The Klebsiella pneumoniae strains used in this experiment were identified using a fully automated microbial identification and susceptibility testing system (BD, phoenix100). The host bacterium P4, verified in preliminary experiments, possesses several virulence genes, including rmp, peg, iuc, and iro. The antibiotic resistance of the remaining K. pneumoniae strains was assessed using the disk diffusion method and PCR, with PCR amplification specifically targeting the KPC gene. The string test was employed to measure the viscosity of K. pneumoniae strains (Shon et al., 2013). Bacteria were inoculated on LB agar plates and incubated at 37°C for 16 h. A single colony was then picked to observe the string formation. A positive result, indicating a hypermucoviscous phenotype, was defined by the formation of a viscous string greater than 5 mm in length. A negative result indicated a non-hypermucoviscous phenotype.

Following the method described by Mohammadi et al. (2023), flowing river water samples were collected from the Han River in Xiangyang, Hubei Province, a tributary of the Yangtze River. The samples were centrifuged at 10,000 rpm for 15 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm syringe filter and stored in 50 mL centrifuge tubes at 4°C. The culture was grown to the logarithmic phase using K. pneumoniae P4 as the host bacterium. Then, mix 1 mL of filtered solution with the host bacteria and incubate overnight in a shaker at 37°C and 160 rpm. After incubation, the mixture was centrifuged at 10,000 rpm for 15 min, followed by filtration to collect the supernatant. A further step involved mixing 100 μL of this supernatant with 100 μL of the host bacterial culture in the logarithmic phase, incubating at 37°C in a shaker at 220 rpm for 15 min. Subsequently, 5 mL of 50°C 0.7–0.8% LB semi-solid medium was added, mixed thoroughly, and quickly poured onto the surface of an LB solid medium. After solidification, the plates were inverted and incubated overnight at 37°C. The double-layer agar plate method (Hyman and Abedon, 2009) was employed to observe the presence of bacteriophage plaques. Upon verification, individual plaques were picked and subjected to multiple rounds of purification to obtain a pure phage. For amplification, 5 mL of purified phage solution was mixed with 5 mL of the host bacterial culture in the logarithmic phase, and a liquid LB medium was added to reach a total volume of 50 mL. This mixture was incubated at 37°C with shaking for 6–8 h. The suspension was then centrifuged at 10,000 rpm for 15 min, followed by filtration through a 0.22 μm pore-size membrane to obtain the supernatant, resulting in an amplified phage solution. The amplified phage solution was aliquoted and stored at −80°C in glycerol for future use.

To examine the phage morphology, 20 μL of the amplified phage suspension was pipetted onto a 200-mesh copper grid and allowed to adsorb naturally for 5–10 min. Excess liquid was removed using a filter paper strip, and the grid was air-dried briefly. A 2% phosphotungstic acid solution (20 μL) was then applied to the grid for negative staining and left for 3–5 min. Afterward, the excess stain was removed with a filter paper strip, and the grid was air-dried under an incandescent lamp. The morphology of the phages was then observed using transmission electron microscopy (HITACHI, HT7700, Japan).

Host range analysis was performed using a panel of 21 K. pneumoniae strains by spot tests as previously described with slight modifications. Briefly, the purified phage stocks were gradient diluted with LB liquid medium and 2 μL of gradient diluted phage concentrate (10²–10⁹ PFU/mL) was added to the tested bacterial lawn and incubated at 37°C for 12 h. The presence of clear plaque on the bacterial lawn indicated that the tested strains were susceptible to the phage (Han et al., 2023). All K. pneumoniae isolates that were sensitive to phage vB_Kp_XP4 in the spot test assay (n = 2) were selected for the determination of the Efficiency of Plating (EOP), following the method described by Khan Mirzaei and Nilsson (2015). The EOP was calculated as the ratio of plaque-forming units (PFU/mL) on a sensitive strain to PFU/mL on the indicator strain. Each combination of bacterial strain and phage dilution was tested in triplicate, and the results are presented as the mean of three observations.

