Efficient screening method and application of bacteriophage

Abstract

The invention relates to the technical field of microbial separation, and provides a method for efficiently screening or separating bacteriophage of a targeted host. The method starts from the sample environment temperature and the bacteriophage infection process, and direct enrichment is carried out without centrifugal membrane passing during sample treatment so as to reduce the loss of potential target bacteriophages; incubating at a temperature close to the ambient temperature of the sample based on the influence of temperature on the lysis activity and lifestyle of the bacteriophage; by adding substances such as CaCl2 into the culture medium, the competence of the host is changed, so that the host is more susceptible to bacteriophage infection. The invention provides an efficient screening method for bacteriophages and develops and applies the efficient screening method for the bacteriophages by improving key processes such as increasing the range of enriched bacteriophages, optimizing the screening temperature and improving the infection sensitivity of hosts.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial separation, and in particular relates to a high-efficiency screening method and application of bacteriophages. Background Art

[0002] The misuse of antibiotics has led to the emergence, prevalence and spread of a large number of drug-resistant bacteria. As the most abundant biological entity on earth, bacteriophages are an important factor affecting the succession of bacterial community structure, and will have a certain impact on ecological functions such as microbial community structure, plant growth, and biogeochemical cycles (TL Brown, et al., 2022; AAPratama and JD van Elsas, 2018; CA Suttle, 1994). In addition, recent studies have found that anaerobic digestion is mainly manifested in the lysis of antibiotic-resistant bacteria by viral communities. There are very few bacteriophages carrying resistance genes, and their reduction of resistance genes is greater than their spread. Therefore, bacteriophages can be used as an important and potential alternative to antibiotics.

[0003] There are certain challenges in the application of phages. On the one hand, it is difficult to screen phages that target specific research hosts. Take Pseudomonas putida as an example. It is a common bacterial species in the natural environment and a rare conditional pathogen. The presence and spread of drug-resistant genes in the natural environment may increase the drug resistance of Pseudomonas putida, thereby threatening human health. Therefore, it is very important to strengthen the screening and mechanism research of phages targeting drug-resistant pathogens such as Pseudomonas putida under laboratory conditions. However, at present, only a few phages have been screened out using the experimental model strain P. putida KT2440 as the host, and the infection rate of the host is not high. On the other hand, the emergence of phage resistance in the target bacteria will affect the effect of phage therapy. Studies have found that bacteria can develop resistance to phages through a variety of mechanisms such as restriction repair and abortive infection (J. Bondy-Denomy et al., 2016). Phage cocktail therapy, i.e. mixing multiple phages, has become the most popular and widely used phage therapy strategy. Studies have shown that bacteria treated with mixed phages have a significantly reduced ability to develop resistance to phages (MT Moghadam et al., 2020). Therefore, a large number of phage species are needed for clinical application research, and a large number of sensitivity tests are required for specific clinical strains to various phages known to infect the bacterial genus. In summary, isolating a large number of new phages and establishing a large-scale phage resource library is a key link in phage application research.

[0004] Although phages exist in many environments, it may not be easy to isolate phages targeting specific hosts. It is particularly difficult to screen phages that are prone to forming biofilm hosts, because the formation of biofilms makes it difficult for microorganisms to be infected by phages. Therefore, it is necessary to improve the traditional phage isolation methods. The isolation and screening of phages generally adopts the double-layer agar plate method. That is, sample collection, enrichment treatment and separation, and plaque formation. Different environments and sample processing processes may affect the isolation of phages. Summary of the invention

[0005] The existing technology has few types of phage screening and isolation for certain specific drug-resistant pathogens, which is not conducive to the application research of phages. In addition, in order to deal with the host's phage resistance, it is necessary to establish a large-scale phage resource library to isolate a large number of new phages. Therefore, it is necessary to improve and optimize the previous phage isolation methods to improve the screening effect of phages with high infection rates on the host.

