Phage for cracking acinetobacter baumannii and application thereof

Abstract

The invention discloses a bacteriophage for cracking acinetobacter baumannii and application thereof, relates to the technical field of bacteriophages, and aims to solve the problem of resource shortage of existing bacteriophages in improvement of acinetobacter baumannii infection. The preservation number of the bacteriophage (Acinetobacter phage) pT2784 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No.46500, and the bacteriophage is a novel bacteriophage. The bacteriophage pT2784 provided by the invention performs targeted lysis on acinetobacter baumannii, has relatively high sterilization efficiency, does not contain virulence genes and drug-resistant genes, is safe and reliable, has good temperature tolerance and pH stability, has strong adaptability to severe environments, and is a promising candidate for bacteriophage treatment and environmental sterilization.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacteriophages, in particular to a bacteriophage for lysing Acinetobacter baumannii and application thereof. BACKGROUND

[0002] Acinetobacter baumannii is ranked the highest level in the WHO priority pathogens list, often causing various infections in hospitalized patients, especially in intensive care unit patients, with the highest mortality rate in hospital-acquired pneumonia and bloodstream infections. Capsular polysaccharide (CPS) is one of the important virulence factors of Acinetobacter baumannii, and almost all clinical Acinetobacter baumannii strains express a thick surface polysaccharide capsule, showing extensive drug resistance to various drugs, disinfectants and environmental pressures, allowing the bacteria to survive on the surface of medical devices for a long time, increasing the risk of hospital outbreaks. Epidemiological data shows that most hospital-acquired Acinetobacter baumannii infections are caused by two globally disseminated clones, called global clones GC1 and GC2, of which ST2_Pasteur in GC2 is the main epidemic clone in China, and ST40_Pasteur is the second most popular clone after ST2_Pasteur, with a continuous increase in clinical isolation rate in the past three years. Under this background, it is urgent to develop new antibacterial strategies targeting threatening branches.

[0003] Multiple studies have shown that phage therapy can improve the clinical outcome of patients with Acinetobacter baumannii infection. However, its wide application still faces multiple challenges: the lack of phage library resources, the rapid generation of bacterial resistance, and other problems restrict clinical translation, highlighting the need to explore new phage resources and analyze resistance mechanisms. SUMMARY

[0004] In view of the above analysis, the present application aims to provide a bacteriophage for lysing Acinetobacter baumannii and application thereof, in order to solve the problem of lack of phage resources in improving Acinetobacter baumannii infection in the prior art.

[0005] The purpose of the present application is mainly realized by the following technical solutions:

[0006] In one aspect, the present application provides a bacteriophage, wherein the bacteriophage is (Acinetobacter phage) pT2784, and the preservation number is CGMCC No. 46500.

[0007] Further, the bacteriophage is a linear double-stranded DNA, and the full-length of the bacteriophage genome is 44335 bp, without virulence genes and drug resistance genes.

[0008] Further, the GeneBank accession number of the whole genome sequence of the bacteriophage is PV550753.

[0009] Further, the bacteriophage belongs to the Myoviridae family.

[0010] Further, the bacteriophage has a stable pH value of 2-11 and a temperature tolerance of 4-65 DEG C.

[0011] Further, the bacteriophage is specific to Acinetobacter baumannii.

[0012] Further, the bacteriophage has a latent period of 5 min, a burst period of 35 min, and a burst size of 193 PFU / cell.

[0013] The application further provides use of the bacteriophage in lysing Acinetobacter baumannii.

[0014] Further, the bacteriophage has a multiplicity of infection of 1:10000-1:100.

[0015] Further, the bacteriophage can be prepared into oral preparations, sprays or injections.

[0016] Compared with the prior art, the application can achieve at least one of the following beneficial effects:

[0017] (1) The bacteriophage pT2784 is isolated from Acinetobacter baumannii T2784 as a host bacterium, and has strong lytic effect on Acinetobacter baumannii. The bacteriophage pT2784 in the application specifically lyses Acinetobacter baumannii. Comparative genomic analysis and phylogenetic analysis show that the bacteriophage has the highest similarity to Acinetobacter baumannii bacteriophage vB_AbaM_IME284 (MH853788.1), with a genomic similarity of 61% (<70%), and is suggested to form a new genus.

