Unraveling
Enterococcus
susceptibility to quaternary ammonium compounds: genes, phenotypes, and the impact of environmental conditions
Authors: Pereira Ana P. , Antunes Patrícia , Bierge Paula , Willems Rob J. L. , Corander Jukka , Coque Teresa M. , Pich Oscar Q. , Peixe Luisa , Freitas Ana R. , Novais Carla ,   from the ESCMID Study Group on Food- and Water-borne Infections (EFWISG)
Published: September 2023

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.