Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms

Abstract

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n ‫؍‬ 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 ؋ 10 8 purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10 10 and 10 12 phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time-and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.

Bacteriophage-derived enzyme that depolymerizes the alginic acid capsule associated with cystic fibrosis isolates ofPseudomonas aeruginosa

“…Biofilms may be dispersed by bacteriophages that carry polysaccharide depolymerases on their surface (Sutherland et al 2004). Pseudomonas aeruginosa phages were found to decrease the viscosity and the molecular weight of purified CF alginate and, although not demonstrated in this study (Hanlon et al 2001), almost certainly carry alginate-degrading enzymes. In the current study, we identify and characterize a phageencoded enzyme that degrades alginate polysaccharides purified from Ps.…”

“…aeruginosa strains were purified essentially according to Hanlon et al (2001). Briefly, large square assay plates (30 · 30 cm) containing tryptone soya agar (TSA) were seeded with sufficient bacteria to produce confluent growth.…”

Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage–antibiotic treatments

“…Similar to antibiotics, extracellular polysaccharides and proteins could prevent phage adsorption to its receptors while metabolically less active cells are less likely to get infected (Abedon, 2017; Labrie, Samson, & Moineau, 2010). However, certain phages have the ability to produce enzymes, which can degrade the extracellular polymeric matrix (Hanlon, Denyer, Olliff, & Ibrahim, 2001; Hughes, Sutherland, Jones, & Rutherford, 1998), while other phages are able to propagate radially through a biofilm (Doolittle, Cooney, & Caldwell, 1996). Surprisingly, little is currently known about phage–antibiotic effects in biofilms.…”

Fluorescence Correlation Spectroscopy To Study Diffusion and Reaction of Bacteriophages inside Biofilms

“…To answer this question, it is necessary to understand that most phages that target bacteria in natural environments are thought to exist in biofilm ecosystems. The reciprocal effects of biofilm structure and the activity of the prophages released have previously been demonstrated for different bacterial species (25,44,57). However, the reaction of biofilms to ubiquitous viral particles in the environment is poorly documented (52).…”