The therapeutic potential of bacteriophages targeting gram-negative bacteria using Galleria mellonella infection model

Prasanth Manohar, Ramesh Nachimuthu, Bruno S. Lopes

Phage therapy is the therapeutic use of bacteriophages to treat highly drug resistant bacterial infections. The current surge in bacteriophage therapy is motivated mainly because of the emergence of antibiotic-resistant bacteria in clinics. This study evaluated the therapeutic potential of three bacteriophages isolated against Escherichia coli ec311, Klebsiella pneumoniae kp235 and Enterobacter cloacae el140 strains using Galleria mellonella. The in vitro activity of three different phages belonging to Podoviridae and Myoviridae families was studied by the double agar overlay method against multi-drug resistant strains. Larval survivability studies were performed to evaluate the potential of phages against infection using G. mellonella. All the three phages were found to have potential to infect the host bacterial strains. For in vivo studies it was observed that E. coli and E. cloacae infected larvae, should be treated with three phage doses (20 μL, 104 PFU/mL) at 6 h interval to achieve 100% survival rate. But in the case of K. pneumoniae, a single phage dose treatment showed promising outcome. When mixed bacterial infections (all three bacterial cultures at 108 CFU/mL) were tested, minimum of four doses of phage cocktail (three phages) at 6 h interval was necessary to recover the larvae. All the results were confirmed by enumerating bacteria from the larvae. Our data shows that although in vitro studies showed high infectivity of phages, for in vivo models multiple phage doses were required for effective treatment.