Stenotrophomonas maltophilia bacteriophage, composition and application thereof

Abstract

The present invention discloses a Stenotrophomonas maltophilia phage, a composition and an application thereof. Relating to the field of biotechnology, the Stenotrophomonas maltophilia phage includes a phage vB_Sma-SZP9, which is taxonomically named Stenotrophomonas phage vB_Sma-SZP9 and has a Latin name of Stenotrophomonas phage. The phage vB_Sma-SZP9 is deposited in the Guangdong Microbial Culture Collection Center, with a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a deposit number of GDMCC No: 64509-B1, and a deposit date of May 16, 2024. The phage of the present invention has a wide host spectrum, strong infection ability, and a wide range of temperature and acid-base tolerance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a Stenotrophomonas maltophilia bacteriophage, a composition and an application thereof. Background Art

[0002] Stenotrophomonas maltophilia is an obligate aerobic Gram-negative non-fermenting bacillus, second only to Pseudomonas aeruginosa and Acinetobacter, a common bacterium widely distributed in water, plants and soil, and can also inhabit the respiratory tract and intestines of humans and animals. It is the only species among the 17 species of Stenotrophomonas that infects humans.

[0003] When the body's resistance is reduced, secondary infection with Stenotrophomonas maltophilia may occur, causing respiratory tract, urinary tract, gastrointestinal tract, skin and soft tissue, bone and joint, eye infections, as well as meningitis, endocarditis and bacteremia.

[0004] Stenotrophomonas maltophilia has multiple resistance mechanisms, including the production of drug-resistant enzymes, the presence of multidrug efflux pumps, SMqnr genes, biofilm mechanisms, etc., which make it resistant to most antibiotics, such as cephalosporins, carbapenems, aminoglycosides, macrolides and β-lactams, which narrows the range of drugs available for clinical treatment.

[0005] Stenotrophomonas maltophilia can form biofilms on moist surfaces that come into direct or indirect contact with patients (with increased resistance to water-soluble disinfectants), such as in respiratory tubes, catheters, intravenous catheters, dialysis equipment, dental equipment, and hospital plumbing systems (sinks and faucets). Compared to planktonic bacteria, bacteria in biofilms can usually withstand antibiotic treatment at concentrations up to 10 to 1000 times higher, which significantly reduces the effectiveness of traditional antibiotic treatment and poses a huge challenge to clinical treatment.

[0006] In this context, there is an urgent need to explore effective strategies to reduce the harm caused by Stenotrophomonas maltophilia. Summary of the invention

[0007] The main purpose of the present invention is to provide a Stenotrophomonas maltophilia phage, a composition and an application thereof, aiming to seek an effective strategy to reduce the harm caused by Stenotrophomonas maltophilia.

[0008] To achieve the above-mentioned purpose, the first aspect of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP9, taxonomically named Stenotrophomonas phage vB_Sma-SZP9, and Latin name Stenotrophomonas phage. The phage vB_Sma-SZP9 is deposited in Guangdong Microbiological Culture Collection Center, with the storage address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64509-B1, and the storage date is: May 16, 2024.

[0009] The second aspect of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP25, taxonomically named Stenotrophomonas phage vB_Sma-SZP25, Latin name Stenotrophomonasphage, the phage vB_Sma-SZP25 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64510-B1, and the storage date is: May 16, 2024.

[0010] The third aspect of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP27, taxonomically named Stenotrophomonas phage vB_Sma-SZP27, Latin name Stenotrophomonasphage, the phage vB_Sma-SZP27 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64511-B1, and the storage date is: May 16, 2024.

[0011] The fourth aspect of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP28, taxonomically named Stenotrophomonas phage vB_Sma-SZP28, Latin name Stenotrophomonasphage, the phage vB_Sma-SZP28 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64624-B1, and the storage date is: May 16, 2024.

[0012] The fifth aspect of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP96, taxonomically named Stenotrophomonas phage vB_Sma-SZP96, Latin name Stenotrophomonasphage, the phage vB_Sma-SZP96 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64625-B1, and the storage date is: May 16, 2024.

[0013] A sixth aspect of the present invention provides a Stenotrophomonas maltophilia phage composition, comprising: at least two of phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96, wherein the taxonomic names of the phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 are Stenotrophomonas phage vB_Sma-SZP9, Stenotrophomonas phage vB_Sma-SZP25, Stenotrophomonas phage vB_Sma-SZP27, Stenotrophomonas phage vB_Sma-SZP28 and Stenotrophomonas phage vB_Sma-SZP96, the Latin name is Stenotrophomonas phage, the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28 and the phage vB_Sma-SZP96 are all deposited in Guangdong Microbiological Culture Collection Center, the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the corresponding deposit numbers are: GDMCC No: 64509-B1, GDMCC No: 64510-B1, GDMCC No: 64511-B1, GDMCC No: 64624-B1 and GDMCC No.: 64625-B1, the preservation date is: May 16, 2024.

[0014] In some embodiments, the titers of the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28, and the phage vB_Sma-SZP96 are all 10 7 -10 10 pfu / mL.

