Identification and characterization of phage protein and its activity against two strains of multidrug-resistant Pseudomonas aeruginosa

Fairoz Al-Wrafy,Sabina Górska,Andrzej Gamian,Ewa Brzozowska,Magdalena Strus,Marek Drab

doi:10.1038/s41598-019-50030-5

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen with a capacity to develop antibiotic resistance, which underlies a larger proportion of hospital-acquired infections and higher morbidity and mortality, compared to other bacterial infections. Effective novel approaches for treatment of infections induced by this pathogen are therefore necessary. Phage therapy represents a promising alternative solution to eradicate antibiotic-resistant pathogens. Here, we investigated phage protein efficacy against multi-drug resistant (MDR) P. aeruginosa PAR21 and PAR50 strains isolated from diabetic foot ulcer patients. The results obtained using spot assay, zymography, spectrophotometry and scanning electron microscopy at low voltage (SEM-LV) indicate that the phage protein, PA-PP, exerts activity against P. aeruginosa PAR50 while having no impact on the PAR21 strain. Using LC-MS-MS/MS and comparative analysis of the peptide molecular mass with the protein sequence database, PA-PP was identified as a member of the serine protease family, a result corroborated by its ability to digest casein. We additionally showed a capacity of PA-PP to digest porin protein on the bacterial outer membrane (OM). Moreover, synergistic activity between PA-PP protein and piperacillin led to higher sensitivity of bacterial cells to this antibiotic. Our collective findings suggest that PA-PP targets porin protein on PAR50 OM, thereby increasing its sensitivity to specific antibiotics. The adverse effects observed on bacterial cells using SEM-LV suggest further roles of this protein that remain to be established.

Highlights

Pseudomonas aeruginosa is an opportunistic pathogen responsible for several acute and chronic infections in humans, including meningitis, abscess, infection of skin, soft tissues, urinary tract, bones and joints and conjunctival erythema in addition to a variety of systemic infections in individuals with genetic diseases as in cystic fibrosis patients (CF), immunocompromised patients, diabetes mellitus patients, and those receiving chemotherapy[1]
The P. aeruginosa PAR21 and PAR50 strains used in this study demonstrated variance in response to antibiotics, with greater resistance of PAR50 than PAR21
The PAR21 strain was resistant to infection by phage whereas PAR50 appeared highly sensitive, as evident from the halo zone observed with the spot assay on the agar plate with or without plaques in the case of P. aeruginosa PAR50 but not PAR21, signifying efficacy of the phage against the PAR50 strain (Supplementary Fig. S1)

Summary

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen responsible for several acute and chronic infections in humans, including meningitis, abscess, infection of skin, soft tissues, urinary tract, bones and joints and conjunctival erythema in addition to a variety of systemic infections in individuals with genetic diseases as in cystic fibrosis patients (CF), immunocompromised patients, diabetes mellitus patients, and those receiving chemotherapy[1]. Regardless of the mechanism of resistance, the prevalence of MDR strains poses a critical medical problem that necessitates further comprehensive investigation to uncover novel approaches for eradicating these pathogens and associated diseases Phages and their components represent a suitable therapeutic solution in view of their ability to target pathogenic bacteria at the site of infection without affecting normal flora and gradual disappearance after the demise of the host in addition to their capacity to influence bacterial biofilms that play roles in the antibiotic resistance[1]. The most notable phage products for therapeutic consideration are phage-encoded peptidoglycan hydrolases (PGH) i.e. endolysins, polysaccharide depolymerases and holin (cell membrane-disrupting protein)[21], in addition to those involved in cell wall synthesis inhibition[20] These proteins exhibit high efficacy against bacterial cells, either killing them or leading to intrinsic changes in their structures, thereby facilitating lysis by other factors. Utilization of a specific phage against MDR P. aeruginosa triggered changes in the efflux pump mechanism critical in antibiotic resistance, which led to increased sensitivity to several antibiotics[33]

Methods

Results

Conclusion