Isolation and Characterization of a Novel Lytic Phage N22 and Its Effect on Drug-Resistant Klebsiella Pneumoniae

Abstract

Background: Klebsiella pneumoniae (KP) infections present a significant clinical challenge and are frequently associated with elevated drug resistance. The use of phage therapy has resurged in response to escalating antibiotic resistance. This study aimed to address the multidrug resistance crisis in intensive care units by exploring the use of ceftazidime/avibactam (CAZ/AVI), a widely used clinical antimicrobial agent, in conjunction with phage therapy.

Materials and methods: We screened a clinical strain of KP from ICU and successfully isolated phage N22 from hospital wastewater. We conducted an in-depth analysis of the physiological and biochemical properties of phage N22 and determined its optimal multiplicity of infection with the clinical KP strain. The inhibitory effects of phage N22 in combination with CAZ/AVI on biofilm formation were investigated. Comparative efficacies of these combinations were evaluated using a Galleria mellonella (G. mellonella) model.

Results: Phage N22 inhibited KP biofilm formation. The impact of varying phage N22 concentrations when used alongside CAZ/AVI was examined, and the combination of phage N22 and CAZ/AVI was more effective against KP than CAZ/AVI alone.

Conclusion: This study provides a preliminary investigation into the effects of combining CAZ/AVI with phage therapy, highlighting its potential significance in developing novel therapeutic strategies for bacterial infections resistant to CAZ/AVI. The findings underscore the importance of advancing highly effective phage agents as alternative treatment modalities for patients with infections refractory to conventional antibiotics.

Keywords: Klebsiella pneumoniae; ceftazidime/avibactam; drug-resistant; phage therapy.

Figure 1

Visual representations of plaques and transmission electron microscopy of phage N22. (A) Plaque formation by phage N22. (B) Transmission electron micrograph of phage N22, with a scale bar indicating 100 nm.

Figure 2

Biological characteristics of phage N22. (A) Optimal multiplicity of infection (MOI) of KP-ASM with phage N22. (B) One-step growth curve of phage N22 on KP-ASM. (C) pH sensitivity: Phage titer of N22 following incubation at various pH values at 37 °C for 1 hour. (D) Thermal stability: Phage titer of N22 measured after 1-hour incubation under different temperature conditions. Data represent the mean of three independent experiments.

Figure 3

Impact of Phage Treatment on Mature Biofilms. (A) The optical density (OD) of the biofilm was measured after incubating well plates for 24 hours with the Staphylococcus aureus model strain ATCC 27853, KP-ASM, KP-ASM combined with phage N22, and LB as a blank control. This experiment was conducted in triplicate. (B) The experiment was also performed using a catheter culture biofilm containing KP-ASM, KP-ASM combined with phage N22, and LB as a control. The biofilm system was connected to a flow rate pump operating at 60 mL/h, and this experiment was similarly conducted in triplicate (****; p < 0.0001, ***; p = 0.0003 and **; p = 0.0017).

Figure 4

Growth characteristics and interaction plots illustrating phage-antibiotic synergy and the growth curve of KP-ASM under various conditions: (A) depicts a heat map representing the combined effect of phage N22 and CAZ/AVI on KP-ASM, the scale corresponds to optical density measurements (OD₅₉₅) at a wavelength of λ = 595 nm, where the depth of the colour represents an increase in the number of bacteria. (B) illustrates growth curves with CAZ/AVI alone (1024 µg/mL), (C) demonstrates growth curves with the combined addition of CAZ/AVI (1024 µg/mL) and phage N22 (10⁷ PFU/mL), (D) displays growth curves in the absence of phage N22 and CAZ/AVI, and (E) shows growth curves with the addition of phage N22 alone (10⁷ PFU/mL).

Figure 5

Survival plot of G.mellonella subjected to various phage-antibiotic combinations. The experimental groups include the PBS control group, the KP-ASM group, the N22 group, the Combination 128 µg/mL group, and the Combination 64 µg/mL group. (A) details the methodology for measuring the length of G.mellonella using alaboratory animal model. (B) illustrates the survival rates of G.mellonella following injection with the specified substances. The Orange line denotes the control group administered with PBS, while the blue line signifies the group treated with KP-ASM. The brown line indicates the group injected with N22, the cyan line represents the group receiving Combination 128 µg/mL, and the purple line corresponds to the group treated with Combination 64 µg/mL. The experiment was conducted in triplicate, and the resulting data were analyzed utilizing GraphPad Prism version 8.0.