title: 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 (10(4) and 10(6) 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.
authors.affiliation: Laboratory of Gene Technology, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Anneleen
authors.initials: A
authors.lastname: Cornelissen
authors.forename: Pieter-Jan
authors.initials: PJ
authors.lastname: Ceyssens
authors.forename: Jeroen
authors.initials: J
authors.lastname: T'Syen
authors.forename: Helena
authors.initials: H
authors.lastname: Van Praet
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Noben
authors.forename: Olga V
authors.initials: OV
authors.lastname: Shaburova
authors.forename: Victor N
authors.initials: VN
authors.lastname: Krylov
authors.forename: Guido
authors.initials: G
authors.lastname: Volckaert
authors.forename: Rob
authors.initials: R
authors.lastname: Lavigne
mesh_terms: D000595:Amino Acid Sequence; D016667:Bacterial Capsules / Q000378:metabolism; D018441:Biofilms; D017124:Conserved Sequence; D016679:Genome, Viral / Q000235:genetics; D006026:Glycoside Hydrolases; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017105:Pseudomonas Phages / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000502:physiology* / Q000821:virology*; D016415:Sequence Alignment; D017376:Viral Tail Proteins / Q000737:chemistry / Q000378:metabolism; D014771:Virion / Q000378:metabolism*
chemical_list: D017376:Viral Tail Proteins; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage