Development of antibacterial drug plus bacteriophage combination assays

Abstract

SynopsisBackgroundThe methods to evaluate the interactions between Phages and antibacterials are unclear. As the laboratory methodologies used to assess conventional antibacterials are well established, we asseassed their efficacy in evaluating phage plus antibacterial.Methods100 multidrug resistantE. colistrains were used with three previously isolated and characterisedE. coliphages of known efficacy. These phages UP17, JK08, 113 were assessed both individually and in a 1:1:1cocktail. In a Phage Microbial Inhibitory Concentration (PmIC) assay, a range of phage concentrations from 101-108were inoculated with 5×105/well bacteria in microtitre plates. The first lysed, clear well was taken as the PmIC. Amikacin(AMI) and meropenem(MERO) MICs were determined by microbroth dilution methods(ISO 2776-1:2019) and in combination AMI and MERO MICs were measured with a fixed Phage concentration of 105/well. MICs were performed in triplicate. Time-Kill curves(TKC) were conducted at fosfomycin concentrations of 133, 50 and 5mg/L with and without phage.ResultsThe PmIC50/90for UP17 were >108/>108; JK08 107/>108; 113 107/>108and the 1:1:1 cocktail 106/>108. AMI MIC50/90were 0.5/>16 and MERO 0.12/>16mg/L. The addition of UP17 to AMI increased AMI MICs >2 fold in 78 strains. Equivalent increases in AMI MIC were seen with 39 strains with JK08, 54 strains with 113 and 45 strains with the cocktails. In contrast, meropenem MICs in the presence of phage were reduced >2 fold in 24 strains with UP17. Equivalent decreases in MERO MIC were seen with 34 strains with JK08, 26 strains with 113 and 29 strains with the cocktails. In TKCs addition of phage suppressed regrowth.ConclusionMicrobroth methodologies based on ISO 2776-1:2019 and TKCs allow the interaction between Phages and antibacterials to be studied. Optimisation may produce laboratory-based methods with translational value.