[0015] In some embodiments, the infection multiplicity of the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28, and the phage vB_Sma-SZP96 is 0.00001-10.

[0016] In a seventh aspect, the present invention provides a phage preparation, comprising a Stenotrophomonas maltophilia phage or a Stenotrophomonas maltophilia phage composition.

[0017] In some embodiments, the pH of the phage preparation is 5-10.

[0018] In the phage preparation, when the phage preparation includes phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96, the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28 and the phage vB_Sma-SZP96 The ratio of the number of live phages vB_Sma-SZP28 to that of the phage vB_Sma-SZP96 is (1-3):(1-3):(1-3):(1-3):(1-3); and / or, the content of the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28 and the phage vB_Sma-SZP96 is greater than or equal to 10 8 pfu / mL.

[0019] The phage preparation further comprises a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises one or more of a stabilizer, a dispersant, a solvent and a filler.

[0020] The dosage form of the phage preparation includes a liquid preparation, a freeze-dried preparation or an oral solid preparation.

[0021] In an eighth aspect, the present invention provides an application of the phage preparation of the seventh aspect for lysing Stenotrophomonas maltophilia.

[0022] In a ninth aspect, the present invention provides a pharmaceutical composition comprising a Stenotrophomonas maltophilia phage or a Stenotrophomonas maltophilia phage composition.

[0023] In the technical scheme of the present invention, phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 have strong infection ability, can quickly kill host bacteria in culture medium, have wide temperature and acid-base tolerance range, and have good inhibition and killing effect on the proliferation of Stenotrophomonas maltophilia. The genome of the phage in the Stenotrophomonas maltophilia phage composition provided by the present invention does not carry virulence genes, drug resistance genes and lysogeny genes, will not cause the spread of virulence genes and drug resistance genes, and is safe to use. The phage composition provided by the present invention has a wide spectrum and can kill more than 90% of drug-resistant Stenotrophomonas maltophilia. The phage composition of the present invention can be used to prepare phage preparations such as sprays and injections, can be used to kill Stenotrophomonas maltophilia in the environment and to treat infections caused by Stenotrophomonas maltophilia in the body, and can effectively solve the treatment difficulties caused by the drug resistance and biofilm of Stenotrophomonas maltophilia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0025] Figure 1 The plaque morphology and electron microscopic images of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZP96;

[0026] Figure 2 is the genome analysis map of bacteriophage vB_Sma-SZP9;

[0027] Figure 3 is the genome analysis map of bacteriophage vB_Sma-SZP25;

[0028] Figure 4 is the genome analysis map of bacteriophage vB_Sma-SZP27;

[0029] Figure 5 is the genome analysis map of bacteriophage vB_Sma-SZP28;

[0030] Figure 6 is the genome analysis map of bacteriophage vB_Sma-SZP96;

[0031] Figure 7 One-step growth curves of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZP96;

[0032] Figure 8 The best infection multiplicity graphs for phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28, and phage vB_Sma-SZP96;

[0033] Fig. 9 The thermal stability diagrams of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28, and bacteriophage vB_Sma-SZP96;

[0034] Fig.10 The pH stability graphs of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28, and bacteriophage vB_Sma-SZP96;

[0035] Fig.11 The host spectrum of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28, bacteriophage vB_Sma-SZP96 and bacteriophage combination;

[0036] Fig.12 It is a time-effect diagram of the bactericidal effect of the phage combination on drug-resistant Stenotrophomonas maltophilia;

[0037] Fig.13 This is a graph showing the effect of the phage combination on the removal of the biofilm of drug-resistant Stenotrophomonas maltophilia.

[0038] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0039] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0040] Stenotrophomonas maltophilia has multiple resistance mechanisms, including the production of drug-resistant enzymes, the presence of multidrug efflux pumps, SMqnr genes, biofilm mechanisms, etc., which make it resistant to most antibiotics, such as cephalosporins, carbapenems, aminoglycosides, macrolides and β-lactams, which narrows the range of available drugs for clinical treatment.

[0041] Among them, Stenotrophomonas maltophilia can form biofilms on wet surfaces that are in direct or indirect contact with patients (with enhanced resistance to water-soluble disinfectants), such as in respiratory tubes, catheters, intravenous catheters, dialysis equipment, dental equipment, and hospital plumbing systems (sinks and faucets). Compared with planktonic bacteria, bacteria in biofilms can usually withstand antibiotic treatments up to 10 to 1000 times the concentration, which significantly reduces the effectiveness of traditional antibiotic treatment and poses a huge challenge to clinical treatment.

[0042] In this context, there is an urgent need to explore new and innovative treatment strategies. As an emerging treatment method, phage therapy has attracted widespread attention. Bacteriophages are a class of viruses that specifically target bacteria and have high host specificity. They infect bacteria and undergo a process of replication and proliferation, eventually causing the bacteria to rupture and die. Unlike traditional antibiotics, phage therapy has little effect on the body's normal microbiome while killing bacteria, thus reducing possible side effects and the risk of drug resistance. This feature gives phage therapy great potential in the treatment of drug-resistant bacterial infections.