[0006] The present invention selects Pseudomonas putida KT2440, which was difficult to screen in previous studies, for research, and selects Escherichia coli K12 to verify the improved method. Starting from the phage infection process, key processes such as enrichment method, incubation temperature and host infection sensitivity are improved and optimized to construct an efficient phage screening method. By integrating sample pretreatment, incubation temperature and improving host infection sensitivity, the screening or separation efficiency of phages can be greatly improved, and phages with high infection rates on the host can be screened out, overcoming the problem that phages that are easy to form biofilm hosts (especially Pseudomonas putida) are difficult to screen, which is helpful for the application of phage therapy.

[0007] In the present invention, P. putida KT2440 was purchased from Beijing Bio-Bowei Biotechnology Co., Ltd. (bio-82074). Escherichia coli E. coli K12 was purchased from Beijing Bio-Bowei Biotechnology Co., Ltd. (bio-04037).

[0008] The present invention provides the following technical solutions:

[0009] 1) Activated strains: Select the target strain for phage isolation, take out at -80℃ and thaw on ice, streak and inoculate on a plate of LB solid culture medium, prepare according to the instructions, and sterilize at 121℃ for 15 minutes for use, culture for 12 hours, take a single colony and streak and purify, purify two to three times and inoculate into LB liquid culture medium, prepare according to the instructions, and sterilize at 121℃ for 15 minutes for use, culture at 30℃ on a shaker for 6-8 hours until the logarithmic phase, and obtain an activated host bacterial solution;

[0010] 2) Sample collection: Fresh domestic sewage or river water rich in various bacteriophages is collected on the same day. Generally, river water replenished with recycled water is used as the sample. The sample environment temperature (such as the river water temperature under natural conditions) is measured when the sample is collected.

[0011] 3) Prepare sample enrichment solution: Take 50 ml of sample, add 5 ml of LB liquid medium, 2 ml of host bacterial solution (add LB liquid medium to the activated host bacterial solution obtained in step 1, adjust the OD600 value to about 1), add CaCl 2 Solution (final solution concentration is 10mM), based on the water sample taken, determine the incubation temperature for enrichment (the incubation temperature is no more than 7°C different from the ambient temperature of the sample source, preferably no more than 6°C different, more preferably no more than 5°C different), for example, if the water sample temperature is 22.3°C, the incubation temperature for enrichment can be selected as 20°C. Oscillate on a shaker at 100rpm, incubate overnight at the above-mentioned enrichment incubation temperature, and take the supernatant as the sample enrichment solution;

[0012] 4) Obtaining phage fluid: centrifuge the sample enrichment fluid obtained in step 3 at 4°C, 10,000 r / min for 10 min, filter through a 0.22 μm filter membrane, and take the supernatant as the phage fluid;

[0013] 5) Screening of phages: Using the double-layer plate verification method, take 1 ml of the phage solution obtained in step 3 and mix it with 200 μl of the activated host bacterial solution obtained in step 1. The same temperature as the incubation temperature for enrichment determined in step 3 is used as the incubation temperature for screening. Incubate the culture in an incubator at this temperature for 20 min. Add about 3.5 ml of LB semi-solid medium cooled to about 40°C (LB liquid medium and add agar in proportion to the corresponding volume. For example, 7.5 g agar is added to 1 L of liquid to obtain LB semi-solid medium. Add CaCl 2 The solution (the final solution concentration is 10 mM) was quickly mixed and poured into a plate prepared with LB solid medium. The plate was incubated overnight at the screening incubation temperature to observe the formation of plaques;

[0014] 6) Phage purification: Select a plate with formed plaques, separate and purify a single plaque, select a single plaque and suspend it in 900 μl of 10-fold diluted SM buffer, sterilize it at 121°C for 15 min, and obtain a phage suspension. Dilute the phage suspension 10 times in sequence. Take 200 μl of the host liquid and add 4 ml of LB semi-solid medium (add CaCl 2 Solution (final solution concentration is 10mM) is added to the double-layer plate, and 2μl of phage suspension and diluted phage solution are respectively titrated on the plate. The plate is incubated at 20°C overnight. Next, a new single phage plaque is picked up and the above operation is repeated until phages of the same size are obtained, indicating that the purification is successful;

[0015] 7) Phage preservation: Purified phages were stored in phage storage buffer (SM buffer) at 4°C and -80°C.

[0016] Specifically, the incubation temperature for enrichment and the incubation temperature for screening are 10°C or 20°C, preferably 20°C.

[0017] Specifically, the method of the present invention is used to screen out bacteriophages with a high infection rate to the host, wherein the high infection rate is higher than 70%.

[0018] Specifically, the present invention provides the following technical solutions:

[0019] 1. A method for efficiently screening or isolating bacteriophages, characterized in that the method comprises the following steps:

[0020] 1) preparing a sample enrichment solution, which includes collecting a sample rich in phages, mixing the sample directly with LB liquid culture medium and host bacterial solution without centrifugation and membrane filtration, and culturing at an enrichment incubation temperature to obtain a sample enrichment solution; this method can reduce the loss of phages located on the surface of particles or colloids, and can greatly improve the screening of host-related phages that are prone to forming biofilms;

[0021] 2) Obtaining phage liquid, which includes taking the sample enrichment liquid obtained in step 1), centrifuging, taking the supernatant and filtering it through a membrane to obtain phage liquid; this step can reduce the change in phage lifestyle caused by the temperature, and further improve the screening efficiency of phage;

[0022] 3) Screening phages, which includes mixing the phage solution obtained in step 2) with the host bacterial solution, adding the solution to LB semi-solid medium and mixing evenly, then pouring the solution into a plate prepared with LB solid medium, and incubating the mixture at a screening incubation temperature;

[0023] The incubation temperature is no more than 7°C different from the ambient temperature of the sample source; the LB liquid medium in step 1) and the LB semi-solid medium in step 3) both contain CaCl 2 , add CaCl 2 This makes the host bacteria more susceptible to phage infection.

[0024] 2. The method described in item 1 is characterized in that the incubation temperature for enrichment is the same as the incubation temperature for screening, and the incubation temperature differs from the ambient temperature of the sample source by no more than 6°C, preferably by no more than 5°C.

[0025] 3. The method described in Item 1 is characterized in that the incubation temperature for enrichment and the incubation temperature for screening are 10°C or 20°C, preferably 20°C.

[0026] 4. The method according to any one of items 1 to 3, characterized in that the CaCl 2 The final solution concentration was 10 mM.

[0027] 5. The method described in any one of items 1 to 4, characterized in that the filter membrane is a 0.22 μm filter membrane.

[0028] 6. The method described in any one of items 1-5 is characterized in that it also includes a step of purifying phages, and the step of purifying phages includes: selecting plaques and suspending them in SM buffer, obtaining a phage suspension, diluting it with SM buffer, and titrating it on a plate prepared with LB solid culture medium.

[0029] 7. The method described in item 1-6 is characterized in that the bacteriophage is a bacteriophage capable of killing or lysing Pseudomonas putida, and the host is Pseudomonas putida, preferably the Pseudomonas putida is P. putida KT2440.

[0030] The present invention has the beneficial effects:

[0031] 1) Compared with the traditional method, this method combines the phage lifestyle and infection process, determines the incubation temperature according to the temperature of the phage living environment, adopts the method of first enrichment and then centrifugation to obtain as many phages as possible, reduces the loss of phages on the surface of particles or colloids, and adds CaCl 2 The host's sensitivity to infection is improved by etc. The screening effect (size of the infection transparent zone) based on this technical solution is 1.4 times higher than that of the traditional method.

[0032] 2) In the traditional method of enrichment and separation, the sample is first centrifuged and filtered through a 0.22μm filter membrane to remove endogenous bacteria and other solid matter, thereby obtaining a possible phage solution for enrichment. However, this process may remove phages on the surface of particles or colloids, especially the targeted phages of hosts that are prone to forming biofilms or colloids (such as Pseudomonas putida), which will reduce the number of phage species obtained in the end, or even lead to phage screening failure. Based on the comparison of the effects of the technical solution of this study and the traditional solution of centrifugation and filtration followed by enrichment, it was found that during the direct enrichment process, plaques were significantly formed on the plate, and the targeted lytic phage of P.putida KT2440 (prone to forming biofilms) was successfully screened. Direct enrichment not only simplified the phage separation process, but also improved the phage screening effect.

