Citric acid plays a dual ‘synergistic/antagonistic’ role with antibiotics in multidrug-resistant Salmonella Typhimurium

Sensitizing drug-resistant bacteria to conventional antibiotics using antibiotic adjuvants is a viable strategy to mitigate bacterial antimicrobial resistance. Here, we tested the ability of NADES (natural deep eutectic solvents) based date seed extracts to reverse antibiotic resistance in penta-resistant Salmonella Typhimurium DT104. Of the tested extracts, NADES-choline chloride-citric acid, lactic acid, and malic acid date seed extracts exhibited significant anti-DT104 activity (FICI <0.5) in the presence of chloramphenicol, highlighting antibiotic synergy. However, when NADES formulations (without date seed extract) were tested as a control, anti-DT104 activity was found specific to the weak acid component of NADES with congruous chloramphenicol synergy (FICI <0.5). Conversely, citric acid presented antagonism with gentamicin in originally susceptible DT104 by promoting growth in the presence of this antibiotic (FICI >4 (256.4)). Penetrance assay was used to characterize whether the DT104 gentamicin survival was due to the formation of a persister or a tolerant population. Penetrance graphs were biphasic, showing the presence of an antibiotic recalcitrant persister population. Since citric acid is a food preservative, we hypothesized that the acid could promote antibiotic recalcitrant persister population in food matrices. When tested in tahini (sesame paste), where citric acid is a preservative, the acid promoted the formation of antibiotic recalcitrant persister population. Therefore, organic acids originally intended to control the Salmonella population activate the production of antibiotic recalcitrant cells in food systems. Since persister reservoirs promote the emergence of antibiotic-resistant mutants, weak acids-based persister formation in food matrices could be a mechanism by which food matrices promote bacterial antibiotic resistance.