[0018] (2) The Acinetobacter baumannii bacteriophage pT2784 isolated in the application is a virulent bacteriophage isolated from nature, and does not contain virulence genes and drug resistance genes, is safe and reliable, has good temperature tolerance and pH stability, and has strong adaptability to harsh environments, and is a promising candidate for bacteriophage therapy and environmental disinfection and killing.

[0019] (3) The application provides a novel bacteriophage pT2784 targeting Acinetobacter baumannii ST40-KL47, which further enriches the resource of Acinetobacter baumannii bacteriophage library. The burst period and burst size of the bacteriophage pT2784 are similar to those of other bacteriophages, but the latent period is much shorter than that of other Acinetobacter baumannii bacteriophages, only 5 min, indicating that pT2784 has higher bactericidal efficiency.

[0020] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the written description and the accompanying drawings.

[0021] Biological Preservation Information:

[0022] The bacteriophage pT2784 of Acinetobacter baumannii of this invention is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 24, 2025. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China, China. The accession number is CGMCC No. 46500. Attached Figure Description

[0023] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0024] Figure 1 This image shows the morphology of plaques formed by bacteriophage pT2784 on a double-layer plate.

[0025] Figure 2 Electron micrograph of bacteriophage pT2784;

[0026] Figure 3 Annotation diagram of the pT2784 phage genome;

[0027] Figure 4 Phage pT2784 phylogenetic diagram;

[0028] Figure 5 A visualization analysis of the evolution of bacteriophage pT2784;

[0029] Figure 6 A bar chart showing the change in phage pT2784 titer with MOI (Multiple of Infection).

[0030] Figure 7 The growth curve of bacteriophage pT2784 is shown in the first step.

[0031] Figure 8 A schematic diagram illustrating the thermal stability of bacteriophage pT2784;

[0032] Figure 9 A schematic diagram illustrating the acid-base stability of bacteriophage pT2784;

[0033] Figure 10 The in vitro lysis curve of bacteriophage pT2784 is shown.

[0034] Figure 11 Survival curves of the large wax moth inoculated with different concentrations of Acinetobacter baumannii;

[0035] Figure 12 A curve showing the therapeutic effect of in vivo injection of phage pT2784 on the large wax moth. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0037] This invention provides a bacteriophage (Acinetobacterphage) pT2784, with accession number CGMCCNo.46500.

[0038] In this invention, bacteriophage pT2784 was isolated from a wastewater sample from Peking University Third Hospital and deposited on July 24, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46500. The bacteriophage pT2784 of this invention was identified as a lytic myotailviridae bacteriophage with an icosahedral head, approximately 54.32 nm in diameter, and a tail of approximately 67.93 nm. It belongs to the Caudoviricetes class and has a titer of up to 10. 9 pfu / mL or higher; the full-length genome of the phage of this invention is 44,335 bp, the GC content is 37.82%, and it does not contain virulence genes or drug resistance genes. It has the highest similarity to vB_AbaM_IME284 (MH853787.1), with a similarity of 61% (<70%), and it is suggested that it be formed into a new genus.

[0039] In this invention, the GeneBank accession number for the bacteriophage whole genome sequence is PV550753.

[0040] In this invention, phage pT2784 can specifically lyse Acinetobacter baumannii, and phage pT2784 can specifically lyse Acinetobacter baumannii ST40-KL47 type. The preferred multiplicity of infection (MLI) of the phage in this invention is 1:10000-1:100, with an optimal MII of 1:100. It also exhibits good temperature tolerance and pH stability, remaining stable at pH values ​​of 2-11 and at temperatures of 4-65°C.

[0041] In this invention, the latency period of bacteriophage pT2784 is 5 minutes, meaning the time from the adsorption of the bacteriophage onto the bacterial surface to the release of the first batch of progeny bacteriophages is 5 minutes. Afterward, it enters the burst phase, which lasts approximately 35 minutes, with a bacteriophage burst rate of 193 PFU / cell (the bacteriophage burst rate is the difference between the final and initial bacteriophage concentrations divided by the initial bacterial concentration). After 40 minutes, it enters the stationary phase.

