Sensitivity of 240 Listeria monocytogenes isolates to common industrial biocides is more dependent on the presence of residual organic matter or biofilm than on genetic determinants

Abstract

Listeria monocytogenes continues to be a problem in ready-to-eat foods. Tolerance and adaptation to biocides have been proposed to contribute to the persistence of L. monocytogenes in the food processing industry. This study aimed to determine if the sensitivity to common biocides varies among 240 L. monocytogenes isolates. The impact of residual organic matter (soils), biofilm, and ability to adapt to biocides was also evaluated. Food and food processing related isolates of L. monocytogenes (n = 240) were whole genome sequenced and assigned to sequence types (n = 53) and clonal complexes (n = 32). Isolates were screened for genetic elements associated with biocide tolerance and tested for their minimum inhibitory concentration (MIC) to benzalkonium chloride (BC), peracetic acid (PAA), sodium hypochlorite (SH) and ethanol (ET). Biocide tolerance under soiled conditions, in biofilm and in a broth suspension test (BST) was further tested for 19 representative isolates. Adaptation to sub-MICs of BC, PAA or SH (18 days, 15 °C) was tested for 15 isolates. MIC for PAA (62 mg/L) showed no variation, while for SH (47–94 mg/L) and ET (4.7–9.4% v/v) values differed 2 × among isolates. For BC, an 8 × difference (0.3–2.5 mg/L) was observed. Eighty-seven of 88 BC tolerant isolates (MIC of 2.5 mg/L) harbored known BC tolerance genes. Soiled conditions increased MIC-values 8–33 × for BC and 8 × for SH but not for PAA and ET. Biofilm increased minimum bactericidal concentrations 4–8 × for BC but not for PAA and SH. Survival of isolates with BC tolerance genes was not significantly (p > 0.05) improved in BSTs with PAA, SH or BC. Lineage I isolates (n = 7 of 19) showed significantly (p < 0.05) better survival than lineage II isolates (n = 12) when exposed to PAA (100 mg/L) in BSTs. Adaptation to BC (8 × MIC increase) occurred exclusively for originally sensitive isolates, while no adaptation to SH and PAA were observed. In conclusion, L. monocytogenes isolates showed limited variation in sensitivity to biocides in MIC assays. However, lineage I isolates were less sensitive to PAA than lineage II isolates in BSTs. The efficacy of SH and BC were affected by residual organic matter and/or biofilm, demonstrating the need for proper cleaning prior to biocide use with PAA being less perturbed.