Serratia marcescens phage and application thereof

Abstract

The invention provides a serratia marcescens phage and application of the serratia marcescens phage, the serratia marcescens phage is serratia marcescens phage WS, the preservation number of the serratia marcescens phage WS is CCTCC NO: M 20241689, and the preservation date of the serratia marcescens phage WS is July 26, 2024. The bacteriophage has the advantages of high splitting specificity, strong splitting capability, good acid and alkali resistance, optimal splitting temperature suitable for human body environment and the like, and is suitable for being used as an active ingredient of various serratia marcescens resisting products such as medicines, biological agents, disinfectants, bactericides, feed additives and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of bacteriophages, and in particular relates to a Serratia marcescens bacteriophage and application thereof. Background Art

[0002] Nosocomial infection refers to infection acquired by inpatients or medical staff in medical institutions, including infection acquired and occurred during hospitalization, infection acquired in the hospital but developed after discharge, and infection induced in the hospital. Nosocomial infection often causes severe respiratory symptoms, urinary tract inflammation, etc., and patients who have undergone surgical operations are more likely to have nosocomial infection. In recent years, nosocomial infections, including those caused by Serratia marcescens, have become increasingly serious around the world. One of the main reasons is that the widespread use of antibiotics has led to a gradual increase in the types of drug-resistant bacteria, and a variety of bacteria have formed resistance mechanisms. The number of drug-resistant strains has increased and the mechanisms are varied. On this basis, environmental cross-infections in hospitals are extremely difficult to prevent and control, and the problem of drug resistance is becoming increasingly serious. Multidrug-resistant bacteria mainly refer to strains that are widely seen in clinical practice and are resistant to three or more antibiotics. At present, clinical drug-resistant strains mainly include Staphylococcus aureus, Acinetobacter baumannii, and Serratia marcescens.

[0003] Studies have shown that the resistance rate of Serratia marcescens to cephalosporin antibiotics, gentamicin, roxithromycin, piperacillin, ampicillin and amoxicillin is at a high level. Due to its multi-drug resistance, the resistance status and resistance mechanism of Serratia marcescens need to be paid attention to. Studies have shown that Serratia marcescens itself is resistant to multiple antibiotics, such as Serratia marcescens has natural resistance to cephalothin. In addition, the extensive abuse of antibiotics in recent years has led to a decrease in the permeability of the outer membrane of Serratia marcescens, which is also an important reason for the emergence of new drug resistance in Serratia marcescens.

[0004] As a multidrug-resistant bacterium, Serratia marcescens causes increasingly serious problems such as hospital-acquired infections and environmental pollution, which need to be solved urgently. However, at present, there is no widely recognized scientific and effective method for the prevention and control of multidrug-resistant bacteria, which has caused a huge public health burden.

[0005] Since phages are relatively specific when infecting a certain type or several highly homologous host bacteria within the same genus, phage therapy is naturally targeted and traceable, and will not directly or quickly inactivate other healthy bacterial communities other than the target pathogens. This is one of its advantages over traditional broad-spectrum antibiotic treatment, and it may become an alternative therapy to antibiotics, thereby becoming a means of prevention and control of multidrug-resistant bacteria including Serratia marcescens.

[0006] The research on bacteriophages and their applications in biomedicine and environmental protection are attracting more and more attention from related fields at home and abroad. However, the current research methods on bacteriophages are mainly to find and discover new bacteriophages directly from epidemic sources, sewage or soil, etc. There are few reports on the extraction and isolation of bacteriophages directly from insect hosts, and the research on developing bacteriophages with medicinal value from medicinal insects is still basically blank. Summary of the invention

[0007] In view of this, the present invention provides a Serratia marcescens phage isolated from the medicinal insect American cockroach. The phage has the advantages of high lysis specificity, strong lysis ability, good acid and alkali resistance, and an optimal lysis temperature suitable for the human body environment.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] On the one hand, the present invention provides a Serratia marcescens phage (Serratia marcescens phage) WS, which was deposited in the China Center for Type Culture Collection on July 26, 2024, address: Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M 20241689.

[0010] The morphology of the Serratia marcescens phage WS is as follows: it has obvious head and tail structures, an overall length of (225±2nm), wherein the head diameter is approximately (110±2nm), presenting a relatively obvious regular polyhedron structure, and the tail is a retractable tail sheath, with a length of approximately (115±2nm), belonging to the Myoviridae family.

