HEK293T, A549, THP-1 and Hela cells were obtained from the American Type Culture Collection. HEK293T, A549 cells and Hela cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) at 37 °C in 5% CO2. THP-1 cells were grown in RPMI-1640 supplemented with 10% FBS and 1% antibiotics (penicillin and streptomycin) at 37 °C in 5% CO2.

A novel lytic phage, named vB_KpnP-6K2 (6K2), was screened from hospital sewage samples using a ST11-KL64 type Kpn as the host bacterium. In brief, sewage samples were centrifuged at 4,000 × g for 10 min. The resulting supernatant was filtered through a 0.22 µm membrane (Millex-GP Filter Unit, Millipore, USA). Subsequently, 5 mL of the filtrate was combined with 5 mL of double-strength LB broth supplemented with host bacteria and incubated overnight at 37 °C. After incubation, the mixture was centrifuged at 4,000 × g for 10 min, and the supernatant was again filtered through a 0.22 µm filter. The filtrate was serially diluted in LB medium, and plaques were observed using the double-layer agar method. The purification procedure was repeated at least three times until homogeneous phage plaques were observed. Finally, SM buffer containing purified phage was centrifuged for 10 min at 4,000× g and filtered through a 0.22 µm syringe-driven filter. The supernatant was added with 10% glycerol and stored at -80 °C (Garcia-Cruz et al., 2023; Abdel-Razek et al., 2025; Mirza et al., 2025).

To determine the optimal multiplicity of infection (MOI) for maximizing phage progeny yield, the host strain Kpn was grown to logarithmic phase and adjusted to a turbidity equivalent to 0.5 McFarland standard (approximately 10⁸ CFU/mL). A volume of 10 mL of the host bacterial suspension at 10⁷ CFU/mL was mixed with 10 μL of serially diluted phage suspensions (approximately 10⁷, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ PFU/mL), as previously described (Zhou et al., 2018). After 6 hours of incubation, the number of progeny phages was quantified using the double-layer agar method.

We evaluated the stability of phage under varying pH conditions and temperatures as previously described (Fang and Zong, 2022). Phage suspensions (100 μL) at a concentration of 10⁹ PFU/mL were mixed with 900 μL of SM buffer adjusted to different pH values ranging from 2 to 13. After incubation at 37 °C for 1 hour, the phage titer was quantified using the double-layer agar method. Thermal stability was assessed by incubating phage suspensions at -20, 4, 37, 50, 60, and 70 °C for 1 hour in a water bath, followed by titer determination as described above. All experiments were conducted in triplicate.

We observed the morphology and the size of phage 6K2 using transmission electron microscopy (TEM) imaging. Lysates of phage 6K2 were centrifuged at 16,000× g for 10 min and supernatants were filtered through 0.22 µm to remove bacteria and cell debris. This procedure yielded a final phage titer of 1 × 10⁹ PFU/mL. We dropped the phage suspension to copper grids for 10 min and then added a drop of phosphotungstic acid (Solarbio, Cat: G1872) for negative staining. We used a Hitachi Transmission Electron Microscope (Hitachi High-Tech; Tokyo, Japan) at an accelerating voltage of 80 kV for TEM.

We assessed the phage adsorption capability by combining 10 mL of the Kpn (10⁸ CFU/mL) with 100 μL of phage suspension (10⁸ PFU/mL) at a MOI of 0.01. Samples were collected at 0-, 3-, 6-, 9-, 12-, and 15-min post-infection and immediately filtered through 0.22 µm membranes to determine the titers of unabsorbed phages. Host bacteria at the mid-log phase were infected with phages at an MOI of 0.01 and adsorbed for 10 min at 37 °C. After removing unabsorbed phages by centrifugation, the infected cells were resuspended in fresh medium to a 10-fold dilution. The diluted culture was incubated at 37 °C with shaking, and samples were collected at indicated time points. Each sample was immediately serially diluted and assayed for PFU using the double-agar overlay method.