[0042] The present invention also provides an application of the above-mentioned bacteriophage in lysing Acinetobacter baumannii.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Unless otherwise specified, the following embodiments are all conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] Example 1

[0047] Isolation and identification of Acinetobacter baumannii phage pT2784

[0048] (1) Phage isolation and identification

[0049] The wastewater samples used in this invention were collected from wastewater at Peking University Third Hospital in 2024.

[0050] Sample preparation: Aliquot the untreated wastewater sample into 50mL centrifuge tubes and centrifuge at 8000rpm for 10min to remove larger solid particles and some bacteria; filter the supernatant through a 0.22μm microporous filter, and store the filtrate in a new 50mL centrifuge tube at 4℃ for later use; mix 1mL of the filtrate with 100uL of overnight culture and 5mL of LB liquid medium, and incubate at 37℃ with shaking at 200rpm / min for 6h, then filter through a 0.22μm filter and collect the filtrate into a new sterile container.

[0051] Phage isolation was performed using the double-layer plate method. 100 μL of host bacterial culture was mixed thoroughly with 5 mL of soft agar and poured onto a bottom agar plate. After solidification, 2 μL of the co-culture filtrate was spotted onto the soft agar, dried, and then incubated overnight at 37°C. The appearance of clear plaques indicated the presence of phages. The clear plaques were removed and resuspended in LB broth for purification using the double-layer plate method. Single clear plaques were picked from the double-layer plates containing phages and purified at least three times until the plaque morphology was consistent. Figure 1 As shown. The purified phage fluid was temporarily stored at 4°C and then stored long-term in 25% glycerol at -80°C.

[0052] (1) Detect phage titer using the double-layer plate method.

[0053] The purified phage solution was serially diluted 10-fold. 100 μL of each phage dilution was mixed thoroughly with 100 μL of host bacterial solution, and then mixed with 5 mL of soft agar. The mixture was poured onto a bottom agar plate, allowed to solidify, and incubated overnight at 37°C. Plates showing 30-300 plaques were selected for plaque counting. Phage titer (PFU / mL) = number of plaques × dilution factor × 10.

[0054] The results showed that the phage plaques exhibited consistent morphology, size, and clarity, displaying typical characteristics of lytic phages. The titer of this phage reached 10. 9 pfu / mL or higher.

[0055] Example 2

[0056] Electron microscopic observation of bacteriophages

[0057] Take 20 μl of phage liquid and drop it onto a carbon-coated copper grid. After the phage has settled fully on the surface of the copper grid, absorb the excess liquid from the side. Negatively stain the phage particles with 2% (wt / vol) uranium acetate and observe and photograph them under a transmission electron microscope at 80 kV.

[0058] The results are as follows Figure 2 As shown, the head of this bacteriophage is icosahedral, with a head diameter of approximately 54.32 nm and a tail diameter of approximately 67.93 nm, belonging to Caudoviricetes.

[0059] Example 3

[0060] Phage host range determination

[0061] The host range of bacteriophages was determined using a dot-matrix assay. Twenty strains of *Acinetobacter baumannii* were prepared. 100 μL of each strain's bacterial suspension was mixed thoroughly with 5 mL of soft agar and poured onto a bottom agar plate. After solidification, 2 μL of the phage solution was dotted onto the surface of each plate and incubated at 37°C for at least 6 hours. The appearance of transparent phage plaques on the double-layer agar surface indicated that the strain was sensitive to the phage (+), otherwise it was insensitive (-).

[0062] The strain T2784 and 19 other Acinetobacter baumannii strains used for phage host range determination were all obtained from the Department of Laboratory Medicine, Peking University Third Hospital. They were isolated from sputum samples of hospitalized patients, identified as Acinetobacter baumannii by mass spectrometry, and stored in 25% glycerol at -80℃. Before the experiment, the bacteria were streaked in three zones on LB solid medium and incubated overnight at 37℃ for recovery. Single colonies were picked and cultured overnight in LB liquid medium at 37℃ and 200 rpm / min to obtain bacterial suspensions.

