The phage cocktail has considerably enhanced the therapeutic value of phage therapy, mainly by circumventing the potential for bacteria to evolve phage resistance. However, the formulation of a cocktail with a broad host range is still a matter of investigation, and the use of phage cocktails as a frontline treatment is far from being met. Here, we consider hospital-acquired infections caused by multi-drug resistant clones of Enterobacter cloacae complex (ECC) and aim to develop a phage cocktail using broad-spectrum phages, each with distinct receptors for strains of the targeted pathogen. Furthermore, we speculate that the overall activity of the phage cocktail can be improved by screening it against a large bank of pathogens and by phage training.
We isolated 25 novel phages across 36 genetically diverse ECC, which were isolated from hospital-acquired infections. About two-thirds of the isolates belonged to four major sequence type (ST114, ST93, ST90 and ST88), with blaIMP-4 being the most common carbapenemase gene. We tested lytic activity of phages against all ECC using plaque assay, which revealed broad-spectrum phages that lysed up to 50% (18/36) of tested strains. Based on these results, we combined three phages, which had 65% coverage across the isolates. All three phages are lytic as observed from genetic and morphological features and have high killing efficiency as shown by the relative efficiency of platting assay and one-step growth curves. To identify receptors, we generated phage resistance mutants against their host of isolation and screened for loss-of-function mutations. Our analysis revealed each phage in the cocktails targeted distinct receptors, including glycosyl-transferase, uridyl-transferase and siderophore receptors. Combining these three characterised phages into a single treatment cocktail has produced a phage cocktail, with broad-spectrum activity, and reduced frequency of phage resistance that has been tailored to combat a nosocomial, multi-drug resistant ECC infections. The cocktail implements concepts of intelligent design and will be used as empirical therapy in the treatment of multidrug-resistant ECC infections.