Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in controlling Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial disease among immunocompromised and chronic cystic fibrosis (CF) patients. We characterized two newly isolated Pseudomonas phages, ϕPA01 and ϕPA02, with different host spectra, and examined their effect as a cocktail with antibiotics against P. aeruginosa, to indicate the possibility of combining a phage cocktail and antibiotics in treating pseudomonal infection. Phages ϕPA01 (66,220 bp) and ϕPA02 (279,095 bp) belong to the genus Pbunalikevirus and Phikzlikevirus, respectively. No virulence or lysogenic associated gene was found in their genomes, thus they are potentially safe for phage therapy. We generated respective phage-resistant strains to investigate cross-resistance between two phages. Slight cross-resistance to ϕPA02 in ϕPA01-resistant strain was observed, while ϕPA02-resistant strain remained susceptible to ϕPA01. A ϕPA01 resistant strain that was cross-resistant to ϕPA02 appeared in round 5 (R5-PA01R), revealed frameshift mutation in phosphoglucomutase (algC), which is important for the synthesis of core lipopolysaccharide (LPS). Knockout of algC was resistant to both phages. Complementation of ΔalgC restored phages' infectivity, suggesting that LPS as host receptor. Phage cocktail suppressed the growth of P. aeruginosa for longer (20 h) hour compared with single phage (8-9 h), further suggesting their potential to be used as a phage cocktail. Furthermore, application of the phage cocktail with ciprofloxacin (0.25 µg/ml) and meropenem (2 µg/ml), managed to suppress the growth of P. aeruginosa up to 96 h. Our results show the potential application of ϕPA01 and ϕPA02 as phage cocktail together with antibiotics for treatment of P. aeruginosa.

Characterization of the depolymerizing activity of commercial lipases and detection of lipase-like activities in animal organ extracts using poly(3-hydroxybutyrate-co-4-hydroxybutyrate) thin film.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the polyhydroxyalkanoate (PHA) copolymers which can be degraded by lipases. In this study, the depolymerizing activity of different known commercial lipases was investigated via microassay using P(3HB-co-92 mol % 4HB) thin film as substrate. Non-enzymatic hydrolysis occurred under conditions in which buffers with pH 12 and 13 were added or temperature of 50 °C and above. Different concentrations of metal ions or detergents alone did not cause the film hydrolysis. The depolymerizing activity of lipases on P(3HB-co-4HB) was optimum in the pH range of 6-8 and at temperatures between 30 and 50 °C. Addition of metal ions and detergents in different concentrations was also shown to cause variable effects on the depolymerizing activity of commercial lipases. Pancreatic extracts from both mouse and chicken showed similar depolymerizing activity as the commercial lipases on the P(3HB-co-4HB) film. The presence of lipolytic enzymes in the organ extracts was confirmed with another lipase activity assay, p-nitrophenyl laurate assay. For the first time this has produced a direct evidence for the involvement of lipase-like enzymes from animal in the degradation of this PHA. Lipase is most likely the enzyme from pancreas that was involved in the degradation.