[0033] 3) Add CaCl 2 It can increase the host's sensitivity to phage infection and help improve the infection efficiency of phage. 2Solution method comparison, adding CaCl 2 The solution improves the screening effect of phages to a certain extent.

[0034] 4) Temperature has a significant impact on the phage lifestyle. Temperature changes may cause phages to change from the original lytic type (can form a transparent circle and achieve separation) to the mild type (do not lyse the host, cannot form a transparent circle, and cannot achieve separation). By improving the incubation temperature of the screening process, the separation of P. putida KT2440 phage, which is difficult to screen in the existing technology, was achieved. Based on this technical solution, the optimal growth temperature of the host and the temperature of different environmental samples (i.e., the phage living temperature) were used as the enrichment incubation temperature for phage separation. It was found that phage screening based on the phage living temperature was the most effective.

[0035] 5) The present invention overcomes the problem that it is difficult to screen phages that are prone to forming biofilm hosts (especially Pseudomonas putida), and successfully screens out phages that are prone to forming biofilm hosts (especially Pseudomonas putida). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The figure is a flow chart of the efficient phage screening method of the present invention.

[0037] Figure 2 This is the phage screening of P.putida KT2440 in different types of water samples at different temperatures (the water temperature when the samples were taken is on the left, and the temperature when phage screening was performed according to the research method is marked below the figure). The first row shows 22.3℃ domestic sewage samples, the second row shows 21.5℃ recycled water replenishment river samples, and the third row shows 9℃ recycled water replenishment river samples. The check mark indicates that the phage was successfully isolated under the corresponding conditions, and the cross mark indicates that the corresponding phage was not isolated.

[0038] Figure 3 The lysis effect of Pseudomonas putida KT2440 P2 at different temperatures under LB conditions. LB is a nutrient-rich condition suitable for the growth of the host; CK is the control concentration of phage and host in the culture medium at 0h of culture; * indicates a significant difference at the t-test p<0.05 level, ** indicates a significant difference at the t-test p<0.001 level.

[0039] Figure 4The lysis effect of phage targeting Pseudomonas putida KT2440 P2 under different environmental conditions; PBS is oligotrophic condition, RW is direct effluent of reclaimed water, and RR is the receiving river of reclaimed water; * indicates significant difference at the t-test p<0.05 level, ** indicates significant difference at the t-test p<0.001 level.

[0040] Figure 5 This is a diagram showing the effect of this research method on phage screening of E. coli K12 compared with different traditional treatment methods. A is the method of this technical solution, that is, enrichment followed by centrifugation, temperature (the enrichment and screening temperature was set at 20°C based on the ambient temperature) and the addition of CaCl 2 ; B is centrifugal filtration followed by enrichment, and the rest is consistent with A method; C's enrichment and screening temperature is 30°C, and the rest is consistent with A method; D is no addition of CaCl 2 , and the rest are consistent with method A.

[0041] Figure 6 This is a comparison chart of phage infection rates of E. coli K12 under different treatments based on ImageJ software. * indicates significant difference at the t-test p<0.05 level, where A is the technical solution method of this aspect, namely, enrichment followed by centrifugation, temperature (the enrichment and screening temperature was set at 20°C based on the ambient temperature in this study) and addition of CaCl 2 ; B is centrifugal filtration followed by enrichment, and the rest is consistent with method A; C is enrichment and screening temperature is 30°C, and the rest is consistent with method A; D is no addition of Cacl 2 , and the rest are consistent with method A.

[0042] Figure 7 The lysis effect of phage targeting E. coli K12 under different environmental conditions. PBS is oligotrophic condition, RW is the direct effluent of reclaimed water, and RR is the receiving river of reclaimed water. DETAILED DESCRIPTION

[0043] Example 1 Screening of P. Putida KT2440-targeting phages at different water sample ambient temperatures

[0044] The samples selected for phage screening in this case came from surface water in the Shahe Reservoir area of ​​Beiyunhe in Changping District, Beijing (a river supplied by recycled water) and domestic sewage effluent from a sewage treatment plant (domestic sewage).

