Genomic characteristics of Listeria monocytogenes isolated during mushroom (Agaricus bisporus) production and processing

Listeria monocytogenes is a foodborne pathogen ubiquitously found in nature and which has been isolated from food and food processing environments. This study aimed to characterize L. monocytogenes strains isolated from the production and processing environments of frozen sliced mushrooms (Agaricus bisporus). An analysis was executed along the mushroom processing chain including one mushroom grower and two mushroom processing factories. A total of 153 L. monocytogenes strains were isolated, which could be grouped in three PCR serogroups, namely, serogroup 1/2a-3a (39.2%), serogroup 1/2b-3b-7 (34.0%) and serogroup 4b-4d-4e (26.8%). A selection of 44 L. monocytogenes strains isolated from the processing environment after cleaning and disinfection (C&D) and from frozen sliced mushrooms was genotyped by whole genome sequencing (WGS), because these strains pose a potential risk for product contamination after C&D and for human consumption. Multilocus sequence typing (MLST) revealed 11 clonal complexes (CCs), with strains belonging to CC1, CC4, CC37 and CC87 being detected in both processing factories. Comparative WGS analysis of the 44 strains showed the presence of Listeria pathogenicity island 1 (LIPI-1) with a disrupted version of actA in all CC1, CC4, CC5, CC59 strains, and all but one CC224 strains. Notably, both inlA and inlB were detected as full-length loci in every strain, except for inlA in a CC6 strain that harbored a three amino acid deletion. LIPI-3 was detected in all CC1, CC4, CC6 and CC224 strains, while LIPI-4 was detected in all CC4 and CC87 strains. In addition, antibiotic susceptibility tests showed susceptibility towards fourteen antibiotics tested. The bcrABC operon was found in one CC5 strain, that showed a higher tolerance towards benzalkonium chloride than any other strain tested with confluent growth till 12.5 mu g/ ml for the CC5 strain compared to 2.5 mu g/ml for the other strains. This study highlights that the ecology of L. monocytogenes in the frozen sliced mushroom production chain is highly diverse, and shows the importance of hygienic measures to control L. monocytogenes along the frozen sliced mushroom production chain.