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

Abstract

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. Enterococcus faecalis and Enterococcus faecium 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 Enterococcus 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 Enterococcus isolates included in this study and 0.5% of publicly available Enterococcus genomes carried qacA/B, qacC, qacG, qacJ, qacZ, qrg, bcrABC or oqxAB 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 E. faecalis and E. faecium isolates (two isolates carrying qacZ ). Modified growth conditions (e.g., 22°C/pH = 5) increased MIC BC /MBC BC (maximum of eightfold and MBC BC = 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, Enterococcus 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. Enterococcus faecium and Enterococcus faecalis 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. E. faecium and E. faecalis 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 Enterococcus 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.

Decoding Klebsiella pneumoniae in poultry chain: unveiling genetic landscape, antibiotic resistance, and biocide tolerance in non-clinical reservoirs

“…Nevertheless, our WGS approach identified a wide range of QAC tolerance genes, underscoring the pressing need for establishing reliable genotypic-phenotypic correlations to elucidate QAC tolerance mechanisms ( Hipólito et al, 2023 ). While several studies have reported that bacterial strains with an elevated MIC or MBC remained susceptible to the in-use BZC concentration ( Vijayakumar et al, 2018 ; Maillard, 2022 ), it is essential to comprehend the environmental factors (e.g., temperature, pH) associated with the expression of these genes ( Gual-de-Torrella et al, 2022 ; Pereira et al, 2023 ) to clarify the environmental, clinical, or veterinary/industrial implications of bacteria with a reduced biocide susceptibility. Further studies are needed to investigate the impact of inappropriate biocide usage or low concentrations, which can act as stressors without killing bacterial pathogens, potentially promoting antimicrobial resistance, and facilitating the transfer of antimicrobial resistance genes ( Maillard, 2022 ; Maillard and Pascoe, 2023 ).…”

“…Each experiment was repeated three times and the MIC/MBC values corresponded to the mean of these determinations. To ensure assay reproducibility, the Enterococcus faecalis ATCC 29212 (without any known QACs tolerance genes) was included as a control strain (MIC BZC and MBC BZC varied between 1 and 4 mg/L; Pereira et al, 2023 ).…”

“…Their versatility and broad-spectrum antimicrobial activity assist in controlling and preventing pathogens dissemination between flocks within poultry facilities ( Chen et al, 2023 ). Genes associated with tolerance to metals and QACs often share the same genetic contexts with antibiotic-resistance genes ( Slifierz et al, 2015 ; Li et al, 2022 ; Pereira et al, 2023 ). Thus, the use of copper-supplemented feed and QAC-based biocides could contribute to co-selection effects ( Webber et al, 2015 ; Kampf, 2018 ; Rebelo et al, 2023 ).…”

DecodingKlebsiella pneumoniaein Poultry Chain: Unveiling Genetic Landscape, Antibiotic Resistance, and Biocide Tolerance in Non-Clinical Reservoirs

“…Each experiment was repeated three times and the MIC/MBC values corresponded to the mean of these determinations. To ensure assay reproducibility, the Enterococcus faecalis ATCC 29212 (without any known QACs tolerance genes) was included as a control strain (MIC BZC and MBC BZC varied between 1-4 mg/L) (Pereira et al, 2023).…”

Current insights into the effects of cationic biocides exposure on Enterococcus spp.

“…Thus, several other factors (e.g., variable temperature or pH, oxygen limitation, starvation, lower or higher bacterial density, bacterial growth phase) that can affect the efficiency of biocides in real application contexts, namely by altering the cytoplasmic membrane or reducing the cell's metabolic activity, are not pondered (Zhang and Rock, 2008;Saito et al, 2014;Ran et al, 2015;Wiegand et al, 2015;Yoon et al, 2015;Gaca and Lemos, 2019;Fox et al, 2022;Maillard and Pascoe, 2024). Recently, we learned that E. faecium and E. faecalis isolates from different epidemiological and clonal backgrounds exhibited decreased susceptibility (MIC and MBC increases of two to eightfold) to BC at 22 • C and/or pH = 5, compared to standard conditions (37 • C; pH = 7.3) (Pereira et al, 2023), confirming the influence of external growth conditions on CBs susceptibility.…”