[0045] The host selected P. putida KT2440 (easy to form biofilm) and first activated the host strain:

[0046] Select the target bacteria for phage isolation, take out at -80℃ and thaw on ice, streak and inoculate on a plate of LB solid culture medium (AOBOX, prepared according to the instructions and sterilized by high pressure at 121℃ for 15 min for later use), culture for 12 hours and take a single colony for streak and purification, purify two to three times and inoculate into LB liquid culture medium (AOBOX), prepare according to the instructions and sterilize by high pressure at 121℃ for 15 min for later use, culture on a shaking table at 30℃ for 6-8 hours until the logarithmic phase, and obtain an activated host bacterial liquid.

[0047] The following steps were used for phage screening:

[0048] 1) Prepare sample enrichment solution: Take 50 ml of sample, add 5 ml of LB liquid medium, 2 ml of host bacterial solution (add LB liquid medium to the above activated host bacterial solution, adjust the OD600 value to about 1), add CaCl 2 Solution (final solution concentration is 10 mM). The enrichment temperature was determined based on the temperature of the water sample, and the cells were shaken on a shaker at 100 rpm at 10°C, 20°C, and 30°C, respectively, and incubated overnight, and the supernatant was taken to obtain the sample enrichment solution.

[0049] 2) Obtaining phage fluid: Centrifuge the sample enrichment fluid obtained in step 1 at 4°C, 10,000 r / min for 10 min, filter through a 0.22 μm filter membrane, and take the supernatant to obtain the phage fluid.

[0050] 3) Screening of phages: Using the double-layer plate verification method, take 1 ml of the phage solution obtained in step 2, mix it with 200 μl of the above-mentioned activated host bacterial solution, and incubate it in an incubator at 10°C, 20°C, and 30°C for 20 min, respectively. Add about 3.5 ml of LB semi-solid medium cooled to about 40°C (LB liquid medium and add agar in proportion. For example, 7.5 g agar is added to 1 L of liquid to obtain LB semi-solid medium, and CaCl is added. 2 The solution (the final solution concentration is 10 mM) was quickly mixed and poured into a plate prepared with LB solid medium (AOBOX). The plate was incubated overnight to observe the formation of plaques.

[0051] The results are as follows Figure 2 As shown, no matter domestic sewage or recycled water replenishment river water samples, when the water sample temperature is about 20℃, the phage can be screened out according to this method, and no plaque formation is observed at 30℃ (7.7℃ or more difference from the water sample temperature), indicating that no phage has been screened out. When the water sample temperature is 9℃, the phage can be screened out at 10℃, and no plaque formation is observed at 20℃ and 30℃ (11℃ or more difference from the water sample temperature), indicating that no phage has been screened out.

[0052] 4) Phage purification: Select a plate with formed plaques, separate and purify a single plaque, select a single plaque and suspend it in 900μl of 10-fold diluted SM buffer (Coolaber, autoclaved at 121℃ for 15min for standby use) to obtain a phage suspension. Dilute the phage suspension 10 times in sequence. Take 200μl of the host activated bacterial solution and add 4ml of LB semi-solid culture medium to a double-layer plate, and take 2μl of the phage suspension and the diluted phage solution and titrate them on the plate prepared with LB solid culture medium. Incubate the plate at 20℃ overnight. Next, pick a new single plaque and repeat the above steps until phages of the same size are obtained, which means the purification is successful.

[0053] 5) Phage preservation: Purified phages were stored in phage storage buffer (SM buffer) at 4°C and -80°C.

