Abstract
Listeria monocytogenes the causative agent of listeriosis is an important public health concern and food safety challenge. Increased tolerance of this bacterium to benzalkonium chloride (BC), an antibacterial agent widely used in industrial settings, is a growing issue. Plasmid pLMST6 harboring the gene of the multidrug efflux pump protein EmrC has been recently linked to enhanced BC tolerance and meningitis due to L. monocytogenes ST6 strains. In this study, occurrence and contribution of this plasmid to BC tolerance was examined using PCR, plasmid curing and transformation, RT-qPCR and proteome analysis, respectively. Furthermore, the substrate specificity of the pLMST6 associated EmrC efflux pump and the impact of the plasmid on L. monocytogenes virulence were investigated. pLMST6 was detected in 7 (1.6%) of 439 L. monocytogenes strains isolated from different sources. A phenotypic role of this plasmid in conferring increased BC tolerance was confirmed by showing that plasmid cure increases BC susceptibility whereas plasmid complementation and transformation increased BC tolerance in different L. monocytogenes genetic backgrounds and L. innocua. RT-qPCR showed that BC stress exposure strongly induces the expression of mRNAs associated with pLMST6 genes for EmrC and a TetR transcription regulator. A full proteome analysis in a plasmid harboring L. monocytogenes strain revealed that the pLMST6 encoded putative TetR family transcription regulator protein is the most upregulated protein in response to BC stress exposure. An investigation into the EmrC efflux pump’s substrate spectrum showed that while pLMST6 confers increased tolerance to other quaternary ammonium compounds (QACs) based disinfectants it has no impact on the sensitivity of L. monocytogenes to non-QAC disinfectants as well as on antibiotics such as ampicillin, tetracycline and gentamicin. A reduction in the survival of zebrafish embryos infected with pLMST6 plasmid harboring L. monocytogenes strains was observed when compared with plasmid cured variants of the same strains suggesting that some pLMST6 harbored genes might contribute to increased virulence capacity. Overall these results confirm the phenotypic contribution of pLMST6 plasmid in promoting and dissemination of BC tolerance in L. monocytogenes as well as provide new insights on different molecular levels of pLMST6 associated genes in response to BC stress.
Highlights
Listeriosis is a severe foodborne infection caused by Listeria monocytogenes, a gram-positive, facultative intracellular bacterium occurring ubiquitously in soil and water
In order to confirm the phenotypic contribution of the pLMST6 plasmid harboring the emrC gene to increased benzalkonium chloride (BC) tolerance, plasmid cured (PCV) and plasmid transformed (PTV) variants were created in two L. monocytogenes strains N12-0935 (CC403) and N12-2082 (CC8) that naturally harbor the pLMST6 plasmid
Growth on such brain heart infusion (BHI)-BC plates could be restored for the N120935PTV and N12-2082PTV strains that were generated by retransforming the plasmid cured variants (PCVs) (N12-0935PCV and N12-2082PCV) of both strains with the pLMST6 plasmid
Summary
Listeriosis is a severe foodborne infection caused by Listeria monocytogenes, a gram-positive, facultative intracellular bacterium occurring ubiquitously in soil and water. Listeria monocytogenes comprises of thirteen serotypes that are subdivided into four main evolutionary genetic lineages and several multilocus sequence typing (MLST) clones, which show variable distribution in food products and processing environments as well as among human and animal clinical listeriosis cases (Orsi et al, 2011; Haase et al, 2014; Maury et al, 2016). Exposure to sublethal BC concentrations for bacteria in food associated environments can lead to development of BC tolerance in pathogens (To et al, 2002). The existence of environmental niches in food processing plants that are difficult to reach for disinfectants leading to exposure of targeted bacterial pathogens such as L. monocytogenes to sublethal concentrations is a major concern
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