Rapid automated system for bacteriophage virulence and antibiotic synergy testing – The ‘phago-antibiogram’

Abstract

Background: The global emergence of multidrug resistant bacteria in the context of desiccating antimicrobial drug pipeline has led to resurgent interest in bacteriophage therapy (BT) to treat serious bacterial infections. Phages are highly specific at the species and strain level and measurement of the bacterial host range plays an important role in utilizing the phage as antimicrobials. We have developed a highly efficient high-throughput method utilizing a liquid handling robot to prepare 96-well plates carrying the targeted organisms in liquid broth and bacteriophage in a gradient across the rows. We also determined antibiotic synergy using the same method adding the antibiotics in fixed concentration to detect phage-antibiotic synergism (PAS). Methods and materials: The 96-well microtiter plates were prepared on Eppendorf epMotion 5075 liquid handling robot for high throughput testing containing LB broth, target bacterial isolates, serial dilutions of phage concentration and fixed concentrations of antibiotics (1/4 MIC). The plates were incubated at 37 °C for 18 h with orbital shaking in SpectraMax iD5 microplate reader with bacterial kinetics monitored every 30 min. Results: The automated plate assay correctly determined the phage virulence in majority of cases. The plaque assay tended to indicate greater phage sensitivity. Continued monitoring of bacterial growth allowed for determining the occurrence of bacterial resistance development over continued phage exposure. Phage combination with imipenem and ciprofloxacin exhibited synergistic activity and delayed the appearance of resistant mutants. Conclusion: Our method produced reproducible results in good agreement with the reference plaque assay for phage virulence determination and can also give valuable data about synergistic activity with different antimicrobial drugs.