A broad host spectrum drug-resistant Acinetobacter baumannii phage and its composition and application

Abstract

The present invention discloses a broad host spectrum resistant Acinetobacter baumannii phage and its composition and application. The Acinetobacter baumannii phage, phage CP‑p‑AB‑21033, has a deposit number of CGMCC 45410; the phage was deposited in the China General Microbiological Culture Collection Management Center on January 9, 2023, and was classified and named as Podoviridae. CP‑p‑AB‑21033 itself has a wide host spectrum and can lyse no less than 31 strains of carbapenem-resistant Acinetobacter baumannii. It has great potential in clinical applications and is a relatively ideal choice. It should not be replaced by others. The phage CP‑p‑AB‑21033 is combined with one or more of the other 7 phages provided by the present invention to form a composition, which broadens the host spectrum and lysis ability, and has broad application potential in the clinical treatment of resistant Acinetobacter baumannii infections and environmental purification.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a broad host spectrum drug-resistant Acinetobacter baumannii phage and a composition and application thereof. Background Art

[0002] Acinetobacter baumannii (Ab) is a conditional pathogen of the Moraxellaceae family and the Acinetobacter genus. It is widely found in water and soil in nature, hospital environment, and human skin, respiratory tract, digestive tract, and urogenital tract.

[0003] Acinetobacter baumannii is one of the main sources of hospital infections, especially in intensive care units. It mainly causes respiratory tract infections, but can also cause bacteremia, urinary tract infections, secondary meningitis, surgical site infections, and ventilator-associated pneumonia. Globally, Acinetobacter baumannii is one of the most drug-resistant pathogens, and is at the top of the World Health Organization's list of superbugs that most urgently need to develop new antibiotics. According to the 2021 annual report of the National Antimicrobial Resistance Surveillance Network (CARSS), the average resistance rate of Acinetobacter baumannii detected in hospitals in my country to carbapenem antibiotics reached 54.3%, and as high as 77.9% in Henan Province. In addition, Acinetobacter baumannii can survive on dry surfaces for more than 25 days. Conventional disinfectants can only inhibit its growth, not kill it, and it tolerates soap. It is the most commonly isolated Gram-negative bacillus from the hands of medical staff, medical devices, and surfaces of objects, which has brought great trouble to hospital infection prevention and control.

[0004] Bacteriophage (phage) is a type of virus that uses specific microorganisms such as bacteria, fungi, actinomycetes or spirochetes as hosts. Similar to other viruses, the head of a bacteriophage is composed of a protein capsid that encapsulates genetic material. Tailed phages also have "tails" of varying lengths and shapes, which are used to specifically identify host bacteria and inject genetic material into the host. Compared with traditional antibiotics, bacteriophages have the advantages of strong bactericidal ability, high specificity, high safety, and low cost. Their bactericidal ability does not depend on the drug resistance of bacteria. Therefore, bacteriophages show great application potential in preventing and treating bacterial infections, especially drug-resistant bacterial infections.

[0005] Phages are undoubtedly a powerful weapon to control drug-resistant bacteria, and their application range is very wide. As early as more than 100 years ago, when phages were first discovered, some European countries had tried to use them to treat diseases such as dysentery. Later, due to the discovery and widespread use of antibiotics, many countries gave up phage therapy. However, with the increasing severity of bacterial resistance, phage therapy has regained attention. Many clinical cases of personalized phage treatment of drug-resistant Acinetobacter baumannii infections have been carried out at home and abroad, demonstrating the great potential of phage therapy.

[0006] At present, phage personalized therapy has not been widely promoted, and there are no phage drugs on the market at home and abroad. Compared with traditional broad-spectrum antibiotics, phage therapy also faces the bottlenecks of strong specificity, narrow bactericidal spectrum and easy bacterial tolerance. Summary of the invention

[0007] The purpose of the present invention is to provide a strain of Acinetobacter baumannii phage and its composition and application. Based on the different lysis spectrum ranges of different natural phages, the bottleneck of the above phage therapy can be broken through to a certain extent through phage combination, which greatly broadens the host spectrum and has broad application potential in clinical treatment of Acinetobacter baumannii infection and environmental purification.

[0008] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0009] The present invention provides a bacteriobacter baumannii phage, wherein the bacteriophage CP-p-AB-21033 has a deposit number of CGMCC No.45410; the bacteriophage was deposited in the China General Microbiological Culture Collection Center on January 9, 2023, and was classified and named as Acinetobacter baumannii phage.

[0010] The present invention also provides a strain of Acinetobacter baumannii phage, the phage CP-p-AB-21011 has a deposit number of CCTCC NO: M 2023404, is deposited in the China Center for Type Culture Collection on March 24, 2023, and is classified and named Acinetobacter baumannii phage CP-p-AB-21011, Acinetobacter baumannii phage CP-p-AB-21011.

[0011] The present invention also provides a strain of Acinetobacter baumannii phage, the phage CP-p-AB-21022 has a deposit number of CGMCC No. 45551, was deposited in the China General Microbiological Culture Collection Center on March 29, 2023, and is classified and named Acinetobacter baumannii phage.

[0012] The present invention also provides a strain of Acinetobacter baumannii phage, the phage CP-p-AB-21023 has a deposit number of CCTCC NO: M 2023061, is deposited in the China Center for Type Culture Collection on January 10, 2023, and is classified and named Acinetobacter baumannii phage CP-p-AB-21023, Acinetobacter baumannii phage CP-p-AB-21023.

