Evaluation of the efficacy of a bacteriophage in the treatment of pneumonia induced by multidrug resistance Klebsiella pneumoniae in mice

Abstract

Multidrug-resistant Klebsiella pneumoniae (MRKP) has steadily grown beyond antibiotic control. However, a bacteriophage is considered to be a potential antibiotic alternative for treating bacterial infections. In this study, a lytic bacteriophage, phage 1513, was isolated using a clinical MRKP isolate KP 1513 as the host and was characterized. It produced a clear plaque with a halo and was classified as Siphoviridae. It had a short latent period of 30 min, a burst size of 264 and could inhibit KP 1513 growth in vitro with a dose-dependent pattern. Intranasal administration of a single dose of 2×10(9) PFU/mouse 2 h after KP 1513 inoculation was able to protect mice against lethal pneumonia. In a sublethal pneumonia model, phage-treated mice exhibited a lower level of K. pneumoniae burden in the lungs as compared to the untreated control. These mice lost less body weight and exhibited lower levels of inflammatory cytokines in their lungs. Lung lesion conditions were obviously improved by phage therapy. Therefore, phage 1513 has a great effect in vitro and in vivo, which has potential to be used as an alternative to an antibiotic treatment of pneumonia that is caused by the multidrug-resistant K. pneumoniae.

Figure 1

Morphology of K. pneumoniae phage 1513. (a) Electron micrograph of phage 1513. (b) Plaque morphologies of phage 1513.

Figure 2

Stability of phage 1513 under different pH (a) or temperature (b).

Figure 3

(a) Phage 1513's one-step growth curve was measured. Bacteriophages showed a latent period of about 30 min and a burst size of 264 PFU mL⁻¹. (b) Lytic ability of phage 1513 in vitro. KP (2 × 10⁹ CFU mL⁻¹) was infected with the phage at different multiplicities of infection (MOI). The optical density at 650 nm was determined at each timepoint. Infection dose of phage: none (▲), 2 × 10¹⁰ PFU mL⁻¹ (▼), 2 × 10⁹ PFU mL⁻¹ (●), and 2 × 10⁸ PFU mL⁻¹ (■).

Figure 4

Efficiency of phage 1513 in vivo. Survival curves of the infected mice treated with either PBS or phages at different multiplicities of infection (MOI). A group of 10 mice were inoculated via the intranasal route with 2 × 10⁸ CFU/mouse K. pneumoniae. Various doses of phage 1513 were administrated intranasally 2 h after infection. Survival curves were compared for significance with the log-rank test of the results obtained from the following parameters: from the phage 1513 (2 × 10⁷ PFU/mouse) treatment group versus those from the control group (P = 0.0058), from the phage 1513 (2 × 10⁸ PFU/mouse) treatment group versus those from the control group (P = 0.0018), and from the phage 1513 (2 × 10⁹ PFU/mouse) treatment group versus those from the control group (P = 0.0002).

Figure 5

Histopathology of KP-infected lung tissue from PBS- or phage 1513-treated mice. A group of 10 mice were inoculated intranasally with 6 × 10⁷ CFU KP/mouse. Additionally, phage 1513 was administered intranasally. The mice were euthanized 24 h after infection, and lung tissues were removed as described above. (a) PBS, without K. pneumoniae; (b) K. pneumoniae, treated with PBS; (c) K. pneumoniae, treated with phage 1513 ((a1), (b1), and (c1) were amplified 40 times; (a2), (b2), and (c2) were amplified 100 times; and (a3), (b3), and (c3) were amplified 200 times).

Figure 6

Body weight loss, bacterial burden, and the phage number present in the lung after phage treatment of K. pneumoniae infection. Groups of 6 mice were inoculated via the intranasal route with 6 × 10⁷ CFU/mouse K. pneumoniae. PBS or phage 1513 (6 × 10⁷ PFU/mouse) was administered intranasally 2 h after infection. The numbers of bacteriophage (a) or bacteria (b) in the lung homogenates were determined. Mice were weighed (c). Bacteria counts and weight loss rate were significantly lower in the lung homogenate from the mice in the phage-treated group than in the control group (P = 0.0022 and P = 0.0087). Bacteriophage counts were significantly higher in the infected group than in the noninfected animals (P = 0.0022).

Figure 7

Cytokine assay of lung tissue after phage treatment of K. pneumoniae infection. Groups of 6 mice were inoculated via the intranasal route with 6 × 10⁷ CFU/mouse K. pneumoniae. PBS or phage 1513 (6 × 10⁷ PFU/mouse) was administered intranasally 2 h after infection. The mice were killed 24 h after infection, and the inflammatory cytokines of the lung homogenate were collected and detected. Cytokine concentrations were remarkably lower in the phage-treated group than in the control group. (a) TNF-α (P = 0.0159). (b) IL-6 (P = 0.0079).