[0063] Table 1 Host range of bacteriophage pT2784

[0064]

[0065]

[0066] As shown in Table 1, phage pT2784 can infect two ST40-KL47 Acinetobacter baumannii strains, including T2784, demonstrating the high specificity of phage pT2784.

[0067] Example 4

[0068] Phage genome sequencing

[0069] Phage DNA was extracted using a λ phage genomic DNA extraction kit (Beijing Leagene Biotechnology Co., Ltd., China). The specific procedures are as follows:

[0070] (1) Take 600 uL of phage fluid into a 1.5 mL EP tube, add 2 uL of RNase I and 1 uL of DNase I, vortex and briefly separate the phage, then incubate in a 37℃ metal bath for 1 h, and then incubate in an 85℃ metal bath for 10 min.

[0071] (2) After removing it from the metal bath, add 24 μL of 0.5 M EDTA solution to the above solution, vortex and detach it briefly; place it in a 65 °C metal bath for 15 min.

[0072] (3) Add 40uL of phage lysis buffer, vortex and briefly separate; place in a 68℃ metal bath for 15min.

[0073] (4) Add an equal volume (600uL) of protein lysis buffer, shake to mix, and centrifuge at 12000rpm at 4℃ for 5min.

[0074] (5) Aspirate the supernatant and transfer it to a new centrifuge tube. Add an equal volume (600uL) of pre-cooled rinsing solution (pre-cooled at -20℃ beforehand), shake well, and place in a -20℃ refrigerator for 1 hour.

[0075] (6) Then centrifuge at 12000 rpm at 4℃ for 10 min and discard the supernatant.

[0076] (7) Add 1 mL of 70% ethanol to the centrifuge tube, centrifuge at 8000 rpm and 4℃ for 8 min, discard the supernatant, and repeat the operation twice.

[0077] (8) Allow to air dry at room temperature. After complete drying, add 30 μL of TE buffer, use Qubit to determine the concentration of extracted DNA, and store in a -20°C refrigerator for later use.

[0078] Both the bacteriophage and bacterial genomic DNA underwent whole-genome next-generation sequencing at Novogene Bioinformatics Technology Co., Ltd. The whole-genome sequencing results of bacteriophage pT2784 are shown below. Figure 3 As shown in Table 2, phage pT2784 has a double-stranded DNA genome of 44,335 bp in length and a GC content of 37.82%. The whole genome is predicted to contain 76 open reading frames (ORFs). BLASTp alignment revealed only 27 functional genes involved in five major processes: phage structure composition, replication and repair, transcription and translation, phage assembly, and host lysis. The remaining ORFs encode proteins with unknown functions and are considered hypothetical proteins. Phage pT2784 does not contain lysogen-related genes, and no virulence or drug resistance-related genes were found, indicating that pT2784 is a lytic phage with significant clinical application value.

[0079] Table 2 Prediction of open reading frames in the pT2784 phage genome

[0080]

[0081]

[0082] BLASTn alignment showed that pT2784 was most similar to Acinetobacter baumannii phage vB_AbaM_IME512 (MH853788.1) listed on the National Center for Biotechnology Information (NCBI), but the sequence coverage was only 57%, and the identity was 91.96%. This indicates that the pT2784 genome contains a large number of sequences completely different from existing Acinetobacter baumannii phages, suggesting it may be a novel phage. To understand its evolutionary status, further research is needed. Figure 5 As shown, a phylogenetic tree was constructed using 11 bacteriophages with high genomic similarity to pT2784. Phage pT2784 and another Acinetobacter baumannii phage, AP22 (NC_017984.1), are on the same branch, indicating their closest phylogenetic relationship. Therefore, pT2784 and AP22 both belong to the class Caudoviricetes. To further verify whether pT2784 is a novel bacteriophage, such as... Figure 4 As shown, we compared the average nucleotide identity of these 12 bacteriophages. The results showed that bacteriophage pT2784 had the highest genomic similarity (61%) to vB_AbaM_IME284 (MH853787.1). According to the International Committee on Taxonomy of Viruses (ICTV, https: / / ictv.global / taxonomy), the threshold for genus classification is 70% nucleotide identity across the genome length; therefore, bacteriophage pT2784 belongs to a new genus.