[0013] The present invention also provides a strain of Acinetobacter baumannii phage, the phage CP-p-AB-21041 has a deposit number of CCTCC NO: M 2023062, is deposited in the China Center for Type Culture Collection on January 10, 2023, and is classified and named Acinetobacter baumannii phage CP-p-AB-21041, Acinetobacter baumannii phage CP-p-AB-21041.

[0014] The present invention also provides a strain of Acinetobacter baumannii phage, the phage CP-p-AB-21042 has a deposit number of CCTCC NO: M 2023403, is deposited in the China Center for Type Culture Collection on March 24, 2023, and is classified and named Acinetobacter baumannii phage CP-p-AB-21042, Acinetobacter baumannii phage CP-p-AB-21042.

[0015] The present invention also provides a strain of Acinetobacter baumannii phage, the phage CP-p-AB-22057 has a deposit number of CGMCC No. 45552, was deposited in the China General Microbiological Culture Collection Center on March 29, 2023, and is classified and named Acinetobacter baumannii phage.

[0016] The present invention also provides a strain of Acinetobacter baumannii phage, the phage CP-p-AB-22059 has a deposit number of CGMCC No. 45553, was deposited in the China General Microbiological Culture Collection Center on March 29, 2023, and is classified and named Acinetobacter baumannii phage.

[0017] The present invention also provides an Acinetobacter baumannii phage composition, which is a combination of the phage CP-p-AB-21033 described in claim 1 and one or more of phage CP-p-AB-21011, phage CP-p-AB-21022, phage CP-p-AB-21023, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057 or phage CP-p-AB-22059.

[0018] As a preferred embodiment, bacteriophage CP-p-AB-21011 comprises the specific nucleotide sequence shown in SEQ ID NO.17; bacteriophage CP-p-AB-21022 comprises the specific nucleotide sequence shown in SEQ ID NO.18; bacteriophage CP-p-AB-21023 comprises the specific nucleotide sequence shown in SEQ ID NO.19; bacteriophage CP-p-AB-21033 comprises the specific nucleotide sequence shown in SEQ ID NO.20; bacteriophage CP-p-AB-21041 comprises the specific nucleotide sequence shown in SEQ ID NO.21; bacteriophage CP-p-AB-21042 comprises the specific nucleotide sequence shown in SEQ ID NO.22; bacteriophage CP-p-AB-22057 comprises the specific nucleotide sequence shown in SEQ ID NO.23; and bacteriophage CP-p-AB-22059 comprises the specific nucleotide sequence shown in SEQ ID NO.24.

[0019] The present invention also provides a preparation for preventing and treating Acinetobacter baumannii, wherein the preparation comprises the Acinetobacter baumannii bacteriophage or the Acinetobacter baumannii phage composition.

[0020] As a preferred embodiment, the preparation is an antibacterial drug and / or an environmental disinfectant.

[0021] The present invention also provides a primer set for detecting Acinetobacter baumannii phage for non-disease diagnosis purposes, wherein the phage includes any one of CP-p-AB-21033, CP-p-AB-21011, CP-p-AB-21022, CP-p-AB-21023, CP-p-AB-21041, CP-p-AB-21042, CP-p-AB-22057 or CP-p-AB-22059, wherein the primer set of CP-p-AB-21011 is a primer pair P-AB011-F / P-AB011-R, the nucleotide sequence of P-AB011-F is shown in SEQ ID NO.1, and the nucleotide sequence of P-AB011-R is shown in SEQ ID NO. NO.2; the primer set of CP-p-AB-21022 is a primer pair P-AB022-F / P-AB022-R, the nucleotide sequence of P-AB022-F is shown in SEQ ID NO.3, and the nucleotide sequence of P-AB022-R is shown in SEQ ID NO.4; the primer set of CP-p-AB-21023 is a primer pair P-AB023-F / P-AB023-R, the nucleotide sequence of P-AB023-F is shown in SEQ ID NO.5, and the nucleotide sequence of P-AB023-R is shown in SEQ ID NO.6; the primer set of CP-p-AB-21033 is a primer pair P-AB033-F / P-AB033-R, the nucleotide sequence of P-AB033-F is shown in SEQ ID NO.7, and the nucleotide sequence of P-AB033-R is shown in SEQ ID NO.8. NO.8; the primer set of CP-p-AB-21041 is a primer pair P-AB041-F / P-AB041-R, the nucleotide sequence of P-AB041-F is shown in SEQ ID NO.9, and the nucleotide sequence of P-AB041-R is shown in SEQ ID NO.10; the primer set of CP-p-AB-21042 is a primer pair P-AB042-F / P-AB042-R, the nucleotide sequence of P-AB042-F is shown in SEQ ID NO.11, and the nucleotide sequence of P-AB042-R is shown in SEQ ID NO.12; the primer set of CP-p-AB-22057 is a primer pair P-AB057-F / P-AB057-R, the nucleotide sequence of P-AB057-F is shown in SEQ ID NO.13, and the nucleotide sequence of P-AB057-R is shown in SEQ ID NO.14; the primer set of CP-p-AB-22059 is the primer pair P-AB059-F / P-AB059-R, the nucleotide sequence of P-AB059-F is shown in SEQ ID NO.15, and the nucleotide sequence of P-AB059-R is shown in SEQ ID NO.16.

