Novel acinetobacter baumannii phage vBAbaSqsb1 and application thereof

Abstract

The invention discloses a novel acinetobacter baumannii phage vBAbaSqsb1 and application thereof, and belongs to the technical field of microorganisms. The bacteriophage is an Acinetobacter baumannii bacteriophage vBAbaSqsb1 which is preserved in the Guangdong Microbiological Culture Collection Center on July 5, 2024, the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 64831-B1, and the preservation address is Institute of Microbiology of Academy of Sciences of Guangdong Province. The bacteriophage can specifically recognize and split the acinetobacter baumannii so as to eliminate infection and overcome the problem of antibiotic resistance of the acinetobacter baumannii. Through discovery and application research of the phage vBAbaSqsb1, the phage vBAbaSqsb1 not only can be used for preparing a phage preparation for preventing and treating diseases caused by acinetobacter baumannii infection, but also can be used for preparing various environmental bactericides for solving potential acinetobacter baumannii in various environments. The bacteriophage vBAbaSqsb1 has the characteristics of safety, no toxic or side effect and the like, so that the problem of drug resistance caused by abuse of antibiotics can be avoided when the bacteriophage vBAbaSqsb1 is applied.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a novel Acinetobacter baumannii phage vB_AbaS_qsb1 and an application thereof. Background Art

[0002] In recent years, the widespread use and even abuse of broad-spectrum antibiotics have led to severe bacterial resistance, and even the emergence of "super bacteria" that are ineffective against any antibacterial drugs. Clinical anti-infection treatment faces huge challenges, and the research and development of new antibiotics has entered a bottleneck period at this stage. Acinetobacter baumannii is a Gram-negative bacterium that is a strictly aerobic, non-flagellated, non-lactose-fermenting conditional pathogen. It is widely distributed in nature and is also one of the common pathogens that cause various complex infections in hospital environments. The most common are pneumonia and sepsis associated with Acinetobacter baumannii. The mortality rate of patients infected with Acinetobacter baumannii in the intensive care unit (ICU) is as high as 54%. Acinetobacter baumannii infection has become one of the risk factors for death in ICU patients. It has been recognized as the main pathogen that causes lung infection, sepsis, meningitis, urinary tract infection and wound infection and leads to patient death. Long-term use of carbapenem antibiotics, combined use of multiple antibiotics, invasive mechanical ventilation, long-term stay in the ICU, and decreased immune function can lead to the production of multidrug-resistant Acinetobacter baumannii. Acinetobacter baumannii is naturally resistant to most antibiotics. If multidrug-resistant strains appear, clinical treatment will be more difficult.

[0003] Bacteriophages are tiny, simple organisms that are mainly composed of nucleic acids and protein shells. Depending on the state of the phage after infecting bacteria, phages can be divided into mild phages and virulent phages. Virulent phages are key tools in phage therapy. Their powerful bacterial lysis ability makes phage therapy a potential treatment for drug-resistant pathogen infections. As a naturally occurring bacterial killer, phages have a unique bactericidal mechanism and can specifically attack drug-resistant bacteria. Summary of the invention

[0004] The purpose of the present invention is to provide a novel Acinetobacter baumannii phage and application thereof.

[0005] The bacteriophage is Acinetobacter baumannii Phage vB_AbaS_qsb1, and the Acinetobacter baumannii Phage has been deposited in Guangdong Microbial Culture Collection Center on July 5, 2024, with the deposit number GDMCC NO: 64831-B1, and the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences.

[0006] The phage was isolated from Qishan Lake Park in Fuzhou City, Fujian Province. Transmission electron microscopy results showed that its head diameter was about 70×80nm, the tail length was about 90nm, and the tail diameter was 20nm. According to the classification rules of the International Committee on Taxonomy of Viruses (ICTV), it belongs to the Siphoviridae family of the Caudovirales order and was named vB_AbaS_qsb1.