[0011] In another aspect, the present invention provides a fungicidal composition, comprising the aforementioned Serratia marcescens bacteriophage WS.

[0012] Preferably, the bactericidal composition further comprises an antibiotic against Serratia marcescens.

[0013] Preferably, the antibiotic is a cephalosporin antibiotic, a quinolone antibiotic, a penicillin antibiotic or a macrolide antibiotic.

[0014] Preferably, the composition further comprises an auxiliary material.

[0015] Preferably, the composition is in liquid, solid or semi-solid form.

[0016] In another aspect, the present invention provides use of the Serratia marcescens bacteriophage WS or the composition in preparing a biological agent for resisting Serratia marcescens infection.

[0017] In another aspect, the present invention provides use of the Serratia marcescens bacteriophage WS or the composition in the preparation of a drug for preventing / treating Serratia marcescens infection.

[0018] In another aspect, the present invention provides use of the Serratia marcescens bacteriophage WS or the composition in the preparation of a disinfectant or bactericide.

[0019] In another aspect, the present invention provides use of the Serratia marcescens bacteriophage WS or the composition in preparing a feed additive.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The optimal growth temperature of Serratia marcescens phage WS is 37°C, and it can maintain a high titer at 4°C, 21°C, and 28°C. 4°C is one of the commonly used storage temperatures for bacteriophages. American cockroaches prefer warm and humid environments and are active at 21-33°C. 28°C is the optimal growth temperature for American cockroaches, and 37°C is close to human body temperature. This allows Serratia marcescens phage WS to maintain a high titer and stably exist in important pathogens, host animals, transmission vectors, and susceptible populations, which has great advantages in its development and utilization as an alternative to antibiotics.

[0022] (2) The pH tolerance of Serratia marcescens phage WS is 5-9, which is consistent with the human environment and ecological environment, providing a prerequisite for its clinical application or environmental protection.

[0023] (3) The phage WS of Serratia marcescens exhibits strong specificity in lysing the corresponding bacteria, which can avoid damage to the probiotic community.

[0024] (4) The WS phage has no obvious incubation period and can rapidly infect bacteria and proliferate to release progeny phages. The optimal multiplicity of infection is 0.01. Even when the multiplicity of infection is 0.00001, the titer of the phage does not decrease significantly. The titer is stable, and the lysis and growth abilities are significantly stronger than those of other phages.

[0025] (5) No antibiotic resistance genes or virulence genes were found in the genome of WS bacteriophage, which can be safely used in humans and the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the WS plaque morphology of Serratia marcescens bacteriophage;

[0027] Figure 2 This is the electron microscopic morphology of Serratia marcescens phage WS;

[0028] Figure 3 is the temperature stability of Serratia marcescens bacteriophage WS;

[0029] Figure 4 is the pH sensitivity of Serratia marcescens phage WS;

[0030] Figure 5 is the optimal infection multiplicity of Serratia marcescens phage WS;

[0031] Figure 6 This is the one-step growth curve of Serratia marcescens phage WS. DETAILED DESCRIPTION

[0032] The present invention is specifically described below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0033] Example 1 Isolation, Preparation and Purification of Serratia marcescens Bacteriophage WS

[0034] 1. Experimental insects and sample processing

[0035] American cockroach samples were obtained from the American cockroach artificial breeding base in Weishan County, Dali City, Dali Bai Autonomous Prefecture, Yunnan Province. Ten American cockroach samples were taken as a group, the whole insects were ground and placed in a 50mL centrifuge tube, and 30mL PSBB buffer was added, numbered WSPA, and placed in a 4℃ refrigerator for culture and preservation to facilitate subsequent phage-related research.

[0036] 2. Host bacteria preparation

[0037] (1) Recovery of Serratia marcescens NZSL6 bacteria: Take out the NZSL6 magnetic bead tube donated by Dali People's Hospital from the -80℃ refrigerator, take a small magnetic bead with an inoculation loop, and inoculate it directly on a 9 cm thick LB plate using the dense streak method. After the inoculation is completed, place the culture dish in a 37℃ constant temperature incubator for 18-24 hours.