[0022] The present invention also provides an application of an Acinetobacter baumannii phage or an Acinetobacter baumannii phage composition in preventing and treating Acinetobacter baumannii pollution.

[0023] The sequence involved in the present invention is as follows:

[0024]

[0025]

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention separates multiple strains of Acinetobacter baumannii phages (a total of 26 strains) from different environments, and uses 110 clinically resistant Acinetobacter baumannii from multiple hospitals in different regions of the country to analyze the lysis spectrum of the 26 isolated Acinetobacter baumannii phages, and finds that two of the Acinetobacter baumannii phages have obvious lysis advantages over other phages. Among them, Acinetobacter baumannii phages CP-p-AB-21033 and CP-p-AB-21011 can lyse 31 and 30 different Acinetobacter baumannii, respectively, and have great potential in clinical applications.

[0028] 2. The optimal infection multiplicity of the Acinetobacter baumannii phages CP-p-AB-21023, CP-p-AB-21041, CP-p-AB-22057 and CP-p-AB-22059 provided by the present invention is 10 -6， Therefore, during the production process, these phages only need a very small amount of mother liquid or seed liquid to amplify a large number of progeny phages, which has a high production efficiency; the titers of phages CP-p-AB-21023 and CP-p-AB-21042 are both over 6×10 9 PFU / mL; the lysis cycles of bacteriophages CP-p-AB-21023, CP-p-AB-21041, CP-p-AB-21042 and CP-p-AB-22057 were all less than 25 min, and these phages could be amplified in large quantities in a short time, indicating that these phages have efficient lysis activity.

[0029] 3. The Acinetobacter baumannii phage provided by the present invention can specifically kill Acinetobacter baumannii, regardless of whether the bacteria contain drug-resistant genes.

[0030] 4. The Acinetobacter baumannii phage composition provided by the present invention broadens the host spectrum and lysis ability, and has broad application potential in the clinical treatment of Acinetobacter baumannii infection and environmental purification.

[0031] 5. The present invention uses 110 clinical strains of Acinetobacter baumannii isolated from different hospitals in different regions, and conducts phage-host interaction analysis to provide an Acinetobacter baumannii phage composition. Compared with a single phage, the phage composition broadens the host spectrum that can be lysed and greatly improves the coverage rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a diagram of the phage purification results of the present invention, wherein A is phage CP-p-AB21011, B is phage CP-p-AB21022, C is phage CP-p-AB21023, D is phage CP-p-AB21033, E is phage CP-p-AB21041, F is phage CP-p-AB21042, G is phage CP-p-AB22057, and H is phage CP-p-AB22059.

[0033] Figure 2 It is the transmission electron microscopy result diagram of the phage of the present invention, wherein A is phage CP-p-AB21011, B is phage CP-p-AB21022, C is phage CP-p-AB21023, D is phage CP-p-AB21033, E is phage CP-p-AB21041, F is phage CP-p-AB21042, G is phage CP-p-AB22057, and H is phage CP-p-AB22059.

[0034] Figure 3 It is the electrophoresis diagram of the phage-specific sequence nucleic acid amplified by PCR of the present invention, A is phage CP-p-AB21011, B is phage CP-p-AB21022, C is phage CP-p-AB21023, D is phage CP-p-AB21033, E is phage CP-p-AB21041, F is phage CP-p-AB21042, G is phage CP-p-AB22057, and H is phage CP-p-AB22059, wherein the left band in Figures AH is the Marker, and the right band is the corresponding phage band.

[0035] Figure 4 This is a graph showing the optimal infection multiplicity result of the Acinetobacter baumannii phage of the present invention, wherein A is phage CP-p-AB21011, B is phage CP-p-AB21022, C is phage CP-p-AB21023, D is phage CP-p-AB21033, E is phage CP-p-AB21041, F is phage CP-p-AB21042, G is phage CP-p-AB22057, and H is phage CP-p-AB22059.

[0036] Figure 5 This is a one-step growth curve of the phage of the present invention, wherein A is phage CP-p-AB21011, B is phage CP-p-AB21022, C is phage CP-p-AB21023, D is phage CP-p-AB21033, E is phage CP-p-AB21041, F is phage CP-p-AB21042, G is phage CP-p-AB22057, and H is phage CP-p-AB22059. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is described in detail below in conjunction with the embodiments. The reagents and biological materials used below are all commercial products unless otherwise specified.

[0038] (1) LB (Luria broth) liquid medium (1 L): peptone 10 g, yeast powder 5 g, NaCl 10 g, add ddH2O to 1 L, adjust pH to 7.0, and sterilize at 121°C for 20 min.

[0039] (2) 0.5% LB semisolid medium (1 L): 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, 5 g of agar powder, add ddH2O to 1 L, adjust the pH to 7.0, and sterilize at 121°C for 20 min.

[0040] (3) 1.5% LB solid medium (1 L): 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, 15 g of agar powder, add ddH2O to 1 L, adjust the pH to 7.0, sterilize at 121°C for 20 min, cool to 50°C, pour into a plate, cool and solidify, and then invert for later use.

