CN109706125B - Bacillus cereus phage composition and application thereof - Google Patents
Bacillus cereus phage composition and application thereof Download PDFInfo
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Abstract
The bacillus cereus phage composition comprises two strains of phage, namely phage phi YSZBA1, with the preservation numbers: CCTCC M2018517, classified and named asBacillus cereusphase phi YSZBA 1; phage Φ YSZBA2, accession number: CCTCC M2018518, classified and named asBacillus cereusphase phi YSZBA 2. According to the invention, a mixed cocktail therapy is used for inoculating a phage mixture into a contaminated soil-vegetable system, directionally infecting and inactivating the repair mode of resistant bacillus cereus contamination in the system and cooperatively removing the resistant genes, after the repair is finished, the resistant bacillus cereus and the resistant genes thereof are greatly reduced, and meanwhile, the functional diversity and stability of the soil ecological environment can be maintained.
Description
Technical Field
The invention belongs to the technical field of repair of soil polluted by antibiotic-resistant pathogenic bacteria, and particularly relates to a bacillus cereus bacteriophage composition and application thereof.
Background
In recent years, due to abuse of antibiotics in medicines and veterinary drugs, shortage of safe treatment technologies of sewage treatment plants and livestock and poultry manure and lack of environmental management, farmland soil-vegetable systems in cities and suburbs of China and many countries worldwide often become high-risk heat point sources for residual and breeding of Antibiotic Resistance Bacteria (ARB) and Resistance Genes (ARGs), and particularly under the promotion action of horizontal transfer or vertical transfer of movable gene elements (plasmids, integron and transposon) in a large amount of environments, the diffusion and propagation risks of some Antibiotic Resistance pathogenic Bacteria are greatly increased. Bacillus cereus is a gram-positive Bacillus with rough surface, soft texture, no capsule, spore production and facultative aerobism, and belongs to the genus Bacillus. Mainly exists in soil, sewage and animal intestinal tracts, and has strong temperature tolerance (10-45 ℃). Bacillus cereus is a conditional pathogen, and can propagate in a large amount in foods (rice, wheaten food and the like) which are not refrigerated properly and deteriorate, and can secrete and produce enterotoxins (vomit enterotoxins and diarrhea enterotoxins), so that the diseases such as nausea, vomit, diarrhea, abdominal pain and the like can be caused, and diseases such as eye diseases, bacteremia, meningitis and the like can be caused in severe cases. The pathogenic bacteria enter a vegetable system mainly through polluted soil, irrigation water, organic fertilizer and the like, and further cause potential harm to human health and safety. Currently, there is less interest in the research of such bacillus cereus and there is also a lack of effective biological risk management techniques. Therefore, there is a need and an urgent need for the development of a technique for targeted inactivation of antibiotic-resistant pathogenic bacteria in soil-vegetable systems by agricultural phage therapy.
Bacteriophages (Phage) are a class of organisms that live by preying on living host bacteria exclusively, and are widely distributed in soil, water, air, and even on the body surface or in the intestinal tract of humans and animals, and their total amount is estimated to be 1031～1032A plurality of; agricultural Phage Therapy (agricultural Phage Therapy) refers to a repair method of separating, screening, purifying and enriching exclusive Phage of host bacteria, screening out Phage with high titer, short lysis period and strong stress resistance, then adding different Phage bacterium liquid mixtures into a contaminated soil-vegetable system, and directionally infecting and inactivating pathogenic bacteria. The advent of Phage Therapy (Phage Therapy) has provided a viable biocontrol technology to solve the above problems.
The main defects of the prior art are as follows: spores of bacillus cereus are easily diffused and spread in soil and attached to vegetables, and improper food treatment of the food by mistake and food poisoning easily occur, so that extremely serious potential threats are brought to human health and ecological safety. Most of the complications are treated by antibiotics, and the bacillus cereus is resistant to the antibiotics due to the excessive use of the antibiotics, however, no effective measure is available for treating the bacillus cereus carrying the antibiotic resistance. Most of the existing pathogenic bacteria pollution phenomena are treated and prevented by adopting antibiotics, however, the frequent use of antibiotics causes a large amount of resistant pathogenic bacteria in soil, so that no effective risk control technology is provided.
