Animals
Sixty-five female SPF Kunming (KM) mice, aged 6 weeks and weighing approximately 22 g, were purchased from Dashuo Laboratory Animal Co., Ltd. in Chengdu, Sichuan, China. They were subjected to a 12-h light/dark cycle for one week prior to the experiment and were provided with clean, adequate feed, drinking water, and bedding, and were housed for 7 days. All animal experiments conducted in this study complied with the guidelines set forth by the Chinese Animal Welfare Committee and received approval from the Animal Welfare and Research Ethics Committee of Northwest A & F University, located in Shaanxi Province, China (approval number DY2022009).
Host P. aeruginosa strains and culture conditions
A total of 167 P. aeruginosa strains and six different strains, including Klebsiella pneumoniae, Escherichia coli, Streptococcus, Pasteurella, Staphylococcus aureus, and Salmonella, were used in this study. All strains were isolated, characterized, and preserved in the Laboratory of Infectious Diseases, School of Animal Medicine, Northwest A&F University. Among them, PAf5, the host bacterium isolated from the lungs of dead Moschus berezovskii (Eartag: 64,675), exhibited significant drug resistance (Additional file 1). In the double-layer plate method, FMD5 and H24 - 1 formed visible phage plaques in combination with the host bacterium PAf5. Moreover, we used PAf5 as the host bacterium to determine the biological characteristics of the two phages. Therefore, PAf5 was also selected as the mice’s bacterial infection model strain. All strains were identified via cetrimide agar (Qingdao Haibo Biological Company; China) as the identification medium and confirmed via 16S rDNA sequencing. After enrichment and amplification in an LB liquid medium, the bacterial suspension was mixed at a 1:1 ratio with 75% glycerol and stored at − 20 °C and − 80 °C.
Isolation and purification of bacteriophages
The isolation and purification of bacteriophages were performed via the double-layer plate method [16]. Numerous mixed faecal effluent samples were collected from different farms in Shaanxi Province and immersed in the saline solution overnight. The next day, the samples were filtered through three layers of gauze and centrifuged (Centrifuge: China Changsha Yingtai TGL16) at 12,000 rpm for 20 min. The supernatant was extracted and passed through a 0.22 μm filter. 3 mL of the filtered supernatant was blended with 1 mL of host bacteria (PAf5) in 10 mL of LB broth, incubated overnight at 37 °C (Constant-temperature shaking incubator: (China Shanghai Nenggong Industrial Co., Ltd), and then centrifuged at 12,000 rpm for 10 min. The supernatant was passed through a 0.22 m filter to obtain the phage enrichment solution. Subsequently, 100 μL of the phage enrichment solution diluted tenfold was mixed with 100 μL of the host bacteria solution and incubated at 37 °C for 10 min. The mixture was combined with the non-solidified upper medium and poured onto the lower culture plate. After solidification, the mixture was incubated at 37 ℃ for 12 h (Constant-temperature incubator:Thermo Fisher Scientific, USA). Once plaque appeared, the single plaque was dissolved in LB broth, incubated at 37 ℃ for 30 min, centrifuged at 12,000 rpm for 10 min, and diluted with the supernatant at a ratio of tenfold, and the above operations were repeated purify the phage. This process was repeated three times to generate a single spot phage isolate.
Transmission electron microscopy
The morphological characteristics of phages FMD5 and H24 - 1 were observed by Transmission Electron Microscopy (TEM) [17]. The specific operation was as follows: 20 μL of phage-enriched droplets with a concentration of 10^8 PFU/mL were placed on copper mesh and incubated for 10 min. The samples were subsequently stained with 2% (w/v) phosphotungstate (Beijing Solarbio Science & Technology Co., Ltd) for 5 min and air-dried. Finally, the morphology of the phage was observed via an HT7700 Transmission Electron Microscope (TEM, Hitachi, Japan), and the images were preserved.
Host spectrum assay
Phage host profiles were determined via the dot-matrix method [18]. In addition to determining the lysing ability of FMD5 and H24 - 1 against 167 strains of P. aeruginosa from different sources, the specificity of FMD5 and H24 - 1 against different species of bacteria such as Klebsiella pneumoniae, Escherichia coli, Streptococcus, Pasteurella, Staphylococcus aureus, and Salmonella, was also determined. The specific methods were as follows: A uniform 100 μL bacterial mixture containing 1 × 10^8 CFU/mL was spread over LB solid medium (Qingdao Haibo Biological Company; China). Next, 3 μL of phage-rich droplets were added to the medium surface, and the mixture was incubated for 12 h at 37 °C. Observe whether bacterial spots appear on the medium to preliminarily assess the range of phage lysis.