[0043] However, single phage therapy may face multiple problems, including bacterial resistance to single phage, coexistence of multiple bacterial strains, and challenges such as phage stability and adaptability. Therefore, it is urgent to explore effective strategies to reduce the harm caused by Stenotrophomonas maltophilia in order to improve the specificity, effectiveness and adaptability of treatment.

[0044] In view of this, the first aspect of an embodiment of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP9, taxonomically named Stenotrophomonas phage vB_Sma-SZP9, Latin name Stenotrophomonas phage, the phage vB_Sma-SZP9 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64509-B1, and the storage date is: May 16, 2024.

[0045] A second aspect of an embodiment of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP25, taxonomically named Stenotrophomonas phage vB_Sma-SZP25, and Latin name Stenotrophomonas phage. The phage vB_Sma-SZP25 is deposited in Guangdong Microbiological Culture Collection Center, with a storage address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a storage number of GDMCC No: 64510-B1. The storage date is May 16, 2024.

[0046] A third aspect of an embodiment of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP27, taxonomically named Stenotrophomonas phage vB_Sma-SZP27, and Latin name Stenotrophomonas phage. The phage vB_Sma-SZP27 is deposited in Guangdong Microbiological Culture Collection Center, with a storage address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a storage number of GDMCC No: 64511-B1. The storage date is May 16, 2024.

[0047] A fourth aspect of an embodiment of the present invention proposes a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP28, taxonomically named Stenotrophomonas phage vB_Sma-SZP28, and Latin name Stenotrophomonas phage. The phage vB_Sma-SZP28 is deposited in Guangdong Microbiological Culture Collection Center, with a storage address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a storage number of GDMCC No: 64624-B1. The storage date is May 16, 2024.

[0048] A fifth aspect of an embodiment of the present invention provides a Stenotrophomonas maltophilia phage, including phage vB_Sma-SZP96, taxonomically named Stenotrophomonas phage vB_Sma-SZP96, and Latin name Stenotrophomonas phage. The phage vB_Sma-SZP96 is deposited in Guangdong Microbiological Culture Collection Center, with a storage address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a storage number of GDMCC No: 64625-B1.

[0049] A sixth aspect of the embodiments of the present invention provides a Stenotrophomonas maltophilia phage composition, comprising: at least two of phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96, wherein the taxonomic names of the phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 are Stenotrophomonas phage vB_Sma-SZP9, Stenotrophomonas phage vB_Sma-SZP25, Stenotrophomonas phage vB_Sma-SZP27, Stenotrophomonas phage vB_Sma-SZP28 and Stenotrophomonas phage vB_Sma-SZP96, the Latin name is Stenotrophomonas phage, the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28 and the phage vB_Sma-SZP96 are all deposited in Guangdong Microbiological Culture Collection Center, the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the corresponding deposit numbers are: GDMCC No: 64509-B1, GDMCC No: 64510-B1, GDMCC No: 64511-B1, GDMCC No: 64624-B1 and GDMCC No.: 64625-B1, the preservation date is: May 16, 2024.

[0050] In the technical scheme of the present invention, in the Stenotrophomonas maltophilia phage composition, phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 have strong infection ability, can quickly kill host bacteria in the culture medium, have a wide temperature and acid-base tolerance range, and have a good inhibitory and killing effect on the proliferation of Stenotrophomonas maltophilia.

[0051] In some embodiments, the titers of the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28, and the phage vB_Sma-SZP96 are all 10 9 -1010 pfu / mL.

[0052] In some embodiments, the infection multiplicity of the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28 and the phage vB_Sma-SZP96 is 0.00001-10; the titer is at least 10 7 pfu / mL. After treatment at 4℃-50℃ for 120 minutes, the 9 ~10 10 The titer of pfu / mL can be maintained at 10 after 120 minutes of treatment at pH = 4 ~ pH = 11. 8 ~10 10 The titer was expressed in pfu / mL.

[0053] In the seventh aspect, the embodiments of the present invention provide a phage preparation, including a Stenotrophomonas maltophilia phage or a Stenotrophomonas maltophilia phage combination. The phage mixed preparation formed by compounding the above five phages not only has a stronger lysis ability, a wider lysis spectrum, and a wider range of applications, which makes up for the limitation of a narrow host spectrum when a single phage is used.

[0054] In some embodiments, the pH of the phage preparation is 5-10.

[0055] In the phage preparation, when the phage preparation includes phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96, the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB The ratio of the number of live phages vB_Sma-SZP28 to that of the bacteriophage vB_Sma-SZP96 is (1-3):(1-3):(1-3):(1-3):(1-3); the content of the bacteriophage vB_Sma-SZP9, the bacteriophage vB_Sma-SZP25, the bacteriophage vB_Sma-SZP27, the bacteriophage vB_Sma-SZP28 and the bacteriophage vB_Sma-SZP96 is greater than or equal to 10 8 pfu / mL.

