Antibacterial effect of a new phage, MIZ-Pa99, in the treatment of Pseudomonas aeruginosa keratitis in an animal model.

BACKGROUND

Pseudomonas aeruginosa is one of the common bacterial agents in keratitis and is resistant to drugs. Due to the difference in the mechanism of action of antibacterial compounds, phage therapy can be a therapeutic candidate to deal with drug-resistant infections. The purpose of this study is to isolate, identify, and investigate the antimicrobial ability of a phage sample to treat keratitis caused by P. aeruginosa.

MATERIALS AND METHODS

The Bacteriophage (phage) MIZ-Pa99 isolated from hospital wastewater in Tehran, Iran was identified by TEM imaging. The presence of phage was confirmed by the spot technique and its titration was evaluated using the double-layer agar culture method. Phage lytic activity was evaluated through host range determination against P. aeruginosa, S. aureus, and E. coli, and biofilm destruction assays. Phage's novelty was determined through whole-genome sequencing. Phage safety was assessed using in vitro cytotoxicity assays and in vivo Draize eye irritation tests in rabbit eyes. Therapeutic efficacy was investigated in a mouse model using phage eye drops, with bacterial load quantified by CFU counts and tissue damage assessed by histopathology. Also, the antibiotic resistance pattern of Pseudomonas clinical isolates was investigated.

RESULTS

MIZ-Pa99 belongs to the Straboviridae family and the Caudoviricetes order. Whole genome analysis of MIZ-Pa99 phage showed that it belongs to Elvirus genus, and has linear and double-stranded DNA with a length 209,183 bp and a GC content of 49.30 %. The anti-biofilm effect of phage was observed on clinical isolates. IC50 of phage (HCE-2) was determined as 10 dilution (9.93 × 10 PFU/ml). The synergistic effect of ciprofloxacin and phage in the form of eye drops was observed in the improvement of keratitis caused by P. aeruginosa.

CONCLUSION

The results obtained in this study indicated that MIZ-Pa99 can be effective in controlling bacterial keratitis infection. However, combined antibiotic and phage treatment has a synergistic effect.