We collected 73 S. maltophilia strains from clinical patient samples: S. maltophilia no. 981, isolated from a blood sample, was designated as the host strain for phage screening; the remaining 72 strains were used for subsequent host range determination experiments. All strains were routinely cultured in LB liquid medium or LB agar medium under a constant temperature of 37°C, and cryopreserved in LB broth supplemented 30% (v/v) glycerol at −80°C for future use.

Genomic DNA of the bacterial strain was extracted using a DNA extraction kit. DNA samples meeting the library construction standards were used to construct libraries with the ALFA-SEQ DNA Library Preparation Kit. These libraries were then sequenced on the Illumina NovaSeq 6000 platform with 150 bp paired-end reads. The raw sequencing data were later subjected to quality control and subsequently assembled de novo using SPAdes v3.13.0 software. The assembled sequences were submitted to the MLST online platform [MLST 2.0](https://cge.food.dtu.dk/services/MLST/) for sequence typing (ST) determination by aligning against seven housekeeping genes (atpD, gapA, guaA, mutM, nuoD, ppsA, recA) (Kaiser et al., 2009).

Phage vB_SmaS_QH16 was isolated and purified from sewage samples of Qinghai Provincial People’s Hospital (36.62°N, 101.78°E; altitude 2,261 m) using the double-layer agar method (Wen et al., 2022). In brief, fresh sewage was initially centrifuged at 5,000 × g for 5 min to remove particulate matter, followed by filtration of the supernatant through 0.22 μm membranes (Millipore, USA). Subsequently, 5 mL filtrate was mixed with an equal volume of logarithmic-phase S. maltophilia no.981 culture (OD₆₀₀ = 0.4–0.6) and incubated at 37°C with 180 rpm agitation overnight for enrichment. The enriched culture was then centrifuged and filtered through 0.22 μm membranes to obtain a phage-enriched lysate. For primary screening, 100 μL of this lysate was combined with host bacterial suspension in 1.5% (w/v) LB semi-solid agar (pre-warmed to 50°C), overlaid onto LB agar plates, and incubated inverted at 37°C for 18–24 h. Discrete plaques were excised using sterile pipette tips, and eluted in phosphate-buffered saline (PBS) buffer. Serial dilutions of phage suspensions were mixed with 100 μL logarithmic-phase S. maltophilia no.981 culture and 5 mL 1.5% LB agar, then overlaid on fresh LB plates. After overnight incubation at 37°C, this purification cycle was repeated until homogeneous plaque morphology was achieved, ensuring phage purity.

The morphology of the phage was observed using transmission electron microscopy (TEM). In brief, 20 µL of phage suspension (≥10⁹ PFU/mL) were applied to a copper grid, and allowed to air-dry for 10 minutes. Excess liquid was then removed using filter paper, and 20 µL of 2% phosphotungstic acid solution was added to the grid for negative staining for 5 minutes. This stain was then removed with filter paper, and the grid was air-dried at room temperature for 30 minutes. Finally, the phage morphology was visualized and photographed using a transmission electron microscope (Hitachi HT7700, Japan) at 80 kV.

The host specificity of phage vB_SmaS_QH16 was evaluated using the double-layer agar spot assay (Wang et al., 2024), as follows: 100 μL of the test bacterial suspension (OD₆₀₀ = 0.4) was mixed thoroughly with 5 mL of preheated 1.5% LB semi-solid agar at 50°C, after which the mixture was immediately poured onto an LB solid agar plate to form a uniform double-layer substrate. After the substrate had solidified completely, 10 μL of phage lysate (titer ≈ 10¹⁰ PFU/mL) was spotted onto the surface of the plate. The plate was then incubated at 37°C for 12–16 hours. Results were determined based on plaque formation on the plate: “++” indicates the formation of clear and distinct phage plaques; “+” indicates weak intensity plaque formation; “−” indicates no phage plaque formation.