The DNA of phage 6K2 was extracted using a Viral Genome Extraction Kit (Tiangen, China) and sent to Chengdu Phagetimes Biotech Co., Ltd., and the genome was sequenced using an Illumina Novaseq 6000. Raw sequencing reads were processed for quality control and adapter trimming using fastp (Chen et al., 2018). This step removed low-quality reads and reads with a high proportion of ambiguous bases (N), yielding high-quality clean reads. De novo assembly of the clean reads was performed with metaSPAdes (Nurk et al., 2017). Based on the assembled contig sequence, specific primers spanning the terminal region were designed to verify the genome is circular or not (Boeckman et al., 2024). The primer sequences used were 6K2-end-F: 5’-CGTATGTCCGGTTGATGACTAC-3’ and 6K2-end-R: 5’- CTATGCACTGACCTGAGGATTAC-3’. Following PCR amplification, the product was purified and its sequence was determined by Sanger sequencing.

The assembled genome was annotated for protein-coding genes and tRNAs using the RAST server (https://rast.nmpdr.org/), and the annotation was further verified by Blastp. Potential virulence factors and antimicrobial resistance genes were identified by screening the genome sequences against the Virulence Factor Database (VFDB) and the ResFinder database, respectively. Finally, the phage genome was visualized using an online Circos tool (https://www.chiplot.online/circos.html).

A549 cells were seeded in 12-well plates at a density of approximately 1×10⁵ cells per well. After 12 hours, the culture medium was replaced with DMEM supplemented with 10% FBS and without antibiotics. The cells were then infected with 5 μL of Kpn containing approximately 1×10⁶ CFU, while the control group received an equivalent volume of sterile LB medium. Following co-culture for 12 and 24 hours, cells were harvested and stained by Cell Cycle and Apoptosis Analysis Kit (Yeasen, 40301ES50) to analyze the apoptosis by flow cytometry (Crowley et al., 2016).

Total RNA was extracted using TRIzol reagent (Rio et al., 2010) and reverse-transcribed to cDNA for qPCR analysis to measure mRNA levels of the indicated genes. The mRNA levels of the tested genes were normalized to 18S rRNA levels. Gene-specific primer sequences were as follows:

Six-week-old male C57BL/6J mice were divided into seven groups (n = 10 per group) and intraperitoneally (IP) injected with 200 μL of Kpn at doses of 5 × 10⁵, 1 × 10⁶, 5 × 10⁶, 1 × 10⁷, 1 × 10⁷, 5 × 10⁷, or 1 × 10⁸ CFU. Control mice received an equal volume of PBS. Survival and body weight changes were monitored over time.

In an independent experiment, six-week-old male C57BL/6J mice were allocated into six groups and intraperitoneally challenged with 200 μL of Kpn at 1 × 10⁸ CFU (n ≥ 5 per group) or with PBS (n =3 per group). 1h later, 6K2 was administered at MOI of 0, 1 and 10. Mice in the MOI = 0 group received an equivalent volume of PBS as control. Survival was recorded every 3 h for all mice. In the Kpn−infected group, blood samples were collected at 1, 6, and 24h post phage administration to quantify bacterial load (CFU/mL) by plate counting and phage titers (PFU/mL) using the double-layer agar method. For mice not infected with Kpn, blood samples were collected solely for monitoring phage titer changes by the double−layer agar assay. All animal experiments were carried out in Jinan Microecological Biomedicine Shandong Laboratory (Jinan, China) according to procedures approved by the institutional ethics committee (Animal testing approval number:2026001).

Statistical significance was calculated using an unpaired Student’s t test (two-tailed). Results are shown as arithmetic means ± SD of at least 3 or more independent measurements. GraphPad Prism 9 was used for statistical analysis.

The bacteriophage 6K2 was isolated from hospital sewage using a ST11-KL64 type Kpn as the host bacterium. Purified phage 6K2 formed clear plaques (the diameter about 4 mm) with translucent halos (the diameter about 3 mm) on double-layer agar plates (Figure 1A). Transmission electron microscopy (TEM) revealed that bacteriophage 6K2 had a polyhedral symmetric head (the head diameter was approximately 60 nm) and a short non-contractile tail (the tail length was 15 nm and the width was 15 nm), which is a typical morphology of the family Podoviridae (Figure 1B).

