Abstract
Biocide use is essential and ubiquitous, exposing microbes to sub-inhibitory concentrations of antiseptics, disinfectants, and preservatives. This can lead to the emergence of biocide resistance, and more importantly, potential cross-resistance to antibiotics, although the degree, frequency, and mechanisms that give rise to this phenomenon are still unclear. Here, we systematically performed adaptive laboratory evolution of the gut bacteria Escherichia coli in the presence of sub-inhibitory, constant concentrations of ten widespread biocides. Our results show that 17 out of 40 evolved strains (43%) also decreased the susceptibility to medically relevant antibiotics. Through whole-genome sequencing, we identified mutations related to multidrug efflux proteins (mdfA and acrR), porins (envZ and ompR), and RNA polymerase (rpoA and rpoBC), as mechanisms behind the resulting (cross)resistance. We also report an association of several genes (yeaW, pyrE, yqhC, aes, pgpA, and yeeP-isrC) and specific mutations that induce cross-resistance, verified through mutation repairs. A greater capacity for biofilm formation with respect to the parent strain was also a common feature in 11 out of 17 (65%) cross-resistant strains. Evolution in the biocides chlorophene, benzalkonium chloride, glutaraldehyde, and chlorhexidine had the most impact in antibiotic susceptibility, while hydrogen peroxide and povidone-iodine the least. No cross-resistance to antibiotics was observed for isopropanol, ethanol, sodium hypochlorite, and peracetic acid. This work reinforces the link between exposure to biocides and the potential for cross-resistance to antibiotics, presents evidence on the underlying mechanisms of action, and provides a prioritized list of biocides that are of greater concern for public safety from the perspective of antibiotic resistance.Significance StatementBacterial resistance and decreased susceptibility to antimicrobials is of utmost concern. There is evidence that improper biocide (antiseptic and disinfectant) use and discard may select for bacteria cross-resistant to antibiotics. Understanding the cross-resistance emergence and the risks associated with each of those chemicals is relevant for proper applications and recommendations. Our work establishes that not all biocides are equal when it comes to their risk of inducing antibiotic resistance; it provides evidence on the mechanisms of cross-resistance and a risk assessment of the biocides concerning antibiotic resistance under residual sub-inhibitory concentrations.
Highlights
Biocides are chemicals which include antiseptics and disinfectants, used to control, eliminate, or reduce the number of undesired organisms (Scenihr and European Commission, 2009)
The adaptation to common disinfectants and antiseptics, and the emergence of cross-resistance to chemicals such as antibiotics, was demonstrated for bacteria exposed to biocides such as benzalkonium chloride (Braoudaki and Hilton, 2004; Bore et al, 2007; Abdelaziz et al, 2019) and chlorhexidine (Braoudaki and Hilton, 2004; Wand et al, 2017; Verspecht et al, 2019) and reviewed elsewhere (Kampf, 2018)
Adaptive laboratory evolution of E. coli in sub-inhibitory and constant concentration of biocides selected for strains resistant to higher concentrations than the ones applied for the evolution experiments
Summary
Biocides are chemicals which include antiseptics and disinfectants, used to control, eliminate, or reduce the number of undesired organisms (Scenihr and European Commission, 2009). Despite their ubiquitous presence, the evolution of biocide resistance is much less studied than that in antibiotics. The adaptation to common disinfectants and antiseptics, and the emergence of cross-resistance to chemicals such as antibiotics, was demonstrated for bacteria exposed to biocides such as benzalkonium chloride (Braoudaki and Hilton, 2004; Bore et al, 2007; Abdelaziz et al, 2019) and chlorhexidine (Braoudaki and Hilton, 2004; Wand et al, 2017; Verspecht et al, 2019) and reviewed elsewhere (Kampf, 2018). The genes allowing bacterial survival in both biocides and antibiotics are transmitted together due to the co-occurrence in the same genetic element such as a plasmid or integron (Chapman, 2003)
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