For bacterial strains that showed positive results in the host range assay, the phage proliferation ability was quantitatively analyzed using the efficiency of plaquing (EOP) (Abdelghafar et al., 2023). In the experiment, 100 μL of bacterial suspension in the logarithmic growth phase (OD₆₀₀ = 0.4–0.6) was thoroughly mixed with an equal volume of phage gradient dilution (10³ to 10⁸ PFU/mL), and plated onto double-layer agar plates. After incubation at 37°C, the number of phage plaques was counted. The EOP value was calculated as the ratio of phage titer on the target bacterial strain to that on the host strain S. maltophilia no.981. The phage proliferation efficiency on different host bacteria was classified into four levels based on the EOP value: high productivity (EOP ≥ 0.5), moderate productivity (0.1 ≤ EOP < 0.5), low productivity (0.001 ≤ EOP < 0.1), and inefficient (EOP < 0.001). To ensure the reliability of the experimental results, all experiments were repeated in triplicate.

The optimal multiplicity of infection (MOI), defined as the ratio of phages to host bacteria during the infection process, was determined by mixing phage vB_SmaS_QH16, S. maltophilia no.981 (concentration of 10⁸ CFU/mL) with different concentrations of phage vB_SmaS_QH16, with MOI values set at 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001. The mixtures were incubated at 37°C with shaking at 180 rpm overnight. Following incubation, the cultures were filtered through a 0.22 μm filter membrane to remove bacterial cells, diluted to an appropriate concentration with PBS buffer, and the phage titer was determined using the double-layer plaque assay. The MOI corresponding to the highest phage titer was defined as the optimal MOI (Zhang et al., 2022). This experiment was independently performed three times to ensure reliability and reproducibility of the results.

The one-step growth curve experiment was performed according to a previously described method (Bai et al., 2022), with necessary optimizations based on preliminary experiments. The procedure was as follows: Phage vB_SmaS_QH16 was mixed with S. maltophilia no.981 (10⁸ CFU/mL) at the optimal MOI and incubated at 37°C for 10 minutes to allow adsorption. Subsequently, the mixture was centrifuged at 4°C and 10,000×g for 5 minutes, after which the supernatant was discarded. The pellet was washed and resuspended in PBS buffer, followed by a second centrifugation (4°C, 10,000×g, 5 minutes). The supernatant was then discarded again, and the washing and centrifugation steps were repeated twice to completely remove unabsorbed free phage particles. Subsequently, the pellet was resuspended in 10 mL of pre-equilibrated LB liquid medium (37°C), and incubated at 37°C with shaking at 180 rpm for 120 minutes. Starting from the beginning of the culture (t = 0), 100 μL samples were taken every 10 minutes. After sampling, the samples were immediately diluted to an appropriate concentration, and the phage titer was determined using the double-layer agar plate method. The experiment was repeated in triplicate, and the average values were used to plot the growth curve. In the one-step growth curve experiment, the latent period is defined as the time interval from phage adsorption to the onset of the first lysis; the burst phase refers to the period during which phages are rapidly released after completing replication; and the burst size is defined as the ratio of the number of progeny phages to the number of initially infected cells.

To evaluate the physicochemical stability of phage vB_SmaS_QH16, its activity retention under various environmental conditions was systematically examined. In brief, phage samples were exposed to water baths at 4°C, 25°C, 37°C, 50°C, 60°C, 70°C, and 80°C for 1 hour to assess thermostability (Han et al., 2021). For pH tolerance, phage suspensions in SM buffer were adjusted to a pH gradient (1–14) and incubated at 37°C for 1 hour, followed by viability testing (Bai et al., 2022). The UV resistance was evaluated by exposing phage suspensions to UV radiation (196.67 ± 28.27 μW/cm²) at a distance of 30 cm for 60 minutes, with sampling every 10 minutes (Ahmad et al., 2024). Organic solvent tolerance was tested by incubating phage suspensions in SM buffer containing 1% (v/v) chloroform at 37°C for 1 hour (Ahmad et al., 2024). Phage titers for all treated samples were determined using the double-layer agar plate method, and all experiments were independently repeated three times.