[0054] The flowchart of the phage efficient screening method of the present invention is as follows Figure 1 The method was used to screen and purify the bacteriophage P.Putida KT2440 P2 targeting Pseudomonas putida. The infection rate and lysis effect were shown in Figure 3 and Figure 4 As shown. Pseudomonas P.Putida KT2440 and the phage P.Putida KT2440 P2 screened to have infection ability at both 20℃ and 30℃ were used to investigate the phage lysis. First, phage liquid and host bacterial suspension were added at a ratio of 0.001, 0.1, 1, 10, and 100, respectively, and 4 mL of LB liquid culture medium was added. Oscillate at 150 r / min for 5 hours at 20°C and 30°C, centrifuge at 10000 r / min for 10 minutes, filter, dilute the filtrate in a continuous gradient, and then take 100μl of the filtrate for double-layer plate coating to determine the phage titer (unit is PFU / mL, that is, the number of phages contained in 1mL of culture medium, phage titer = number of plaques × dilution factor × 10), and the multiplicity of infection (MOI) with the highest titer is the optimal MOI. In addition, by diluting and coating, counting the number of changes in host bacterial colonies, the optimal bactericidal ratio was obtained. After determination, the optimal MOI of P.Putida KT2440 and P2 was determined to be 0.1.

[0055] Then, add phage solution and host bacteria (1 × 10 8cfu / mL) into 4mL LB liquid medium and cultured aerobically at 20°C and 30°C for 5 hours. LB liquid medium is a nutrient-rich condition suitable for the growth of the host. CK is the control concentration of phage and host in the culture medium at 0h of culture. The cultured bacterial solution was centrifuged at 10000 r / min for 10 minutes and then filtered through a 0.22 μm filter membrane. The filtrate was diluted in a continuous gradient and the double-layer plate method was used to determine the change in phage titer. In addition, the change in host bacterial concentration was counted by dilution and coating, and plotted as Figure 3 The results showed that P. Putida KT2440 P2 exhibited a strong lysis effect at both 20°C and 30°C, which was 3 log higher than the initial concentration.

[0056] Phage solution and host bacteria (1 × 10 6 cfu / mL) into 4mL of liquid culture medium under different environmental conditions, and cultured aerobically at 30°C for 5 hours. The PBS condition is to use PBS (Solarbio, 10×PBS buffer diluted to one time with deionized water) at 120°C, sterilize it under high pressure for 15 minutes, and then use it as a liquid culture medium; RW is the use of reclaimed water discharged from the reclaimed water plant that meets the national standards (for example, see GB / T 18921-2019) as a liquid culture medium; RR is the use of water from the river that receives reclaimed water, which is sterilized under high pressure and used as a liquid culture medium. The blank is when no phage liquid is added, but host bacteria are added. The cultured bacterial solution was centrifuged at 10000 r / min for 10 minutes and then filtered through a 0.22 μm filter membrane. The filtrate was diluted in a continuous gradient, and the phage titer change was determined by the double-layer plate method. In addition, the changes in host bacteria concentration were counted by dilution and coating, and plotted as Figure 4 The results showed that a certain lysis effect was observed in both PBS and the receiving river (RR) of recycled water. The concentration of host bacteria was significantly reduced under the direct effluent condition of recycled water (RW) (p<0.05).

[0057] Example 2 Phage screening results of this research method compared with traditional different treatment methods for E. coli K12

[0058] Surface water from the Shahe Reservoir area of ​​Beiyunhe, Changping District, Beijing was collected for E. coli K12 targeted phage screening. The steps described in Example 1 were referred to, and variable design was performed for enrichment temperature, enrichment method and host sensitivity state. The difference in phage infection effect between the present invention and the traditional method was compared:

[0059] 1) When preparing the sample enrichment solution and enriching the bacteriophage, according to this technical solution, based on the water sample temperature of 21.5°C, the enrichment temperature was set to 20°C, and the enrichment method of first enrichment and then centrifugation was adopted, and Cacl was added.2 Enhance the sensitivity of the host bacteria. Set the temperature to 30℃, centrifuge through a 0.22μm filter before enrichment, and do not add CaCl 2 Three variables, except for the variables, the rest of the steps are consistent with the technical plan, shake on a shaker at 100rpm, and culture overnight; the next day, the obtained bacterial solution is centrifuged at 4℃, 10000r / min for 10min, filtered through a 0.22μm filter membrane, and the supernatant is taken, which is the phage solution. Take 1ml of phage solution, mix it with 200μl of host activated bacterial solution, and culture it in a determined enrichment temperature incubator for 20min, add about 3.5ml of semi-solid culture medium cooled to about 40℃ (add CaCl 2 Solution (final solution concentration is 10mM)), mix quickly, pour into the plate prepared with LB solid medium, incubate the plate at 20℃ overnight (about 12h), and observe the formation of plaques ( Figure 5 ).

