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

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.