The genomic DNA of phage vB_SmaS_QH16 was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany), and sequencing was performed on the DNBSEQ-T7 platform with PE150 reads. Raw sequencing data were quality-controlled and filtered using fastp (v0.23.2) to obtain high-quality clean reads for use in de novo genome assembly with metaSPAdes (v3.15.4) (Nurk et al., 2017). Gene prediction and functional annotation were performed using Prokka (v1.14.6) (Seemann, 2014), and the annotation results were compared against the NCBI Non-redundant Protein Database (NR) using BLASTp to obtain homologous sequence information. Virulence and antibiotic resistance genes were identified using the VFDB (Virulence Factor Database) (Liu et al., 2021) and ResFinder (v4.1) (Bortolaia et al., 2020m), respectively. The phage lifestyle was predicted using the phage AI platform, version 1.0.2 (Tynecki et al., 2020). A genomic circular map was generated using the online tool Proksee (https://proksee.ca/) (Grant et al., 2023) to visualize the distribution of gene functions.

To analyze genomic relatedness, the whole-genome sequence was compared using the NCBI BLASTn algorithm to identify clusters of highly homologous phage sequences. Subsequently, the VIRIDIC online platform (https://rhea.icbm.uni-oldenburg.de/VIRIDIC/) (Moraru et al., 2020) was applied to perform average nucleotide identity (ANI) analysis of the selected sequences. The phylogenetic analysis of phage vB_SmaS_QH3 was performed using VICTOR (Meier-Kolthoff and Göker, 2017) (https://ggdc.dsmz.de/victor.php#), while its taxonomic status was validated through maximum-likelihood phylogenetic tree construction of highly conserved proteins (e.g., terminase large subunit and major capsid protein) using MEGA11 (Tamura et al., 2021)(https://www.megasoftware.net/).

The lysogenic efficiency of phage vB_SmaS_QH16 was evaluated in a dynamic antibacterial experiment (Chen et al., 2023). The experiment was set up with an MOI gradient (MOI = 100, 10, 1, 0.1, 0.01, 0.001, 0.0001), as follows: 100 μL of host bacterial suspension in the logarithmic growth phase (10⁶ CFU/mL) and 100 μL of gradient-diluted phage suspension (10⁸–10² PFU/mL) were inoculated into a 96-well plate to achieve the predetermined MOI. An equal volume of bacterial suspension with no phage infection was used as a control. The reaction system was placed in a microplate reader (Sunrise, Tecan, Switzerland) for constant-temperature shaking incubation at 37°C. The OD₆₀₀ absorbance was automatically measured every 30 minutes for continuous monitoring over 24 hours. By analyzing the real-time growth curves, the rate of absorbance decrease relative to the control group was calculated for each MOI group to characterize the immediate lytic kinetics of the phage.

G. mellonella larvae (provided by Huide Biotechnology Co., Ltd., Tianjin, China) were used as an animal model to evaluate the in vivo anti-infective efficacy of the target phage (Li et al., 2023). Healthy fifth-instar larvae (weight: 273 ± 31 mg, length: 2.72 ± 0.13 cm) were selected, with 10 larvae per group. Before injection, the injection site was disinfected with a swab containing 75% ethanol. The host bacteria (S. maltophilia no.981) were cultured to the logarithmic growth phase, washed three times with PBS, and resuspended to the desired concentration. To determine the appropriate infectious dose, 10 μL of bacterial solution at different concentrations (1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, and 1×10⁹ CFU/mL) was injected into the posterior left leg of the larvae using a micro-injector (Gaoge, Shanghai, China). Following injection, the larvae were placed in petri dishes lined with sterile filter paper, and incubated at 37°C and 70% humidity in the dark. The survival status of the larvae was observed and recorded every 8 hours for up to 72 hours to assess the impact of different infectious doses on larval survival. G. mellonella larvae were considered dead when they showed no response to touch (Wei et al., 2016).