The main causes of defects are: in recent years, the academia gradually recognizes that the soil-vegetable system is a source and a sink for accumulation occurrence of antibiotic resistant bacteria and resistant genes, and the novel resistant pathogenic bacteria and genes seriously threaten the health of human bodies and the safety of ecological environment through the transmission effect of a food chain; however, the existing research has less attention to the bacillus cereus and often neglects the potential pathogenic risk. Almost no report is made on the technology for removing the antibiotic-resistant bacillus cereus in the soil-vegetable system, so that the research and development work of a biological targeting inactivation technology for pertinently reducing and eliminating the accumulation risk of the antibiotic-resistant bacillus cereus in the soil-vegetable system is urgently needed.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the defects of the prior art, the invention provides a phage composition and application thereof in inactivating bacillus cereus, in particular to a bioremediation technology for removing bacillus cereus carrying antibiotic resistance and related resistance genes thereof. According to the method, two phage strains with high sensitivity, strong attack ability and short cracking time are screened out by separating and purifying phage using bacillus cereus as host bacteria, a mixed cocktail therapy is used for inoculating a phage mixture into a contaminated soil-vegetable system, a repair mode of directionally infecting and inactivating resistant bacillus cereus pollution in the system to cooperatively remove resistance genes is adopted, after repair is finished, the resistant bacillus cereus and the resistance genes thereof are greatly reduced, and meanwhile, the functional diversity and stability of the ecological environment of the soil can be maintained, so that the method is an environment-friendly bioremediation technology.
The technical scheme is as follows: a phage composition comprises two phage, wherein the phage is preserved in China center for type culture Collection in 2018, 8 and 1 months
The preservation number is: CCTCC M2018517, named as Bacillus cereus phase
Bacteriophage
The preservation number is: CCTCC M2018518, named as Bacillus cereus phase
The preservation address is Wuchang Lojia mountain in Wuhan city, Hubei province, and the China center for type culture preservation of Wuhan university.
The application of the phage composition in targeted inactivation of antibiotic-resistant pathogenic bacteria in a soil-vegetable system.
The application of the phage composition in preparing antibiotic-resistant pathogenic bacteria products in a targeted inactivated soil-vegetable system.
The working principle of the invention is as follows: 1. the bacteriophage is a micro organism which survives specific 'predation' host bacteria and can be divided into a lytic type and a lysogenic type; 2. the virulent phage can identify host bacteria cell membrane surface receptor protein in the environment migration process, the tail can be specifically adsorbed onto the cell membrane, the nucleic acid injects self DNA into the host bacteria through the hollow tail to execute the invasion process, then the phage DNA can quickly finish self nucleic acid replication and protein synthesis by utilizing the nucleic acid base pair and energy substances in the host, and then a large number of progeny phage are assembled and propagated in the bacteria, and cell wall muramidase is released, so that the host bacteria are cracked and die, the internal structure of the bacteria is damaged, and finally the cracking and releasing process is finished; 3. the 'cocktail' therapy means that two or more phages are mixed and then inoculated into the polluted soil for deep inactivation of pathogenic bacteria; 4. the phage selected by the phage therapy is selected as an optimal strain by simulating high titer, short cracking period and strong stress resistance according to the in-situ polluted soil environment conditions (temperature, pH and the like); the method can target and track host bacteria corresponding to the plant in the environment, deeply inactivate resistant pathogenic bacteria in a soil-vegetable system and prevent secondary 'rebound' of the pathogenic bacteria; 5. the length of the bacteriophage is about 20-200 mu m, which is equal to hundreds and one thousandth of bacteria, part of the bacteriophage can be transmitted into the vegetable body along with the penetration of the plant root and the transpiration of the leaf surface in a soil-vegetable system, resistant pathogenic bacteria in the vegetables are synchronously tracked and inactivated, and the propagation and diffusion of the bacteriophage are prevented and controlled to indirectly block the propagation of the bacteriophage through a food chain to influence the health of a human body; 6. the selected phage is derived from soil and finally returned to the soil without any transformation, the environment is friendly, and the ecological risk after the phage therapy is applied is evaluated to ensure the diversity and stability of the microbial ecological function.