[0038] (2) Preparation of bacterial suspension of Serratia marcescens NZSL6: Use an inoculation loop to pick up a monoclonal NZSL6 strain, fully dissolve it in a centrifuge tube containing LB liquid culture medium, shake it gently to make a bacterial suspension, and culture it in a shaker at 220 rpm and 37°C for 8-10 h until the logarithmic growth phase.

[0039] (3) Determination of bacterial concentration of Serratia marcescens NZSL6: Take 3 mL of NZSL6 bacterial suspension in a turbidity tube, so that the volume of the bacterial suspension is about 2 / 3 of the volume of the turbidity tube, and measure its McFarland concentration and record it. Take 3 mL of LB liquid culture medium in a turbidity tube, and also measure its concentration and record it. Final host bacteria concentration = McFarland concentration of bacterial suspension - McFarland concentration of LB liquid culture medium. The above concentration determination should be repeated three times to take the average value.

[0040] 3. Screening, purification and potency determination

[0041] (1) Preliminary screening: Take 5 mL of WSPA sample with PSBB buffer added, filter it with a 0.22 μm filter membrane, add liquid culture medium, appropriate amount of SM liquid and 100 μL of Serratia marcescens 18, number it, shake it thoroughly and mix it, place it in a constant temperature air bath shaking incubator at 220 rpm and 37 °C for overnight culture, and observe whether the liquid in the centrifuge tube changes from turbid to clear, so as to preliminarily judge whether the sample contains bacteriophages. Then use the double-layer agar plate method for plating, filter the proliferated WSPA sample with a 0.22 μm filter membrane, add 100 μL of NZSL6 bacterial suspension, mix it thoroughly and slowly pour it into a 9 cm thin solid plate. In the control group, no filtrate sample is added, only 100 μL of NZSL6 bacterial suspension is taken and placed in a centrifuge tube containing 6 mL of semi-solid culture medium, and it is fully manually mixed and then plated. After paving, place the culture dish in a 37°C constant temperature incubator for overnight culture and observe whether clear and transparent plaques appear.

[0042] (2) Purification: After culturing plaques by the double-layer plate method, observe the plaques that appear on the plate, select a single transparent plaque that appears in the culture dish, and use an inoculation loop to pick up the selected plaque and a small amount of colonies around it along with the agar part, put it into a centrifuge tube containing 6mL of liquid culture medium, and culture it overnight in a constant temperature air bath shaking incubator at 220rpm and 37℃. The next day, filter it with a 0.22μm filter membrane and plate it. Repeat the above operation 5 to 6 times, and the purification is complete.

[0043] (3) Titer determination: Use the multiple dilution method to dilute and then use the double-layer agar plate method to determine the titer. Take several 1.5 mL sterile centrifuge tubes and label them according to the dilution multiple. -1 , 10 -2 ……10 -n Add 900 μL of LB liquid medium to each sterile centrifuge tube, and take 100 μL of the phage filtrate after proliferation and dilute it at a dilution factor of 10. -1 After being thoroughly mixed with a vortex mixer, 100 μL of the mixture was taken and diluted to 10 -2 Place the phage in a sterile centrifuge tube and perform gradient dilutions to the required dilution multiples according to the above method. After the filtered phage proliferation liquid is diluted 10 times, 10 μL of the phage dilution liquid of each gradient is taken, mixed with 100 μL of the host bacterial liquid, and then plated. After culturing in a 37°C incubator for 6-8 hours, the plaques are counted. Three parallel plates are plated for each dilution, and the results are averaged.

[0044] Phage titer (PFU / mL) = number of phages × dilution factor × 100.

[0045] 4. Results

[0046] The plaques formed by the phages were uniform in size, with a diameter of (0.63±0.06) mm, good growth status, clear edges, and clear and transparent. The titer could reach 1.08×10 9 PFU / mL, and its phage plaque morphology is as follows Figure 1 Shown is Serratia marcescens phage WS.

[0047] The deposit number of Serratia marcescens phage WS is: CCTCC NO: M20241689.