To evaluate the therapeutic effect of the phage, larvae were initially infected with bacteria, and then treated with phage injections (Li et al., 2023). S. maltophilia no.981 was subsequently resuspended and diluted to 1×10⁸ CFU/mL, after which 10 µl of cell resuspension was injected into the posterior left leg. One hour post-infection, 10 μL of phage suspension at different MOI values (100, 10, 1, and 0.1) was injected into the opposite side of the bacterial injection site. The larvae were then placed in petri dishes with sterile filter paper, and incubated at 37°C and 70% humidity in the dark. Survival status was recorded every 8 hours for 72 hours to assess the therapeutic effects of different phage doses.

In the prophylactic phage treatment model, the phage was injected into G. mellonella larvae 1 hour prior to bacterial infection (Li et al., 2023). In brief, 10 μL of phage suspension at different MOI values (100, 10, 1, and 0.1) was injected into the posterior left leg of the larvae. After 1 hour of incubation, 10 μL of S. maltophilia no.981 (1×10⁸ CFU/mL) was injected into the opposite side of the phage injection site. The larvae were then placed in petri dishes with sterile filter paper and incubated at 37°C and 70% humidity in the dark. Survival status was recorded every 8 hours for 72 hours to evaluate the prophylactic effects of different phage doses. Positive control larvae were infected with S. maltophilia no.981 and treated with PBS. Negative controls included larvae injected with PBS alone and those injected with phage at an MOI of 100 without bacterial challenge. All experiments were repeated in triplicate.

The mouse infection model of S. maltophilia no.981 was established as per literature methods (Han et al., 2022). The logarithmic growth phase bacteria were harvested, washed twice with PBS, and adjusted to a concentration of 5×10⁸ CFU/mL. In this study, SPF BALB/c mice (3–5 weeks old, 15–19 grams) were purchased from Beijing Weilitonghua Laboratory Animal Technology Co., Ltd. The Ethics Committee of Qinghai Provincial People’s Hospital approved the animal study protocol (2024-047-01). All mice received an intraperitoneal injection of cyclophosphamide (125 mg/kg) on day 4 and an intraperitoneal injection of cyclophosphamide (125 mg/kg) and dexamethasone (12.5 mg/kg) on day 1 before infection to induce immunosuppression.

Immunocompromised mice were randomly divided into six groups (13 mice per group): Group I (negative control), Group II (positive control), and treatment groups (Group III–VI). Details are as follows: Group I mice received 40 μL of PBS intranasally, followed by intranasal administration of phages (2×10⁸ PFU/mouse) 6 hours later; Group II mice were infected intranasally with 2×10⁷ CFU/mouse of bacterial suspension and then given 40 μL of PBS intranasally 6 hours post - infection (hpi); Group III–VI mice were also infected intranasally with 2×10⁷ CFU/mouse of bacterial suspension, and 6 hpi, they received phages intranasally at different MOIs: Group III, 2×10⁸ PFU/mouse (MOI=10); Group IV, 2×10⁷ PFU/mouse (MOI=1); Group V, 2×10⁶ PFU/mouse (MOI=0.1); Group VI, 2×10⁵ PFU/mouse (MOI=0.01). Mouse survival was monitored daily for 7 days. At 30 hours post-phage treatment, three mice were randomly selected from each group. Their lung tissues were collected. One part was fixed for H&E staining to evaluate histopathological changes, and the other part was homogenized and plated to quantify viable bacteria (Pu et al., 2022).

