Characterization of Disinfectant and Phage Resistance Determinants of Listeria monocytogenes

Environmental contamination of food plays a key role in transmission of Listeria monocytogenes. Adaptations such as ability to grow under refrigeration, biofilm formation, disinfectant resistance, and resistance to phage are crucial to the survival of L. monocytogenes in the environment. Here we have investigated some of the adaptations that may contribute towards environmental persistence of this pathogen. These include disinfectant (quaternary ammonium compounds, QAC) resistance, temperature-dependent resistance to phage among epidemic clone II strains, triphenylmethane dye reduction and resistance to heavy metals such as cadmium. Using in silico analysis, we have analyzed the prevalence of cadAC efflux systems associated with cadmium resistance among different Listeria genomes. Of the three distinct cadAC types known to date in L. monocytogenes, two (cadA1C1 and cadA2C2) were harbored by plasmids and were encountered in diverse strains of L. monocytogenes and (in the case of cadA2C2) non-pathogenic Listeria spp. Large plasmids from two different epidemic-associated strains of L. monocytogenes harbored cadA2C2 together with a drug efflux system (bcrABC), and both determinants were associated with a putative IS1216 transposon. Plasmid-cured derivatives of these strains were susceptible both to cadmium and to BC. Genetic analyses revealed that cadA2C2 was sufficient for restoring resistance to cadmium to a plasmid-cured derivative of L. monocytogenes H7550. Furthermore, bcrABC was sufficient for restoring resistance to high levels of QAC to the plasmid-cured strain. The transcription of bcrABC was temperature-dependent, with higher transcript levels at 37°C than at 25°C or below. The bcrABC system was encountered among L. monocytogenes strains of different serotypes, origin (environmental or clinical) and genotypes and was detected more frequently in strains harboring the cadmium resistance cassette cadA2C2 (alone or together with cadA1C1) than in those harboring cadA1C1 alone. bcrABC was found in several different genetic arrangements, mostly on plasmids, but we also identified L. monocytogenes strains in which bcrABC appeared to have translocated into the chromosome. Transcriptional analysis revealed that transcription of cadA2C2 and bcrABC was independent, and induced upon exposure to the respective compounds (cadmium and BC, respectively). On the other hand, bcrABC was co-transcribed with the downstream gene tmr, encoding a putative triphenylmethane reductase. We showed that tmr mediated decolorization (reduction) of toxic dyes such as crystal violet (CV) and that CV could also induce bcrABC. Analysis of a panel of strains of diverse serotypes revealed that those harboring tmr tended to also harbor bcrABC and cadA2C2 (alone or together with cadA1C1), suggesting the presence of a co-selected multi-resistance unit that includes cadA2C2, bcrABC, and tmr. In previous studies, a gene (ORF 2753) was found to be responsible for the ability of L. monocytogenes epidemic clone II (ECII) to be resistant to phage when propagated at temperatures <25°C. Here we have shown that ORF2753 corresponded to a putative restriction endonuclease that was a component of a type II restriction-modification (RM) system (LmoH1) that is specific for GTATCC (N6/5). This RM system is present on a chromosomal genomic region unique to ECII strains. Transcriptional analysis revealed that the putative restriction endonuclease was expressed significantly more at ≤25°C than at 37°C, potentially accounting for the temperature-dependent phage resistance of ECII strains, while ORF2754 (putative methyltransferase) was expressed at all tested temperatures; this was in agreement with the resistance of the DNA from cells grown at either 25°C or 37°C to BfuI, specific for GTATCC (N6/5). Given that genetic basis for distinct adaptive attributes were elucidated in an outbreak strain (H7550), it would be of further interest to investigate the contribution of these determinants towards virulence of this pathogen. Such details would provide new avenues to control the incidence of listeriosis.