Therapy of Experimental Pseudomonas Infections with a Nonreplicating Genetically Modified Phage

Abstract

Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection. An export protein gene of the P. aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of P. aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.

Efficacy of Bacteriophage Therapy against Gut-Derived Sepsis Caused by Pseudomonas aeruginosa in Mice

“…Reports have revealed that a single treatment with phage leads to recovery in mice with infections caused by E. coli (34), P. aeruginosa (12), methicillin-resistant Staphylococcus aureus (24), and vancomycin-resistant Enterococcus faecium (6). In the present study, we also found that a single treatment with phage strain KPP10 prevented the death of mice after gutderived sepsis and intraperitoneal infection with P. aeruginosa.…”

“…The therapeutic efficacy of phage therapy against infectious diseases caused by P. aeruginosa (12), Staphylococcus aureus (including methicillin-resistant S. aureus) (24), Escherichia coli (3), Enterococcus faecium (including vancomycin-resistant Enterococcus) (6), and Streptococcus pneumoniae (16) has been shown in experimental animal models. However, the models used in those studies were simple models of infection that did not closely resemble the pathophysiologies of diseases in humans.…”

“…However, intraperitoneal and intravenous administration of KPP10 may be more likely to result in adverse effects caused by increased levels of endotoxins. One study found that the levels of endotoxin released after the administration of phages were 30-to 60-fold higher in lytic phage-treated mice than in non-lytic phage-treated mice (12). Because of the possibility of adverse effects, we believe that oral administration of phage strain KPP10 is preferable.…”

Genome sequence comparison and superinfection between two related Pseudomonas aeruginosa phages, D3112 and MP22

“…They will also provide essential information for the development of new phage vectors and therapeutic agents to control infection by P. aeruginosa (Darzins & Casadaban, 1989a;Groisman & Casadaban, 1987;Hagens et al, 2004). Plaque formation on P. aeruginosa lysogens.…”

“…The emergence of new P. aeruginosa strains and the persistence of the existing antibiotic-resistant clinical isolates has led to an urgent need to explore alternative strategies for managing P. aeruginosa-mediated infections. Among these, bacteriophages have emerged as potentially attractive alternative anti-infection modalities (Sulakvelidze et al, 2001), as exemplified by the success of a genetically modified filamentous phage (Pf3R) in controlling experimental P. aeruginosa infections (Hagens et al, 2004).…”