[0083] The released clean sequencing data were assembled using SPAdes software (v3.13.0). Similar phages were identified by comparing them with other phages in the GenBank database using the Basic Local Alignment Search tool (BLASTn). Domain amino acid sequence homology alignment was performed using the MAFFT online tool (http: / / mafft.cbrc.jp / alignment / server), and phylogenetic tree visualization was achieved using FastTree software (http: / / www.microbesonline.org / fasttree / ). Sequence similarity between phages was analyzed using VRIDIC (https: / / rhea.icbm.uni-oldenburg.de / viridic / ). Open reading frame (ORF) prediction and annotation were performed using the online RAST website (https: / / rast.nmpdr.org / rast.cgi), and the annotation results were corrected using NCBI's protein BLAST (BLASTp) tool. Complete genome annotation loops for the phages were plotted using the CGview online website (https: / / proksee.ca / ). The ResFinder (https: / / cge.cbs.dtu.dk / services / ResFinder / ) and VirulenceFinder (https: / / cge.cbs.dtu.dk / services / VirulenceFinder) were used to detect the absence of drug resistance and virulence genes in the phage genome.

[0084] Example 5

[0085] Optimal multiplicity of infection experiment

[0086] The multiplicity of infection (MOI) refers to the ratio of the number of bacteriophages to the number of bacteria when a bacteriophage infects a bacteriophage.

[0087] Adjust the concentration of the overnight cultured host bacterial culture to 1×10⁻⁶. 8Prepare CFU / mL for later use, and dilute the phage solution to different concentrations. Mix 100 μL of phage solution and 100 μL of host bacterial solution at MOIs of 0.0001, 0.001, 0.01, 0.1, 1, and 10, respectively, and add the mixture to 5 mL of liquid culture medium. Incubate at 37°C with shaking for 6 h. Filter the incubated liquid through a 0.22 μm filter. Continuously dilute the resulting phage solution 10-fold. Mix 100 μL of the appropriately diluted phage solution with 100 μL of the host bacterial solution and plate it onto a double-layer plate. Calculate the phage titer for different MOI groups using the double-layer plate method. The MOI with the highest phage titer is the optimal multiplicity of infection (MOI). Repeat the experiment three times.

[0088] like Figure 6 As shown, phage pT2784 grew well across a wide range of MOIs. However, at MOIs of 0.01 and 0.1, the number of phages proliferated by pT2784 was significantly higher than at other MOIs, reaching 1.07 × 10⁻⁶. 9 PFU / mL and 1.05*10 9 The optimal MOI is 0.01, meaning that the number of progeny phages is maximized when the phage and host bacteria are co-incubated at a ratio of 1:100.

[0089] Example 6

[0090] One-step growth curve of bacteriophage pT2784

[0091] (1) Mix 100uL of phage fluid with 100uL of host bacterial fluid at the optimal MOI and incubate at 37°C for 15 min.

[0092] (2) After centrifuging at 12000 rpm for 5 min, discard the supernatant, wash the precipitate 3 times with PBS, and resuspend it in LB liquid medium for the last time, and transfer it to a 100 mL Erlenmeyer flask containing 50 mL of LB liquid medium.

[0093] (3) Immediately take 1 mL of liquid at this point; this is the "0 min" sample. Then, place the conical flask back into the shaker at 37°C for incubation. Afterward, take 1 mL of liquid at 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 minutes, filter the liquid through a 0.22 μL filter membrane, and determine the phage titer using the double-layer plate method and plot a one-step growth curve. Repeat the experiment three times.

[0094] like Figure 7As shown, the latency period of bacteriophage pT2784 is approximately 5 minutes, meaning that the time from the adsorption of pT2784 to the release of the first batch of progeny phages is only 5 minutes. Afterward, it enters the burst phase, lasting approximately 35 minutes. The phage burst rate is calculated as the difference between the final and initial phage concentrations divided by the initial bacterial concentration, which is 193 PFU / cell. After 40 minutes, it enters the stationary phase.

