Identification and preclinical efficacy evaluation of two lytic bacteriophages targeting highly virulent and multidrug-resistant Klebsiella pneumoniae

Abstract

Background: The emergence of MDR K. pneumoniae poses a critical challenge in treating respiratory-associated pneumonia. Bacteriophages are promising antibiotic alternatives with unique features. This study aimed to isolate new bacteriophages from the hospital environment and investigate their therapeutic potential and mechanisms.

Methods: We employed plaque assays, transmission electron microscopy, and whole-genome sequencing to systematically characterize the biological properties, morphology, and genomic profiles of the phages in parallel. The bacteriostatic curve, biofilm staining quantification, and biofilm inhibition rate assay were employed to evaluate the in vitro lytic efficacy of the phage. More importantly, we established the murine pneumonia infection models through nasal instillation, assessed the therapeutic potential of the phage in vivo by observing pathological morphology via HE staining, detecting pro-inflammatory cytokine levels via qPCR and ELISA, and monitoring bacterial load changes in lung tissue through PCR analysis.

Results: Phages vB_KpnP_XY3 and vB_KpnP_XY4, taxonomically classified as Siphoviridae, demonstrated broad temperature (4-60 °C), pH (4-11) tolerance, chloroform resistance, latent periods of 40/35 min, and burst sizes of 340/126 PFU/cell. Both genomes contained circular dsDNA genomes (47,466 bp/50,036 bp) without virulence or antibiotic resistance genes. The bacterial concentration markedly decreased at 2 h post-treatment, reaching its biological nadir by 6 h. Concurrent biofilm assays demonstrated 80% biofilm inhibition and rapid bacterial clearance. In murine pneumonia models, both phage monotherapy and phage-antibiotic combinations significantly reduced bacterial loads compared with antibiotics alone (P < 0.05), concurrently attenuating inflammation (IL-1β/IL-6/TNF-a. P < 0.0001) and restoring alveolar architecture with reduced necrosis.

Conclusion: The phages vB_KpnP_XY3 and vB_KpnP_XY4 demonstrated robust environmental adaptability. Its antibacterial effect is related to its specific biofilm dissolution performance in vivo and in vitro. These findings provide strong evidence for the precise phage treatment of MDR K. pneumoniae infections.

Keywords: Bacteriophage therapy; Biofilm degradation; Histopathology; Lytic bacteriophage; Multidrug-resistant Klebsiella pneumoniae.

Fig. 1

Morphological and genomic characterization of phages vB_KpnP_XY3 and vB_KpnP_XY4. Plaque morphology of vB_KpnP_XY3 (A) and vB_KpnP_XY4 (B) on K. pneumoniae host lawn. Transmission electron microscopy (TEM) images of vB_KpnP_XY3 (C) and vB_KpnP_XY4 (D). The genomic map of vB_KpnP_XY3 (E) and vB_KpnP_XY4 (F). Phylogenetic trees reconstructed via the neighbor-joining method (MEGA 11.0 software, 1,000 bootstrap replicates) on the basis of major capsid protein sequences, demonstrating the evolutionary relationships of vB_KpnP_XY3 (G) and vB_KpnP_XY4 (H) with related phages. Scale bars: 100 nm (C, D)

Fig. 2

Biological properties of phages vB_KpnP_XY3 and vB_KpnP_XY4. Optimal MOI of vB_KpnP_XY3 (A) and vB_KpnP_XY4 (B). Thermal stability of vB_KpnP_XY3 (C) and vB_KpnP_XY4 (D). PH stability of vB_KpnP_XY3 (E) and vB_KpnP_XY4 (F). Chloroform stability assays for vB_KpnP_XY3 (G) and vB_KpnP_XY4 (H). One-step growth curves of vB_KpnP_XY3 (I) and vB_KpnP_XY4 (J). The data are presented as the means ± SD (n = 3 independent experiments). (A) ^***P < 0.0001, vs. 10-1; (B) ^***P < 0.0001, vs. 10-2.