[0041] Example 1: Acinetobacter baumannii phage isolation

[0042] Sewage samples were collected from the sewage outlet of a hospital in Shanghai. 20 mL of sewage samples were centrifuged at 4 ° C and 12000 rpm for 10 minutes. The supernatant was filtered with a 0.22 μm filter membrane and the filtrate was set aside. 9 mL of supernatant filtrate was added with 1 mL of 10-fold LB liquid culture medium and 0.1 mL of phage logarithmic phase host bacteria liquid, and placed at 37 ° C for overnight culture. The next day, centrifuged at 4 ° C and 12000 rpm for 10 minutes, and the supernatant was filtered with a 0.22 μm filter membrane to sterilize, forming a stock solution containing phages, i.e., phage suspension. Different clinical drug-resistant strains were streaked and inoculated on 1.5% LB solid culture medium. After culturing at 37 ° C overnight, a single clone was picked and inoculated in 5 mL of LB liquid culture medium. After shaking and culturing at 37 ° C for 8 hours, it was used as a host bacteria culture for standby use. Take 0.3mL of the above-mentioned spare host bacteria culture and 3mL of 0.5% LB semi-solid medium, mix them evenly, and spread them on 1.5% LB solid medium. After drying, divide the agar plate into 2 areas, take 10μL of the above-mentioned phage suspension and drop it on one area, and drop an equal amount of PBS on the other area as a control. After drying naturally, place it in a 37℃ incubator and observe whether there are plaques formed in the phage drop area. If there are plaques, it proves that phages exist.

[0043] Example 2: Purification of Acinetobacter baumannii phage

[0044] The phage plaques in Example 1 were picked up and placed in a sterile EP tube containing 0.5 mL PBS, and placed at 4°C overnight to release the phages to obtain a phage extract. The phage extract was diluted 10 times in a row using the double-layer plate method, and 10 -2 , 10 -4 , 10 -6 Add 0.3 ml of logarithmic phase host bacterial solution to 0.1 ml of diluent, add 3 ml of 0.5% LB semi-solid medium at about 55°C, evenly spread on the pre-prepared solid LB plate, and invert and culture at 37°C for 5-10 hours to obtain a double-layer plate with plaques. Pick a single transparent, uniform-sized, and regular-edged plaque into a sterile EP tube filled with 0.5 mL of PBS to obtain a phage extract. Repeat the above steps 3 times to obtain a purified phage suspension for later use.

[0045] Example 3: Cluster analysis and lysis spectrum analysis of Acinetobacter baumannii phage and bacteria

[0046] According to the lysis correspondence between phage and host, we can perform cluster analysis on phage and bacteria respectively. Different objects clustered in the same cluster have great similarities, and objects in different clusters have great differences. The specific analysis process is as follows:

[0047] (1) First, 110 clinically resistant Acinetobacter baumannii strains from multiple hospitals in different regions of the country and 26 Acinetobacter baumannii phages isolated from different sewage were used for lysis spectrum analysis. The specific operation is as follows:

[0048] 1) Take 0.3 ml of the logarithmic phase bacterial solution of Acinetobacter baumannii, add 3 ml of 0.5% LB semi-solid medium at about 55°C, and evenly spread it on the pre-prepared solid 1.5% LB solid medium;

[0049] 2) Then, each plate was divided into two areas, 2 μL of the reserved phage in Example 2 was dripped into one area, and an equal amount of PBS was dripped into the other area as a control, and after the droplets dried, they were inverted and cultured at 37°C for 12 hours. Phage plaques are empty plaques formed by phage lysis on the plate where bacteria grow, and are classified as present (+) or absent (blank) according to the presence or absence of phage plaques.

[0050] (2) The lysis spectrum results were statistically analyzed and classified into large and transparent (LT), small and transparent (ST), large and turbid (LO), small and turbid (SO), and no plaque (NP) according to the plaque morphology;

[0051] (3) Convert the symbol of the plaque transparency into a numerical value (0 means no plaque, 1 means blurred plaque, 2 means small and transparent plaque, and 3 means large and transparent plaque);

[0052] (4) Use RStudio software to load the R package functions for drawing;

[0053] (5) Analyze the generated clustering results.

[0054] Experimental results: The results are shown in Table 1, which is the lysis spectrum results of 26 strains of Acinetobacter baumannii phage. According to the lysis spectrum and cluster analysis results, phage CP-p-AB-21033 with a relatively broad host spectrum and complementary to each other was selected and combined with one or more strains of phage CP-p-AB-21011, phage CP-p-AB-21022, phage CP-p-AB-21023, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057 or phage CP-p-AB-22059.

[0055] Single phage CP-p-AB-21033, phage CP-p-AB-21011, phage CP-p-AB-21022, phage CP-p-AB-21023, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057 and phage CP-p-AB-22059 can lyse 31, 30, 12, 14, 12, 18, 14 and 9 strains of Acinetobacter baumannii, respectively; among them, the "cocktail" combination of phage CP-p-AB-21033 and one or more strains of the other 7 phages can lyse up to 96 strains of Acinetobacter baumannii, and the coverage rate can reach 87.3% (96 / 110). Compared with single phage, "cocktail" phage broadens the spectrum of hosts that can be lysed and greatly improves the coverage rate.

[0056] Table 1

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] Note: "+" represents bacteria that were lysed by phage, and a space represents bacteria that could not be lysed by phage.

