Development of antibacterial drug plus bacteriophage combination assays

Background The methods to evaluate the interactions between Phages and antibacterials are unclear. As the laboratory methodologies used to assess conventional antibacterials are well established, we assessed their efficacy in evaluating phage plus antibacterial. Methods 100 multidrug resistant E. coli strains were used with three previously isolated and characterised E. coli phages 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 -108 were inoculated with 5x105/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. Results The PmIC50/90 for UP17 were >108/>108; JK08 107/>108; 113 107/>108 and the 1:1:1 cocktail 106/>108. AMI MIC50/90 were 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. Conclusion Microbroth 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. ### Competing Interest Statement APM holds research grants/activities with Merck, Shionogi, InfectoPharm, GSK, Roche, Bioversys, and Nosopharm. Other authors declare no competing interests.