Adaptation to Extremely High Antibiotic Levels (“Hyper”-resistance) Impacts the Stress Tolerance of Listeria monocytogenes and Salmonella spp

Introduction: The overuse of antibiotics in animal agriculture may lead to development of multi-drug resistant pathogens and possibly impart cross-tolerance to environmental stresses encountered during food processing.

Purpose: (i) To assess the innate antibiotic resistance of Listeria monocytogenes and Salmonella spp. isolates from meat and dairy products, feeds or food-contact surfaces, and (ii) to evaluate the thermo-, acid- and alkaline-tolerance of selected strains, following adaptation to the maximum achievable Minimum Inhibitory Concentration (i.e., 36-800-fold higher than the innate MIC) of selected antibiotics.

Methods: Using the disk diffusion method, thirty strains of each pathogen were exposed to ampicillin, amoxicillin/clavulanic acid, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, erythromycin, rifampicin, streptomycin, tetracycline. Antibiotics which showed the highest variation in the inhibition zone of L. monocytogenes (rifampicin, streptomycin, ampicillin) and Salmonella (rifampicin, streptomycin) were selected for further investigation. For each organism, three strains of low (20.5-30.5 mm), intermediate (16.5-26.0 mm) and high (0-15 mm) innate resistance to each of the selected antibiotics were exposed to gradually increasing concentrations of the same agent, selecting for “hyper-resistant” (HR) cells. The HR and the corresponding parental strains were exposed in triplicates to:  (i) 60^oC in capillary tubes, (ii) TSB of pH 3.5 (lactic acid), and (iii) TSB of pH 10.5 (NaOH). 

Results: Contrary to L. monocytogenes, Salmonella isolates from meat products showed higher (P < 0.05) innate antibiotic-dependent resistance than other isolates. Rifampicin-HR strains of both pathogens were more sensitive (P < 0.05) to all lethal conditions, compared to the parental strains. Ampicillin- or streptomycin-HR Salmonella strains showed similar or lower sensitivity to lethal stresses than parental strains. In contrast, streptomycin-HR strains of L. monocytogenes showed 2.0 to 4.1 log CFU/g higher (P < 0.05) survival than the parental strains, after 45 minutes exposure to pH 3.5, indicating potential utilization of cells efflux pumps for both antibiotics and protons disposal from the cytoplasm. 

Significance: Adaptation of L. monocytogenes to high concentrations of antibiotics may induce cross-protection to acid but not to heat or alkaline lethal conditions. Such risk is limited for Salmonella.