[0056] The phage preparation further comprises a pharmaceutically acceptable excipient, wherein the pharmaceutically acceptable excipient comprises one or more of a stabilizer, a dispersant, a solvent and a filler.

[0057] The dosage form of the phage preparation includes a liquid preparation, a lyophilized preparation or an oral solid preparation. The phage preparation of the present invention can be administered via aerosol inhalation, intravenous, intra-arterial, intraperitoneal, catheter, skin, oral, external and rectal routes. It is used to prevent or treat infection of Stenotrophomonas maltophilia in humans or farmed animals by spraying, injection or oral administration. The phage preparation includes an environmental disinfectant, which is used to improve the environment and reduce the content of Stenotrophomonas maltophilia on the surface of the medium. The phage preparation can be used to disinfect Stenotrophomonas maltophilia in infected organs through an in vitro organ perfusion system.

[0058] It should be noted that the phage composition preparation provided by the present invention may use the above-mentioned phage or phage composition as the sole active ingredient. It may also contain other phages other than the phage of the present invention, which can be used in combination with the phage of the present invention to obtain an effect of preventing and controlling Stenotrophomonas maltophilia that is equivalent to or better than that of the phage of the present invention.

[0059] It should be noted that in the environmental disinfectant provided by the present invention, the concentration of each bacteriophage is not less than 10 8 pfu / mL, and the environmental disinfectant also contains other active ingredients for inhibiting or eliminating Stenotrophomonas maltophilia in the environment.

[0060] The present invention also provides the use of the environmental disinfectant in the disinfection of hospital, community health center, nursing home, farm environment and object surface. The application method of the environmental disinfectant includes but is not limited to the disinfection and decontamination of water distribution system, medical facilities, breeding facilities, public and private facilities or other environmental surfaces in the form of liquid immersion, spraying, and combined use with aqueous carriers, which can effectively control the activity and growth of Stenotrophomonas maltophilia.

[0061] In an eighth aspect, an embodiment of the present invention provides an application of the phage preparation of the seventh aspect for lysing Stenotrophomonas maltophilia.

[0062] In a ninth aspect, an embodiment of the present invention provides a pharmaceutical composition comprising a Stenotrophomonas maltophilia phage or a Stenotrophomonas maltophilia phage composition.

[0063] It can be seen from the above technical solutions that the present invention has at least the following advantages and positive effects compared with the prior art:

[0064] (1) In the technical solution of the present invention, the genomes of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZ96 do not carry virulence genes, drug resistance genes and lysogenic genes, will not cause the spread of virulence genes and drug resistance genes, and are safe to use.

[0065] (2) In the technical scheme of the present invention, bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZP96 have strong infection ability, can quickly kill host bacteria in the culture medium, have a wide range of temperature and acid-base tolerance, can be used as sprays and injections, have good inhibitory and killing effects on the proliferation of Stenotrophomonas maltophilia, and have good therapeutic and protective effects on organisms (humans and animals) infected with multidrug-resistant Stenotrophomonas maltophilia.

[0066] (3) In the technical scheme of the present invention, the combination of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZP96 has a broad spectrum and can kill more than 90% of drug-resistant Stenotrophomonas maltophilia. The phage cocktail has the advantage of enhancing the therapeutic effect of phages.

[0067] (4) The phage composition of the present invention is 7 pfu / mL-10 9 The pfu / mL titer can effectively eliminate the biofilm of Stenotrophomonas maltophilia, and can overcome the treatment difficulties of Stenotrophomonas maltophilia resistance and biofilm.

[0068] In the examples of the present application, the bacteriophage vB_Sma-SZP9, taxonomically named Stenotrophomonas phagevB_Sma-SZP9, and Latin name Stenotrophomonas phage, is deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number: GDMCC No: 64509-B1, and the deposit date is May 16, 2024. The bacteriophage vB_Sma-SZP25, taxonomically named Stenotrophomonas phage vB_Sma-SZP25, and Latin name Stenotrophomonas phage, is deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number: GDMCC No: 64510-B1, and the deposit date is May 16, 2024. The bacteriophage vB_Sma-SZP27, taxonomically named Stenotrophomonas phage vB_Sma-SZP27, and Latin name Stenotrophomonas phage, is deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number: GDMCC No: 64511-B1, and the deposit date is May 16, 2024. The bacteriophage vB_Sma-SZP28, taxonomically named Stenotrophomonas phage vB_Sma-SZP28, and Latin name Stenotrophomonas phage, is deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number: GDMCC No: 64624-B1, and the deposit date is May 16, 2024. The bacteriophage vB_Sma-SZP96, taxonomically named Stenotrophomonas phage vB_Sma-SZP96, and Latin name Stenotrophomonas phage, is deposited in Guangdong Microbiological Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with the deposit number: GDMCC No: 64625-B1, and the deposit date is May 16, 2024.

[0069] The experimental methods in the following examples without specific experimental conditions are usually carried out under conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used are commercially available unless otherwise specified.