[0066] Example 4: Expansion culture of Acinetobacter baumannii phage

[0067] Experimental method: Take 40 mL of LB liquid culture medium, add 0.4 mL of logarithmic phase host bacterial solution, place it in a 37°C shaker at 200 rpm and culture it for 1-2 hours until the logarithmic phase, then add 0.05 mL of the purified phage suspension in Example 2, continue to place it in a 37°C shaker at 200 rpm and culture it until the liquid in the test tube becomes clear, and then centrifuge and filter it for use.

[0068] The double-layer plate method was used to detect the phage titer: the phage suspension was diluted 10 times in a gradient, 0.1 ml of the phage dilution in each gradient was taken and mixed with 0.3 ml of the host bacterial solution, and then spread on a double-layer agar plate. The plates were cultured at 37°C for about 5-10 hours, and the plaques were counted on each agar plate. The plates with about 30-300 plaques were selected, and the initial phage concentration was calculated according to the dilution multiple, which was the phage titer (phage titer (pfu / ml) = dilution multiple × number of plaques × 10).

[0069] Test results: Figure 1 It is a diagram of the phage purification results of the present invention, wherein A is phage CP-p-AB21011, B is phage CP-p-AB21022, C is phage CP-p-AB21023, D is phage CP-p-AB21033, E is phage CP-p-AB21041, F is phage CP-p-AB21042, G is phage CP-p-AB22057, and H is phage CP-p-AB22059.

[0070] like Figure 1 As shown in the figure, bacteriophage CP-p-AB-21011 can form clear plaques in agar medium without halos, with clear and regular edges, a diameter of about 4 mm, and a titer of 1.4×10 9 pfu / mL, which is a typical lytic phage.

[0071] Bacteriophage CP-p-AB-21022 can form clear plaques in agar medium, surrounded by a halo, with clear and regular edges, a diameter of about 4 mm, and a titer of 5.2×10 9 pfu / mL, which is a typical lytic phage.

[0072] Bacteriophage CP-p-AB-21023 can form clear plaques in agar medium, surrounded by a halo, with clear and regular edges, a diameter of about 5 mm, and a titer of 6.0×10 9 pfu / mL, which is a typical lytic phage.

[0073] Bacteriophage CP-p-AB-21033 can form clear plaques in agar medium, with no halo around, clear and regular edges, a diameter of about 4 mm, and a titer of 4.3×10 9 pfu / mL, which is a typical lytic phage.

[0074] Bacteriophage CP-p-AB-21041 can form clear plaques in agar medium, with no halo around them, clear and regular edges, a diameter of about 3 mm, and a titer of 4.5×10 9pfu / mL, which is a typical lytic phage.

[0075] Bacteriophage CP-p-AB-21042 can form clear plaques in agar medium, with no halo around them, clear and regular edges, a diameter of about 2 mm, and a titer of 6.4×10 9 pfu / mL, which is a typical lytic phage.

[0076] Bacteriophage CP-p-AB-22057 can form clear plaques in agar medium, surrounded by a halo, with clear and regular edges, a diameter of about 3-4 mm, and a titer of 4.6×10 9 pfu / mL, which is a typical lytic phage.

[0077] Bacteriophage CP-p-AB-22059 can form clear plaques in agar medium, surrounded by a halo, with clear and regular edges, a diameter of about 3-4 mm, and a titer of 2.4×10 9 pfu / mL, which is a typical lytic phage.

[0078] Example 5: Transmission electron microscopy observation of Acinetobacter baumannii phage

[0079] Experimental method: Take the phage suspension in Example 4 for electron microscopy observation, drop 10ul of the purified phage suspension in Example 2 on a copper sheet, let it precipitate naturally for 10 minutes, absorb the excess liquid with filter paper, add a drop of 2% phosphotungstic acid (PTA, 2% w / v) for staining for 1-2 minutes, and observe using a transmission electron microscope after drying at room temperature.

[0080] Test results: Figure 2 It is a transmission electron microscopy result diagram of the phage of the present invention, wherein A is phage CP-p-AB-21011, B is phage CP-p-AB-21022, C is phage CP-p-AB-21023, D is phage CP-p-AB-21033, E is phage CP-p-AB-21041, F is phage CP-p-AB-21042, G is phage CP-p-AB-22057, and H is phage CP-p-AB-22059.

[0081] Observation results such as Figure 2 As shown:

[0082] Bacteriophage CP-p-AB-21011 has a regular icosahedral head with a diameter of about 60nm and a short, non-contractile tail of about 8nm. According to the "Taxonomy of Viruses - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-21011 belongs to the family of short-tailed viruses.

[0083] Bacteriophage CP-p-AB-21022 has a regular icosahedral head with a diameter of about 55nm and a short, non-contractible tail of about 10nm. According to the "Virus Classification - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-21022 belongs to the Podoviridae family.

[0084] Bacteriophage CP-p-AB-21023 has a regular icosahedral head with a diameter of about 55nm and a short, non-contractible tail of about 15nm. According to the "Virus Classification - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-21023 belongs to the Podoviridae family.

[0085] Bacteriophage CP-p-AB-21033 has a regular icosahedral head with a diameter of about 60nm and a short, non-contractible tail with a length of about 15nm. According to the "Virus Classification - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-21033 belongs to the Podoviridae family.

