-level resistance that does not generate resistance to QACs at the concentrations applied in the food industry. However, dilution in the environment and biodegradation result in QAC concentration gradients. As a result, the microorganisms are frequently exposed to subinhibitory concentrations of QACs. Therefore, the low-level resistance to QACs in L. monocytogenes may contribute to its environmental adaptation and persistence. In fact, in certain cases, the relationship between low-level resistance and the environmental persistence of L. monocytogenes in different food production chains has been previously established. The resistant strains would have survival advantages in these environments over sensitive strains, such as the ability to form biofilms in the presence of increased biocide concentrations.

The aim of this study was to investigate the basis of the putative persistence of Listeria monocytogenes in a new industrial facility dedicated to the processing of ready-to-eat (RTE) Iberian pork products. Quaternary ammonium compounds, which included benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance to disinfectants was associated with persistence. Of the 14 isolates obtained from product contact and non-product contact surfaces, only five different pulsed-field gel electrophoresis (PFGE) types were identified during the 27-month study period. Two of these PFGE types (S1 and S10-1) were previously identified to be persistent and BAC-resistant (BAC ^r ) strains in a geographically separate slaughterhouse belonging to the same company. The remaining three PFGE types, which were first identified in this study, were also BAC ^r . Whole-genome sequencing and in silico multilocus sequence typing (MLST) analysis of five BAC ^r isolates of the different PFGE types identified in this study showed that the isolate of the S1 PFGE type belonged to MLST sequence type 31 (ST31), a low-virulence type characterized by mutations in the inlA and prfA genes. The isolates of the remaining four PFGE types were found to belong to MLST ST121, a persistent type that has been isolated in several countries. The ST121 strains contained the BAC resistance transposon Tn 6188 . The disinfection-resistant L. monocytogenes population in this RTE pork product plant comprised two distinct genotypes with different multidrug resistance phenotypes. This work offers insight into the L. monocytogenes subtypes associated with persistence in food processing environments.

Contamination routes of Listeria monocytogenes were examined for 3 years in an Iberian pork-processing plant that produced high-quality ready-to-eat meat products. Molecular subtypes of L. monocytogenes were determined by polymerase chain reaction–based serotyping and pulsed-field gel electrophoresis (PFGE) restriction analysis. A total of 541 L. monocytogenes isolates were recovered from the environment and equipment (n = 165), carcasses (n = 28), raw products (n = 321), and dry-cured products (n = 27). Only 29 different PFGE types were identified, 3 of which were repeatedly found to be persistent types and accounted for 73% of the isolates. One PFGE type dominated (45% of the isolates) and was mostly recovered from intermediate manufactured products and the environment of the manufacturing area. L. monocytogenes persistence appeared strongly linked to the manufacture of products and not to its sustained entrance with the raw material. Some clones were found to survive in the manufacturing area for 3 years. Controlling the contamination of raw ingredients, improving the compartmentalization, and changing the cleaning protocols resulted in reduced prevalence rates of L. monocytogenes on products; two persistent PFGE types were eliminated from the processing plant, although eradication of other adapted strains has not been achieved.

This study was intended to investigate the effect of contact with concentrations close to the minimum inhibitory concentration (MIC) (0.5, 1, and 1.5 MIC; MIC of planktonic cells was determined using a microdilution broth method) of sodium hypochlorite (SHY) or benzalkonium chloride (BZK) during the process of formation of biofilm (24 h), upon the architecture and viability of the biofilms formed by four L. monocytogenes isolates of molecular serotype 1/2a: S2-1 (BZK-susceptible strain; MIC _BZK = 3.0 ppm), S2-2 (BZK-resistant strain qacH positive; MIC _BZK = 13 ppm), CDL 69 (BZK-resistant strain bcrABC positive; MIC _BZK = 10 ppm), and S2 ^BAC (BZK-resistant laboratory mutant of S2-1, with multidrug resistance phenotype; MIC _BZK = 9 ppm). Images were examined through confocal laser scanning microscopy after staining with SYTO 9 and Propidium Iodide. Biovolume values in the observation field (14,161 μm ² ) in the absence of biocides ranged from 103,928.3 ± 6,730.2 μm ³ (S2 ^BAC ) to 276,030.9 ± 42,291.9 μm ³ (S2-1). Exposure to SHY at 0.5 MIC reduced ( p < 0.05) the biovolume of biofilms formed by S2-1 and S2 ^BAC and did not modify ( p > 0.05) the biovolume of biofilms by S2-2 and CDL 69. Exposure to sub-MICs of BZK decreased ( p < 0.05; S2-1) or enhanced ( p < 0.05; S2-2, CDL 69 and S2 ^BAC ) biofilm development. Exposure to biocides at 1 or 1.5 MIC inhibited biofilm formation. This study provides clear evidence that BZK at sub-MICs can enhance the biofilm-forming ability of BZK-resistant L. monocytogenes strains. Because biofilms contribute to the persistence of bacteria throughout the food chain and represent a major source of food contamination, our findings suggest the importance of avoiding sub-MICs of disinfectants in food-handling environments.