[0048] Example 2 Electron Microscopic Observation of Serratia marcescens Phage WS

[0049] Proliferate phage to a titer of 1×10 8 PFU / mL, filter and store in a refrigerator at 4℃ for later use; take 500μL of host bacteria for double-layer agar plating, culture in a 37℃ constant temperature incubator for 2h, take 50μL of phage filtrate and drop it on the plate, 5 drops per plate, two plates for each phage, and place it in a 37℃ constant temperature incubator for overnight culture after the phage suspension is dried. The next day, scrape the semi-solid layer in the transparent area of ​​the plate, add 4mL SM buffer, and place it in a 37℃, 220rpm constant temperature air bath shaking incubator for 4h to make the phage free in the SM buffer. Centrifuge the shaken liquid at 4℃, 3000rpm for 3min to let the agar sink to the bottom, absorb 1mL, and filter the supernatant with a bacterial filter membrane; centrifuge the filtrate at 4℃, 6000rpm for 3min, absorb the supernatant and filter it with a bacterial filter membrane, repeat this step once, and the resulting liquid is the sample for electron microscopy observation.

[0050] The Serratia marcescens phage WS showed obvious head and tail structures under electron microscopy, with an overall length of (225±2nm), of which the head diameter was about (110±2nm), showing a relatively obvious regular polyhedron structure, and the tail was a retractable tail sheath, about (115±2nm) long, belonging to the Myoviridae family. Its morphology is as follows Figure 2 shown.

[0051] Example 3 Gene sequencing of Serratia marcescens bacteriophage WS

[0052] 1. Phage proliferation

[0053] After propagating the host bacteria NZSL6, use the newly propagated host bacteria to propagate phages. Add 20mL LB liquid culture medium, 1mL host bacteria in the logarithmic growth phase, and 2mL phage to a 50mL centrifuge tube, and propagate a total of 4 tubes. Put them together in a constant temperature air bath shaking incubator at 37°C and 220rpm and culture until the liquid is clear. Use a 0.22μm diameter filter membrane to filter into two new 50mL centrifuge tubes (about 40mL per tube) to obtain the propagated phage.

[0054] 2. Preparation of crude phage particles

[0055] Preheat the water bath at 37℃, add 8μL DNaseⅠ (DNA lyase) and 6μL RNase A (RNA lyase) to the proliferated phage, and place it in a 37℃ water bath for reaction for 30min. Add 2.5mL EDTA (pH=8,c=20mmol / L). Add NaCl (2.4g), shake and dissolve, then place in an ice bath for 1h, take it out and place it in a centrifuge at 4℃, centrifuge at 7000rpm for 10min, and take 40mL of supernatant. Add 4g PEG8000 to the supernatant to a final concentration of 10%, shake it manually until it is completely dissolved, and place it in an ice bath overnight to fully precipitate it. The next day, place it in a centrifuge set to 4℃, centrifuge at 12000rpm for 45min, discard the supernatant, and invert it on a clean absorbent paper for 3-5min. The white solid on the tube wall is the phage precipitate.

[0056] 3. Extraction of bacteriophage DNA

[0057] (1) Prepare a 1.5 mL sterile centrifuge tube. Use a pipette to draw up 1 mL of lysis buffer. Gently resuspend the phage pellet to dissolve it. Transfer the liquid to a 1.5 mL sterile centrifuge tube using a pipette.

[0058] (2) Preheat the water bath to 70°C, add 200 μL SDS (100 μL 20% SDS per 500 μL) to the eluted liquid, and immediately and gently mix by inverting 4 to 6 times. Place the mixture in a preheated 70°C water bath, incubate for 10 min, and then cool on ice.

[0059] (3) Add 200 μL of impurity precipitation solution of equal volume to SDS, mix gently by manual inversion 4 to 6 times, and centrifuge at 4°C and 12,000 rpm for 10 min.

[0060] (4) After centrifugation, gently remove the tube and place it on a flat surface to stand for 5 minutes to make the supernatant clear. Take the supernatant and gently transfer it to a new centrifuge tube. Add an equal volume of LB binding solution and gently vortex to mix. Warm the LB binding solution in advance. After preheating the water bath to 37°C, place the LB binding solution in a 37°C water bath and heat it for 1-2 minutes and mix it.

[0061] (5) Add the above liquid to the same AC adsorption column multiple times, put it into a centrifuge, set the temperature to 4°C, and centrifuge at 12,000 rpm for 60 seconds. After centrifugation, discard the waste liquid in the collection tube.

[0062] (6) Add 700 μL of rinsing solution WB with anhydrous ethanol and centrifuge at 12,000 g for 60 seconds. Discard the waste liquid. Repeat this step twice.