De novo assembly of the 6K2 genome produced a single contig of 40,328 bp, which featured terminal repeats of 181 bp at both ends. To determine whether this structure represented a linear genome with redundant termini or a circular conformation, we performed PCR with primers spanning the repeat junction. Sanger sequencing of the amplicon confirmed a circular genome structure. The complete, circularized double-stranded DNA genome comprises 40,147 bp with a GC content of 53.05% (Figure 1C; see Additional file 1 for sequence verification details). Phage 6K2 has 48 predicted open reading frames (ORFs), with 32 ORFs (68.1%) annotated with predicted functions (Additional file 2). The annotated genes of 6K2 were categorized into different functional groups, including phage infection, DNA replication and regulation, phage capsid assembly, DNA packaging and host lysis (Figure 1C). In addition, no homologs of known virulence factors or antibiotic resistance genes were identified in the 6K2 genome, suggesting its potential suitability for clinical applications.

The phylogenetic relatedness of 6K2 with similar phages was determined based on genome-wide sequence similarities calculated by tBLASTx with the use ViPTree (https://www.genome.jp/viptree/). 6K2 was clustered with Klebsiella phage vB Kpn16-P1, vB Kpn16-P3, vB Kp IME531 and Kp 11 (Figure 2). Based on these results and the viral classification provided by the International Committee on Taxonomy of Viruses (ICTV), it was concluded that 6K2 belongs to the family of Autographiviridae, within the genus of Przondovirus.

To determine the efficiency of phage 6K2 in killing the Kpn, we examined its inhibitory effects at varying doses. Complete inhibition of Kpn by 6K2 was observed at MOIs ranging from 0.001 to 10 (Figure 3A). During the first 2 hours post-infection, the group of 6K2 (MOI≥0.1) effectively suppressed Kpn growth. After this initial period, however, bacterial loads in groups with an MOI <0.1 also became suppressed, resulting in no significant difference compared to the higher MOI groups. The results of pH and thermal stability showed that 6K2 has a wide pH tolerance, which is ranging from 4 to 12 (no significant differences among the four groups tested at pH 6, pH 7, pH 8, and pH 10) (Figure 3B). Although with minimal loss in titer, 6K2 remained active between 4 °C and 50 °C. However, the phage titer of 6K2 decreased by 5 log and 7 log after incubation of 1 hour at 60 °C and 70 °C, respectively (Figure 3C). No viable phage particles were detected following treatment at 80 °C (data not show). This suggests that phage 6K2 is less tolerant to the temperatures above 60 °C. The one-step growth curve analysis demonstrated the latent period of 6K2 lasted for about 40 min and followed by a burst period lasted for about 80 min (Figure 3D). And the burst size was calculated as 13.6 PFU per host cell. The optimal multiplicity of infection (MOI) was 0.01 of 6K2 vs Kpn (Figure 3E). Phage adsorption measurements showed that about 80% phages absorbed to host cells at 3 min and almost all phages absorbed to host cells after 6 min (Figure 3F).

Infections caused by Kpn encompass a broad spectrum of clinical manifestations, including renal impairment in kidney transplant recipients, pulmonary infections leading to pneumonia, bloodstream and intra-abdominal infections resulting in sepsis (Yang Y. et al., 2021; Zhang et al., 2021; Zheng et al., 2022; Tang et al., 2024; Yang YY. et al., 2024). Therefore, we assessed the ability of Kpn and its culture supernatant to induce inflammatory responses in various cell types. Our results demonstrated that Kpn and its culture supernatant treatment only promote IL1B, TNFA and IL6 expression in THP-1 (Figure 4A). During the initial phase of an infection, resident cells release chemokines that recruit diverse immune cells to the site of infection, where they exert their effector functions (Borish and Steinke, 2003; van der Vorst et al., 2015). Furthermore, we also assessed the chemokines expression induced by Kpn and its culture supernatant. Treatment with Kpn or its supernatant upregulates chemokine expression in THP-1 and A549 cells, but not in HEK293T or HeLa cells (Figures 4B-E). Notably, CXCL5 and CXCL10 induction was absent in THP-1 cells (Figure 4C), while CXCL2 and CXCL12 were not induced in A549 cells (Figure 4D).