[0007] A series of in vitro tests showed that the phage vB_AbaS_qsb1 had good lysis ability against Acinetobacter baumannii and good stability, and maintained a high titer in the range of 10℃ to 60℃ and had good activity in the range of pH 1 to 11. Further extraction of the phage genome and full gene analysis and comparison revealed that this was a new phage strain that had not been reported before. After amplification, the phage titer could reach 10 11 PFU / mL, with good application prospects. A series of in vivo experiments in mice showed that the survival rate of mice after tracheal instillation of high-dose phage liquid was 100%, no strong immune response was induced, and the safety was good. A mouse pneumonia model was constructed and phage administration was used for treatment, and the phage protection rate could reach 80%.

[0008] The present invention also provides the use of the bacteriophage in preparing an antibacterial preparation, wherein the antibacterial preparation is an Acinetobacter baumannii antibacterial preparation.

[0009] The present invention also provides a phage preparation, which comprises the Acinetobacter baumannii phage vB_AbaS_qsb1.

[0010] The present invention also provides the use of the bacteriophage preparation in preparing a medicine for preventing or treating a disease caused by Acinetobacter baumannii infection.

[0011] The present invention screens a highly active and virulent Acinetobacter baumannii phage vB_AbaS_qsb1 from the natural environment. The phage can specifically identify and lyse Acinetobacter baumannii to eliminate infection and overcome the problem of antibiotic resistance of Acinetobacter baumannii. The discovery and application research of phage vB_AbaS_qsb1 can not only be used to prepare phage preparations for preventing and treating diseases caused by Acinetobacter baumannii infection, but also can be used to prepare various environmental bactericides to solve potential Acinetobacter baumannii in various environments. The safety and non-toxic side effects exhibited by phage vB_AbaS_qsb1 enable its application to avoid the problem of drug resistance caused by the abuse of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The plaques and transmission electron microscopy images of bacteriophage vB_AbaS_qsb1.

[0013] Figure 2 The optimal infection multiplicity (A) and one-step growth curve (B) of bacteriophage vB_AbaS_qsb1.

[0014] Figure 3 The temperature stability (C) and pH stability (D) of bacteriophage vB_AbaS_qsb1.

[0015] Figure 4 Verification of the nucleic acid type of the bacteriophage vB_AbaS_qsb1 genome;

[0016] (a) 0.7% agarose gel electrophoresis of vB_AbaS_qsb1 genome; M: Takaraλ-Hind IIIDNALadder; 1-vB_AbaS_qsb1 genome; 2-DnaseI treated; 3-RnaseA treated;

[0017] (b) Verification of RNaseA activity: 4-RnaseA treated; 5-RNA sample.

[0018] Figure 5 Verification of the genome size of bacteriophage vB_AbaS_qsb1; M: Bio-RAD DNA Size Standards-5kb ladder; 1-vB_AbaS_qsb1 genome.

[0019] Figure 6 This is the complete genome map of bacteriophage vB_AbaS_qsb1.

[0020] Figure 7 This is the NCBI alignment result of bacteriophage vB_AbaS_qsb1.

[0021] Figure 8 To verify the safety of bacteriophage vB_AbaS_qsb1.

[0022] Fig. 9 Dose verification for the mouse Acinetobacter baumannii pneumonia model.

[0023] Fig.10 Phage therapy efficacy in a mouse model of Acinetobacter baumannii pneumonia. DETAILED DESCRIPTION

[0024] In the following example, Acinetobacter baumannii ioag01 can be obtained from the research published by the inventor team of this application, see the literature for details: LIN J, JIANG W, SHI Y, et al. Metagenomic Sequencing Revealed the Potential Pathogenic Threats of Banknotes[J]. ACS Omega, 2021, 6(5): 3499-507. Example 1 Isolation, screening and identification of bacteriophages

[0025] 1. Isolation and culture of bacteriophage

[0026] (1) Preparation of host bacterial suspension

[0027] After streaking Acinetobacter baumannii ioag01 on an LB solid culture medium plate, the plate was cultured at 37°C overnight. The next day, a single colony was picked and inoculated into LB liquid culture medium, and the plate was cultured at 37°C and 220 rpm overnight to obtain a host bacterial culture.

