Topically Applied Bacteriophage to Control Multi-Drug Resistant Klebsiella pneumoniae Infected Wound in a Rat Model

Abstract

(Background): Multi-drug-resistant Klebsiella pneumoniae (MDR-KP) has steadily grown beyond antibiotic control. Wound infection kills many patients each year, due to the entry of multi-drug resistant (MDR) bacterial pathogens into the skin gaps. However, a bacteriophage (phage) is considered to be a potential antibiotic alternative for treating bacterial infections. This research aims at isolating and characterizing a specific phage and evaluate its topical activity against MDR-KP isolated from infected wounds. (Methods): A lytic phage ZCKP8 was isolated by using a clinical isolate KP/15 as a host strain then characterized. Additionally, phage was assessed for its in vitro host range, temperature, ultraviolet (UV), and pH sensitivity. The therapeutic efficiency of phage suspension and a phage-impeded gel vehicle were assessed in vivo against a K. pneumoniae infected wound on a rat model. (Result): The phage produced a clear plaque and was classified as Siphoviridae. The phage inhibited KP/15 growth in vitro in a dose-dependent pattern and it was found to resist high temperature (˂70 °C) and was primarily active at pH 5; moreover, it showed UV stability for 45 min. Phage-treated K. pneumoniae inoculated wounds showed the highest healing efficiency by lowering the infection. The quality of the regenerated skin was evidenced via histological examination compared to the untreated control group. (Conclusions): This research represents the evidence of effective phage therapy against MDR-KP.

Keywords: Gram-negative; Klebsiella pneumoniae; antibiotics; bacteriophage; bioinformatics; immunohistochemical (IHC); in vivo; multi-drug resistance (MDR); phage characterization; phage isolation; phage therapy; wound healing; wound infection.

Figure 1

Transmission electron microscopic image of phage ZCKP8.

Figure 2

Phylogenetic relationships between ZCKP8 phage and BLASTN top-matched phages. The closely related phage is Klebsiella phage ZX4 (GenBank Acc. No. NC 054654.1).

Figure 3

The ZCKP8 genome map. Only CDSs with known functions are labeled in their positions. Those representing hypothetical proteins are not labeled.

Figure 4

These plots represent the in vitro activity of phage ZCKP8 at 37 °C. These panels show bacterial counts and phage titers of K. pneumoniae KP/15 infected with ZCKP8 at (A) MOI of 0.1; (B) MOI of 1; (C) MOI of 10. MOI: Multiplicity of infection; CFU: Colony-forming unit; PFU: Plaque forming unit.

Figure 5

These diagrams represent the stability of phage ZCKP₈ at different temperatures (A), at different pH values (B), and under UV lamp (C). PFU: Plaque forming unit.

Figure 6

Morphometric examination of full-thickness excision wounds in rats at a fixed focal distance by Millimetre (mm) using digital photographs.

Figure 7

The wound healing percentage from day 0 to the 17th day in the four groups.

Figure 8

Histological analysis of wounded skin section from different rat groups at the 17th-day post wounding. Hematoxylin and eosin (H&E) staining, ×100, Masson Trichrome stain, ×100 and IHC by using α-SMA, and CD-45, ×400 showed in the non-infected, non-treated group complete skin wound healing and remodeling, the infected non-treated group shows epidermal ulceration and crust formation, early inflammatory phase of wound healing and a minimal amount of fibrosis, denoting delayed wound healing. While the infected groups treated with phage suspension and phage embedded in gel show, a complete wound healing and remodeling with adequate amount of fibrosis and epidermal re-epithelialization in the latter group denoting complete maturation of the healing process.