Has the advantages that: 1. the method comprises the following steps of (1) targeted inactivation of resistant pathogenic bacteria in the polluted soil and synchronous reduction of abundance of related resistance genes; 2. the bacteriophage cocktail therapy has the advantages of low cost, convenient storage, convenient transportation, simple and convenient use and operation, accurate inactivation, high broad spectrum, prevention of rebound after recovery and easy popularization; 3. the phage is from soil and returns to soil, has positive promotion effect on the diversity and stability of the ecological functions of soil microorganisms, and is environment-friendly. The method has wide application prospect for the remediation work of the soil of the antibiotic resistance pathogenic bacteria and resistance gene polluted site in the farmland soil in China.
Drawings
FIG. 1 is a Bacillus cereus phage
Double-layer plate plaque-raising pattern;
FIG. 2 is a Bacillus cereus phage
Double-layer plate plaque-raising pattern;
FIG. 3 is a Bacillus cereus phage
Transmission electron microscopy images of;
FIG. 4 is a Bacillus cereus phage
Transmission electron microscopy images of;
FIG. 5 is a Bacillus cereus phage
The growth curve of (a);
FIG. 6 is a Bacillus cereus phage
The growth curve of (a);
FIG. 7 is a bacteriophage
And
mixing the bacteriostatic effect graphs of the 'cocktail' pair;
FIG. 8 is a graph showing the effect of inactivating pathogenic bacteria in a contaminated soil-vegetable system in a beam cow farm fecal accumulation area of Nanjing, Jiangsu province, when potatoes are planted in the contaminated soil by using the technical scheme of the present invention;
FIG. 9 is a graph showing the effect of inactivating pathogenic bacteria in a contaminated soil-vegetable system in a beam cow farm fecal accumulation area in Nanjing, Jiangsu province, when carrot is planted in the contaminated soil by using the technical scheme of the present invention;
FIG. 10 is a verification diagram of the inactivation effect of pathogenic bacteria in a contaminated soil-vegetable system of the excrement accumulation area of the beam cow farm in Nanjing City of Jiangsu province when lettuce is planted in the contaminated soil by using the technical scheme of the invention.
Detailed Description
The following detailed description does not limit the technical solutions of the present invention in any way, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the scope of the present invention.
The phage phi YSZBA1 is a prophase-preserved phage which is special for 'predatory' bacillus cereus, and the preservation number is as follows: CCTCC M2018517, date of deposit: 8/1/2018. Bacteriophage
The spherical head and a long tail can be seen, the length of the head is about 60nm, the transverse diameter is about 70nm, and the length of the tail is about 220 nm; the plaque is clear and transparent, has neat edges and is a circular spot without a halo, and the diameter of the plaque is about 1-2 mm; incubation period 40min, outbreak period 70 min; the optimal multiplicity of infection (MOI) is 0.1. Phage, according to the ninth report of the International Commission on viral Classification
Belonging to the family of Long-tailed bacteriophages (Siphoviridae bacteriophages).
The phage phi YSZBA2 is a prophase-preserved phage which is special for 'predatory' bacillus cereus, and the preservation number is as follows: CCTCC M2018518, date of deposit: 8/1/2018. Bacteriophage
Regular polyhedral heads and contracted long tails can be seen, with the head length being about 90nm, the transverse diameter being about 80nm, and the tail length being about 190 nm; the plaque is a clear and transparent circular spot with neat edge and no halo, and the diameter is about 2-3 mm; incubation period 20min, outbreak period 55 min; the optimal multiplicity of infection (MOI) was 0.01. Phage, according to the ninth report of the International Commission on viral Classification
Belonging to the family of Long-tailed bacteriophages (Siphoviridae bacteriophages).