[0028] (2) Collection and processing of environmental samples

[0029] On August 31, 2023, lake water samples from Qishan Lake Park in Fuzhou City were collected in centrifuge tubes, then centrifuged at maximum centrifugal force at 4°C for 10 min, the supernatant was passed through a 0.22 μm filter membrane, and the filtrate was collected for later use.

[0030] (3) Isolation and purification of bacteriophage

[0031] 1. Propagation of phage in filtrate

[0032] The host bacterial solution obtained in step (1) was centrifuged at 10000g and 4°C for 1 min to collect the bacterial precipitate. Then, 5 mL of 2×LB liquid culture medium was added to resuspend the bacterial precipitate, and then 5 mL of the lake water filtrate in step (2) was added to it. After sufficient mixing, the mixture was cultured at 37°C and 220 rpm until the liquid was clear. The clarified co-culture solution was dispensed into centrifuge tubes, centrifuged at 4°C at maximum centrifugal force for 10 min, and the supernatant was collected.

[0033] 2. Verification of the co-culture solution by drip method

[0034] Take 1 mL of host bacterial solution and 15 mL of LB semi-solid medium and mix thoroughly, then quickly pour it onto the LB solid medium that has been plated in advance to make a double-layer plate. After the semi-solid medium solidifies, spot 5 μL of the co-culture supernatant in (1) above on the surface of the semi-solid medium, then culture overnight at 37°C. The next day, observe the formation of plaques, and perform subsequent phage isolation on the supernatant sample (i.e., phage stock solution) that is positive by the spot method.

[0035] 3. Isolation and purification of bacteriophage

[0036] The double-layer plate method was used to separate and purify the phage. First, the phage stock solution was diluted tenfold to the appropriate concentration, and then 500μL of the phage dilution solution was mixed with 500μL of the host bacterial solution and incubated at 37℃ for 10min. The entire mixture was added to 15mL of LB semi-solid medium, mixed and poured onto the pre-plated solid medium, sealed with a sealing film, and inverted and cultured at 37℃ overnight. The next day, a single plaque with clear edges was picked up and added to 500μL of SM buffer, vortexed and left to stand at 4℃ for more than 6h to extract the phage. 500μL of the host bacterial solution was added to the phage leaching solution, and co-cultured at 37℃ overnight to proliferate the phage, thus completing the first round of purification. The next day, the phage proliferation solution was centrifuged at 20000g and 4℃ for 10min, and the supernatant was collected. The supernatant was taken and repeated step 3 3-5 times to complete the phage purification.

[0037] After purification, 1 mL of phage proliferation liquid was added to 5 mL of host bacterial liquid, and cultured at 37°C and 220 rpm until the bacterial liquid became clear, completing the phage expansion culture.

[0038] 4. Phage titer determination and seed preservation

[0039] The phage culture solution was centrifuged at 20000g and 4℃ for 10min, the supernatant was filtered through a 0.22μm filter membrane, and the filtrate was collected. The phage titer in the culture solution was then determined by the double-layer plate method. The phage solution was diluted tenfold to the appropriate concentration, and then 500μL of the phage dilution solution was mixed with 500μL of the host bacterial solution and incubated at 37℃ for 10min. The entire mixture was added to 15mL LB semi-solid culture medium, mixed well, and poured onto the pre-plated solid culture medium, sealed with a sealing film, and inverted at 37℃ overnight. The plaques were counted the next day, and the phage titer was calculated according to the following formula.

[0040] (Unit: PFU / mL)

[0041] Phages were preserved with PEG-4000 at a final concentration of 10 mg / mL. High titer phage solution was mixed evenly with an equal volume of 20 mg / mL PEG-4000, sealed with a sealing film, and stored at -80°C.

