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Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action.
BIOCIDE
(2022)
Antimicrobial resistance (AMR) is a global health problem with the environment being an important compartment for the evolution, selection and transmission of AMR. These processes are impacted by pollution with antibiotics. However, biocides used as disinfectants and material preservatives are major pollutants by far excceding the market for antibiotics in terms of mass. Our work shows that biocides have the potential to affect evolutionary processes towards AMR by increasing the rates of de-novo mutation and conjugation. These effects depend on the species and biocidal substance. Importantly, chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations in E. coli. Moreover, our results show a connection between the RpoS-mediated general stress and the RecA-linked SOS response with increased rates of mutation and conjugation, but not for all biocides. Furthermore, our work highlights the potential of biocides to contribute to selection and transmission of AMR. We show that the application of biocides, especially QAC disinfectants, leads to the rapid evolution of tolerance (i.e. increased survival) in adaptive laboratory evolution (ALE) experiments. The evolved tolerant strains have a selective advantage in the presence of environmentally-relevant concentrations of antibiotics, which could lead to the stabilization of biocide tolerance in environments where biocides and antibiotics co-occur (e.g. wastewater, animal stables). ALE experiments with biocide tolerant strains indicate a decreased evolvability of resistance to antibiotics. Taken together, our work shows the importance of assessing the contribution of biocides on evolution, selection and transmission of AMR in the environment.
Development of a laboratory method to assess resistance development of microorganisms to biocides
(2021)
Bacteria are exposed to biocides through surface disinfection or by antimicrobial surfaces. These stressful environments provide a strong selective pressure for bacteria to adapt. Here, we describe the development of a laboratory method to assess adaption of bacteria by resistance development in response to surface disinfection and antimicrobial surfaces.
Development of a laboratory method to assess resistance development of microorganisms to biocides
(2021)
Bacteria are exposed to biocides through surface disinfection or by antimicrobial surfaces. These stressful environments provide a strong selective pressure for bacteria to adapt. Here, we describe the development of a laboratory method to assess adaption of bacteria by resistance development in response to surface disinfection and antimicrobial surfaces.
Pseudomonas aeruginosa is an opportunistic pathogen of considerable medical importance, owing to its pronounced antibiotic tolerance and association with cystic fibrosis and other life-threatening diseases. The aim of this study was to highlight the genes responsible for P. aeruginosa biofilm tolerance to antibiotics and thereby identify potential new targets for the development of drugs against biofilm-related infections. By developing a novel screening approach and utilizing a public P. aeruginosa transposon insertion library, several biofilm-relevant genes were identified. The Pf phage gene (PA0720) and flagellin gene (fliC) conferred biofilm-specific tolerance to gentamicin. Compared with the reference biofilms, the biofilms formed by PA0720 and fliC mutants were completely eliminated with a 4-fold-lower gentamicin concentration. Furthermore, the mreC, pprB, coxC, and PA3785 genes were demonstrated to play major roles in enhancing biofilm tolerance to gentamicin. The analysis of biofilm-relevant genes performed in this study provides important novel insights into the understanding of P. aeruginosa antibiotic tolerance, which will facilitate the detection of antibiotic resistance and the development of antibiofilm strategies against P. aeruginosa.