A lytic bacteriophage vB_KpnP-6K2 inhibits ST11-KL64 Klebsiella pneumoniae induced cell death and inflammatory response

Abstract

Introduction: The global dissemination of multidrug-resistant Klebsiella pneumoniae (Kpn) underscores the critical demand for alternative therapeutics such as bacteriophages. This study characterizes a novel bacteriophage, vB_KpnP-6K2 (6K2), isolated against a clinically relevant ST11-KL64 Kpn strain, and evaluates its potential for therapeutic application.

Methods: Phage 6K2 was morphologically examined by transmission electron microscopy and genomically analyzed via whole-genome sequencing. Its stability across pH and temperature ranges, adsorption kinetics, and burst size were determined in vitro. The inflammatory response to Kpn infection was assessed in HEK293T, A549, Hela, and THP-1 monocytic cells by measuring cytokine and chemokine expression, while cell death was evaluated in A549 lung epithelial cells. The therapeutic efficacy of 6K2 was tested in a lethal murine systemic infection model, where a single intraperitoneal dose was administered one-hour post-bacterial challenge. Survival, bacterial clearance, and phage kinetics in blood were monitored.

Results: Phage 6K2 exhibits a polyhedral head and short tail, classifying it within the Podoviridae family (Autographiviridae family, Przondovirus genus). Its double-stranded DNA genome comprises 40,147 bp. The phage demonstrated stability across a broad pH (4-12) and temperature (4-50°C) range, rapid adsorption, and a burst size of 13.6 PFU/cell. In vitro, Kpn infection significantly upregulated inflammatory mediators in THP-1 cells and induced death in A549 cells; both responses were potently inhibited by 6K2 treatment. In the murine infection model, a single dose of 6K2 achieved 100% survival, accompanied by rapid clearance of bacteremia and high initial phage titers in the blood.

Discussion: These findings highlight the promising potential of bacteriophage 6K2 as an effective therapeutic agent against multidrug-resistant Kpn infections. The phage not only suppresses bacterial load but also mitigates infection-associated inflammatory responses and cellular damage. The complete rescue in a lethal systemic infection model underscores it's in vivo efficacy and supports further development of phage-based strategies for combating resistant bacterial infections.

Keywords: Klebsiella pneumoniae; bacteriophage; bloodstream infections; cell death; inflammatory response.

Figure 1

Morphology of phage 6K2. (A) Plaques formed by phage 6K2 on Kpn host using the double agar overlay method. (B) Transmission electron microscopy (TEM) image of phage 6K2. The head diameter was approximately 60 nm, with a tail measuring 15 nm in length and 15 nm in width. Scale bar: 100 nm. (C) Genomic maps of phage 6K2. Genome was annotated using the RAST server. ORF positions are represented by colored bars on concentric rings, with the specific ring (inner or outer) indicating the direction of transcription. Functional categories are distinguished by color, as detailed in the legend. The circular genome map was generated using an online Circos tool (https://www.chiplot.online/circos.html).

Figure 2

Phylogenetic tree of 6K2 and relative bacteriophages based on the complete genome sequences. The tree was constructed using the ViPTree. The red star indicates the phage 6K2.

Figure 3

Characterization of phage 6K2. (A) The bacteriolytic activity of 6K2 in vitro. (B) pH stability detection of 6K2. (C) Temperature stability of 6K2. (D) One-step growth curve of 6K2. (E) The optimal multiplicity of infection. (F) Absorption rate of 6K2.

Figure 4

Inflammatory responses in various cell types induced by Kpn. (A) HEK293T, A549, Hela, THP-1 cells were treated with Kpn and its culture supernatant for 12h. the cells were collected for qPCR assay to detect the IL1B, TNFA, and IL6 expression. (B–E) HEK293T, A549, Hela, THP-1 cells were treated with Kpn and its culture supernatant for 12h. the cells were collected for qPCR assay to detect the CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL10, and CXCL12 expression. mock: cells were treated with an equal volume of LB medium.

Figure 5

6K2 Reduces cytokines and chemokine expression during Kpn infection. (A)Kpn and THP-1 cells were co-cultured in RPMI 1640 supplemented with 10% FBS at 37 °C for 6 h, and the culture supernatant was subsequently imaged. (B–D)Kpn and THP-1 cells were co-cultured under the same conditions in the presence or absence of 6K2 for 12 h, after which cells were harvested for qPCR to analysis the cytokines (B) and chemokine (C, D) expression. mock: cells were treated with an equal volume of LB medium and SM buffer.

Figure 6

6K2 promotes antiviral innate immunity and inflammatory response activation. (A-C) THP-1 cells were treated with heat-killed Kpn and bacteriophage 6K2, while control groups received equivalent volumes of LB medium and PBS, respectively. After 12 hours, cells were harvested, and the expression of inflammatory cytokines (IL1B, TNFA) (A), chemokines (CXCL3, CXCL8) (B), and interferon pathway antiviral genes (IFNB, ISG54) (C) was analyzed by qPCR.

Figure 7

6K2 inhibits cell death induced by Kpn infection. (A–D)Kpn and A549 cells were co-cultured under the same conditions in the presence or absence of 6K2 for 6 h, after which the following performants were conducted. (A) imaging was conducted to record the changes in the co-culture system. (B) the CFU of Kpn was analyzed. (C) morphological changes of A549 were conducted by microscopic examination. (D) A549 apoptosis were analyzed by flow cytometry. (E) Quantification of A549 apoptosis rates.

Figure 8

Prompt phage treatment rescues mice with systemic Kpn infection. (A) Survival of mice after a single IP injection with the indicated inoculum (CFU) of Kpn. n = 10. (B) The weight of surviving mice was determined every day. (C) Survival of mice infected IP with Kpn (1×10⁸ CFU) followed by IP treatment with phage (MOI = 1, 10) 1h later. Equivalent volumes of PBS were injected in lieu of phage as control. For each group, For the Kpn-infected groups, n ≥ 5; for the groups injected with bacteriophages only, n = 3. (D) Blood was collected via the tail vein at 1, 6, and 24h post-phage treatment to determine the CFU of Kpn in the blood. (E, F) Blood samples were collected from the tail vein at 1, 6, and 24 h after phage administration to quantify phage 6K2 PFU in the bloodstream of phage-only control mice (E) and Kpn-infected mice (F).