Optimal multiplicity of infection (OMOI) and one-step growth curve assay
The multiplicity of infection (MOI) is the ratio of phage to host bacteria. The protocol as follows: the host bacterial suspension was cultivated at the logarithmic phase, and seven groups were designed with MOIs of 100, 10, 1, 0.1, 0.01, 0.001, and 0.0001. One milliliter of phage and one milliliter of host bacteria were added to 10 mL of LB liquid culture medium and incubated at 37 °C in a constant-temperature water bath shaker for 4 h. The mixture was centrifuged at 6000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter. The filtrate was collected, and its titer was determined via the double-layer plate method. Each dilution was replicated three times for each sample, and the results were recorded. The one-step growth curve enabled the observation of the growth law of the phage and the calculation of its incubation period and burst quantity. The measurement of the one-step growth curve was based on a modification of previous test methods [19]. The specific procedure was as follows: the ratio of phage to host bacteria was set as OMOI, 1 mL of each to 10 mL of LB liquid culture medium was added, the mixture was incubated at 37 °C for 5 min, and the mixture was centrifuged at 6000 rpm for 10 min. The supernatant was discarded, and the precipitate was retained. Subsequently, 10 mL of liquid LB culture medium was added to resuspend the mixture, which was subsequently placed in a 37 °C constant-temperature water bath shaker incubator for cultivation. Samples were taken every 5 min within the first 30 min, and then every 10 min until the phage titer stabilized. After each sampling, the samples were centrifuged at 12,000 rpm for 5 min. The supernatant was collected, and a tenfold dilution was performed. The phage titer of each sample was subsequently determined via the double-layer plate method. There groups of parallel samples were prepared for each dilution, and the results were recorded. A one-step growth curve graph was drawn with time on the horizontal axis and the logarithm of the phage titer on the vertical axis.
Temperature, pH, chloroform, and UV stability determinations
To explore the influences of diverse conditions on the titer of phages, the stability of phages under various unfavourable conditions was assessed to facilitate better preservation and application of phages. 500 μL of the phage-rich solution with a known titer was extracted from a centrifuge tube, and the phages were exposed to 1 h of treatment at temperatures of − 20 ℃, 4 ℃, 37 ℃, 50 ℃, 60 ℃, and 70 ℃ before being restored to room temperature (Constant Temperature Water Bath: Gongyi Yuhua Instrument Co., Ltd., China). The titer was calculated by employing the double-layer plate culture method to determine the tolerance of the phages to different temperatures. Similarly, 5 mL of the known titer phage enrichment solution was obtained from each sample and placed in 10 mL centrifuge tubes. The pH of the phage enrichment mixture was adjusted to 2, 4, 6, 8, 10, 12, and 14 by employing hydrochloric acid and sodium hydroxide, and the mixture was placed in a water bath at 37 °C for 1 h. After this procedure, the pH was changed back to neutral, and the double-layer plate method was used to calculate the titer.
Furthermore, 500 μL of the known titer phage enrichment solution was mixed with 500 μL of chloroform. After 2 min of vigorous reaction, the mixture was centrifuged at 12,000 rpm for 5 min. The phage count in the supernatant was determined via the double-layer plate culture method. In the control group, a sterile equivalent amount of Phosphate Buffered Saline (PBS) was used to treat the phage. The differences in the phage titer after chloroform or PBS treatment were compared. Finally, 5 mL of the known titer phage enrichment solution was placed in a 60 mm culture dish and uniformly exposed to ultraviolet light. The phage titer was calculated every 20 min within 2 h via the double-layer plate culture method. The phage titer was recorded every 20 min. Each experiment was replicated three times.
Phage DNA extraction, whole-genome sequencing and analysis
The phage was concentrated via the Polyethylene Glycol (PEG) precipitation approach. DNaseI and RNaseA were added to the phage enrichment solution to reach a final concentration of 1 μg/mL and incubated in a 37 °C water bath for 30 min. Thereupon, 1.76 g of NaCl was added, thoroughly mixed and dissolved, followed by incubation in an ice bath for 1 h. Centrifugation was performed at 12,000 rpm for 10 min at 4 °C. Subsequently, 3 g of PEG8000 was added to the supernatant, thoroughly mixed and dissolved, followed by incubation in an ice bath overnight. The next day, the phage concentrate was acquired through centrifugation at 12,000 rpm for 10 min at 4 °C, elimination of the supernatant, and resuspension of the precipitate in 500 μL of SM buffer. The genome of the phage-concentrated solution was extracted via the TIANamp Virus DNA/RNA Kit (China Tiangen Biochemical Technology Co., Ltd.). The phage DNA bands were detected by 1% agarose gel electrophoresis at 120 V for 30 min (Electrophoresis Apparatus: Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd., China), and then sent to MeiGe Gene Technology Co., Ltd. in Guangdong, China, for second-generation sequencing on the Illumina Nova Seq PE150 platform.
