Large-scale phenotypic and genomic analysis of Listeria monocytogenes reveals diversity in the sensitivity to quaternary ammonium compounds but not to peracetic acid

Abstract

Listeria monocytogenes presents a significant concern for the food industry due to its ability to persist in the food processing environment. One of the factors contributing to its persistence is decreased sensitivity to disinfectants. Our objective was to assess the diversity of L. monocytogenes sensitivity to food industry disinfectants by testing the response of 1,671 L. monocytogenes isolates to quaternary ammonium compounds (QACs) and 414 isolates to peracetic acid (PAA) using broth microdilution and growth curve analysis assays, respectively, and to categorize the isolates into sensitive and tolerant. A high phenotype-genotype concordance (95%) regarding tolerance to QACs was obtained by screening the genomes for the presence of QAC tolerance-associated genes bcrABC, emrE, emrC, and qacH. Based on this high concordance, we assessed the QAC genes' dissemination among publicly available L. monocytogenes genomes (n = 39,196). Overall, QAC genes were found in 23% and 28% of the L. monocytogenes collection in this study and in the global data set, respectively. bcrABC and qacH were the most prevalent genes, with bcrABC being the most detected QAC gene in the USA, while qacH dominated in Europe. No significant differences (P > 0.05) in the PAA tolerance were detected among isolates belonging to different lineages, serogroups, clonal complexes, or isolation sources, highlighting limited variation in the L. monocytogenes sensitivity to this disinfectant. The present work represents the largest testing of L. monocytogenes sensitivity to important food industry disinfectants at the phenotypic and genomic level, revealing diversity in the tolerance to QACs while all isolates showed similar sensitivity to PAA.

Importance: Contamination of Listeria monocytogenes within food processing environments is of great concern to the food industry due to challenges in eradicating the isolates once they become established and persistent in the environment. Genetic markers associated with increased tolerance to certain disinfectants have been identified, which alongside other biotic and abiotic factors can favor the persistence of L. monocytogenes in the food production environment. By employing a comprehensive large-scale phenotypic testing and genomic analysis, this study significantly enhances the understanding of the L. monocytogenes tolerance to quaternary ammonium compounds (QACs) and the genetic determinants associated with the increased tolerance. We provide a global overview of the QAC genes prevalence among public L. monocytogenes sequences and their distribution among clonal complexes, isolation sources, and geographical locations. Additionally, our comprehensive screening of the peracetic acid (PAA) sensitivity shows that this disinfectant can be used in the food industry as the lack of variation in sensitivity indicates reliable effect and no apparent possibility for the emergence of tolerance.

Keywords: Listeria monocytogenes; disinfectants; food industry; peracetic acid; quaternary ammonium compounds.

Fig 1

Distribution of the L. monocytogenes isolates by (A) country of isolation, (B) isolation source, and (C) year. In (B), countries with <1% isolates are not included in the graph (Romania [n = 16], UK [n = 10], Turkey [n = 3], Russia [n = 3], Belgium [n = 1], China [n = 1], Finland [n = 1], and France [n = 1]). In (C), 220 isolates with an unknown year of isolation are not shown in the graph.

Fig 2

Mid-rooted maximum likelihood tree constructed from 2.19 Mb core-genome alignment using IQ-TREE with 1,000 ultrafast bootstraps and GTR+G nucleotide substitution model. The color of the branches represents bootstrap support values, from 50 (red) to 100 (blue). Roman numbers represent the L. monocytogenes phylogenetic lineages.

Fig 3

Distribution of the MIC values of (A) benzalkonium chloride, (B) Mida SAN 360 OM (cQAC), and (C) DDAC for the 1,671, 163, and 251 L. monocytogenes isolates tested, respectively. In (A), at MIC = 1.25 mg/L, the distribution of the isolates is as follows: qacH = 4, emrE = 2, and no gene = 2. Dashed lines represent the cut-off values established for this study and divide the isolates into sensitive and tolerant for each disinfectant.

Fig 4

Overall distribution of the percentage change in area under the curve (ΔPAUC) for peracetic acid-induced effect on the growth of 414 L. monocytogenes isolates displaying a normal distribution with a mean ΔPAUC of 7.06% (±7.3 SD).

Fig 5

Peracetic acid effect on ΔPAUC comparisons between L. monocytogenes isolates grouped according to (A) isolation source, (B) phylogenetic lineage, (C) PCR serogroup, and (D) clonal complex. Groups not sharing a letter are significantly (P < 0.05) different. “Other CCs” category includes CCs with ≤5 isolates.

Fig 6

Distribution of the bcrABC, emrC, emrE, and qacH harboring L. monocytogenes isolates, including tolerant isolates with no known QAC gene according to (A) CC and (B) isolation source.

Fig 7

(A) Prevalence of stress resistance genes among the benzalkonium chloride (BC)-tolerant (n = 388) and sensitive (n = 1,283) isolates. (B) Plasmid-harboring BC-sensitive and -tolerant L. monocytogenes isolates divided by isolation source. Asterisks indicate significant differences determined with the Pearson’s chi-squared association test with P < 0.05. Details on the stress resistance gene screening are given in Table S5.

Fig 8

Distribution of bcrABC, emrC, emrE, and qacH genes (A) globally and (B) in Europe in L. monocytogenes sequencing runs deposited in ENA as of 29 April 2021. The pie charts indicate the proportion (rate) of the genes in each of the country in which at least one of the genes was present, and their size reflects the rate of each gene per 1,000 genomes. The pie sizes represent the number of QAC-tolerant L. monocytogenes isolates.