[0042] 2. Determination of biological characteristics of bacteriophage

[0043] (1) Transmission electron microscopy observation of bacteriophage morphology

[0044] Method: Take 20 μL high titer phage solution (10 11 PFU / mL) to the surface of the copper mesh, let it stand for 10 minutes to allow the phage to adsorb, and use filter paper to absorb the excess phage liquid. Then add 20μL of 4% tungsten phosphate to the center of the copper mesh for negative staining for 90s, use filter paper to remove the excess staining liquid, and observe the phage morphology under a transmission electron microscope at an accelerating voltage of 100kV after drying overnight.

[0045] Results: Figure 1 As shown in the figure, transmission electron microscopy (TEM) results show that the head of qsb1 is about 70×80nm, the tail is about 90nm long, the tail diameter is about 20nm, and no short tail morphology is found under the transmission electron microscope. According to the classification rules of the International Committee on Taxonomy of Viruses (ICTV), the phage was identified as belonging to the family of Caudovirales and named vB_AbaS_qsb1.

[0046] (2) Determination of optimal infection multiplicity of phage

[0047] Methods: First, the bacterial concentration was quantified by turbidimetry, and the bacterial concentration was adjusted to 10 using LB liquid medium. 8 CFU / mL. The titer of the phage suspension was determined by the double-layer plate method, and then 500 μL of bacterial suspension was mixed with 500 μL of phage suspension at infection multiplicity of 0.01, 0.01, 1, 10, and 100, and co-cultured at 37°C for 6 hours. The co-culture solution was then centrifuged at 20000g for 10 minutes at 4°C, the supernatant was collected and the phage titer in the supernatant was determined by the double-layer plate method, and the determination was repeated 3 times, and the results were averaged.

[0048] Results: The optimal infection multiplicity of phage is Figure 2 As shown in (A), the optimal infection multiplicity of phage and bacteria is 0.1, at which the phage proliferation efficiency is the highest, and the phage titer reaches 1.91×10 within 6 hours. 9 PFU / mL.

[0049] (3) Phage one-step growth curve determination

[0050] Methods: First, the bacterial concentration was quantified by turbidimetry, and the bacterial concentration was adjusted to 10 using LB liquid medium. 8 CFU / mL. The titer of phage suspension was determined by double-layer plate method. 5 mL of phage liquid and 5 mL of host bacterial liquid were fully mixed according to the optimal infection multiplicity, incubated at 37°C for 10 min to allow phage adsorption, and then centrifuged at 20,000g, 4°C for 1 min. The unadsorbed phage in the supernatant was discarded, and the precipitate was washed with 5 mL of LB liquid medium. After centrifugation, the bacterial precipitate was resuspended in 10 mL of LB liquid medium and kept in a constant temperature incubator at 37°C for 4 h. In the first 2 h, the co-culture solution was blown and mixed every 20 min, and 500 μL of the co-culture solution was collected and added to a 1.5 mL EP tube. In the next 2 h, the co-culture solution was blown and mixed every 30 min, and 500 μL of the co-culture solution was collected and added to a 1.5 mL EP tube. A total of 11 samples were taken. Each sample collected was centrifuged at 20,000g, 4°C for 10 min, and the supernatant was collected in a new sterile 1.5 mL EP tube. The phage titer in the supernatant was then determined by the double-layer plate method, and the determination was repeated 3 times, and the results were averaged. The one-step growth curve of phage was drawn with the infection time as the horizontal axis and the phage titer as the vertical axis.

[0051] Results: The one-step growth curve of phage is as follows Figure 2 As shown in (B), the lysis cycle of vB_AbaS_qsb1 is about 120 minutes long, the incubation period of qsb1 is short (less than 20 minutes), the burst period is 20 minutes after infection, the number of phages increases sharply within 20 to 120 minutes, and reaches a plateau at 120 minutes. According to the results, the burst amount of the phage is calculated according to the following formula to be about 24 PFU·cell -1 This indicates that the phage has a short incubation period, a short lysis period, and high lysis activity, and is suitable for phage therapy.

