Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 146, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/58636
Keywords
Immunology and Infection; Issue 146; Antimicrobial synergy; antibiotic synergy; synergy; antimicrobial resistance; colistin resistance; carbapenem-resistant Enterobacteriaceae; time-kill synergy; checkerboard array; automation; inkjet printing
Categories
Funding
- Eunice Kennedy Shriver National Institute of Child Health and Human Development pediatric infectious diseases research training grant [T32HD055148]
- National Institute of Allergy and Infectious Diseases training grant [T32AI007061]
- Boston Children's Hospital Office of Faculty Development Faculty Career Development fellowship
- National Institute of Allergy and Infectious Diseases career development award [1K08AI132716]
- National Institute of Allergy and Infectious Diseases of the National Institutes of Health [R33 AI119114]
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As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing. The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.
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