[0063] (7) Place the adsorption column AC back into the empty collection tube and centrifuge it at 12,000 g for 2 min to remove as much rinse solution as possible. Place it on a clean paper to dry.

[0064] (8) Take out the adsorption column and place it in a clean centrifuge tube. Add 60 μL of elution buffer EB, which has been preheated in a 50°C water bath, to the middle part of the adsorption membrane. Leave it at room temperature for 2 min. Place it in a centrifuge and set the speed to 12,000 g for 1 min.

[0065] (9) Add the obtained liquid to the adsorption column again, leave it at room temperature for 1 min, put it in a centrifuge and set the speed to 12000g for 1 min. Store the obtained DNA at -80℃.

[0066] 4. Agarose gel electrophoresis

[0067] Prepare 1× electrophoresis buffer, take 20mL of 50× electrophoresis buffer and add 980mL of pure water, mix well. Prepare agarose gel (1.5%), add 4μL of dye Gold View, shake gently to mix and pour into the gel tank. Add 50mL of electrophoresis buffer to the electrophoresis tank, take out the comb in the gel and put the gel into the tank so that the liquid surface is submerged. Take 1μL Loading Buffer and 5μL DNA in a centrifuge tube, blow and mix, add sample, mark, and add marker. Electrophoresis: Set the voltage (100V) and time (45min) to start electrophoresis, and save the results after completion. Send qualified DNA to the sequencing company for whole genome sequencing using Illumina HiSeq.

[0068] After sequencing, the nucleotide sequence of Serratia marcescens phage WS is shown as SEQ ID NO:1.

[0069] The virulence genes and antibiotic resistance genes in the WS genome were detected through the antibiotic resistance gene website and the virulence and anti-virulence gene website, and no related antibiotic resistance genes and virulence genes were found.

[0070] Example 4 Study on the host spectrum of Serratia marcescens bacteriophage WS

[0071] Take out the strain magnetic bead tube for host spectrum experiment from -80℃ refrigerator and thaw at room temperature. After thawing, take a small magnetic bead and inoculate it directly on the 9cm thick plate by dense streaking method. After inoculation, place the culture dish in a constant temperature incubator with appropriate temperature for 18-24h to observe its growth. After all bacteria are successfully revived, seal the plate and store it in a 4℃ refrigerator for standby. Draw lines on the 9cm solid culture dish to divide it into zones, and mark the time, strain and temperature (21℃, 28℃, 37℃). Take a ring of bacteria with an inoculation loop and inoculate it in the corresponding area by dense streaking method. Each strain is inoculated 9 times, and 3 temperatures are repeated 3 times to form 3 parallels. After all strains are inoculated, drop 5μL of phage liquid in the center of each inoculated colony. After drying, place it in a constant temperature incubator at 21℃, 28℃ and 37℃ for 6h according to the temperature marked on the plate. Take the host bacteria as a control, observe the lysis morphology and degree of other strains, and judge whether they are lysed. If the control group has clear and transparent plaques that are the same as those in the host bacteria group, it is judged that it is completely lysed. If the plaques are less transparent than the host bacteria group, it is judged that it is lysed, and the degree of lysis is weaker than that of the host bacteria. If no plaques appear at all, it is judged that it is not lysed.

[0072] A total of 47 strains were tested for host spectrum experiments, including Shigella, Yersinia pseudotuberculosis, Yersinia enterocolitica, Serratia marcescens and other clinical strains, of which 17 were Serratia marcescens. Except for Serratia marcescens, the remaining strains were not lysed by the Serratia marcescens phage WS, which has a narrow host spectrum. Among the 17 strains of Serratia marcescens, 14 were lysed, with a lysis rate of 86.25%, which has strong lysis specificity.

