Isolation of bacteriophages against Klebsiella pneumoniae and in vivo activity

Klebsiella pneumoniae is a bacterial pathogen able to induce severe healthcare-associated or community-acquired infections in humans and animals. The constant emergence of antibiotic resistant strains reinforces the need to find alternatives to antibiotic treatments. The use of bacteriophages is a promising approach. The aim of this study was to isolate bacteriophages directed against K. pneumoniae strains and to test their efficacy in a murine model. Bacteriophages against five different K. pneumoniae (2 of capsular type K1 and K2 and 1 undetermined) were isolated and purified from waste water collected in Paris area. The morphology of plaques (zones of bacterial killing) was recorded and several of them were purified three times by successive replating. Phage titers were determined by serial dilutions on their respective hosts as well as on 18 other Klebsiella strains to identify their host range. Kinetics of bacterial lysis were monitored during 15h at 3 multiplicities of infection, in triplicates. For in vivo experiment, a total of 10 mice were inoculated with 200 µl of K. pneumoniae (4.6E+07 CFU) by oral gavage and the level of K. pneumoniae in fecal samples was monitored for 10 days. Five mice did not receive any treatment and 5 other mice received a cocktail of three bacteriophages (8E+07 PFU) at day 4 post-inoculation. A total of 54 bacteriophages were isolated and purified with titers ranging from 2E+5 to 3.6E+10 PFU/ml. The host range study showed that bacteriophages against K. pneumoniae have a specificity related to the capsular type of their bacterial host. Lysis kinetics of bacteria suggested that different phages were isolated. Despite difficulties with the murine intestinal model, evidence was obtained that bacteriophages are able to reduce intestinal carriage. Our results show that bacteriophages isolated against K. pneumoniae are specific for a given capsular type, although further studies are necessary to provide more details on this capsular specificity and its molecular determinants. To fully address the in vivo potential of phages, a reliable mouse model of intestinal carriage of K. pneumoniae strains needs to be established.