[0070] The present invention is described in detail below in conjunction with specific embodiments:

[0071] Example 1

[0072] (1) Amplification, titer determination and concentration of Stenotrophomonas maltophilia phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96.

[0073] Phage amplification: Phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 were all isolated and obtained by our laboratory from the sewage of Shenzhen Third People's Hospital. Take out Stenotrophomonas maltophilia Sma1, Sma3 and Sma7 stored at -80 ℃, streak on LB non-antibiotic plates, and culture at 37 ℃ overnight. Pick single colonies into 3 mL LB medium and culture at 37 ℃ 200 rpm overnight; transfer the overnight bacterial solution to fresh 10 mL LB at a ratio of 1:20, culture at 37 ℃ 200 rpm for 2 h, add vB_Sma-SZP9 phage solution to Sma1, add vB_Sma-SZP25, vB_Sma-SZP27 and vB_Sma-SZP28 phage solutions to Sma3, and add vB_Sma-SZP96 phage solution to Sma7; the ratio of phage to host bacteria is about (0.001-10:1), with 0.1:1 being the best, and culture at 37 ℃ 150 rpm for 2 h until the bacterial solution becomes clear, and the phage amplification solution is obtained;

[0074] Double-layer plate method for detecting phage titer: The above phage suspension was diluted 10 times in a gradient, 0.1 mL of phage dilution in each gradient was taken and mixed with 0.3-0.4 mL of host bacterial solution, and then spread on double-layer agar plates. Incubate at 37 °C overnight, count the plaques on each agar plate, and select plates with about 20-200 plaques. The initial phage concentration was calculated according to the dilution multiple, and the phage titer was obtained (phage titer (pfu / mL) = dilution multiple × number of plaques × 10). The results are as follows: Figure 1 As shown:

[0075] Phage vB_Sma-SZP9 can form clear plaques in agar medium, with no halo around them, clear and regular edges, a diameter of about 1.0 mm, and a titer of 1.23×10 10 pfu / mL, typical lytic phage;

[0076] The phage vB_Sma-SZP25 can form clear plaques in agar medium, with no halo around, clear and regular edges, and a diameter of about 1.0 mm. The titer reaches 9.08×10 9 pfu / mL, typical lytic phage;

[0077] Phage vB_Sma-SZP27 can form clear plaques in agar medium, with no halo around them, clear and regular edges, and a diameter of about 1.0 mm. The titer reaches 1.08×10 10 pfu / mL, typical lytic phage;

[0078] The phage vB_Sma-SZP28 can form clear plaques in agar medium, with no halo around, clear and regular edges, and a diameter of about 1.5 mm. The titer reaches 7.83×10 9 pfu / mL, typical lytic phage;

[0079] The phage vB_Sma-SZP96 can form clear plaques in agar medium, with no halo around, clear and regular edges, and a diameter of about 1.5 mm. The titer reaches 3.33×10 9 pfu / mL, which is a typical lytic phage.

[0080] Phage concentration: The amplified phage crude suspension was concentrated with polyethylene glycol (PEG8000). The specific steps were as follows: NaCl solid with a final concentration of 1 M was added to the enrichment solution, stirred to dissolve, and ice-bathed for 1 h to promote the separation of phage and corresponding host bacterial fragments; centrifuged at 11,000 g for 10 min to remove cell debris, added 10% PEG (v / v) to the supernatant and allowed to stand at 4 °C overnight; centrifuged at 11,000 g at 4 °C for 10 min, removed the supernatant, gently resuspended the precipitate with an appropriate amount of SM buffer, and filtered with a 0.22 μm filter membrane for sterilization to obtain phage concentrate.

[0081] (2) Morphological observation of bacteriophage

[0082] Transmission electron microscopy observation of bacteriophages: Phages were observed by TEM using the phosphotungstic acid (PTA) negative staining method. The phage concentrated particles were dropped onto the Formvar carbon support film, left to dry for 10 min, and excess liquid was removed with paper. 10 μL of PTA was added to the membrane for staining for 10 min, and deionized water was added to wash away excess PTA, excess liquid was removed with paper, and the sample was dried again for 10 min. The morphology of the phage was observed under TEM. The results are shown in Figure 2. Figure 1 shown.

[0083] The morphology of bacteriophage vB_Sma-SZP9 is: the head is a 24-hedron structure, the head length is about 100 nm, and the tail length is about 138 nm. It is a long-tailed phage. According to the latest classification and naming of bacteriophages, through whole genome sequence analysis, bacteriophage vB_Sma-SZP9 belongs to the Kyanoviridae bacteriophage family.

[0084] The morphology of bacteriophage vB_Sma-SZP25 is: the head is a 24-hedron structure, the head length is about 100 nm, and the tail length is about 150 nm. It is a long-tailed phage. According to the latest classification and naming of bacteriophages, through whole genome sequence analysis, bacteriophage vB_Sma-SZP25 belongs to the Kyanoviridae bacteriophage family.