All data were analyzed using GraphPad Prism 10.1.2. Continuous data are presented as the mean ± SD. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was performed to assess intergroup differences. Survival curves were constructed using the Kaplan-Meier method, and differences in survival rates between groups were evaluated using the Log-rank (Mantel-Cox) test. Statistical significance was defined as P < 0.05, with significance levels indicated as follows: NS not significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

A novel phage was successfully isolated from a hospital sewage sample using S. maltophilia no.981 as the host bacterium. On double-layer agar plates, this phage formed transparent plaques with a diameter of 1 to 2 mm ( Figure 1A ), featuring clear edges and no “halo” effect, potentially indicative of a lytic life cycle. TEM observations revealed that phage vB_SmaS_QH16 exhibited typical morphological characteristics of tailed phages: the head was icosahedrally symmetrical with a diameter of 54.36 ± 1.68 nm, while the tail constituted a non-contractile, elongated tube with a length of 166.97 ± 6.37 nm, terminating in several tail fibers ( Figure 1B ). Based on the morphological characteristics and the 2024 ICTV classification criteria, vB_SmaS_QH16 was classified within the class Caudoviricetes (NCBI:txid3237192).

MLST analysis of 73 S. maltophilia strains revealed 28 distinct sequence types (STs), with 21 strains not matching any specific ST, indicating at least 29 distinct lineages, and highlighting high genetic diversity ( Table 1 and Supplementary Table 1 ). Phage vB_SmaS_QH16 lysed 35 strains (47.95%), covering 13 STs (ST4, ST8, ST115, ST138, ST185, ST208, ST212, ST223, ST365, ST517, ST518, ST644, ST1139) and 13 untyped strains. The EOP results showed significant variation in lysis efficiency, with EOP values ranging from <0.001 to 1.375 ± 0.163. Classification revealed 12 strains (16.44%) as high-producers (EOP ≥ 0.5), 4 (5.48%) as moderate-producers (0.1 ≤ EOP < 0.5), 1 (1.37%) as low-producers (0.001 ≤ EOP < 0.1), and 18 (24.66%) as inefficient (EOP < 0.001). Notably, three strains (4.11%) exhibited an EOP > 1, with the highest showing a 37.5% higher plaque efficiency than the host S. maltophilia no.981, indicating superior proliferation in the target host.

In the MOI experiment for phage vB_SmaS_QH16, the highest progeny phage titer was achieved at an MOI of 0.01, reaching 5.73×10¹⁰ PFU/mL. Thus, the optimal MOI for phage vB_SmaS_QH16 was determined to be 0.01 ( Figure 2A ).

For the one-step growth curve at this MOI, the phage titer remained stable, with no significant changes during the first 40 minutes post-infection. However, from 40 to 100 minutes, the titer increased rapidly, indicating active phage replication and release. The latent period was approximately 40 minutes, while the lysis phase lasted 60 minutes, with an average burst size of approximately 37.69 PFU/cell ( Figure 2B ).

Phage vB_SmaS_QH16 exhibited specific stability across various physicochemical conditions. Thermostability testing revealed no significant titer change (P > 0.05) after 1 hour of incubation at 4 to 50°C, with peak activity retained. Although the titer gradually decreased at higher temperatures, it only dropped by 3 log10 at 80°C, indicating good thermal stability ( Figure 3A ). In pH tolerance experiments ( Figure 3B ), the phage retained high activity between pH 3.0 and 11.0, with significant titer reductions at pH 2 and 12 (P < 0.0001), and complete inactivation at extreme pH values of 1, 13, and 14. The phage was highly sensitive to UV radiation, with significant inactivation after 10 minutes, and nearly complete loss of activity after 30 minutes ( Figure 3C ). Chloroform treatment had no significant effect on phage titer, suggesting minimal or no lipid content in the phage capsid ( Figure 3D ) (Mäntynen et al., 2019).

