Low-Level Tolerance to Antibiotic Trimethoprim in QAC-Adapted Subpopulations of Listeria monocytogenes

Abstract

Between January and July 2021, there were as many as 30 recalls in the U.S. due to potential Listeria monocytogenes contamination from a variety of food products including muffins, kimchi, chicken salad, ready-to-eat chicken, smoked fish, mushrooms, queso fresco cheese, ice cream, turkey sandwiches, squash, and other foods. A contaminated food chain can serve as a potential vehicle for transmitting antibiotic resistant bacteria since there is a slow emergence of multi-drug antibiotic resistance in L. monocytogenes. Biocides are essential for safe food processing, but they may also induce unintended selective pressure at sublethal doses for the expression of antibiotic resistance in L. monocytogenes. To better understand the sources of such slow emergence of antibiotic resistance through biocide residues present in the food environments, we are working on the role of sublethal doses of commonly used biocides in defined broth and water models for understanding L. monocytogenes adaptation. We recently published the development of low-level tolerance to fluoroquinolone antibiotic ciprofloxacin in quaternary ammonium compound (QAC) adapted subpopulations of L. monocytogenes (Microorganisms 9, 1052). Of the six different antibiotics tested to determine heterologous stress adaptation in eight strains of L. monocytogenes, trimethoprim was the second one that exhibited low-level tolerance development after continuous exposure (by three approaches) to sublethal concentrations of QAC against actively growing planktonic cells of L. monocytogenes. When adapted to daily cycles of fixed or gradually increasing sublethal concentrations of QAC, we observed three main findings in eight L. monocytogenes strains against trimethoprim: (a) 3 of the 8 strains exhibited significant increase in short-range minimum inhibitory concentration (MIC) of trimethoprim by 1.7 to 2.5 fold in QAC-adapted subpopulations compared to non-adapted cells (p < 0.05); (b) 2 of the 8 strains exhibited significant increase in growth rate in trimethoprim (optical density (OD) by 600 nm at 12 h) by 1.4 to 4.8 fold in QAC-adapted subpopulations compared to non-adapted cells (p < 0.05); and (c) 5 of the 8 strains yielded significantly higher survival by 1.3-to-3.1 log CFU/mL in trimethoprim in QAC-adapted subpopulations compared to the non-adapted control (p < 0.05). However, for 3/8 strains of L. monocytogenes, there was no increase in the survival of QAC-adapted subpopulations compared to non-adapted control in trimethoprim. These findings suggest the potential formation of low-level trimethoprim tolerant subpopulations in some L. monocytogenes strains where QAC may be used widely. These experimental models are useful in developing early detection methods for tracking the slow emergence of antibiotic tolerant strains through food chain. Also, these findings are useful in understanding the predisposing conditions leading to slow emergence of antibiotic resistant strains of L. monocytogenes in various food production and food processing environments.

FepR as a Central Genetic Target in the Adaptation to Quaternary Ammonium Compounds and Cross-Resistance to Ciprofloxacin in Listeria monocytogenes

“…Different types of selective pressure (such as antibiotics, biocides, or heavy metals) may play a role in the prevalence of antibiotic resistance of Lm in the food chain. Exposure to disinfectant biocides such as the QACs BC and DDAC, extensively used in the food processing industry, has the potential to promote cross-resistance to CIP, as indicated by an increasing number of studies ( Rakic-Martinez et al, 2011 ; Yu et al, 2018 ; Guérin et al, 2021 ; Kode et al, 2021 ; Bland et al, 2022 ). These biocides may co-select for strains resistant to CIP, due to shared mechanisms of resistance (cross-resistance) ( Kampf, 2019 ).…”

“…The development of bacterial resistance to QACs ( Lunden et al, 2003 ; Yu et al, 2018 ) and the subsequent effects on antibiotic susceptibility have been reported in several studies ( Rakic-Martinez et al, 2011 ; Yu et al, 2018 ; Kode et al, 2021 ; Bland et al, 2022 ). We recently demonstrated a link between QAC exposure and reduced susceptibility to a fluoroquinolone antibiotic in Lm , using a panel of 28 strains from different sources and different clonal complexes (CC) ( Guérin et al, 2021 ).…”

Antimicrobial Tolerance in Salmonella: Contributions to Survival and Persistence in Processing Environments

“…However, the nutrient-rich poultry processing environment with organic residue from poultry carcasses and water pH can influence the dissociation of hypochlorous acid, thus reducing the amount of free chlorine that reacts with bacterial populations in the environment. This has led to many studies that examined the ability of Salmonella and other food-borne pathogens to handle sublethal chlorine stress and the reaction such stressor(s) induce [77][78][79][80][81][82]. Many authors have reported diverse changes in tolerance in different Salmonella strains after sublethal chlorine exposure as expressed by higher antimicrobial minimum inhibitory concentrations (MIC's) compared to the initial MIC before exposure [76,77,[83][84][85][86].…”

“…The efficacy of QACs to inactivate Salmonella has been previously described, but some factors including improper or prolonged storage and exposure to sublethal concentrations could negatively impact efficacy resulting in the selection of strains with reduced susceptibility. The acquired tolerance to QACs in Salmonella and other food-borne pathogens through sublethal exposure has been evaluated, and studies have reported different responses including changes in the MIC after sublethal exposure, cell growth, and morphology [81,82,104,105]. Mangalappalli-Illathu et al (2008) [104] reported an adaptive response and survival of both planktonic and biofilms of Salmonella Enteritidis that were exposed to benzalkonium chloride.…”

Listeria monocytogenes – How This Pathogen Survives in Food-Production Environments?

“…Increased tolerance of L. monocytogenes to disinfectants (biocides) has been deeply studied in relation to quaternary ammonium compounds (QACs), such as benzalkonium chloride (BC), widely applied in food production and health care environments as well as in households due to their effectiveness, low toxicity, and non-corrosive properties ( Gerba, 2015 ; Kode et al, 2021 ). QACs are usually used in concentrations ranging from 200 ppm to 400 ppm on food-contact surfaces; however, some formulations also have 1,000 ppm concentrations ( Aryal and Muriana, 2019 ).…”