[0085] The morphology of bacteriophage vB_Sma-SZP27 is: the head is a 24-hedron structure, the head length is about 100 nm, and the tail length is about 142 nm. It is a long-tailed phage. According to the latest classification and naming of bacteriophages, through whole genome sequence analysis, bacteriophage vB_Sma-SZP27 belongs to the Kyanoviridae bacteriophage family.

[0086] The morphology of bacteriophage vB_Sma-SZP28 is: the head is a 24-hedron structure, the head length is about 104 nm, and the tail length is about 146 nm. It is a long-tailed phage. According to the latest classification and naming of bacteriophages, through whole genome sequence analysis, bacteriophage vB_Sma-SZP28 belongs to the Kyanoviridae bacteriophage family.

[0087] The morphology of bacteriophage vB_Sma-SZP96 is: the head is a 24-hedron structure, the head length is about 100 nm, and the tail length is about 138 nm. It is a long-tailed phage. According to the latest classification and naming of bacteriophages, through whole genome sequence analysis, bacteriophage vB_Sma-SZP96 belongs to the Kyanoviridae bacteriophage family.

[0088] (3) Phage genome extraction and sequencing

[0089] Take 1 mL of phage concentrate, add 6 μL of DNase I (PROMEGA) with a final concentration of 6 U / mL and 6 μL of RNase (TAKARA) with a final concentration of 50 μg / mL, and react at 37 °C for 1 h to remove exogenous nucleic acids. Then add 20 μL 2M ZnCl2, mix well and react at 37 °C for 5 min. After centrifugation at 10,000g for 1 min, discard the supernatant and keep the precipitate. Then resuspend the precipitate with 500 μL TES buffer, react at 60 °C for 15 min, add 20 μL proteinase K, react at 37 °C for 90 min, add 60 μL 3M potassium acetate after the reaction is completed, mix well, ice bath for 15 min, then add an equal volume of phenol chloroform isoamyl alcohol (25:24:1), invert to mix, centrifuge at 10,000g for 5 min, take the supernatant (this step is repeated 3 times), add an equal volume of chloroform isoamyl alcohol (24:1), invert to mix, centrifuge at 10,000g for 5 min, take the supernatant (this step is repeated 2 times), add an equal volume of isopropanol to the supernatant, precipitate overnight, centrifuge at 10,000g for 5 min, discard the supernatant and keep the precipitate. The precipitate was washed twice with 70% ethanol, centrifuged at 10,000 g for 5 min, the supernatant was removed, the white precipitate was air-dried, and finally the nucleic acid was dissolved with 20 μL of sterile water and sent to BGI for whole genome sequencing. Figure 2 shown.

[0090] Depend on Figures 2 to 6 It can be seen that the full length of the genomes of bacteriophages vB_Sma-SZP9, vB_Sma-SZP25, vB_Sma-SZP27, vB_Sma-SZP28 and vB_Sma-SZP96 are 159670bp, 160057bp, 159891bp, 159981bp and 160628bp respectively, and they do not carry resistance genes, virulence genes or lysogenic genes.

[0091] The head fiber protein sequences and tail fiber protein sequences of bacteriophage vB_Sma-SZP9, vB_Sma-SZP25, vB_Sma-SZP27, vB_Sma-SZP28 and vB_Sma-SZP96 are shown in Tables 1-5 below.

[0092] Table 1 vB_Sma-SZP9 lyase, tail protein and tail fiber protein sequences

[0093]

[0094] Table 2 vB_Sma-SZP25 lyase, tail protein and tail fiber protein sequences

[0095]

[0096] Table 3 vB_Sma-SZP27 lyase, tail protein and tail fiber protein sequences

[0097]

[0098] Table 4 vB_Sma-SZP28 lyase, tail protein and tail fiber protein sequences

[0099]

[0100] Table 5 vB_Sma-SZP96 lyase, tail protein and tail fiber protein sequences

[0101]

[0102] (4) One-step growth curve determination of bacteriophage

[0103] Take the host bacteria culture prepared in Example 1 and transfer it to fresh culture medium at a ratio of 1:100 and culture until the logarithmic phase (OD 600 nm=0.5); Take 1 mL of host bacteria cultured to the logarithmic growth phase, and mix them with vB_Sma-SZP9, vB_Sma-SZP25, vB_Sma-SZP27, vB_Sma-SZP28 and vB_Sma-SZP96 phage solutions at a ratio of MOI=0.1, react in a 37 ℃ incubator for 5 minutes, centrifuge at 10 000 rpm at room temperature for 1 min, and keep the precipitate. Pipette 1 mL of fresh LB medium and blow the precipitate, centrifuge at 10 000 rpm at room temperature for 30s, and blow again after keeping the precipitate (the blowing and centrifugation steps are repeated at least 3 times). Finally, resuspend the precipitate with 10 mL of fresh LB medium, and incubate the resuspended test tube in a 37 °C shaker 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. Select a 0.22 μm standard disposable filter membrane for filtration, and place the filtrate in a medical refrigerator. Dilute the filtrate obtained at each time in multiples, measure the titer, and repeat the experiment 3 times. Statistical data obtained from the experiment, and draw a one-step growth curve. Observe and calculate the incubation period and outbreak period of the bacteriophage. The results are as follows: Figure 7As shown: The incubation periods of phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 infecting the host were 5 min, 10 min, 0 min, 5 min and 10 min, respectively, and the lysis periods were 90 min, 50 min, 60 min, 50 min and 30 min, respectively.

