Development of antibacterial drug + bacteriophage combination assays

Abstract

The best methods to study the interactions between phages and antibacterials are unclear. As laboratory methodologies used to assess conventional antibacterials are established, we assessed their ability to evaluate phage plus antibacterial. The efficacy of three characterized Escherichia coli phages (UP17, JK08, 113) were tested on a 100 MDR E coli. strains. The phages were assessed individually and in a 1:1:1 cocktail. In a phage microbial inhibitory concentration (PmIC) assay, a range of phage concentrations from 101 to 108 were inoculated with 5 × 105 bacteria/well in 96-well microtitre plates. The first fully lysed well was taken as the PmIC. Amikacin and meropenem MICs were determined by ISO 2776-1:2019 methods alone and in combination with a fixed phage concentration of 105/per well. Time–kill curves (TKCs) were conducted at fosfomycin concentrations of 133, 50 and 5 mg/L, with and without phage. The PmIC50/90 values, in plaque-forming units (pfu)/mL, for individual phage titre were >108/>108 for UP17, 107/>108 for JK08, 107/>108 for 113 and 106/>108 for the 1:1:1 phage cocktail, all with a standard deviation (SD) of <0.05. Amikacin and meropenem MIC50/90 (SD) values were 2/8 mg/L (<0.05–9.2) and 0.12/8 mg/L (<0.05–8.7), respectively. The addition of UP17 to amikacin increased amikacin MICs >2-fold in 78 strains, with equivalent trends in 39 strains with JK08, 54 strains with 113 and 45 strains with phage cocktail. Meropenem MICs in the presence of phage were reduced >2-fold in 24 strains with UP17. Equivalent decreases were seen with 34 strains with JK08, 26 strains with 113 and 29 strains with the cocktail. In TKCs, the addition of phage suppressed regrowth. Microbroth methodologies based on ISO 2776-1:2019 lend themselves to be viable options for phage assessment, alone or in combination with antibiotics. The reproducibility of PmIC and familiarity of the methodology described allows for laboratory validation for phage and antibiotic combinations.