[0086] Bacteriophage CP-p-AB-21041 has a regular icosahedral head with a diameter of about 60nm and a short, non-contractile tail with a length of about 15nm. According to the "Taxonomy of Viruses: The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-21041 belongs to the Podoviridae family.

[0087] Bacteriophage CP-p-AB-21042 has a regular icosahedral head with a diameter of about 68nm and a long and retractable tail with a length of about 160nm. According to the "Taxonomy of Viruses - The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-21042 belongs to the Myoviridae family.

[0088] Bacteriophage CP-p-AB-22057 has a regular icosahedral head with a diameter of about 60nm and a short, non-contractile tail with a length of about 10nm. According to the "Taxonomy of Viruses: The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-22057 belongs to the Podoviridae family.

[0089] Bacteriophage CP-p-AB-22059 has a regular icosahedral head with a diameter of about 60nm and a short, non-contractile tail with a length of about 10nm. According to the "Taxonomy of Viruses: The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2015, bacteriophage CP-p-AB-22059 belongs to the Podoviridae family.

[0090] Example 6: Genome extraction, sequencing and specificity verification of Acinetobacter baumannii phage

[0091] Experimental method: (1) Take 10 mL of each of bacteriophage CP-p-AB-21011, bacteriophage CP-p-AB-21022, bacteriophage CP-p-AB-21023, bacteriophage CP-p-AB-21033, bacteriophage CP-p-AB-21041, bacteriophage CP-p-AB-21042, bacteriophage CP-p-AB-22057 and bacteriophage CP-p-AB-22059 in Example 4, add DNase I (10 U / μg) and RNase A (10 U / μg) with a final concentration of 1 μg / mL, mix thoroughly and incubate at 37°C for 30 min;

[0092] (2) Add EDTA to a final concentration of 25 mM and incubate at 65°C for 10 min (to inactivate DNase);

[0093] (3) At the beginning of this step, the nucleic acid was extracted using the M5λ phage genomic DNA rapid extraction kit; (4) After verification by 1% agarose gel nucleic acid electrophoresis, the concentration and purity were determined using NanoDrop-300, and the product was sent to Shanghai Paisono Biotechnology Co., Ltd. for sequencing.

[0094] (5) The genome sequences of Acinetobacter baumannii phage CP-p-AB-21011, phage CP-p-AB-21022, phage CP-p-AB-21023, phage CP-p-AB-21033, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057, and phage CP-p-AB-22059 were analyzed through the website, and specific nucleotide sequences were selected and primers were designed. The primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0095] (6) Construct a PCR reaction system. Table 2 shows the PCR system for amplifying the specific sequences of Acinetobacter baumannii phage CP-p-AB-21011, phage CP-p-AB-21022, CP-p-AB-21023, phage CP-p-AB-21033, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057 and phage CP-p-AB-22059. The PCR reaction program is: 94°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 90 s (32 cycles in total), and 72°C final extension for 10 min. Take 5 μL of PCR product for 1% agarose gel nucleic acid electrophoresis and observe the results. The remaining PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0096] The results of whole genome sequencing showed that the complete genome size of Acinetobacter baumannii phage CP-p-AB-21011 was 40150bp; the complete genome size of Acinetobacter baumannii phage CP-p-AB-21022 was 40330bp; the complete genome size of Acinetobacter baumannii phage CP-p-AB-21023 was 40435bp; the complete genome size of Acinetobacter baumannii phage CP-p-AB-21033 was 40997bp; the complete genome size of Acinetobacter baumannii phage CP-p-AB-21041 was 40847bp; the complete genome size of Acinetobacter baumannii phage CP-p-AB-21042 was 99661bp; the complete genome size of Acinetobacter baumannii phage CP-p-AB-22057 was 40792bp; and the complete genome size of Acinetobacter baumannii phage CP-p-AB-22059 was 41060bp.

[0097] Table 2

[0098] system Volume (μl) 2x Taq Mix 25 F 1 R 1 Template 1 Water 22 Total 50

[0099] PCR verification was performed using the primer pair 1 shown in Table 3: P-AB011-F / P-AB011-R, with the genome of Acinetobacter baumannii phage CP-p-AB-21011 as template. The results are shown in Figure 3 As shown in A, Figure 3 A is the electrophoresis diagram of the PCR-amplified phage-specific sequence nucleic acid of the present invention, wherein: Figure 3The right band in A represents the amplification of the phage-specific sequence nucleic acid band using the primer pair: P-AB011-F / P-AB011-R, and the left band is the marker. The primers can amplify the corresponding specific target band, and the band size is consistent with the expected size, proving that the primers can specifically verify the Acinetobacter baumannii phage CP-p-AB-21011. According to the above operation, different primers were used to verify the specific sequence nucleic acid electrophoresis results of phage CP-p-AB-21022, CP-p-AB-21023, phage CP-p-AB-21033, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057 and phage CP-p-AB-22059, respectively. Figure 3 B-3E shows that all of them can obtain results consistent with the expected band size. Among them, the specific nucleotide sequence amplified by the primers of bacteriophage CP-p-AB-21022 is shown in SEQ ID NO.18; the specific nucleotide sequence amplified by the primers of bacteriophage CP-p-AB-21023 is shown in SEQ ID NO.19; the specific nucleotide sequence amplified by the primers of bacteriophage CP-p-AB-21033 is shown in SEQ ID NO.20; the specific nucleotide sequence amplified by the primers of bacteriophage CP-p-AB-21041 is shown in SEQ ID NO.21; the specific nucleotide sequence amplified by the primers of bacteriophage CP-p-AB-21042 is shown in SEQ ID NO.22; the specific nucleotide sequence amplified by the primers of bacteriophage CP-p-AB-22057 is shown in SEQ ID NO.23; the specific nucleotide sequence amplified by the primers of bacteriophage CP-p-AB-22059 is shown in SEQ ID NO.24.