The genome of phage vB_SmaS_QH16 (GeneBank accession number: PP921744) comprised a linear dsDNA molecule of 43,500 bp with a GC content of 67.49%. A total of 64 ORFs were predicted, with a coding density of 95.93%, while no tRNA genes were detected ( Supplementary Table 2 ). Of these ORFs, 46 were on the forward strand and 18 on the reverse strand. In total, fifty-one ORFs use ATG as the start codon, while 13 use GTG. The total length of all ORFs reached 41,730 bp, averaging approximately 652 bp each, accounting for 95.93% of the entire genome. No tRNA, virulence, antibiotic resistance, or integrase genes were predicted in the vB_SmaS_QH16 genome ( Figure 4 ). Structural Proteins, Replication, Packaging, Lysis, and otFunctional annotation of the ORFs against non-redundant protein databases using BLASTp revealed that these ORFs could be broadly classified into five functional modules: hers. Of the 64 ORFs, 36 are hypothetical proteins, while the remaining 28 are functional genes. Specifically, 9.34% (6/64) of the ORFs encode structural proteins essential for phage morphology and assembly, such as the major capsid protein (ORF2), virion morphogenesis protein (ORF5), major tail protein (ORF7), and additional structural proteins (ORF6, ORF12, ORF13). Overall, 6.25% (4/64) of the ORFs are involved in phage replication and DNA processing, including the DNA-binding protein (ORF21), exonuclease (ORF23), ERF superfamily protein (ORF24), and DnaD-like primosome initiator (ORF26). 10.93% (7/64) of the ORFs are associated with DNA packaging, tail and capsid assembly, such as the tape measure chaperone (ORF8), tape measure protein (ORF10), tail assembly protein (ORF17), Mor-like transcriptional activator (ORF38), terminase large subunit (ORF49), portal protein (ORF50), and capsid morphogenesis protein (ORF51). A further 7.81% (5/64) of the ORFs are involved in host lysis, including endolysin (ORF39), holin/antiholin class IV (ORF40), holin/antiholin class I (ORF41), i-spanin (ORF42), and o-spanin (ORF43). Finally, 9.38% (6/64) of the ORFs have functions that are not fully understood, or may participate in multiple stages of the phage life cycle, such as RusA-like resolvase/endonuclease (ORF27), DNA methylase (ORF44), FtsK-like DNA translocase (ORF45), 5-formyltetrahydrofolate cyclo-ligase (ORF52), and putative membrane proteins (ORF56, ORF64). Additionally, there are 37 hypothetical proteins whose functions remain unclear, requiring further research to uncover their potential importance.

Whole-genome alignment revealed that phage vB_SmaS_QH16 shares 52–53% sequence coverage with closely related phages StM171 (MZ611865.1), HXX_Dennis (ON711490.1), and Suso (MZ326866.1) (BLASTn, E-value = 0) ( Supplementary Table 3 ). However, its maximum similarity was only 79.37%, far below the species-level classification threshold (95% ANI), suggesting that it likely represents a novel species. Further analysis using VIRIDIC for ANI calculations revealed that the ANI value between vB_SmaS_QH16 and its closest relative, HXX_Dennis, was only 59.5% ( Figure 5A ), well below the genus-level threshold of 70% ANI (ICTV 2023), indicating the need to classify this phage as a new species within a novel genus.

Phylogenetic analysis using the VICTOR tool demonstrated significant differences in tree topology stability across different distance formulas (D0, D4, D6). The D4 formula yielded the highest branch support (average 88%), outperforming D0 (50%) and D6 (53%). Thus, D4 was selected for further analysis. OPTSIL clustering further identified four species clusters, two genus clusters, and two family clusters ( Figure 5B ). The phylogenetic tree showed that vB_SmaS_QH16 forms a distinct evolutionary branch, supporting its differentiation at the family, genus, and species levels. This further reinforces its classification as a member of a new genus within the class Caudoviricetes. Additionally, single-gene phylogenetic trees for the terminase large subunit ( Figure 5C ) and major capsid protein ( Figure 5D ) confirmed that vB_SmaS_QH16 is most closely related to phages StM171 (MZ611865.1), HXX_Dennis (ON711490.1), and Suso (MZ326866.1) within a larger clade. In summary, vB_SmaS_QH16 is a novel phage that can be classified within the class Caudoviricetes.