The genome sequences of the phages were assembled using Megahit v1.1.2 [20] software, and gene prediction for the phage genome was performed using Prokka [21] software. The number and length of the predicted genes were subsequently statistically analyzed. Rapid Annotations using Subsystems Technology (RAST) software was used for the rapid annotation of all potential open reading frames (ORFs) within the phage genome, and the BlastP program was utilized on National Center for Biotechnology Information (NCBI) to compare the annotated amino acid sequences and predict their possible functions. tRNAscan-SE software was used to search for tRNAs within the genome. The Phage Scope online tool was utilized for whole-genome comparative analysis and to search for potential toxin genes and resistance genes. On the basis of the results of protein function annotation, CGview online software was used to generate a full genomic map of the phage. MEGA X was used to construct a phylogenetic tree of the conserved genes of the FMD5 and H24 - 1 phages [22].
Determination of the bacteriophage inhibition curve in vitro
To explore the bactericidal efficacy of the phages in vitro, the inhibitory bactericidal curve of the phages was determined through shaking culture at 37 °C. The MOIs of the phage were set at 10, 1, 0.1, 0.01, and 0.001. One milliliter of the phage enrichment mixture and the host bacteria were added to 10 mL of LB liquid culture medium. In the control group, one mL of the phage liquid was replaced with 1 mL of LB mixture culture medium, while the other conditions remained unchanged. The mixture was incubated at 37 °C under shaking conditions. Three samples were taken from each tube at 0 h, and then samples were collected every hour for 15 h, after which the OD630 (Optical Density at 630 nm) variation of the host bacteria was measured, and an LB liquid culture medium was used as the blank control. The in vitro inhibitory curve of the phage was plotted on a graph with time on the horizontal axis and OD630 on the vertical axis. The experiment was replicated three times.
Therapeutic trials in infected animal models
To assess the therapeutic efficacy of the two phages against P. aeruginosa infection in mice, a mouse sepsis model was established via intraperitoneal injection of the host bacteria. First, the half-lethal dose (LD50) of the host bacterium PAf5 was determined. Thirty mice were randomly divided into six groups, one of which received 100 μL of PBS as a control. The remaining five groups were injected intraperitoneally with 100 μL of PAf5 bacterial suspensions at concentrations of 5 × 10^6 CFU/mL, 5 × 10^7 CFU/mL, 5 × 10^8 CFU/mL, 5 × 10^9 CFU/mL, and 5 × 10^10 CFU/mL. The mice were continuously monitored for 7 days, and the mortality of the mice was documented.
Before conducting the formal phage therapy trial, the safety of the phages was evaluated by dividing 10 mice into two groups, with each group receiving intraperitoneal injections of FMD5 and H24 - 1 phages for 7 consecutive days. In the course of the formal treatment, the mice were randomly allocated into 5 groups, each consisting of 5 mice. Among these groups, 4 groups were intraperitoneally administered 100 μL of PAf5 bacterial suspension at the LD50 concentration as the experimental group, while 1 group was designated the blank control group. Two hours after bacterial colonization, phage therapy was initiated. Within the experimental group, two subgroups were intraperitoneally inoculated with 100 μL of FMD5 (10^9 PFU/mL) or H24 - 1 (10^9 PFU/mL) phages.Moreover, one subgroup was administered 100 μL of an equiproportional mixture of the two phages. Another subgroup was injected with 100 μL of PBS as the control group. After 24 h of phage therapy, blood samples were collected from each group, and the experiment was terminated via isoflurane anesthesia. The bacterial load in the blood was determined, and a Mouse TNF-α ELISA Kit (Qianzhou City Ruixin Biotechnology Co., Ltd., China) alongbwith a Mouse IL- 1β ELISA Kit (Shanghai Yuanju Bio-Tech Center, China) were used to measure the contents of the inflammatory factors TNF-α and IL- 1β in 1 mL of blood from each group of mice. In each group of mice, approximately 0.1 g of each of the lungs, kidneys, livers, and spleens was obtained and ground by adding 1 mL of sterile saline(Grinding instrument: MP Biomedicals, USA). The samples were subsequently spread on NAC plates via the dilution smear plate method. The bacterial load and phage load of each organ were calculated. Pathological tissue sections were taken from the lungs, kidneys, livers, and spleens. HE staining was employed to observe the tissue lesions under a microscope (Microscope: Motic China Group CO., LTD).
Statistical analysis
All the experiments were replicated three times to ensure the reproducibility of the results. The data presented in this study were statistically analyzed via Microsoft Excel 2021 and GraphPad Prism 8.0 software. Comparison of all the columns was done through t-tests, and P values less than 0.05 were considered astatistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant difference).