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Biocides, including disinfectants and antimicrobial surfaces (AMCs), are important to prevent the spread of pathogens and antimicrobial resistant bacteria via surfaces. However, concerns have been raised about the evolution and selection of resistance against disinfectants and AMCs. In turn, resistance against disinfectants and AMCs can be associated to antibiotic resistance due to cross-resistance and co-resistance. We need to understand the mechanisms and risks of disinfectants and AMCs for resistance and cross-resistance evolution to optimize their application and safeguard their long-term efficacy. We used adaptive laboratory evolution (ALE) experiments based on repeated exposure of bacteria to disinfectants. Our results show that repeated disinfection of E. coli with benzalkonium chloride in suspension results in a 2000-fold increase in survival within 5 exposure cycles. Adaption is linked to the initial presence of persister cells highly tolerant to benzalkonium chloride. We used the same approach to develop standardizable ALE experiments to determine resistance evolution to AMCs. The results highlight rapid adaptation of E. coli and P. aeruginosa towards copper surfaces. Moreover, there are multiple situations in the clinic or in the environment in which biocides and antibiotics co-occur and in which combination effects can shape their antimicrobial activity or their selective effects. Our work with P. aeruginosa shows prevalent combination effects of biocides and antibiotics, ranging from synergy to antagonism and resulting in the selection for or against antibiotic resistant strains. The combination effects are dependent on the biofilm mode-of-growth, manifesting in apparent differences in the structural arrangement of antibiotic sensitive and resistant strains in biofilms exposed to combinations. Furthermore, biocides affect rates of mutation and horizontal gene transfer, thereby having a potential facilitating effect on resistance evolution. Taken together, our work shows that the role of biocides as potential drivers of resistance evolution and selection deserves further study and regulative action.
Biocides used as material preservatives are in contact with various environments during direct application or passive leaching from protected materials [1]. Antimicrobial resistance (AMR) is a global health problem and the environment is an important compartment for the evolution and transmission of AMR [2,3]. Soil is an environment with a large reservoir of natural microbial communities and antimicrobial resistance (AMR) genes. Those natural microbial communities are frequently exposed to biocides used as material preservatives. Previous studies have shown that antibiotics, metals and pesticides affect the underlying processes of resistance evolution and spread; namely de novo mutagenesis and horizontal gene transfer by conjugation and transformation in microbial communities. However, it is unknown if active substances used in material preservatives are involved in these processes. We show that biocides used as material preservatives affect rates of mutation and conjugation in microorganism in a species- and substance-dependent manner, while rates of transformation are not directly affected. Our data highlights the importance of assessing the contribution of material preservatives on AMR evolution and spread in the environment.
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.
We investigated the selection dynamics between a benzalkonium chloride (BAC)-tolerant Escherichia coli strain (S4) and a sensitive wild type under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the opposite was observed at all ciprofloxacin concentrations investigated.Furthermore, we assessed the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type.