emrE Gene Located on the Listeria Genomic Island 1 Encodes for an Efflux Pump That Contributes to Listeria monocytogenes Tolerance to Quaternary Ammonium Compounds

Introduction: Contamination of deli meats with Listeria monocytogenes (Lm) resulted in 57 cases of Listeriosis and 24 deaths in 2008 in Canada. Sequencing of the strains implicated in the outbreak (08-5578 and 08-5923) revealed the presence of a novel putative efflux pump (EmrE_Lm) located on an uncharacterized 50 kb island, LGI1.

Purpose: To investigate the role of LGI1 encoded emrE in Lm tolerance to antimicrobials.

Methods: Non-polar emrE_Lm deletion mutant (ΔemrE_Lm) was created in Lm 08-5578 using allelic exchange and pKSV7 vector. Isolates were exposed to antimicrobials [benzalkonium chloride (BAC), E-San, ciprofloxacin, chloramphenicol, erythromycin, gentamicin, tetracycline, triclosan] and acriflavine dye, and their minimum inhibitory concentrations (MICs) were assessed with agar and microbroth dilution methods. Real-time reverse transcription PCR was used to measure transcript levels of emrE_Lm, lde, mdrL, sigB, and putative LGI1 regulators (lmo1851, lmo1861) in Lm 08-5578 following 1 h exposure to BAC (10 μg/ml). Differences in the lag phase (LP; h), growth rate (GR; ΔOD₆₀₀/h), and maximum optical density (MOD; OD₆₀₀) of the parent and ΔemrE_Lm (t-test), and genetically similar strains with (n = 8) and without LGI1 (n = 8; Mann-Whitney) were assessed at sub-lethal QACs concentrations (24 h, 30°C). 

Results: Increased transcription of lmo1861 (82.4-fold), emrE (49.6), sigB (4.1), and lmo1851 (2.3) was observed in the presence of BAC. Deletion of the emrE gene resulted in longer LP (P < 0.0001), and slower GR (P < 0.05) at sub-lethal QACs concentrations, and 2 - 3 times lower MICs; no change in MICs to other tested antimicrobials was observed. Shorter LP (P < 0.05), faster GR (P < 0.001), and higher MOD (P < 0.05) were seen for strains possessing LGI1 than those without LGI1. 

Significance: These data confirm the role of a novel Lm efflux pump, EmrE, in Lm tolerance to QACs. Since QAC sanitizers are commonly used in the food chain, there is a concern Lm strains possessing emrE_Lm will have a survival advantage in food processing environments.