Crystal violet staining, MTT assays, and acridine orange fluorescence staining were conducted to evaluate the total biofilm mass, metabolic activity, and three-dimensional structure, respectively, to investigate the effects of phage vB_SmaS_QH16 on S. maltophilia no.981 biofilms. During biofilm formation, phage treatment significantly reduced biofilm mass (P < 0.0001). The inhibition rate revealed a concentration-dependent increase as the MOI decreased from 100 to 0.01, rising from 65.02% to 89.61%, indicating better inhibition at lower phage concentrations ( Figure 6A ). This trend was confirmed by MTT assays, in which metabolic activity inhibition correlated with biofilm mass reduction across the different MOI groups ( Figure 6C ). Fluorescence microscopy further showed that the control group had dense three-dimensional biofilm structures, whereas the treated groups exhibited significant structural disintegration, with mesh-like or discrete distributions and a marked reduction in bacterial aggregation ( Figure 6E ). In experiments targeting mature biofilms, phage treatment led to a significant reduction in biofilm mass (P < 0.0001) ( Figure 6B ), with MTT results confirming a corresponding decrease in metabolic activity that positively correlated with phage concentration ( Figure 6D ). Structural analysis further revealed that treated biofilms exhibited disrupted three-dimensional architecture, forming loose, net-like morphologies with reduced bacterial density ( Figure 6E ). Overall, vB_SmaS_QH16 demonstrated significant efficacy at inhibiting biofilm formation and eradicating mature biofilms, with distinct concentration-dependent effects: lower concentrations were more effective at preventing biofilm formation, while higher concentrations were better suited to clearing established biofilms.

As shown in Figure 7A , the growth curve of the S. maltophilia no.981 control group in LB liquid medium exhibited a typical “S” shape. Following the addition of phage vB_SmaS_QH16, all phage-treated groups demonstrated significant antibacterial effects, with inhibition rates at 24 hours of 59.61% (MOI 100), 67.77% (MOI 10), 68.77% (MOI 1), 76.63% (MOI 0.1), 81.12% (MOI 0.01), 83.56% (MOI 0.001), and 80.66% (MOI 0.0001) ( Figure 7B ). However, the different concentration groups exhibited dynamic heterogeneous growth patterns: in the high-concentration groups (MOI 100–0.1), bacterial growth was completely suppressed in the initial stage (0–10 hours), indicating phage dominance. However, after 10 hours, a gradual increase in bacterial concentration was observed across the groups, indicating the emergence and proliferation of phage-resistant mutants. In the low-concentration groups (MOI 0.01–0.0001), a transient increase in bacterial concentration occurred during the initial stage (0–9 hours), reflecting a temporary advantage of the host bacteria. Subsequently, between 9 and 11 hours, an inflection point in concentration was observed, marked by a significant decrease in bacterial load, indicating that the phage established a secondary infection advantage through the lytic cycle. Notably, all treated groups began to show an increase in bacterial concentrations after 16 hours, suggesting a dynamic balance between resistant mutants and residual phages, forming a periodic pattern of “resistance escape-phage inhibition.”