[0052] Phage burst = plateau phage titer / initial infection host bacteria concentration (unit: PFU·cell -1 )

[0053] (4) Phage temperature stability determination

[0054] Methods: The titer of phage suspension was determined by double-layer plate method and the titer of phage suspension was adjusted to 1×10 7 PFU / mL. Take 180μL of phage liquid and add it to the PCR tube, and set the temperature gradient (10℃, 20℃, 40℃, 60℃, 80℃, 100℃) in the PCR instrument and keep it warm for 30min, 60min, and 90min respectively. After the insulation, take 100μL of phage liquid and use the double-layer plate method to determine the titer. Repeat the measurement 3 times and take the average value of the results.

[0055] Results: Phage temperature stability Figure 3 As shown in (C), vB_AbaS_qsb1 has good stability between 10℃ and 60℃, and the phage titer basically does not change. With the increase of treatment time, the phage titer does not change much at temperatures before 60℃ (the overall change is within 5%). When treated at 60℃ for 90min, the phage titer decreases slightly. At 80℃ and 100℃, the phage completely loses its activity in a short time. This shows that the phage has a wide temperature tolerance range and is relatively stable at 60℃ and below.

[0056] (5) Phage pH stability determination

[0057] Methods: The titer of phage suspension was determined by double-layer plate method and the titer of phage suspension was adjusted to 1×10 7 PFU / mL. Take 100μL of phage liquid and add it to 900μL of SM buffer with different pH values ​​(1, 3, 5, 7, 8, 9, 11, 13), and keep it at 4℃ for 1h. After the insulation, add appropriate amount of NaOH or HCl to the samples with pH 1 and 13 to adjust the pH to 7 so that the bacteria can grow normally. The pH of the other samples can grow normally, so no additional treatment is required. Finally, the double-layer plate method is used to determine the titer of the phage liquid. The experiment is repeated 3 times and the results are averaged.

[0058] Results: The pH stability of phages was as follows: Figure 3 As shown in (D), vB_AbaS_qsb1 has good stability in the pH range of 1 to 11, and the pH is significantly reduced only when pH = 1. The phage activity is completely lost at pH = 13. The results show that the phage has a strong tolerance to acid and alkali.

[0059] 3. Analysis of the complete genome of bacteriophage vB_AbaS_qsb1

[0060] (1) Extraction of phage genome

[0061] Method: The phage genome was extracted by phenol-chloroform extraction, and then an appropriate amount of sterile ddH 2 O resuspend the nucleic acid pellet and measure the nucleic acid concentration using Nanodrop2000.

[0062] (2) Identification of phage nucleic acid types

[0063] Method: Take 200 ng of vB_AbaS_qsb1 phage genome into sterile PCR tubes, add 1 μL 200 μg / mL RNaseA to one tube and add sterile ddH 2O to a total volume of 10 μL, then incubate at 28°C for 30 min, add 1 μL DNaseI to another tube and add sterile ddHO 2 O to a total volume of 10 μL, and then incubate at 37°C for 15 minutes. After the incubation, perform 0.7% agarose gel electrophoresis: weigh 0.14g agarose and dissolve it in 20ml 1×TAE solution, add 1μL 5mg / mL ethidium bromide (EB), mix thoroughly and pour into the gel plate, and place it in the electrophoresis tank after solidification. Then take 1.5μL λ-Hind IIIDNAMarker, 200ng genomic sample and the above enzyme treatment solution and mix with 6×Loading buffer and load the sample, electrophoresis at 80V for 30-45min, and observe the electrophoresis and band position under the gel imager after the electrophoresis is completed.

[0064] Results: The results are as follows Figure 4 As shown, the vB_AbaS_qsb1 phage genome was treated with RnaseA and DnaseI and then electrophoresed. The genome band did not change after RnaseA treatment, which was consistent with the genome electrophoresis result. However, after DnaseI treatment, the genome band disappeared, indicating that the phage genome nucleic acid type was DNA.

