Bacteriophage as a potential therapy to control antibiotic-resistant Pseudomonas aeruginosa infection through topical application onto a full-thickness wound in a rat model

Abstract

Background: Antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa) is one of the most critical pathogens in wound infections, causing high mortality and morbidity in severe cases. However, bacteriophage therapy is a potential alternative to antibiotics against P. aeruginosa. Therefore, this study aimed to isolate a novel phage targeting P. aeruginosa and examine its efficacy in vitro and in vivo.

Results: The morphometric and genomic analyses revealed that ZCPA1 belongs to the Siphoviridae family and could infect 58% of the tested antibiotic-resistant P. aeruginosa clinical isolates. The phage ZCPA1 exhibited thermal stability at 37 °C, and then, it decreased gradually at 50 °C and 60 °C. At the same time, it dropped significantly at 70 °C, and the phage was undetectable at 80 °C. Moreover, the phage ZCPA1 exhibited no significant titer reduction at a wide range of pH values (4-10) with maximum activity at pH 7. In addition, it was stable for 45 min under UV light with one log reduction after 1 h. Also, it displayed significant lytic activity and biofilm elimination against P. aeruginosa by inhibiting bacterial growth in vitro in a dose-dependent pattern with a complete reduction of the bacterial growth at a multiplicity of infection (MOI) of 100. In addition, P. aeruginosa-infected wounds treated with phages displayed 100% wound closure with a high quality of regenerated skin compared to the untreated and gentamicin-treated groups due to the complete elimination of bacterial infection.

Conclusion: The phage ZCPA1 exhibited high lytic activity against MDR P. aeruginosa planktonic and biofilms. In addition, phage ZCPA1 showed complete wound healing in the rat model. Hence, this research demonstrates the potential of phage therapy as a promising alternative in treating MDR P. aeruginosa.

Keywords: Bacteriophage; Biofilm; Immunohistochemical (IHC); In vivo; Multi-drug resistant (MDR); Phage characterization; Phage isolation; Pseudomonas aeruginosa; Wound infection.

Fig. 1

Transmission electron microscopic image of phage ZCPA1

Fig. 2

The physical stability of the phage ZCPA1. A Phage viability at different temperatures, B phage viability at different pH values, and C phage viability under UV light (λ = 253 nm). ns stands for no significance, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 3

Phylogenetic relationships between phage ZCPA1 and BLASTn top-matched phages. The closely related phage is Pseudomonas phage vB_PaeS_PcyII-40_PfII40a isolate genome (GenBank Acc. No. LT608331.1)

Fig. 4.

A One-step growth curve of ZCPA1 at MOI 1. IC indicates phage infective centers, and PFU indicates plaque forming unit. B Phage killing curve at different MOIs over 240 min

Fig. 5

The impact of different MOIs on biofilms clearance. ns stands for no significance, *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 6

The nontreated and noninfected wound (positive control; group 1), nontreated infected wound (negative control; group 2), infected wound treated with gentamicin antibiotic (group 3), infected wound treated with multiple phage doses (group 4), and the infected wound treated with single phage dose (group 5) from day zero till day 17. A Morphometric analysis of full-thickness excision infected wounds in rats by millimeter (mm) at a fixed focal distance. B Percentage of wound closure throughout the 17 days. C Enumeration of bacteria in the wound for the four groups throughout 14 days

Fig. 7

Representative histopathological images of wound tissues on day 17. A Hematoxylin and eosin (H&E) staining, ×100, Masson Trichrome stain, ×100, and B IHC by using α-SMA and CD-45, ×400 (skin adnexa; white arrow, skin ulceration; black arrow, epidermis; green arrow, blood vessels; red arrow, myofibroblasts; blue arrow, lymphocytes; yellow arrow)