Utilizing Automated Microscopy Analysis to Detect Heteroresistant Subpopulations among P. aeruginosa susceptible To Ciprofloxacin
Heteroresistance is defined as resistance to certain antibiotics expressed by a subset of a microbial population that is generally considered to be susceptible by traditional in-vitro susceptibility testing. Various factors may subsequently lead to the proliferation of the resistant sub-population and the emergence of a fully resistant microbial strain. Detecting heteroresistance is challenging with standard culture based methodologies. Here we have utilized Multiplexed Automated Digital Microscopy (MADM) to demonstrate heteroresistant growth of ciprofloxacin-susceptible Pseudomonas aeruginosa in the presence of 2 μg/mL ciprofloxacin.
A total of 35 clinical P. aeruginosa isolates with MIC < 2 by to frozen broth micro-dilution (BMD) testing per CLSI standards were evaluated in this study. A 0.5 McFarland aliquot of each isolate was diluted 200-fold then introduced into independent flowcells of a disposable multichannel cassette. Electrokinetic concentration immobilized cells on the transparent lower surface of each flowcell channel (5 min). Immobilized cells were challenged with a single concentration of ciprofloxacin (2 μg/mL) prepared in cation-adjusted Mueller Hinton broth with 0.85% agar. Automated microscopy with image analysis software scanned and analyzed growth rates from changes in the mass of each progenitor cell as it grew into a clone of daughter cells (4.5 h). A growth control was also performed. A plot of clone intensity vs time was constructed for each MIC result defined by BMD.
A time-lapse plot of individual growing clones of ciprofloxacin-susceptible P. aeruginosa exhibited a surprising degree of growth rate dispersion at MIC ≥0.5, with a large percentage of those clones demonstrating continued growth after 4 hours in the presence of 2 μg/mL ciprofloxacin. See Figure.
MADM was able to demonstrate heteroresistant growth of susceptible P. aeruginosa in the presence of 2 μg/mL ciprofloxacin. Conventional susceptibility testing does not adequately characterize minority clone expression. Further studies are required to evaluate the clinical implications of heteroresistance in an era in which rates of antimicrobial resistance are increasing alarmingly worldwide.
Accelerate Diagnostics: Collaborator, Grant Investigator and Scientific Advisor, Research support
A. Shamsheyeva, Accelerate Diagnostics, Inc: Employee, Salary
B. Turng, Accelerate Diagnostics, Inc: Employee, Salary
D. Howson, Accelerate Diagnostics, Inc: Consultant, Consulting fee