Biocide-Tolerant Listeria monocytogenes Isolates from German Food Production Plants Do Not Show Cross-Resistance to Clinically Relevant Antibiotics

Abstract

Contamination of food during processing is recognized as a main transmission route of Listeria monocytogenes To prevent microbial contamination, biocides are widely applied as disinfectants in food processing plants. However, there are concerns about the development of antimicrobial resistance in foodborne pathogens due to widespread biocide usage. In our study, 93 L. monocytogenes isolates from German food production facilities were (i) tested for biocide and antibiotic susceptibility using broth microdilution assays, (ii) analyzed for links between reduced biocide susceptibility and antibiotic resistance, and (iii) characterized by whole-genome sequencing, including the detection of genes coding for biocide tolerance, antibiotic resistance, and other virulence factors. Fifteen L. monocytogenes isolates were tolerant to benzalkonium chloride (BAC), and genes conferring BAC tolerance were found in 13 of them. Antibiotic resistance was not associated with biocide tolerance. BAC-tolerant isolates were assigned to 6 multilocus sequence type (MLST) clonal complexes, and most of them harbored internalin A pseudogenes with premature stop codons or deletions (n = 9). Our study demonstrated a high genetic diversity among the investigated isolates including genotypes that are frequently involved in human infections. Although in vitro adaptation studies to biocides have raised concerns about increasing cross-resistance to antibiotics, our results do not provide evidence for this phenomenon in field isolates.IMPORTANCE Foodborne pathogens such as L. monocytogenes can persist in food production environments for a long time, causing perennial outbreaks. Hence, bacterial pathogens are able to survive cleaning and disinfection procedures. Accordingly, they may be repeatedly exposed to sublethal concentrations of disinfectants, which might result in bacterial adaptation to these biocides. Furthermore, antibiotic coresistance and cross-resistance are known to evolve under biocide selection pressure in vitro Hence, antimicrobial tolerance seems to play a crucial role in the resilience and persistence of foodborne pathogens in the food chain and might reduce therapeutic options in infectious diseases.

Keywords: Listeria monocytogenes; antibiotic resistance; biocide susceptibility; virulence factors.

FIG 1

MIC (black bars) and MBC (white bars) distributions of 93 L. monocytogenes isolates. Arrows mark MIC₉₅ (black) and MBC₉₅ (white) values representing tentative ECOFFs. conc., concentration; ECOFF, epidemiological cutoff; BAC, benzalkonium chloride; GDA, glutaraldehyde; IPA, isopropanol; NaClO, sodium hypochlorite; PAA, peracetic acid; APD, biocidal product containing bis(3-aminopropyl)dodecylamine.

FIG 2

Minimum-spanning tree based on core genome MLST (cgMLST) allelic profiles of 93 L. monocytogenes isolates from food production environments in Germany. Each circle represents an allelic profile derived from sequence analysis of 1,701 cgMLST target genes. The size of each circle corresponds to the number of isolates. Numbers on the connecting lines illustrate numbers of target genes with differing alleles in a pairwise comparison. Isolates with reduced biocide susceptibility are color-coded as specified in the legend. Allelic patterns belonging to identical MLST clonal complexes (CC) are shaded in gray. BAC, benzalkonium chloride; GDA, glutaraldehyde; IPA, isopropanol; NaClO, sodium hypochlorite; PAA, peracetic acid; APD, biocidal product containing bis(3-aminopropyl)dodecylamine.

FIG 3

Distribution of biocide tolerance and virulence genes. Listeria monocytogenes isolates with reduced susceptibility to biocides are highlighted in yellow. The occurrence of genetic determinants is shown in black. Deletions and truncations of inlA are shaded green, whereas a full-length inlA is brown. BAC, benzalkonium chloride; GDA, glutaraldehyde; IPA, isopropanol; NaClO, sodium hypochlorite; PAA, peracetic acid; APD, biocidal product containing bis(3-aminopropyl)dodecylamine; SSI, stress survival islet; LIPI, Listeria pathogenicity island; LGI, Listeria genomic island; CC, clonal complex.