After 6 hours incubation of Kpn and THP-1 at 37 °C, cell culture medium (RPMI 1640 cell culture medium with 10% FBS) exhibited acidification. Treatment with 6K2 significantly alleviated the medium acidification induced by Kpn infection (Figure 5A). Acidification of the cell culture medium serves as an indicator of Kpn proliferation. In order to determine 6K2 on the inflammation and chemokine expression induced by Kpn infection. qPCR assay was performed, our results demonstrated that 6K2 markedly suppressed the IL1B and TNFA expression significantly but not IL6 (Figure 5B). Moreover, 6K2 suppressed the expression of chemokines (CXCL1, CXCL2, and CXCL3) following Kpn infection (Figure 5C). In contrast, our data demonstrate that 6K2 potentiated the upregulation of CXCL10 in response to Kpn infection (Figure 5D).

To further investigate whether 6K2 inhibits inflammation by clearing Klebsiella pneumoniae infection or exerts direct anti-inflammatory effects, we treated THP-1 cells with heat-killed Kpn to induce inflammation. We found that 6K2 did not suppress the expression of inflammatory cytokines and chemokines induced by heat-killed Kpn. Moreover, 6K2 itself could also induce the expression of inflammatory cytokines and chemokines (Figures 6A, B). To determine whether phage 6K2 acts as a viral agent that triggers interferon-mediated antiviral immune responses, we performed qPCR analysis and observed that 6K2 induced the expression of IFNB and interferon-stimulated gene ISG54 (Figure 6C).

Previous studies have demonstrated that Kpn infection could induce cell death by promoting apoptosis, pyroptosis, and autophagy (Wang et al., 2018; Jiang et al., 2022; Wang et al., 2023; Wei et al., 2023). Therefore, we further investigated the impact of Kpn infection on cell death. Initially, co-culture of Kpn with A549 cells was found to promote acidification of the culture medium, while 6K2 inhibited this Kpn-induced acidification (Figure 7A). Further examination of the Kpn in the co-culture system revealed that 6K2 significantly reduced Kpn proliferation (Figure 7B). Microscopic observation of A549 cell morphology showed that Kpn induced cell rounding, fragmentation, and death, whereas 6K2 markedly alleviated these morphological changes and reduced cell death (Figure 7C). To further elucidate the mode of Kpn-induced A549 cell death in the co-culture system, flow cytometry analysis was performed. The results indicated that Kpn-induced A549 cell death did not occur via apoptosis (Figures 7D, E).

To determine whether 6K2 could protect against lethal Kpn infection in vivo, we first established a murine model of bloodstream infection using Kpn. Our results showed that mice administered Kpn at doses exceeding 1×10⁸ CFU exhibited 100% mortality within 24 hours, while a dose of 5×10⁷ CFU led to approximately 70% mortality within two days, accompanied by a transient decrease in body weight, followed by gradual recovery (Figures 8A, B). Based on these findings, mice were infected with 1×10⁸ CFU of Kpn and treated with 6K2. During the mouse challenge and treatment experiment, the clinical symptoms of the mice were observed every 3h. The results demonstrated that all 6K2-treated mice survived, whereas all control mice died within 18h (Figure 8C). Blood samples collected at 1, 6, and 24h post 6K2 administration revealed that the bacterial load of Kpn in the blood of 6K2-treated mice fell below the detection limit as early as 6 hours after treatment (Figure 8D). Furthermore, quantification of 6K2 phage in the blood indicated high phage titers within 6 hours of treatment, which subsequently decreased by 24 hours (Figures 8E, F).