| dc.description.abstract | Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has been difficult to eradicate from hospital settings due to its ability to form biofilm and colonize biotic and abiotic surfaces. Phages may represent an alternative to antibiotic therapy and prophylaxis for biofilm-forming bacteria, but the interaction between phages and encapsulated bacteria such as A. baumannii is not well understood. Recently, bacteriophage Petty was isolated against A. baumannii strain AU0783. Analysis of its genome suggested that it encoded a depolymerase that degrades the host capsular exopolysaccharide (EPS). The gene was cloned and the purified protein was shown to reproduce the halo phenotype observed around phage Petty’s plaque, degrade purified host EPS, and affect biofilm formation of 5 different A. baumannii isolates. It is our aim to determine the cellular requirements for Petty to infect the host cell by characterizing the EPS of bacterial mutants of AU0783 that are resistant to phage infection. We isolated 42 mutants of AU0783 resistant to Petty and determined the titer of the phage. The capsule of these mutants and the wild-type strain was observed and its thickness measured. The ability of the mutants to produce biofilm was assessed using a microtiter plate assay. The EPS of 8 mutant strains was purified; the ability of the phage’s depolymerase to degrade EPS and produce reducing ends was measured using a colorimetric assay and the effect of the enzyme on the viscosity of EPS solutions was established. No plaques were observed under the given conditions in 91% of the Petty-resistant mutants isolated (EOP <10-7). The titer of the phage on the remainder of the mutant strains was reduced 3 to 7 orders of magnitude, depending on the mutant. Eight mutants were selected for further studies, based on biofilm production and on capsule formation compared to the wt strain AU0783. Purified EPS isolated from mutants B11, D6, and E8 was unable to be degraded by the depolymerase of Petty, as judged by colorimetric assay, and by the viscosity of the capsular polysaccharide when treated with the enzyme. The EPS viscosity of mutant D14 was reduced when treated with active depolymerase, but the halo phenotype was not reproduced on a bacterial lawn. Studies aimed at finding phage host range mutants that would infect these selected strains are underway. The differences between these phages and the mutant strains will give insight on the mechanism of phage infection of encapsulated A. baumannii. | en |