Fig. 3

Whole genome sequencing of the the K. pneumoniae strains Kpn32416. (A) The genomic map. (B) Annotation of drug resistance genes. (C)The table of partial high-toxicity genes

Fig. 4

Genotypic and phenotypic profiling of the K. pneumoniae strains Kpn32416 and Kpn31109. Antimicrobial susceptibility testing of the host bacteria Kpn32416 (A) and Kpn31109 (B). (C) Serotyping analysis of strain Kpn32416. (D) Antibiotic resistance gene profile of Kpn32416. (E) Virulence-associated genes identified in Kpn32416. (F) Serotyping analysis of strain Kpn31109. (G) Antibiotic resistance gene profile of Kpn31109. (H) Virulence-associated genes identified in Kpn31109. MD102 DNA marker (100–1200 bp. key bands highlighted at 700 bp). Full-length gel electrophoresis images are provided in Fig. S2

Fig. 5

In vitro bactericidal and biofilm-inhibitory activities of the phages. (A) Time-dependent lytic activity of the phage vB_KpnP_XY3 against its host strain at MOIs ranging from 100 to 0.001 over 7 h. (B) Lytic kinetics of phage vB_KpnP_XY4 at MOIs of 10 to 0.001 were monitored for 5 h. Biofilm inhibition rates (%) induced by vB_KpnP_XY3 (C. MOIs: 1–0.001) and vB_KpnP_XY4 (D. MOIs: 10–0.001). Biofilm biomass quantification by crystal violet staining for vB_KpnP_XY3 (E. MOIs: 1–0.001) and vB_KpnP_XY4 (F. MOIs: 10–0.001). Comparative lytic efficacy of individual phages (vB_KpnP_XY3, vB_KpnP_XY4, vB_KpnP_XY5) and their cocktails over short-term (G. 12 h) and extended (H. 4 days) incubation periods. (I) In vitro antimicrobial and antimicrobial effects of phages and antibiotics alone and in combination. Data are representative of three independent experiments (mean ± SD). ( ^**P < 0.01, ^***P < 0.0001

Fig. 6

In vivo therapeutic efficacy of phages against K. pneumoniae-induced pneumonia. (A) Experimental workflow for phage therapy administration. (B) Serum IL-6 levels were quantified via ELISA at 6 h, 12 h, 24 h, and 48 h post infection. (C) Histopathological evaluation of lung tissues stained with H&E at 6, 12, 24, and 48 h post infection. Representative micrographs (10× and 100× magnifications) demonstrate the following tissue architecture: nuclei (dark blue) and cytoplasmic proteins (pink). The data are expressed as the means ± SD (n = 3 mice per group). ^*P < 0.05, ^**P < 0.01, ^***P < 0.0001 versus untreated infection controls (two-way ANOVA with Tukey’s post-hoc test)

Fig. 7

Therapeutic efficacy of phage-antibiotic combinations (PACs) against K. pneumoniae - induced pneumonia in vivo. (A) Experimental workflow for PAC administration. (B) Histopathological assessment of lung tissues stained with H&E at 48 h post infection. Representative micrographs (10×, 100×, and 400× magnifications) illustrate the following tissue morphology: nuclei (dark blue) and cytoplasmic components (pink)

Fig. 8

Anti-inflammatory effects of phage therapy in K. pneumoniae-induced pneumonia. mRNA expression levels of the pro-inflammatory cytokines (A) IL-1β, (B) IL-6, and (C) TNF-α in lung tissues at 48 h post infection, as quantified by qRT-PCR. Protein levels of (D) IL-1β, (E) IL-6, and (F) TNF-α in lung homogenates at 24 h post infection, as measured via ELISA. Protein levels of (G) IL-1β, (H) IL-6, and (I) TNF-α in lung homogenates at 48 h post infection, as measured via ELISA. (^***P < 0.0001, Ctrl vs. Kpn. ^##P < 0.01, ^###P < 0.0001, Kpn + XY3 vs. Kpn, Kpn + Cock vs. Kpn, Kpn + GM vs. Kpn, Kpn + GM + XY3 vs. Kpn. ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.0001, Kpn + XY3 vs. Kpn + GM, Kpn + Cock vs. Kpn + GM, Kpn + GM + XY3 vs. Kpn + GM). The values are expressed as the means ± SD (n = 3 mice/group)

Fig. 9

Quantitative assessment of the pulmonary bacterial burden in K. pneumoniae Kpn32416-infected mice. (A) K. pneumoniae load in lung tissues quantified via the strain-specific genetic marker Kp-khe (PCR amplification). MD102 DNA marker (100–1200 bp. key band highlighted at 700 bp). (B) Total bacterial burden assessed by universal 16 S rRNA gene amplification. DL15000 DNA marker (250–15,000 bp. key band highlighted at 2500 bp). Full-length gel electrophoresis images are provided in Fig. S3

Fig. 10

Evaluation of the hepatotoxic, splenotoxic, and nephrotoxic effects of phages in a murine model. Histopathological analysis of the livers, spleens, and kidneys, which were observed at 48 h after phage treatment and captured under an optical microscope (100× magnification). The tissues were stained with hematoxylin-eosin. The nucleic acids, including components such as the nucleus, were stained dark blue, and the proteins, including components such as the cytoplasm, were stained pink