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

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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.

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Bacteria, culture media and growth condition

Standard strain P. aeruginosa PAO1 was used in all experiments, unless otherwise stated. All experiments were conducted in Luria–Bertani (LB) broth (10 g polypeptone, 10 g sodium chloride and 5 g yeast extract per liter) at 37 °C, with shaking at 120 rpm, unless otherwise stated.

Isolation and preparation of phage stock

Two lytic phages, ϕPA01 and ϕPA02, were isolated from sewage influent obtained from the municipal wastewater treatment plant in Tokyo using P. aeruginosa PAO1 as the host by double layer agar plating method. Phages were

Morphology of phage ϕPA01 and ϕPA02

Based on the morphology of ϕPA01 and ϕPA02 observed (Fig. 1A–D), they were classified to the Myoviridae family. Both phages displayed a capsid head connected to a long contractile tail (Fig. 1B and D, indicated by black arrows). ϕPA02 showed a distinct morphology with a large capsid with the diameter and height of 121 ± 3 nm and 134 ± 4 nm, respectively. This feature is similar to reported giant phages (37). The relaxed tail fiber of ϕPA02 was 206 ± 7 nm long and 25 ± 1 nm wide. Meanwhile, the

Genomic characterization of novel phages

A well characterized phage is one of the key factors determining the success of a phage therapy (18). Phages from the genera Pbunalikevirus and Phikzlikevirus were able to control the growth and biofilm formation of P. aeruginosa in vitro (42) and in vivo (43,44) in previous studies. Genomic analysis has revealed the absence of integrase, genes associated with toxin and virulence in ϕPA01 and ϕPA02, suggesting that they are potentially safe candidates for phage therapy (18). Previous study

Acknowledgments

We are thankful to Professor Longzhu Cui, Dr. Shinya Watanabe and Dr. Kitaro Kiga (Jichi Medical Hospital University, Department of Bacteriology) for kindly supporting us in conducting the experiment using the clinical strains collection from Jichi Medical University Hospital. S.P. Ong received scholarship from Ministry of Education, Culture, Sports, Science and Technology of Japan during her post graduate study. All authors declared that there are no conflicts of interest in this article. This

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