[0100] Table 3

[0101]

[0102]

[0103] Example 7: Determination of the optimal multiplicity of infection (MOI) of Acinetobacter baumannii phage

[0104] The multiplicity of infection (MOI) is the ratio of the number of bacteriophages to the number of bacteria.

[0105] Experimental method: (1) Take the host bacterial culture prepared in Example 1 and transfer it to the logarithmic phase (OD 600 nm=0.5), and adjust the concentration to 1×10 8cfu / mL for later use; dilute the phage culture solution in Example 4 to 1×10 9 pfu / mL, 1×10 8 pfu / mL, 1×10 7 pfu / mL, 1×10 6 pfu / mL, 1×10 5 pfu / mL, 1×10 4 pfu / mL, 1×10 3 pfu / mL, 1×10 2 pfu / mL and 1×10 1 pfu / mL standby;

[0106] (2) According to the multiplicity of infection, 1 , 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 100 μL of the reserved phage dilution, 100 μL of the host bacterial culture and 5 mL of LB liquid medium were mixed evenly and cultured in a shaker at 37°C and 200 rpm for 5 h;

[0107] (3) Take 1 ml of lysate and centrifuge at 8000 rpm for 10 min at 4°C. Collect the supernatant and filter through a 0.22 μm filter membrane.

[0108] (4) Take 100 μL of the filtrate in step (3) and dilute it 10 times in a series;

[0109] (5) Take 100 μL of the diluent and mix it with 300-400 μL of the bacterial suspension. After standing for 5-10 min, add it to a 0.5% semi-solid culture medium cooled to about 55°C, mix it, spread it on a double-layer plate, and invert it in a 37°C constant temperature incubator for overnight culture;

[0110] (6) Observe the plate and calculate the phage titer. Repeat the experiment three times and draw a bar graph.

[0111] Test results: The results are as follows Figure 4 As shown, Figure 4is the optimal infection multiplicity of the Acinetobacter baumannii phage of the present invention, and the optimal infection multiplicity of Acinetobacter baumannii phage CP-p-AB-21011, phage CP-p-AB-21022, phage CP-p-AB-21023, phage CP-p-AB-21033, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057 and phage CP-p-AB-22059 are 10, respectively. -5 , 10 -5 , 10 -6 , 10 -4 , 10 -6 , 10 -5 , 10 -6 , 10 -6 , the titer can reach 1.4×10 9 , 5.2×10 9 , 6.0×10 9 , 4.3×10 9 , 4.5×10 9 , 6.4×10 9 , 4.6×10 9 , 2.4×10 9 PFU / mL.

[0112] Example 8: One-step growth curve assay of Acinetobacter baumannii phage

[0113] Test method: One-step growth curve can reflect the replication characteristics of bacteriophage, including latent period and outbreak period. The specific method is as follows:

[0114] (1) Take the host bacterial culture prepared in Example 1 and transfer it to fresh culture medium at a ratio of 1:100, and add CaCl2 with a final concentration of 5 mmol / L to culture the bacteria to the logarithmic phase (OD 600 nm=0.5);

[0115] (2) Take 1 mL of the host bacteria cultured to the logarithmic growth phase, add the phage culture solution prepared in Example 4 at a ratio of MOI = 0.1, mix well, and incubate in a 37° C. incubator for 5 min (to allow the bacteria to adsorb to the phage);

[0116] (3) Centrifuge at 4°C, 10,000 rpm for 1 min and discard the supernatant (unadsorbed phage);

[0117] (4) Take 1 mL of fresh LB liquid to resuspend the precipitate (mutually adsorbed bacteria and phage particles);

[0118] (5) Repeat steps (3) and (4), resuspending and washing at least twice;

[0119] (6) Finally, resuspend the pellet with 10 mL of fresh LB medium and incubate the resuspended test tube in a shaker at 37 °C at 200 rpm for 90 min. Samples were taken every 5 min (0, 5, 10, 15, 20, 25, 30 min) for the first 30 min, and then every 10 min (40, 50, 60, 70, 80, 90 min), taking out 600 μL of the culture mixture each time.

[0120] (7) Centrifuge at 4°C, 10,000 rpm for 1 min, collect the supernatant, and filter through a 0.22 μm filter membrane;

[0121] (8) Take 100 μL of the phage filtrate from step (6) and dilute it 10 times in a series;

[0122] (9) Take 100 μL of the diluent and mix it with 300 μL-400 μL of the bacterial suspension. After standing for 5-10 min, add it to a 0.5% semi-solid culture medium cooled to about 55°C, mix it, spread it on a double-layer plate, and invert it in a 37°C constant temperature incubator for overnight culture;

[0123] (10) Observe the plate and calculate the phage titer. Repeat the experiment three times and draw a one-step growth curve.