[0065] (3) Identification of phage genome size

[0066] Methods: Pulse field electrophoresis was used to verify the size of the phage genome. A 0.85% pulse field electrophoresis gel was prepared and mixed thoroughly, then poured into a gel plate and placed in a pulse field electrophoresis tank after solidification. Then DNA Size Standards-5kbladder and an appropriate amount of genome sample (mixed with 6× Loading buffer in advance) were loaded and electrophoresed at 80V for 14h. After the electrophoresis, staining was performed in TBE Buffer containing ethidium bromide for 30min, and the electrophoresis and band positions were observed under a gel imager.

[0067] Results: The results are as follows Figure 5 As shown, the phage genome has a band near the 49.8 kbp band that is slightly larger than the marker.

[0068] (4) Assembly and annotation of phage genomes

[0069] Methods: The phage genome was constructed using the Nextera library construction kit with an insert fragment of about 500 bp. After the library was quality checked by capillary electrophoresis, it was sent to a third-party sequencing company for second-generation high-throughput sequencing based on the Illumina Novaseq X Plus sequencing platform and PE-150 mode. After the sequencing results were returned, the sequencing data were quality controlled using Fastqc (https: / / github.com / s-andrews / FastQC) and Trim Galore (https: / / github.com / FelixKrueger / Trim Galore). The quality-controlled data were spliced ​​without parameters using megahit (https: / / github.com / voutcn / megahit) software to obtain contigs. The contigs were assembled into scaffolds using metaSPAdes (https: / / anaconda.org / bioconda / spades). The scaffolds were compared and visualized with the original sequencing data using IGV (https: / / github.com / igvteam / igv). Prokka (https: / / github.com / tseemann / prokka), RAST (https: / / rast.nmpdr.org / ), and GeneMarks (https: / / exon.gatech.edu / GeneMark / genemarks.cgi) were used to annotate the assembled phage sequences. Finally, CGView online service (https: / / proksee.ca) was used to circle the phage genome and visualize the information contained in the genome. This step helped us to intuitively display the structure and functional regions of the genome.

[0070] Results: The vB_AbaS_qsb1 genome is 54713bp long, with a C+G content of 39%. There are 94 CDSs, of which 23 CDSs are located on the positive strand, which means that the transcription direction of these CDSs is consistent with the direction of the genome reference sequence. The other 71 CDSs are located on the negative strand, and their transcription direction is opposite to the direction of the genome reference sequence. Among the 94 CDSs, there are 28 CDSs with known functions, 2 tRNAs, and 64 hypothetical proteins with unknown functions. The results are as follows: Figure 6The whole genome sequence of vB_AbaS_qsb1 was uploaded to NCBI for Blast. The comparison results showed that the phage with the highest similarity to vB_AbaS_qsb1 was vB_AbaS_TRS1 (NC.031098.1), but the comparison coverage was only 20%. Therefore, vB_AbaS_qsb1 was confirmed to be a new type of Acinetobacter baumannii phage (Acinetobacter baumannii Phage), and the phage was deposited in Guangdong Microbial Culture Collection Center, with a deposit date of July 5, 2024, a deposit number of GDMCC NO: 64831-B1, and a deposit address of Institute of Microbiology, Guangdong Academy of Sciences.

[0071] 4. Establishment of the mouse model of Acinetobacter baumannii lung infection and phagosome therapy

[0072] (1) Phage safety assessment:

[0073] Methods: Ten 6-8 week old male BALB / C mice were divided into two groups. After the mice were anesthetized with 2.5% Avertin, 30 μL of 10 9 PFU / mL phage solution, and the mice in the blank control group were dripped with an equal volume of normal saline, and then housed in normal cages for 7 days and the status of the mice was observed.

[0074] Conclusion: If Figure 8 As shown, no clinical symptoms were found in all mice during the 7-day observation period, and the survival rate of all mice was 100% (Note: the curves of the phage-treated group and the blank control group overlap). This proves that after the mice were dripped with high-dose phage fluid, there was no adverse symptoms such as severe immune response in the mice, and the phage was safe.