[0073] Table 1 Lysis of Serratia marcescens phage WS at different temperatures

[0074]

[0075]

[0076] Example 5 Study on the Temperature Stability of Serratia marcescens Bacteriophage WS

[0077] Proliferate phages and filter. Take 6 1.5mL Ep tubes and add 1mL of phage liquid to each. Number them according to the corresponding temperature (4℃, 21℃, 37℃, 50℃, 60℃, 70℃). Preheat the water bath to the corresponding temperature, and then put the 6 numbered Ep tubes into their corresponding water baths, and take them out after 1 hour. Add 100μL of NZSL6 bacterial suspension to 3 centrifuge tubes containing 10mL semi-solid culture medium, shake slowly until mixed, and then pour them into 3 square plates with a length and width of 13cm and shake gently until mixed. After solidification, take 100μL of each of the 6 tubes of phage after 1 hour in the water bath, add them to 1.5mL Ep tubes with 900μL liquid culture medium for dilution, and continuously dilute to 10-6, take 5μL of the diluted phage and drop it in the center of the square to measure its titer. After the plate is dried, it is placed in a 37°C incubator for incubation. At the end of the experiment, the plate is placed in a 37°C incubator for incubation for 6 hours and the results are observed. The plaques are counted, the average value is taken and recorded, and the titer at each temperature is calculated and recorded. The experimental results are plotted using GraphPadPrism8, with temperature as the horizontal axis and the logarithm of the phage titer as the vertical axis to draw a bar graph.

[0078] The temperature stability experiment of Serratia marcescens phage WS showed that Figure 3 As shown in the figure, although the phage titer is little affected by temperature at 4℃-50℃, it can be seen that the titer of the phage increases slowly with the increase of temperature before 37℃; after 37℃, as the temperature increases, the titer of the Serratia marcescens phage WS gradually decreases, and the titer of the phage decreases sharply at 60℃; when the temperature rises to 70℃, the titer of the phage is 0, and the phage is inactivated. It can be seen that the optimal lysis temperature of the Serratia marcescens phage WS is 37℃, and it loses its activity at 70℃.

[0079] Example 6 Study on WSpH sensitivity of Serratia marcescens bacteriophage

[0080] Prepare PBS solution with a pH value of 0-14 and multiply the phage to a titer of 1×10 8PFU / mL. Take 16 1.5mL sterile centrifuge tubes and mark them, and take 1mL of PBS liquid of different pH and add it to 1.5mL Ep tubes, and add 10μL of phage liquid to 15 Ep tubes containing pH liquid. Add 1mL LB liquid culture medium and 10μL phage liquid to the Ep tube of the control group. After placing the 16 sterile centrifuge tubes in a 37℃ constant temperature incubator for 1h, use the doubling dilution method and the drip method to determine the titer. The titer determination method is the same as the temperature stability determination experiment. At the end of the experiment, the plate was placed in a 37℃ constant temperature incubator for 6h, and the plaques were counted, the average value was taken and recorded, and the titer of each pH was calculated and recorded. The experimental results were plotted using GraphPad Prism 8, and a bar graph was drawn with temperature as the horizontal axis and the logarithm of the phage titer as the vertical axis.

[0081] The pH sensitivity experiment of Serratia marcescens bacteriophage WS showed that Figure 4 As shown in the figure, after the Serratia marcescens phage WS reacted with different pH buffers for 1 hour, when the pH value was ≤4 or pH value ≥10, the phage lost its activity and the titer was 0; when the pH was 5-9, the Serratia marcescens phage WS still had activity after the phage reacted with the pH buffer for 1 hour; when the pH value was between 5-7, the titer of the phage gradually increased with the increase of pH; when pH = 8, the titer of the phage was the highest; it can be considered that the optimal pH environment for the phage is when the pH is 8. When the pH value exceeds 8, the titer of the phage continues to decrease with the increase of pH until it reaches 0.

[0082] Example 7 Study on the Optimal Multiplicity of Infection of Serratia marcescens Phage WS

[0083] Proliferate the phage and its host bacteria NZSL6 bacteria to make the phage titer reach 1×10 8 PFU / mL, the concentration of bacterial suspension reaches 1×10 7CFU / mL. After the bacterial suspension was mixed and the bacterial concentration was fixed, 7 15mL centrifuge tubes were taken, and the phage filtrate was diluted 10 times by the doubling dilution method, and then mixed according to the multiplicity of infection (phage titer / host bacteria concentration) of 0.00001, 0.0001, 0.01, 0.1, 1, 10. The mixed solution was cultured in a constant temperature air bath shaking incubator at 37°C and 220rpm for 6 hours, and then taken out and filtered with a 0.22μm filter membrane. Take 7 1.5mL sterile centrifuge tubes, mark them according to the multiplicity of infection (0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10), add 100μL host bacteria and 10μL phage filtrate corresponding to the mark, mix them thoroughly, let them stand for 5-10min, and determine the titer by double-layer plate method. Three parallel plates are laid for each ratio and the average is calculated. After the experiment, the plate is placed in a 37℃ incubator for 6h, the plaques are counted and the average is recorded, and the titer of each infection gradient is calculated and recorded. The experimental results are plotted using GraphPadPrism 8, with MOI as the horizontal axis and the logarithm of the phage titer as the vertical axis to draw a bar graph.