[0060] 2) Use ImageJ software to process the experimental plate, calculate the proportion of the phage infection area (the plaque area in the plate or the transparent area formed by phage infection) to the plate area, and measure the phage infection effect as a percentage. Figure 6 As shown, the phage screening effect of the method based on this research scheme is better than that of the traditional method (p<0.05). The formation of bacterial lawns and transparent plaques can be clearly seen, and the infection rate of the host (average higher than 70%) is also significantly higher than the infection rate screened by the traditional method (40%~50%).

[0061] The bacteriophage E.coli K12 P4 was obtained by screening and purification using the method.

[0062] Example 3 Evaluation of phage lysis effect

[0063] E. coli K12 and the phage E. coli K12 P4 screened in Example 2 were used to investigate the phage lysis. First, the phage solution and host bacterial suspension were added at a ratio of 0.001, 0.1, 1, 10, and 100, respectively, according to the optimal phage infection multiplicity MOI (the number of phages infected per cell, usually calculated as the ratio of the number of phages to host cells), and 4 mL of LB liquid culture medium was added. Oscillate at 30°C 150 r / min for 5 hours, centrifuge at 10000 r / min for 10 minutes, filter, and dilute the filtrate in a continuous gradient. Then take 100 μl of the filtrate for double-layer plate coating to determine the phage titer (in PFU / mL, that is, the number of phages contained in 1 mL of culture medium, phage titer = plaque number × dilution factor × 10), and the highest infection multiplicity (MOI) is the optimal MOI. In addition, the optimal bactericidal ratio was obtained by dilution coating and counting the number of changes in host bacterial colonies. After measurement, it was determined that the optimal MOI of E.coli K12 P4 was 0.001.

[0064] Phage solution and host bacteria (1 × 10 8 cfu / mL) into 4mL of liquid culture medium under different environmental conditions, and cultured aerobically at 30°C for 5 hours. The PBS condition is to use PBS (Solarbio, 10×PBS buffer diluted to one time with deionized water) at 120°C, sterilize it under high pressure for 15 minutes, and then use it as a liquid culture medium; RW is the use of reclaimed water discharged from the reclaimed water plant that meets the national standards (for example, see GB / T 18921-2019) as a liquid culture medium; RR is the use of water from the river that receives reclaimed water, which is sterilized under high pressure and used as a liquid culture medium. The blank is when no phage liquid is added, but host bacteria are added. The cultured bacterial solution was centrifuged at 10000 r / min for 10 minutes and then filtered through a 0.22 μm filter membrane. The filtrate was diluted in a continuous gradient, and the phage titer change was determined by the double-layer plate method. In addition, the changes in host bacteria concentration were counted by dilution and coating, and plotted as Figure 7 The results showed that the phage activity in PBS (oligotrophic environment) was very weak, but in the simulated natural environment (recycled water direct effluent (RW) and recycled water receiving river (RR)), it showed a strong lysis effect. 7 ~10 8 PFU / mL, the concentration of host bacteria was significantly reduced, indicating that the phage had a higher lytic activity.

[0065] References:

[0066] 1. T. L. Brown, O. J. Charity, and E. M. Adriaenssens, Ecological andfunctional roles of bacteriophages in contrasting environments: marine,terrestrial and human gut, Curr. Opin. Microbiol., vol. 70, p. 102229, Dec.2022, doi: 10.1016 / J.MIB.2022.102229.

[0067] 2. A. A. Pratama and J. D. van Elsas, The ‘Neglected’ Soil Virome –Potential Role and Impact, Trends Microbiol., vol. 26, no. 8, pp. 649–662,Aug. 2018, doi: 10.1016 / J.TIM.2017.12.004.

[0068] 3. C. A. Suttle, The significance of viruses to mortality in aquaticmicrobial communities, Microb. Ecol., vol. 28, no. 2, pp. 237–243, Sep. 1994,doi: 10.1007 / BF00166813 / METRICS.