The resistance gene tetW refers to the relevant tetracycline resistance gene carried on a plasmid in the bacillus cereus cell.
The soil for potting is prepared by adding pathogenic bacteria with the same concentration into collected original soil, inoculating Bacillus cereus into soil sample to final concentration of 107cfu/g, 10 kg/pot were filled in flower pots (upper diameter. times.lower diameter. times.height: 26.8 cm. times.19.78 cm. times.28 cm).
Example 1:
1. separation and purification of Bacillus cereus phage
The soil sample to be tested is collected from polluted soil around a beam dairy farm excrement accumulation pool in Nanjing City of Jiangsu province. Basic physicochemical properties of soil: 23.8 percent of sand grains, 45.4 percent of soil grains, 31.8 percent of clay grains, 7.7 percent of pH and 1.7 g/kg of total nitrogen-11.7 g.kg of water-soluble nitrogen-11.3 g.kg of total phosphorus-117.5 g/kg of total potassium-1，CEC 19.4cmol·kg-1。
Adding 10g of fresh soil sample into 100mL of sterile water, performing shaking culture at 30 ℃ and 250rpm for 5h, centrifuging at 10000 rpm for 5min, sterilizing the supernatant with a 0.22 mu m filter membrane, adding 9mL of filtrate and 1mL of bacillus cereus suspension which grows to logarithmic phase into 40mL of 3 XLB liquid culture medium, and adding calcium chloride solid until the final concentration of the solution is 1 mmol.L-1Shaking-culturing at 30 deg.C and 250rpm for 12h, centrifuging the obtained culture solution at 10000 rpm for 5min, and sterilizing with 0.22 μm filter membrane to obtain bacteriophage stock solution; screening phages by adopting a double-layer plate method, purifying, uniformly mixing 100 mu L of filtrate with 100 mu L of bacillus cereus suspension, standing for 20min at room temperature, adding 5mL of 0.75% semisolid LB agar culture medium, uniformly mixing, flatly spreading and pouring onto an LB solid plate, culturing at 30 ℃ for 10-12 h, observing plaques, picking a single clear and transparent plaque with a clear edge into LB liquid containing host bacteria when the plaque (the diameter of the plaque in figure 1 is about 1-2mm, and the diameter of the plaque in figure 2 is about 2-3mm), and culturing at 30 ℃ and 250rpm for 8 h; centrifuging at 10000 rpm for 5min, sterilizing with 0.22 μm filter membrane to obtain two phage, respectively naming
) And storing in SM buffer solution at low temperature of 4 ℃.
2. Microbiological characterization of Bacillus cereus phage
For bacteriophage
And (5) observing by using an electron microscope. Dropping the phage stock on a copper net, negatively dyeing with 2% phosphotungstic acid with pH 7.0 for 90s, absorbing the excessive dye solution with filter paper, drying, and observing the form under Hitachi H-7650 type transmission electron microscope. Bacteriophage
The spherical head and the long tail can be seen, the length of the head is about 60nm, the transverse diameter is about 70nm, and the length of the tail is about 220 nm; phage, according to the ninth report of the International Commission on viral Classification
Belonging to the family of Long-tailed bacteriophages (Siphoviridae bacteriophages); bacteriophage
Regular polyhedral heads and contracted long tails can be seen, with the head length being about 90nm, the transverse diameter being about 80nm, and the tail length being about 190 nm; the plaque is a clear and transparent circular spot with neat edge and no halo, and the diameter is about 2-3 mm; phage, according to the ninth report of the International Commission on viral Classification
Belonging to the family of Long-tailed bacteriophages (Siphoviridae bacteriophages).