The bactericidal activity of phage vB_Kp_XP4 was assessed by determining its time-killing curves. Phage solutions (500 μL) were mixed with 500 μL of host bacterial culture in the logarithmic phase at varying multiplicities of infection (MOIs) of 10, 1, 0.1, 0.01, 0.001, and 0.0001. The mixtures were incubated at 37°C with shaking at 220 rpm for 1 h. After incubation, the cultures were centrifuged at 10,000 rpm for 8 min, and the supernatant was filtered. The supernatant was then serially diluted, and the phage titer was determined using the double-layer agar plate method. The MOI with the highest phage titer was considered the optimal MOI for this phage. The experiment was repeated three times to ensure accuracy.

Following the method described by Mohammadi et al. with slight modifications (Feng et al., 2023), the phage suspension was mixed with the host bacterial culture at the optimal multiplicity of infection (MOI) and incubated at 37°C for 15 min. Following incubation, the mixture was subjected to immediate centrifugation at 10,000 rpm, and the supernatant was discarded. The pellet was washed multiple times with LB liquid medium and then resuspended in LB medium. The suspension was incubated in a shaker at 37°C and 180 rpm. Samples (300–400 μL) were collected at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min. Each sample was immediately centrifuged at 10,000 rpm, and the supernatant was filtered through a 0.22 μm pore-size membrane. The filtrate was serially diluted in an LB liquid medium, and 100 μL of each dilution was mixed with 100 μL of host bacterial culture. The mixtures were incubated for 15 min and then plated using the double-layer agar plate method. The plates were inverted and incubated overnight at 37°C to observe plaque formation and calculate the phage titer. Three parallel experiments were conducted for each time point. Burst size was calculated using the following formula: (titer after burst—titer at T0)/ (added phage—titer at T0). The curve of phage titer changes was constructed based on the phage titer at each time point. Perform Gram staining on the P4 strain both before and after bacteriophage treatment within a 12-h period. Bacterial morphology was observed using an optical microscope at 1,000× magnification.

Phage stability under different temperatures was assessed by placing the phage suspension in metal baths at 4°C, 10°C, 20°C, 30°C, 37°C, 40°C, 50°C, 60°C, 70°C, and 80°C for 60 min. For pH stability, the pH of the solutions was adjusted from 2 to 11 using HCl and NaOH. Four mL of phage solution (approximately 10⁸ PFU/mL) was incubated at each pH level for 60 min. Phage solutions were then added to host bacteria cultures at an MOI of 0.1, while control groups received LB medium. These were placed in a shaker at 220 rpm and 37°C. Bacterial OD630 was measured at 30-min intervals to monitor changes in bacterial growth. Measurements were taken continuously for 3 to 12 h, with each group tested in triplicate.

Galleria mellonella larvae model was used to assess the potential in vivo efficacy of phage against K. pneumoniae. The methods for larval injection and incubation were carried out with reference (Han et al., 2023; Li et al., 2023). The experimental procedures are as follows: the larvae selected were 25 ± 5 mm in length, 300 ± 50 mg in weight, with high activity and no visible black spots on the surface. All injections were performed using a Hamilton syringe into the left or right hind leg. Larvae were considered dead when they did not respond to touch. In vivo experiment, the larvae were divided into 12 groups, with 10 larvae randomly selected per group: (i)10 μL PBS injection, (ii) 10 μL of 10⁷ CFU/mL strain P4, (iii) 10 μL of 10⁸ CFU/mL strain P4 injection, (iv) 10 μL of 10⁸ CFU/mL bacteriophage, (v) injection of 10 μL of PBS in the right leg (symmetrical position) at 0 h, (vi) injection of 10 μL of PBS in the right leg after 1 h; (vii-ix) MOI = 0.1 (P4 = 10⁸ CFU/mL), 1 (P4 = 10⁷ CFU/mL), 10 (P4 = 10⁷ CFU/mL), with 10 μL of the corresponding concentration of strain P4 injected into the left hind leg, while 10 μL of bacteriophage with titers of 10⁸, 10⁷, or 10⁸ PFU/mL was injected into the right hind leg, respectively; for MOI = 0.1, 10 μL of 10⁸ CFU/mL strain P4 was injected into the left hind leg in three groups of larvae, followed by 10 μL of 10⁷ PFU/mL bacteriophage in the right hind leg at 1 h, 2 h, and 4 h intervals. After completing the above procedures, all larvae were incubated at 37°C and monitored for mortality every 2 h for a total of 60 h. Survival curves were generated using GraphPad Prism v.10.1 and the survival rates were analyzed using Kaplan–Meier and log-rank test. Differences with p < 0.05 were considered statistically significant.

