Development of a rapid fluorescent assay to detect cationic antimicrobial tolerance based on Gram-negative bacterial membrane integrity
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Date
2020
Authors
Gregorchuk, Branden S.J.
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Abstract
Bacterial cationic antimicrobials (CA) encompass a wide spectrum of molecules that can be separated into two main CA categories: antibiotics which can be used parenterally and biocides which are administered topically to inhibit cell growth. CAs possess a mechanism of action that disrupts bacterial cell membrane integrity. Previous studies have shown that bacteria with increased tolerance to biocide CAs can provide these organisms cross-tolerance/resistance to other antimicrobials. CA biocide tolerance is not routinely tested due to a lack of recognized international standards. When biocide CA tolerance is tested, measurements are based on minimal inhibitory concentration values collected using methods which are laborious and time-consuming, highlighting the need for a rapid assay. In this thesis, I describe the development of a rapid fluorescent dye membrane integrity assay (RFDMIA), which is able to distinguish between biocide CA susceptibility and tolerance within 30 minutes utilizing a fluorescent 96-well microplate assay format. To validate this assay, we examined various Gram-negative Enterobacterial species (Escherichia, Klebsiella and Shigella spp.) as well as Pseudomonas and Acinetobacter species exposed to two major biocide CAs classes (quaternary ammonium compounds (QACs) and bisbiguanides) and two CA antibiotic classes (polymyxins and aminoglycosides). The RFDMIA assay was validated using QACs and confirmed that QAC tolerance and susceptibility could be distinguished in E. coli when exposed to increasing concentrations of biocide. RFDMIA was also able to predict QAC and bisbiguanide tolerance in all tested isolates except for P. aeruginosa, likely due to its inherent fluorescent properties. Scanning electron microscopy was used to visualize the membrane disruptive capacities of both biocide CA classes, revealing that QAC-adapted replicates had a novel biocide “dependant” phenotype and that all biocide CA adapted isolates displayed altered morphology after a 30 minutes of biocide exposure. Lastly, we examined if the RFDMIA assay could predict antibiotic CA resistance to colistin, tobramycin, and gentamicin with similar bacterial species. RFDMIA could not predict antibiotic CA susceptibilities, potentially due to the slower and diverse mechanisms of action of these antibiotics. Together, this project has developed a new assay to rapidly and accurately detect of biocide CA tolerance in a high throughput assay.
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Keywords
Fluorescent emission, Membrane integrity, Biocide, Cationic antimicrobial, Antiseptic, Disinfectant, Quaternary ammonium compound, Benzalkonium, Cetrimide, Antimicrobial resistance, Colistin, Chlorhexidine
Citation
Antimicrobial Agents and Chemotherapy