[0104] (5) Determination of optimal infection multiplicity of phage

[0105] 1 mL of phage (titer 10 9 pfu / mL) was diluted 10-fold with LB broth, and 0.1 mL of each dilution and 0.1 mL of host bacterial proliferation solution (10 pfu / mL) were taken according to the MOI value of 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001. 8 pfu / mL) was added to 5 mL of LB broth and cultured at 37°C with shaking at 200 rpm for 5 h. The culture was centrifuged at 10,000 rpm for 5 min and the titer was measured using the double-layer plate method. The combination with the highest titer was the optimal multiplicity of infection. Figure 8 shown.

[0106] Depend on Figure 8 It can be seen that phage vB_Sma-SZP27 and phage vB_Sma-SZP28 have strong lysis ability from MOI 10 to 0.00001, and the titer is 10 9 -10 10 pfu / mL; the optimal infection multiplicity of phage vB_Sma-SZP9 was 10, 1, 0.1 and 0.01, and the titer could reach 1.75×10 9 , 2.75×10 9 , 1.02×10 10 and 5.25×10 8 pfu / mL; the optimal infection multiplicity of phage vB_Sma-SZP25 was 10, 1, 0.1, 0.01, 0.001 and 0.0001, and the titer could reach 5.33×10 9 , 1.01×10 11 ,7.83×10 10 , 6.25×10 9 ,3.92×10 9 and 6.25×10 8pfu / mL; the optimal infection multiplicity of phage vB_Sma-SZP96 was 10, 1, 0.1, 0.01, 0.001 and 0.0001, and the titer could reach 5.80×10 9 8.00×10 9 9.00×10 9 9.60×10 9 ,7.90×10 9 and 4.60×10 8 pfu / mL.

[0107] (6) Determination of bacteriophage thermal stability

[0108] Take several sterile 2 mL EP tubes, add 500 μL of phage respectively, incubate at 4℃, 25℃, 30℃, 37℃, 50℃, 60℃, 70℃ and 80℃ for 2 h, and use the double agar method to determine the phage titer after incubation at different temperatures. Fig. 9 shown.

[0109] Depend on Fig. 9 It can be seen that the titer of the phage treated at 50 ℃ for 2 h was equivalent to that at 4 ℃, indicating that phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 have excellent high temperature resistance.

[0110] (7) Determination of pH stability of bacteriophage

[0111] 100 μL of phage was mixed with 900 μL of pH buffer solution with pH=3-12, and incubated in a 37℃ water bath for 2 hours. The titer of phage at different pH was determined using the double-layer agar method. Fig.10 shown.

[0112] Depend on Fig.10 It can be seen that phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 still maintain high titer after being exposed to pH in the range of 4-11 for 2 h, indicating that these five phages have a wide acid-base tolerance range.

[0113] (8) Determination of phage host spectrum

[0114] In this example, a total of 138 clinically resistant Stenotrophomonas maltophilia from the Third People's Hospital of Shenzhen were detected. 150 μL of logarithmically growing clinically resistant Stenotrophomonas maltophilia was taken into a 15 mL centrifuge tube, and 15 mL of 0.5% AgarLB at 55 °C was added. After mixing, it was immediately poured into a square plate, and the cover was opened in a biosafety cabinet and allowed to solidify. The square plate and the 96-well plate containing the phage library of the Third People's Hospital of Shenzhen were placed in an automatic pipetting workstation for high-throughput pipetting. The bacterial plate was then cultured overnight at 37 °C, and the screening results were photographed and recorded using a colony imager. The results are shown in the figure. Fig.11 shown.

[0115] Depend on Fig.11 It can be seen that phage vB_Sma-SZP9, phage vB_Sma-SZP25, phage vB_Sma-SZP27, phage vB_Sma-SZP28 and phage vB_Sma-SZP96 can form clear plaques against 47.10%, 55.80%, 51.45%, 48.55% and 47.10% of clinical drug-resistant Stenotrophomonas maltophilia, respectively, and 2 1.02%, 16.66%, 15.22%, 14.49% and 15.94% of the clinically resistant Stenotrophomonas maltophilia formed fuzzy plaques, the five-strain phage cocktail formed clear plaques against 81.88% of the clinically resistant Stenotrophomonas maltophilia and fuzzy plaques against 11.60% of the clinically resistant Stenotrophomonas maltophilia, indicating that the five-strain phage combination has a good broad spectrum.

[0116] (9) Bactericidal assay of phage combination in liquid culture medium

[0117] The bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZP96 were mixed at a quantitative ratio (pfu / mL) of 1:1:1:1:1 to make the phage titer of the composition 10 10 pfu / mL and stored at 4°C for future use. 600 ≈0.2, and 200 μL of phage composition (10 7 pfu / mL, 10 8 pfu / mL and 10 9 pfu / mL) and cultured statically at 37 °C. OD was measured every 15 min. 600 The results are as follows Fig.12 shown.