[0075] (2) Establishment of mouse lung infection model

[0076] Methods: Thirty 6-8 week old male BALB / C mice were divided into five groups, four in the bacterial attack group and one in the control group. They were injected with 200 mg / kg and 150 mg / kg of cyclophosphamide 4 days and 1 day before modeling, respectively. After the mice were anesthetized, 3×10 9 CFU, 3×10 8 CFU, 3×10 7 CFU, 3×10 6 The mice in the blank control group were instilled with an equal volume of normal saline into the lungs, and then housed in normal cages for 7 days and the condition of the mice was observed. Then the median lethal dose for mice was used as the subsequent modeling dose.

[0077] Conclusion: The survival of mice is Fig. 9 During the 7-day observation period, the high-concentration bacterial solution drip group (3×10 9 CFU / mouse) mice had a mortality rate of 100%, and the medium and high concentration bacterial liquid instillation groups (3×10 8 CFU / mouse) mortality rate exceeded 80%, and the medium concentration bacterial solution drip group (3×10 7 CFU / mouse) mice mortality rate was only 20%, and the low concentration bacterial solution drip group (3×10 6 CFU / mouse) and the blank control group (Control) had a 0% mortality rate. Based on the mortality rate of mice, it was shown that we had successfully established acute pneumonia in mice, so we finally selected 3×10 8 The dose of CFU / mouse was used as the modeling dose for the mouse lung infection model.

[0078] (3) Phage therapy in mice with acute pneumonia

[0079] Methods: Twenty-five 6-8 week old male BALB / C mice were divided into five groups, three phagosome treatment groups and two control groups. They were injected with 200 mg / kg and 150 mg / kg of cyclophosphamide 4 days and 1 day before modeling, respectively. After anesthesia, all mice were instilled with 3×10 8 ioag01 bacterial solution. One hour after infection, mice in the phage treatment group were instilled with 3×10 9 PFU (high-dose treatment group), 3×10 8 PFU (medium dose treatment group), 3×10 7 PFU (low-dose treatment group) of phage vB_AbaS_qsb1, the model control group mice were instilled with an equal volume of saline into the lungs 1 hour after the infection, and the blank control group mice were instilled with an equal volume of saline into the lungs 1 hour after the instillation of saline. Then they were caged normally for 7 days and the mice were observed.

[0080] Results: The survival of mice within 7 days was as follows Fig.10 As shown. Among all the phage-treated mice, the medium-dose treatment group, the low-dose treatment group and the model-treated Hangzhou Ah-Zhu mice all had a high mortality rate (reaching 80%), and there was a significant difference between the survival rates of mice in these groups and those in the blank control group. The mortality rate of mice in the high-dose treatment group was only 20%, no mice died in the blank control group, and there was no significant difference in the survival rate between the high-dose treatment group and the blank control group. This shows that high-dose phage treatment can effectively alleviate the symptoms of lung infection in mice and significantly improve the survival rate of mice.

Claims

1. A bacteriophage, It is characterized in that The bacteriophage is Acinetobacter baumannii phage ( Acinetobacter Phage baumannii )vB_AbaS_qsb1, the Acinetobacter baumannii phage ( Acinetobacter baumannii Phage ) was deposited in Guangdong Microbiological Culture Collection Center on July 5, 2024, with the deposit number GDMCC NO: 64831-B1, and the deposit address is Institute of Microbiology, Guangdong Academy of Sciences.

2. Use of the bacteriophage according to claim 1 in the preparation of an antibacterial preparation, It is characterized in that The antibacterial preparation is an Acinetobacter baumannii antibacterial preparation.

3. A bacteriophage preparation, It is characterized in that The phage preparation comprises the Acinetobacter baumannii phage as claimed in claim 1 ( Acinetobacter baumannii Phage )vB_AbaS_qsb1.

4. Use of the phage preparation according to claim 3 in the preparation of a medicament for preventing or treating a disease caused by Acinetobacter baumannii infection.

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