To assay bacteriostatic activity, a gradient concentration of host bacteria (1×10⁵–1×10⁹ CFU/mL) was injected to determine the optimal bacterial concentration for the G. mellonella infection model ( Supplementary Figure 1 ). The results revealed a significant positive correlation between the lethal effect of the host bacteria and the concentration. The group injected with 1×10⁹ CFU/mL exhibited 100% mortality within 16 hours; however, this rapid lethality was not conducive to experimental observation. The group injected with 1×10⁸ CFU/mL achieved progressive 100% mortality within 48 hours, meeting the requirement for controllable experimental progression ( Figure 8A ). Injections of 1×10⁷, 1×10⁶, and 1×10⁵ CFU/mL induced mortality with decreasing incidence rates of 26.67%, 13.33%, and 10%, respectively, with significant delays. Balancing lethal efficiency and the observation window, 1×10⁸ CFU/mL was selected as the standard infection concentration. In the experimental system, the survival rate of larvae in the PBS-treated group remained at 100%, with no abnormal pigmentation observed on the larval surface, confirming the non-toxicity of the buffer. To ensure experimental consistency, host bacteria were washed three times with PBS and resuspended, while phages were diluted using the same buffer to eliminate potential interference from residual culture medium.

This study evaluated the protective effects of phage vB_SmaS_QH16 against G. mellonella infection using therapeutic intervention and prophylactic protection models. In the therapeutic model, injecting phages at different multiplicities of infection (MOI, 0.1–100) following host bacterial infection significantly improved larval survival rates (P < 0.001). During the 72-hour observation period, survival rates exhibited a non-monotonic dose-dependent relationship. The highest survival rate (80.00%) was achieved at an MOI of 10, followed by 100 (70.00%) and 1 (66.67%). The survival rate at MOI = 0.1 was significantly reduced to 40.00%, contrasting sharply with the PBS control group, which exhibited no survival ( Figure 8B ). In the prophylactic intervention model, pre-injection of phages prior to bacterial challenge demonstrated superior protective effects. Survival rates at MOI = 10 remained at 80.00%, while the rates at MOI = 100, 1, and 0.1 were 76.67%, 73.33%, and 56.67%, respectively. Notably, MOI = 10 achieved the highest protective efficiency in both models, indicating that this MOI may represent an optimal intervention threshold in vivo ( Figure 8C ). Safety validation further confirmed that larvae injected with phage alone (without bacterial challenge) exhibited 100% survival and no observable abnormal phenotypes, indicating no toxicity of the phage to the host. Throughout the experiment, PBS was used as the buffer system, and the 100% survival rate of the PBS control group further validated the reliability of the experimental setup.

Survival analysis revealed that all mice in the positive control group died within 3 days post-infection, whereas those in the negative control group survived throughout the experiment. In infected mice treated with varying concentrations of phages, mortality occurred during the early phase (days 1–2), but survival rates stabilized from day 3 onward. Compared with the positive control group, the phage-treated groups (MOI 10, 1, 0.1, and 0.01) demonstrated a significant enhancement in 7-day survival rates, with improvements of 50% (P < 0.05), 50% (P < 0.05), 60% (P < 0.01), and 20% (P > 0.05), respectively ( Figure 9A ). Bacterial load quantification demonstrated that 30 hours post - phage treatment, the pulmonary burden of S. maltophilia no. 981 in the positive control group reached 10⁶ CFU/g. In contrast, mice treated with higher phage titers (MOI = 10, 1, and 0.1) showed a marked reduction in bacterial loads to approximately 10³ CFU/g (P < 0.0001). Even at the lowest phage concentration (MOI = 0.01), the bacterial load was significantly reduced to 10⁴ CFU/g (P < 0.0001) ( Figure 9B ).

Gross examination of murine lung tissues revealed normal pulmonary morphology in the negative control group. In contrast, the positive control group exhibited marked pulmonary swelling with multiple hemorrhagic foci. All phage-treated groups (MOI=10, 1, 0.1, 0.01) showed varying degrees of congestion and edema, with only minor hemorrhagic spots observed in the MOI=0.01 group. Notably, the overall pathological severity in all treatment groups was substantially milder than in the positive controls ( Figure 10A ). Histopathological examination further confirmed that the positive control group’s lung parenchyma had multiple scattered bacterial colonies along with lymphocytic and neutrophilic infiltration. In contrast, higher-titer phage treatment (MOI = 10, 1, 0.1) significantly alleviated these pulmonary pathological changes ( Figure 10B ).