Development of a polymicrobial checkerboard assay as a tool for determining combinatorial antibiotic effectiveness in polymicrobial communities

Abstract

AIMSTo establish a methodology for identifying the effects of combinatorial antibiotic treatment on individual members of a polymicrobial community. METHODS AND RESULTSBoth gentamicin and ceftazidime were diluted to concentrations ranging from 0.06 g ml-1 to 128 g ml-1. An equal ratio polymicrobial community of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, and Acinetobacter baumannii was inoculated into the combined antibiotics in the checkerboard and incubated for 18 hours in static conditions. After incubation, visible turbidity of the overall community was recorded, and bacteria from the wells were diluted to 10-3 and then spot plated on selective and differential media. After 24 hours, colony-forming unit (CFU) counts were obtained for each species. CONCLUSIONSVisible turbidity is not truly indicative of cell viability, and the polymicrobial community can decrease the antibiotic susceptibility of P. aeruginosa, rendering the clinically-established beneficial combination of gentamicin and ceftazidime ineffective. SIGNIFICANCEPrevious checkerboard methodology which focuses on using visible turbidity to determine monomicrobial antibiotic susceptibility fails to account for polymicrobial cooperation that has been shown to reduce antibiotic efficacy. Our new methodology could be implemented in clinical microbiology laboratories with minimal impact on the overall time for diagnosis.