The T7-Related Pseudomonas putida Phage φ15 Displays Virion-Associated Biofilm Degradation Properties

Abstract

Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a ‘T7-like virus’ with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (104 and 106 pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections

“…In a previous study (unpublished data), we found that the plaques of the bacteriophages F770/05, F510/08 and F1245/ 05 were surrounded by growing opaque halo zones, which could be related to the presence of a virion-associated exopolysaccharide depolymerase (Cornelissen et al, 2011). This and related enzymes have been found to enhance the biofilm-eradicating activity of bacteriophages compared with non-depolymerase-producing bacteriophages (Hughes et al, 1998).…”

Morphology, Physiological Characteristics, and Complete Sequence of Marine Bacteriophage ϕRIO-1 Infecting Pseudoalteromonas marina

“…There is no indication from the sequence analysis that RIO-1 can be temperate; hence, we do not believe that the turbid zones come from that source. Other precedented causes of turbid lysis zones include phage-borne or phage-produced enzymes that attack host extracellular polymers (56), feeding of hosts by phage metabolites (57), or lysis inhibition related to superinfection (58).…”

Personalized Therapeutic Cocktail of Wild Environmental Phages Rescues Mice from Acinetobacter baumannii Wound Infections

“…Interestingly, immediately surrounding the AB-Army1 plaque, there was a large zone of AB5075 that was phenotypically distinct from AB5075 in the adjacent lawn. Halos of this kind implicate a diffusible phage-encoded protein causing an enzymatic change to the surface of the bacteria, a well-recognized phenomenon of phages that infect capsular bacteria (37)(38)(39)(40)(41)(42)(43)(44). An efficiency-of-plating assay showed that when a dilution series from 10 10 to 10 2 of AB-Army1 was spotted on an overlay plate of AB5075, AB-Army1 could infect at a concentration as low as 10 2 PFU per ml.…”