Understanding Bacterial Resistance and Dissemination: The Impact of Biocide Priming
Background: Public awareness to infection control, for example the current covid-19 pandemic, can cause a rise in the use of biocidal products. Uncontrolled use of biocides, especially at sub-inhibitory concentrations has led to growing concerns that their selective pressure may favour the prevalence of less susceptible bacterial strains and encourage the expression and dissemination of antibiotic resistance genes. Aims: To investigate the priming effects of sub-inhibitory concentrations of biocides on antibiotic resistance in bacteria and to understand the resistance mechanisms activated by these effects. Methodology: Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were exposed to sub-inhibitory concentrations of biocides (hydrogen peroxide, chlorhexidine, glutaraldehyde and benzalkonium chloride). Minimum inhibitory concentrations of antibiotics and biocides were determined before and after exposure to low concentrations of biocides to test for changes in biocide tolerance and cross resistance to antibiotics. Efflux pump inhibitors (thioridazine and chlorpromazine) were used to investigate efflux as a mechanism. Known antibiotic resistance genes were sequenced to look for any mutations and Quantitative Realtime PCR was used to compare their regulation and expression between parent and primed strains. Growth assays were used to investigate if there were fitness costs attached to the antibiotic cross resistance observed. Results: Stable cross-resistance to antibiotics was observed in bacterial strains, with no observed increased tolerance to biocides. Six-fold increases in MIC to cephalothin and four-fold to ceftriaxone and ampicillin were observed in hydrogen peroxide primed E. coli and thioridazine increased the susceptibility of E. coli to cephalothin and cefoxitin. The ompF porin gene was downregulated by 30.5-fold after exposure to hydrogen peroxide and a further 37.9-fold when grown in 4 mg/l cephalothin compared to parent strain. Chlorhexidine primed S. aureus showed a four-fold increase in MIC to oxacillin, both thioridazine and chlorpromazine increased susceptibility to oxacillin. Sequence analysis of norA and the norA promoter region showed a single adenine to thymine change in codon 313, corresponding to isoleucine to phenylalanine change in position 313 in the coding region, but no change in the promoter region. Gene expression revealed a seven-fold upregulation in norA efflux gene. Glutaraldehyde primed P. aeruginosa showed a four-fold increased MIC to sulphatriad and an eight-fold increased MIC to ciprofloxacin. No increased expression of the efflux gene mexJ and its regulator mexL was observed, suggesting efflux may not be the responsible mechanism of cross-resistance observed in P. aeruginosa. There was no observable fitness cost as a result of the cross-resistance to antibiotics in the strains studied. Conclusion: The widespread and uncontrolled use of biocides may exacerbate the ongoing antibiotic resistance that is seen in clinically relevant bacteria such as E. coli, S. aureus and P. aeruginosa. Sub-inhibitory use of biocides may trigger the activation of protective response in bacteria that can lead to antibiotic cross-resistance. The results shown in EcH2O2 and PaGTA support the theory that not all antibiotic resistant phenotypes come at a cost to fitness because bacteria sometimes have trade-offs and other beneficial mutations that compensates for or offset imbalances that may occur from a selective resistance. The reduced survival of SaCHG in water shows the oxacillin résistance came at a cost.
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