Statistical analysis and plotting were performed using GraphPad Prism version 10.1 software. Student’s t-test was used for both intra-group and inter-group comparisons, with the significance level set at p ≤ 0.05.

Phage-sensitive strains were tested for antibiotic susceptibility using the disk diffusion method, including imipenem, meropenem, ertapenem, levofloxacin, ceftazidime, ciprofloxacin, and amikacin. Strains resistant to more than three classes of antibiotics were defined as multi-drug resistant (Table 1). The results of capsule typing for the Klebsiella pneumoniae strains, along with the amplification of KPC and NDM genes and the outcomes of the string test, are presented in Table 2.

Using the hypervirulent K. pneumoniae strain P4 as the host, a bacteriophage was isolated and purified from Han River water and named Klebsiella phage vB_Kp_XP4. Serial dilutions of the phage stock solution (≈10⁹ PFU/mL) were prepared, and within 12 h of incubation at 36°C on double-layer agar plates, plaques with a diameter of approximately 2 mm formed. These plaques exhibited a halo with multiple semi-transparent layers around them. The plaque morphology at different dilutions (10⁻³, 10⁻⁶, 10⁻⁷, 10⁻⁸) is shown in Figures 1A,a–d. Over time, the halo gradually expanded, with the plaque and halo diameters reaching up to 15 mm at 24 h under ambient conditions (Figures 1A,e) and 28 mm at 36 h (Figures 1A,f).

Transmission electron microscopy (TEM) revealed the morphological characteristics of phage vB_Kp_XP4. The phage displayed a typical icosahedral head structure with a diameter of approximately 55 ± 2 nm and a non-contractile tail measuring about 168 ± 10 nm in length (Figure 1B). Based on these morphological features, phage vB_Kp_XP4 was classified as a tailed phage with a flexible, non-contractile tail.

The lytic spectrum and efficiency of phage vB_Kp_XP4 were evaluated across 21 K. pneumoniae strains. The phage exhibited a lytic rate of 9.5% (2 out of 21 strains). Virulence genes iucA1, iucA2, iroB1, iroB2, prmpA, prmpA2, and peg-344 were amplified in phage-sensitive strains using PCR. Strains positive for all these genes were defined as ‘high-virulence strains’ (Table 1). These results suggest that phage vB_Kp_XP4 has therapeutic potential against infections caused by hypervirulent or multidrug-resistant K. pneumoniae. The EOP analysis showed that when the bacteriophage-to-bacteria ratio is approximately 0.1, phage vB_Kp_XP4 demonstrates extremely high efficiency in lysing the P4 strain, with fewer than 10 colonies growing (EOP ≈ 1). In contrast, the lysis rate for strain 10 is only 0.51 ± 0.08. Detailed information is presented in Table 2.

Klebsiella phage vB_Kp_XP4 was tested for its bactericidal activity against its host, K. pneumoniae P4, at various MOIs. At an MOI of 0.1, the phage titer reached approximately 10⁸ PFU/mL, significantly higher than in other groups (Figure 2A).

The parameters of phage reproduction, including but not limited to the latent period and changes in phage quantity during the growth cycle, are valuable for the practical application of phages. Monitoring changes in phage load (Figure 2B) indicated that phage vB_Kp_XP4 has a relatively short latent period of approximately 10 min. When co-cultured with the host bacteria for 10 to 50 min, the phage titer increased rapidly, and the bacterial suspension transitioned from turbid to clear. After 50 min of co-culture, the phage concentration peaked at 10¹⁴PFU/mL, entering a plateau phase where the suspension remained relatively clear. The average burst size of this phage was about 387 phages/cell. However, after 100 min, a noticeable decline in phage concentration was observed, and the bacterial suspension gradually became turbid. Under an optical microscope at 1,000× magnification, the morphology of the P4 strain changed after treatment with bacteriophage vB_Kp_XP4. Macroscopically, we observed that the staining intensity in image “a” is higher compared to image “b.” Microscopically, individual bacterial cells appeared smaller after phage treatment than before. This suggests that phages may have disrupted the capsule structure of Klebsiella pneumoniae. (Figure 2C). However, this morphological change was not observed in strain 10.