[0084] The titer of the phage and host bacteria at different ratios was measured by the optimal multiplicity of infection (MOI) experiment, such as Figure 5 As shown, there is no significant difference in the titer of the phage under different infection ratios, but when the ratio of WS phage to host bacteria is 0.01 (i.e., host bacteria: phage = 100:1), the titer of the phage is the highest. Therefore, the MOI of WS phage to its host bacteria (NZSL6) is 0.01.

[0085] Example 8 Study on the one-step growth curve of Serratia marcescens bacteriophage WS

[0086] According to the MOI, the ratio of phage to host bacteria was adjusted, and 500 μL of phage solution and bacterial suspension adjusted to the optimal infection multiplicity ratio were taken, mixed thoroughly with a vortex mixer, and placed in a constant temperature air bath shaking incubator at 37°C and 220rpm for reaction and adsorption for 10 minutes, and centrifuged at 4°C and 12,000rpm for 3 minutes, washed 3 times, centrifuged for 1 minute, and the precipitate was resuspended in LB liquid culture medium preheated at 37°C, fully shaken and mixed, and sampled at time 0, with a sampling volume of 500 μL. After sampling, the mixed solution was quickly placed in a constant temperature air bath shaking incubator at 37°C and 220rpm for culture. Since the phage proliferates rapidly and has a strong lysis ability, samples were taken every 5 minutes from 0min to 20min, and every 10 minutes from 20min to 90min. The sample was centrifuged at 4°C, 12,000 rpm for 3 minutes in a low-temperature high-speed centrifuge, filtered, and the supernatant was taken and the titer at this time was determined by the doubling dilution method. Three parallel experiments were performed at each time. At the end of the experiment, the plate was placed in a 37°C incubator for 6 hours, the plaques were counted and the average value was recorded, and the titer at each time point was calculated and recorded. The experimental results were plotted using GraphPad Prism 8, with the time as the horizontal axis and the logarithm of the phage titer as the vertical axis to draw a line graph.

[0087] The one-step growth curve of Serratia marcescens phage WS is shown in Figure 6 As shown in the figure, the phage strain has a strong lysis ability and no obvious incubation period; the number of phage progeny increases rapidly within 20 minutes, indicating that its lysis period is 20 minutes; from 20 minutes to 50 minutes, the number of phage progeny of this strain increases slowly, and after 50 minutes, the phage titer of this strain is concentrated at nearly 1×10 8 PFU / mL.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bacteriophage against Serratia marcescens, characterized in that: The bacteriophage is Serratia marcescens phage WS, with a preservation number of CCTCC NO: M 20241689 and a preservation date of July 26, 2024.

2. A bactericidal composition, characterized in that: The composition comprises the bacteriophage according to claim 1.

3. The composition according to claim 2, characterized in that The composition also includes an antibiotic against Serratia marcescens.

4. The composition according to claim 3, characterized in that The antibiotics are cephalosporin antibiotics, quinolone antibiotics, penicillin antibiotics or macrolide antibiotics.

5. The composition according to any one of claims 2 to 4, characterized in that The composition further includes auxiliary materials.

6. The composition according to claim 5, characterized in that The composition is in liquid, solid or semi-solid form.

7. Use of the bacteriophage according to claim 1 or the composition according to any one of claims 2 to 6 in the preparation of a biological agent for preventing Serratia marcescens infection.

8. Use of the bacteriophage according to claim 1 or the composition according to any one of claims 2 to 6 in the preparation of a medicament for preventing / treating Serratia marcescens infection.

9. Use of the bacteriophage according to claim 1 or the composition according to any one of claims 2 to 6 in the preparation of a disinfectant or bactericide.

10. Use of the bacteriophage according to claim 1 or the composition according to any one of claims 2 to 6 in the preparation of a feed additive.

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