[0095] Example 7

[0096] pH and temperature stability of bacteriophage pT2784

[0097] (1) Prepare a buffer solution with a hydrogen ion concentration index (pH) of 1-12 using 1M hydrochloric acid and 1M sodium hydroxide.

[0098] (2) Mix 100 μl of phage fluid with a potency of 1×10⁸ PFU / mL with 900 μl of buffer solution with different pH values ​​and incubate at 37°C for 1 h.

[0099] (3) Place 1 mL of 108 PFU / mL phage solution in an environment of 4℃, 26℃, 37℃, 50℃, 65℃, 75℃, 80℃ and 85℃ respectively and incubate for 1 h.

[0100] (4) The phage titer was determined under different pH and temperature conditions using the double-layer plate method. The experiment was repeated 3 times.

[0101] like Figure 8 As shown, the titer of bacteriophage pT2784 remained above the 8th power from 4 to 65°C, but decreased significantly at 75°C and completely lost its activity at 85°C. Figure 9 As shown, the activity of bacteriophage pT2784 is highly stable under pH conditions from 2 to 11, but is drastically inactivated at pH 1 and 12.

[0102] Example 8

[0103] In vivo and in vitro therapeutic trials of bacteriophage pT2784

[0104] (I) In vitro lysis curve of bacteriophage pT2784

[0105] (1) The experiment was divided into a bacterial control group (containing only bacterial culture), a bacteriophage pT2784 group (bacteriophage and culture were mixed at MOI = 100, 10, 1, 0.1, 0.01), and an LB control group.

[0106] (2) Dilute the overnight cultured host bacterial culture to 1×10⁻⁶. 6CFU / mL, bacterial and phage solutions were added to 96-well plates according to different groups. The bacterial control group received only 200 μL of bacterial solution, the phage group received 100 μL of phage solution at different concentrations according to different MOIs, and the LB control group received only 200 μL of LB liquid medium. Each group had at least three replicates. The 96-well plates were incubated at 37°C.

[0107] (3) Every hour, the 96-well plate was removed from the 37°C incubator, and the absorbance (optical density, OD) at a wavelength of 600 nm, i.e., the OD600 value, was measured using a microplate reader to monitor the inhibitory effect of the bacteriophage on bacterial growth. The experiment was repeated 3 times.

[0108] like Figure 10 As shown, in the first 6 hours, phage pT2784 completely inhibited the growth of host bacteria, with absorbance values ​​close to 0 at 600 nm. However, after 6 hours, regardless of whether the MOI was 100 or 0.01, the absorbance values ​​began to increase, but remained far lower than those of the isolated bacterial group. This indicates the possible emergence of bacteria resistant to phage pT2784.

[0109] (II) In vivo therapeutic experiment with bacteriophage pT2784

[0110] 1. Calculation of bacterial concentration

[0111] (1) Take 1 mL of overnight cultured host bacteria into a 1.5 mL EP tube, centrifuge at 10000 rpm for 2 min, discard the supernatant, resuspend in PBS buffer, and repeat the above operation 3 times.

[0112] (2) Dilute the host bacterial solution 10 times, take 100uL of the appropriate dilution of bacterial solution, spread it on a plate, dry it and then incubate it at 37℃ overnight.

[0113] (3) Select plates with 30-300 single colonies for bacterial counting. Bacterial concentration (CFU / mL) = number of single colonies × dilution factor × 10.

[0114] 2. Determination of bacterial inoculation dosage

[0115] (1) In this experiment, the larvae of the large wax moth were used as an infection model to evaluate the minimum lethal dose of the host bacterium T2784. The large wax moth larvae were purchased from Tianjin Huiyude Biotechnology Co., Ltd. The larvae with a body length of about 2-3 cm, a weight of about 300 mg, milky white body without gray marks, and vigorous activity were selected. The larvae were thawed at 37℃ for 30 min before the experiment.

