Unraveling susceptibility to quaternary ammonium compounds: genes, phenotypes, and the impact of environmental conditions.

Quaternary ammonium compounds (QACs) have been extensively used in the community, healthcare facilities, and food chain, in concentrations between 20 and 30,000 mg/L. and are ubiquitous in these settings and are recognized as nosocomial pathogens worldwide, but QACs' activity against strains from diverse epidemiological and genomic backgrounds remained largely unexplored. We evaluated the role of isolates from different sources, years, and clonal lineages as hosts of QACs tolerance genes and their susceptibility to QACs in optimal, single-stress and cross-stress growth conditions. Only 1% of the isolates included in this study and 0.5% of publicly available genomes carried or genes, shared with >60 species of Bacillota, Pseudomonadota, Actinomycetota, or Spirochaetota. These genes were generally found within close proximity of antibiotics and/or metals resistance genes. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of benzalkonium chloride (BC) and didecyldimethylammonium chloride ranged between 0.5 and 4 mg/L (microdilution: 37°C/20 h/pH = 7/aerobiosis) for 210 and isolates (two isolates carrying ). Modified growth conditions (e.g., 22°C/pH = 5) increased MIC/MBC (maximum of eightfold and MBC = 16 mg/L) and changed bacterial growth kinetics under BC toward later stationary phases in both species, including in isolates without QACs tolerance genes. In conclusion, are susceptible to in-use QACs concentrations and rarely carry QACs tolerance genes. However, their potential gene exchange with different microbiota, the decreased susceptibility to QACs under specific environmental conditions, and the presence of subinhibitory QACs concentrations in various settings may contribute to the selection of particular strains and, thus, require a One Health strategy to maintain QACs effectiveness. IMPORTANCE Despite the increasing use of quaternary ammonium compounds (QACs), the susceptibility of pathogens to these antimicrobials remains largely unknown. and are susceptible to in-use QACs concentrations and are not main hosts of QACs tolerance genes but participate in gene transfer pathways with diverse bacterial taxa exposed to these biocides. Moreover, QACs tolerance genes often share the same genetic contexts with antibiotics and/or metals resistance genes, raising concerns about potential co-selection events. and showed increased tolerance to benzalkonium chloride under specific environmental conditions (22°C, pH = 5), suggesting that strains might be selected in settings where they occur along with subinhibitory QACs concentrations. Transcriptomic studies investigating the cellular mechanisms of adaptation to QACs tolerance, along with longitudinal metadata analysis of tolerant populations dynamics under the influence of diverse environmental factors, are essential and should be prioritized within a One Health strategy.