[0118] Depend on Fig.12It can be seen that the combination of five Stenotrophomonas maltophilia phages can quickly kill different clinically resistant Stenotrophomonas maltophilia (numbered Sma69, Sma70, Sma72 and Sma79) under different infection multiplicity conditions.

[0119] (10) Determination of the ability of phage combinations to remove biofilm

[0120] 200 μL LB suspension of clinical drug-resistant Stenotrophomonas maltophilia (Sma69, Sma70, Sma72 and Sma79) (10 8 cfu / mL) were added to 96-well plates and incubated at 37 °C for 24 h and 48 h to form mature biofilms for 24 h and 48 h. After washing three times with PBS, 200 μL of phage composition (10 7 pfu / mL, 10 8 pfu / ml and 10 9 pfu / mL) and placed at 37 °C for 4 h. An equal volume of LB was added as a negative control. Biofilm removal rate = 100% × (OD595 of the experimental group - OD595 of the control group) 595 ) / control group OD 595 The results are as follows Fig.13 shown.

[0121] Depend on Fig.13 It can be seen that under different titer conditions, the phage combination of five strains of Stenotrophomonas maltophilia has a good removal effect on the biofilms of clinically resistant Stenotrophomonas maltophilia Sma69, Sma70, Sma72 and Sma79.

[0122] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A Stenotrophomonas maltophilia phage, characterized in that Including bacteriophage vB_Sma-SZP9, taxonomically named Stenotrophomonas phage vB_Sma-SZP9, Latin name Stenotrophomonas phage, the bacteriophage vB_Sma-SZP9 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64509-B1, and the storage date is: May 16, 2024.

2. A Stenotrophomonas maltophilia phage, characterized in that Including bacteriophage vB_Sma-SZP25, taxonomically named Stenotrophomonas phage vB_Sma-SZP25, Latin name Stenotrophomonas phage, the bacteriophage vB_Sma-SZP25 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64510-B1, and the storage date is: May 16, 2024.

3. A Stenotrophomonas maltophilia phage, characterized in that Including bacteriophage vB_Sma-SZP27, taxonomic name Stenotrophomonas phage vB_Sma-SZP27, Latin name Stenotrophomonas phage, the bacteriophage vB_Sma-SZP27 is preserved in Guangdong Microbiological Culture Collection Center, the preservation address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is: GDMCC No: 64511-B1, and the preservation date is: May 16, 2024.

4. A Stenotrophomonas maltophilia phage, characterized in that Including bacteriophage vB_Sma-SZP28, taxonomically named Stenotrophomonas phage vB_Sma-SZP28, Latin name Stenotrophomonas phage, the bacteriophage vB_Sma-SZP28 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64624-B1, and the storage date is: May 16, 2024.

5. A Stenotrophomonas maltophilia phage, characterized in that Including bacteriophage vB_Sma-SZP96, taxonomically named Stenotrophomonas phage vB_Sma-SZP96, Latin name Stenotrophomonas phage, the bacteriophage vB_Sma-SZP96 is deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the storage number is: GDMCC No: 64625-B1, and the storage date is: May 16, 2024.

6. A Stenotrophomonas maltophilia phage composition, characterized in that: include: at least two of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZP96, wherein the taxonomic names of bacteriophage vB_Sma-SZP9, bacteriophage vB_Sma-SZP25, bacteriophage vB_Sma-SZP27, bacteriophage vB_Sma-SZP28 and bacteriophage vB_Sma-SZP96 are Stenotrophomonas phage vB_Sma-SZP9, Stenotrophomonas phage vB_Sma-SZP25, Stenotrophomonas phage vB_Sma-SZP27, Stenotrophomonas phage vB_Sma-SZP28 and Stenotrophomonas phage vB_Sma-SZP96, Latin name is Stenotrophomonas phage, the phage vB_Sma-SZP9, the phage vB_Sma-SZP25, the phage vB_Sma-SZP27, the phage vB_Sma-SZP28 and the phage vB_Sma-SZP96 are all deposited in Guangdong Microbiological Culture Collection Center, the storage address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the corresponding storage numbers are: GDMCC No: 64509-B1, GDMCC No: 64510-B1, GDMCC No: 64511-B1, GDMCC No: 64624-B1 and GDMCC No: 64625-B1, and the storage date is: May 16, 2024.

7. A bacteriophage preparation, characterized in that: The method comprises the Stenotrophomonas maltophilia phage according to any one of claims 1 to 5 or the Stenotrophomonas maltophilia phage composition according to claim 6.

8. The bacteriophage preparation according to claim 7, characterized in that The pH value of the phage preparation is 5-10.

9. A pharmaceutical composition, characterized in that The method comprises the Stenotrophomonas maltophilia phage according to any one of claims 1 to 5 or the Stenotrophomonas maltophilia phage composition according to claim 6.

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