Pathogenic K. pneumoniae is distributed across various environmental conditions. Therefore, the ability of phages to control these pathogens under different conditions is crucial for their practical application. The tolerance of phage vB_Kp_XP4 at different titers (10⁸ and 10¹²PFU/mL) was tested across a range of temperatures (4°C, 37°C, 40°C, 50°C, 60°C, 70°C, and 80°C). The results showed that phage vB_Kp_XP4 reached its highest titer at 50°C after 1 h. At 70°C for 1 h, phages with a titer of 10⁸ PFU/mL were completely inactivated, while phages with a titer of 10¹²PFU/mL partially survived, and even at 80°C for 1 h, a small amount of phages with this titer still survived (Figure 3).

The impact of different pH levels on the phage’s ability to inhibit its host bacteria was analyzed, as shown in Figure 4. The results indicated that at pH ≤ 3, the growth of host bacteria in both the experimental and control groups was significantly inhibited, suggesting that both phage and bacterial growth are restricted by highly acidic conditions. Within a pH range of 4 to 11, the growth of host bacteria in the experimental group (MOI of 0.1) was significantly inhibited, indicating that the phage exhibits antibacterial activity across this broad pH spectrum. Additionally, it was observed that after 12 h, the concentration of host bacteria increased markedly, and the difference between the experimental and control groups diminished. This suggests that the phage was unable to completely eradicate the host bacteria, leading to a relative equilibrium between the two over time.

Galleria mellonella larvae model was used to assess the efficacy of phage vB_Kp_XP4 against strain P4 in vivo. At 60 h, the survival rate of larvae injected with only PBS and phage was both 90%, while the survival rate of larvae injected with strain P4 (10⁸) was 0%, and those injected with strain P4 (10⁷) had a survival rate of 10%. The P4 + PBS group had a survival rate of 20%, while the groups injected with P4 + phage at MOI = 0.1, 1, and 10 had survival rates of 40, 80, and 50%, respectively. Notably, at MOI = 0.1, the lower survival rate was related to the higher concentration of P4 in this group (Figure 9A). It can be observed that the phage treatment groups performed significantly better than the untreated group. Larvae injected with strain P4 (10⁸) all died within 20 h, and those injected with PBS at 1 h post-injection died within 36 h; when MOI = 0.1, larvae injected with phage at 1 h had a survival rate of 20% at 60 h; larvae injected with phage at 2 h all died by 45 h; larvae injected with phage at 4 h all died within 22 h (Figure 9B). It is evident that under the same infection conditions, the earlier phage treatment is administered, the longer the survival time of Galleria mellonella larvae, and the more pronounced the therapeutic effect. Additionally, larvae injected with phage alone still had a high survival rate, demonstrating the safety of phage therapy in this model. The morphology of the Galleria mellonella larvae after treatment with phage vB_Kp_XP4 is shown in Figure 9C.

In conclusion, bacteriophages and their protein products have garnered significant interest globally as potential therapies to reduce or replace antibiotic use. This study successfully isolated a novel Klebsiella phage vB_Kp_XP4, and characterized its biological properties and genomic features. In vivo experiments demonstrated its therapeutic effect on a Galleria mellonella infection model, with high safety and efficacy, making it a promising candidate for phage therapy and a potential synergist in combination with antibiotics, offering additional options for antimicrobial treatment. Furthermore, phage protein products, such as lytic enzymes and endolysins, possess properties that assist in bacterial lysis. Future in vitro and in vivo experiments could explore the antibacterial effects of phage suspensions, protein synthesis products, and various combinations with antibiotics.