Detection of fluoroquinolone resistance and efflux pumps activity by flow cytometry

Abstract

Fluoroquinolones are bactericidal drugs which have been widely used due to their great activity and wide spectrum namely against gram negative bacteria like Enterobacteriacea. Multidrug resistance is a rising health concern worldwide and increased AcrAB-TolC efflux pump expression has been documented in association with resistance to fluoroquinolones. The classic susceptibility methods are based on growth in the presence of antimicrobial drugs which takes at least 24 to 48 hours and empiric therapy usually is used to overcome this delayed answer. A rapid assay was created to determine the susceptibility of gram negative bacteria to ciprofloxacin and levofloxacin; facing a resistant phenotype, another protocol was developed to detect the presence of efflux pump over-expression. These are rapid and accurate protocols based on flow cytometry that demonstrated great advantages for clinical Microbiology. Sixty two resistant and susceptible clinical isolates of Enterobacteriaceae were tested for ciprofloxacin and fifty three were tested for levofloxacin, previously evaluated by Vitek2®. Genetically modified E. coli K12 with AcrAB-TolC efflux system inactivated, over-expressed and wild-type were used as efflux controls. For susceptibility profile, the cells were incubated with antimicrobial breakpoints according to CLSI, then fixed with ethanol 70% (v/v) and stained with SYBR-Green I, a nucleic acid probe. CFU assays were performed before flow cytometric analysis. For efflux activity study thirty resistant strains were tested. Bacteria were diluted in PBS supplemented with glucose and subinhibitory concentrations of ciprofloxacin and stained with Ethidium Bromide. In parallel the same strains were incubated with chlorpromazine, a pump inhibitor, and the protocol repeated. Flow cytometric analysis was performed in FL1 (520 nm) and FL3 (600 nm) for susceptibility phenotype and efflux determination, respectively. In the susceptibility test, susceptible strains showed a decrease in the fluorescence intensity compared to the control; conversely, resistant strains maintained approximate values, even after incubation with high concentrations of the drugs. Correlation between conventional CFU assay and flow cytometry was successfully achieved. In the efflux assay, a comparison between the fluorescence intensity with and without chlorpromazine was done. When it decreased there was AcrAB-TolC over-expression, if the values maintained there wasn’t. Flow cytometry demonstrated to be an excellent approach to evaluate the resistance to fluoroquinolones and the responsibility of efflux pumps on such resistance.

Date of Award	2014
Original language	English
Awarding Institution	Universidade Católica Portuguesa
Supervisor	Cidália Pina Vaz (Supervisor)