[0116] (2) Dilute the washed host bacterial culture to 5×10⁸, 2.5×10⁸, 1×10⁸, 5×10⁷, 2.5×10⁷, and 1×10⁷ CFU / mL, respectively.

[0117] (3) Use a microsyringe to take 5 μL of bacterial solution or PBS buffer of different concentrations and slowly inject it into the body of the larvae of the large wax moth from the left side of the last pair of abdominal legs. Place it in a 37°C constant temperature incubator to incubate in the dark.

[0118] (4) Record the survival status of the large wax moth larvae every 12 hours until 72 hours (the large wax moth larvae are considered dead if there is no reaction when touched on the tail and the body turns black). Plot the survival curve.

[0119] (5) The experiment was set up with PBS group and bacterial suspension groups of different concentrations, with 10 large wax moth larvae in each group. The concentration suitable for observing the therapeutic effect of bacteriophage was selected as the subsequent inoculation dose.

[0120] To further investigate the therapeutic effect of bacteriophage pT2784 on Acinetobacter baumannii in vivo, we constructed a large wax moth infection model. First, we analyzed the survival status of large wax moths inoculated with different concentrations of Acinetobacter baumannii, such as... Figure 11 As shown, the results revealed 5×10 7 A dose of CFU / mL resulted in significant mortality of the large wax moth within 72 hours.

[0121] 3. Bacteriophage pT2784 therapeutic experiment

[0122] (1) Based on the inoculation dose determined in the above steps, dilute the washed host bacterial solution to a concentration of 5 × 10⁻⁶. 7 CFU / mL, dilute the phage solution to 5×10 9 PFU / mL;

[0123] (2) Use a microsyringe to take 10 μL of 5 × 10 7 CFU / mL bacterial solution was slowly injected into the body of the larvae of the large wax moth from the left side of the last pair of abdominal legs, and then placed in a 37°C constant temperature incubator for cultivation in the dark.

[0124] (3) One hour after infection, the larvae of the large wax moth were taken out of the incubator and the phage fluid was slowly injected into the body of the larvae from the right side of the last pair of abdominal legs according to MOI=100, 1, 0.01. After that, the larvae were placed in a constant temperature incubator at 37°C to be cultured in the dark.

[0125] (4) Record the survival status of the larvae of the large wax moth every 12 hours until 72 hours, and plot the survival curve.

[0126] like Figure 12As shown, the survival rate of *Heliotropium indicum* injected with bacteriophage after infection was no lower than that of *Heliotropium indicum* injected with PBS, with better treatment efficacy observed under high MOI (MOI 100). Furthermore, a group injected with only bacteriophage was established, and the survival rate of *Heliotropium indicum* was consistent with that of the PBS-only group, both being 100%. This indicates that phage therapy is safe and demonstrates the potential of bacteriophages as an alternative antibacterial agent.

[0127] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A bacteriophage, characterized in that, The bacteriophage is (Acinetobacter phage) pT2784, and the preservation number is CGMCC No. 46500.

2. The bacteriophage of claim 1, characterized in that, The bacteriophage is linear double-stranded DNA, the full length of the bacteriophage genome is 44335 bp, and the bacteriophage does not contain virulence genes and drug resistance genes.

3. The bacteriophage of claim 1, wherein, The GeneBank accession number of the whole genome sequence of the bacteriophage is PV550753.

4. The bacteriophage of claim 1, wherein, The bacteriophage belongs to the Myoviridae family.

5. The bacteriophage of claim 1, wherein, The bacteriophage is stable in the pH range of 2-11 and the temperature range of 4-65℃.

6. The bacteriophage of claim 1, wherein, The bacteriophage is specific for Acinetobacter baumannii.

7. The bacteriophage of claim 1, wherein, The latent period of the bacteriophage is 5 min, the burst period is 35 min, and the burst size is 193 PFU / cell.

8. The bacteriophage according to any one of claims 1 to 7 for use in the lysis of Acinetobacter baumannii.

9. Use according to claim 8, characterized in that, The multiplicity of infection of the bacteriophage is 1:10000-1:

100.

10. Use according to claim 9, characterized in that, The bacteriophage can be used to prepare oral preparations, sprays or injections.

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