[0124] Test results: The results are as follows Figure 5 As shown, Figure 5 This is a one-step growth curve of the phage of the present invention. The incubation periods of Acinetobacter baumannii phage CP-p-AB-21011, phage CP-p-AB-21022, phage CP-p-AB-21023, phage CP-p-AB-21033, phage CP-p-AB-21041, phage CP-p-AB-21042, phage CP-p-AB-22057 and phage CP-p-AB-22059 infecting the host bacteria are 0 min, 5 min, 0 min, 10 min, 10 min, 10 min, 5 min, 5 min, respectively, and one lysis cycle is 90 min, 40 min, 20 min, 30 min, 25 min, 20 min, 20 min, 30 min, respectively; wherein, one lysis cycle includes a latent period, an outbreak period and a plateau period; the above results indicate that these phages can be amplified in large quantities in a short period of time, indicating that these phages have efficient lysis activity.

[0125] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. A bacteriophage of Acinetobacter baumannii, characterized in that: The bacteriophage CP-p-AB-21033 has a deposit number of CGMCC 45410; the bacteriophage was deposited in the China General Microbiological Culture Collection Center on January 9, 2023, and is classified as Podoviridae; or, The bacteriophage CP-p-AB-21022 has a deposit number of CGMCC 45551 and was deposited in the China General Microbiological Culture Collection Center on March 29, 2023, and is classified as Podoviridae; or, The bacteriophage CP-p-AB-21023 has a deposit number of CCTCC M 2023061, was deposited in the China Center for Type Culture Collection on January 10, 2023, and is classified as Podoviridae; or, The bacteriophage CP-p-AB-21041 has a deposit number of CCTCC M 2023062, was deposited in the China Center for Type Culture Collection on January 10, 2023, and is classified as Podoviridae; or, The bacteriophage CP-p-AB-22057 has a deposit number of CGMCC 45552, which was deposited in the China General Microbiological Culture Collection Center on March 29, 2023, and is classified as Podoviridae; or, The bacteriophage CP-p-AB-22059 has a deposit number of CGMCC 45553 and was deposited in the China Center for the Preservation of General Microbiological Cultures on March 29, 2023. It is classified and named as Podoviridae.

2. A bacteriophage composition of Acinetobacter baumannii, characterized in that: The composition is composed of multiple strains of bacteriophage CP-p-AB-21033, bacteriophage CP-p-AB-21022, bacteriophage CP-p-AB-21023, bacteriophage CP-p-AB-21041, bacteriophage CP-p-AB-22057 and bacteriophage CP-p-AB-22059; the above strains of bacteriophage are as described in claim 1.

3. A bacteriophage composition of Acinetobacter baumannii, characterized in that: The composition is composed of multiple strains of bacteriophage CP-p-AB-21033, bacteriophage CP-p-AB-21011, bacteriophage CP-p-AB-21022, bacteriophage CP-p-AB-21023, bacteriophage CP-p-AB-21041, bacteriophage CP-p-AB-21042, bacteriophage CP-p-AB-22057 and bacteriophage CP-p-AB-22059; The bacteriophage CP-p-AB-21011 has a deposit number of CCTCC M 2023404, was deposited in the China Center for Type Culture Collection on March 24, 2023, and is classified as Podoviridae; The bacteriophage CP-p-AB-21042 has a deposit number of CCTCC M 2023403, was deposited in the China Center for Type Culture Collection on March 24, 2023, and is classified as the Myoviridae family; The remaining phage strains are as described in claim 1.

4. The Acinetobacter baumannii phage composition according to claim 3, characterized in that: The composition is a combination of bacteriophage CP-p-AB-21033 and one or more of bacteriophage CP-p-AB-21011, bacteriophage CP-p-AB-21022, bacteriophage CP-p-AB-21023, bacteriophage CP-p-AB-21041, bacteriophage CP-p-AB-21042, bacteriophage CP-p-AB-22057 and bacteriophage CP-p-AB-22059.

5. The Acinetobacter baumannii phage composition according to claim 3 or 4, characterized in that: The bacteriophage CP-p-AB-21011 comprises the specific nucleotide sequence shown in SEQ ID NO.17; the bacteriophage CP-p-AB-21022 comprises the specific nucleotide sequence shown in SEQ ID NO.18; the bacteriophage CP-p-AB-21023 comprises the specific nucleotide sequence shown in SEQ ID NO.19; the bacteriophage CP-p-AB-21033 comprises the specific nucleotide sequence shown in SEQ ID NO.20; the bacteriophage CP-p-AB-21041 comprises the specific nucleotide sequence shown in SEQ ID NO.21; the bacteriophage CP-p-AB-21042 comprises the specific nucleotide sequence shown in SEQ ID NO.22; the bacteriophage CP-p-AB-22057 comprises the specific nucleotide sequence shown in SEQ ID NO.23; and the bacteriophage CP-p-AB-22059 comprises the specific nucleotide sequence shown in SEQ ID NO.

24.

6. A preparation for preventing and treating Acinetobacter baumannii, characterized in that: The preparation comprises the Acinetobacter baumannii phage of claim 1 or the Acinetobacter baumannii phage composition of any one of claims 2 to 5.

7. The preparation according to claim 6, characterized in that The preparation is an antibacterial drug and / or an environmental disinfectant.

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