[0069] 4. J. Zhang et al., Viral Communities Contribute More to the Lysis ofAntibiotic-Resistant Bacteria than the Transduction of Antibiotic ResistanceGenes in Anaerobic Digestion Revealed by Metagenomics, Environ. Sci.Technol., vol. 58, no. 5, pp. 2346–2359, Feb. 2024, doi: 10.1021 / ACS.EST.3C07664 / ASSET / IMAGES / LARGE / ES3C07664_0006.JPEG.

[0070] 5. J. Bondy-Denomy et al., Prophages mediate defense against phageinfection through diverse mechanisms, ISME J., vol. 10, no. 12, pp. 2854–2866, Dec. 2016, doi: 10.1038 / ISMEJ.2016.79.

[0071] 6. M. T. Moghadam et al., How Phages Overcome the Challenges of DrugResistant Bacteria in Clinical Infections, Infect. Drug Resist., vol. 13, pp.45–61, Jan. 2020, doi: 10.2147 / IDR.S234353.

[0072] 7. Brauer, Age et al. 2024. “Isolation and Characterization of aPhage Collection against Pseudomonas Putida.” Environmental Microbiology 26(6): e16671. https: / / onlinelibrary.wiley.com / doi / full / 10.1111 / 1462-2920.16671(August 29, 2024).

[0073] 8. Buchfink, Benjamin, Chao Xie, and Daniel H. Huson. 2015. “Fast andSensitive Protein Alignment Using DIAMOND.” Nat. Methods 12(1): 59–60.

[0074] 9. Pärnänen, Katariina et al. 2018. “Maternal Gut and Breast MilkMicrobiota Affect Infant Gut Antibiotic Resistome and Mobile GeneticElements.” Nature Communications 2018 9:1 9(1): 1–11. https: / / www.nature.com / articles / s41467-018-06393-w (May 11, 2024).

[0075] The above are only specific embodiments of the present disclosure and are not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A method for efficiently screening or isolating bacteriophages, characterized in that: The method comprises the following steps: 1) preparing a sample enrichment solution, which includes collecting a sample rich in phages, directly mixing the sample with LB liquid culture medium and host bacterial solution without centrifugation and membrane filtration, and culturing at an enrichment incubation temperature to obtain a sample enrichment solution; 2) obtaining a phage solution, which includes taking the sample enrichment solution obtained in step 1), centrifuging it, taking the supernatant and filtering it through a membrane to obtain a phage solution; 3) Screening phages, which includes mixing the phage solution obtained in step 2) with the host bacterial solution, adding the solution to LB semi-solid medium and mixing evenly, then pouring the solution into a plate prepared with LB solid medium, and incubating the mixture at a screening incubation temperature; The incubation temperature is no more than 7°C different from the ambient temperature of the sample source; the LB liquid culture medium in step 1) and the LB semi-solid culture medium in step 3) both contain CaCl2.

2. The method according to claim 1, characterized in that The incubation temperature for enrichment is the same as the incubation temperature for screening, and the incubation temperature is no more than 6°C different from the ambient temperature of the sample source, preferably no more than 5°C different.

3. The method according to claim 1, characterized in that The incubation temperature for enrichment and screening is 10°C or 20°C, preferably 20°C.

4. The method according to any one of claims 1 to 3, characterized in that The final solution concentration of CaCl2 is 10 mM.

5. The method according to any one of claims 1 to 4, characterized in that The filter membrane is a 0.22 μm filter membrane.

6. The method according to any one of claims 1 to 5, characterized in that The method further comprises the step of purifying the phage, wherein the step of purifying the phage comprises: selecting the phage plaques and suspending them in SM buffer, obtaining the phage suspension, diluting the phage suspension with SM buffer, and titrating the phage suspension on a plate prepared with LB solid medium.

7. The method according to claims 1 to 6, characterized in that The bacteriophage is a bacteriophage capable of killing or lysing Pseudomonas putida, and the host is Pseudomonas putida, preferably the Pseudomonas putida is P. putida KT2440.

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