Multiplicity of Infection (MOI), also known as phage titer, refers to the ratio of phage to host bacteria count before Infection occurs. Two phage strains are obtained based on the above operation, 100 μ L phage filtrate is taken, and 100 μ L log-phase host bacterial suspension is added according to the infection complex numbers of 100: 1, 10: 1, 1: 100, 1: 1000 and 1: 10000 respectively. Shaking and culturing for 5h at 30 ℃. Phage titers were determined for each group by the double-plate method (table 1). Bacteriophage
The optimal multiplicity of infection is 0.1, bacteriophage
The optimal multiplicity of infection is 0.01.
TABLE 1 determination of optimal multiplicity of infection
The phage one-step growth curve was determined. According to the optimal multiplicity of infection, 500. mu.L of phage and 500. mu.L of host bacterial suspension are added into 9mL of LB liquid medium, cultured with shaking at 37 ℃ and 150rpm, sampled every 10min, centrifuged, filtered and the phage titer is determined by the double-layer plate method. Bacteriophage
Incubation period of 40min, outbreak period of 70min (fig. 5); bacteriophage
The incubation period of (1) was 20min and the outbreak period was 55min (FIG. 6).
Example 2:
and (3) verifying the bacteriostatic effect of the single bacteriophage and the composition thereof on the bacillus cereus in the water phase.
Set up 4 sets of test treatments. Control (treatment 1): 100. mu.L of Bacillus cereus (tetW) in logarithmic phase was added to 100mL of LB liquid medium, and 100. mu.L of the strain was inoculated according to the optimum MOI value
Inoculation of a Single phage
Bacteriostatic effect on host bacteria (treatment 2): based on the control group, 100. mu.L of the suspension was inoculated at the optimum MOI value
Oscillating and mixing uniformly; inoculation of a Single phage
(treatment 3): based on the control group, 100. mu.L of phage was inoculated at the optimal MOI value
Oscillating and mixing uniformly; inoculation of 1/2 phage
And 1/2 bacteriophage
(treatment 4): based on the control group, 50. mu.L of the suspension was inoculated at the optimum MOI value
And 50. mu.L
And after each group of treatment is uniformly shaken and mixed, shaking culture is carried out at 37 ℃ and 150rpm, sampling is carried out once every 2h, bacteria of bacillus cereus (tetW) are counted, and a bacteriostatic effect graph is drawn according to the number of the bacillus cereus. From the results of FIG. 7, it can be seen that the number of Bacillus cereus in the four groups after 12 hours of culture in aqueous phase was: 3.2X 1010cfu·g-1、 1.8×108cfu·g-1、7.6×107cfu·g-1、4.6×106cfu·g-1The abundance of the carried relevant tetracycline resistance gene tetW is respectively as follows: 1.6X 1011copies·g-1、8.3×108copies·g-1、3.3×108copies·g-1、2.6×107copies·g-1. The phage inoculated singly or in mixture has obvious inhibition effect on bacillus cereus and resistance gene tetW thereof (p)<0.05). The number of bacillus cereus in the three groups of treatments inoculated with phage was reduced by 2.1, 2.6, 3 compared to CK.9 orders of magnitude, tetW decreases by 2.4, 2.9, 3.9 orders of magnitude. The mixed phage treatment (treatment 4) had the most significant effect on the removal of pathogenic bacteria and resistance genes (P)<0.05). Provides effective theoretical basis and technical support for developing the application of phage therapy in a contaminated soil-vegetable system.
Example 3:
soil for the test pot culture was collected from contaminated soil around a beam dairy farm excrement accumulation pond in Nanjing City of Jiangsu province. The crop is potato (Solanum tuberosum), agricultural science and academy of Jiangsu province. Basic physicochemical properties of soil: 23.8 percent of sand grains, 45.4 percent of soil grains, 31.8 percent of clay grains, 7.7 percent of pH and 1.7 g/kg of total nitrogen-11.7 g.kg of water-soluble nitrogen-11.3 g.kg of total phosphorus-117.5 g/kg of total potassium-1， CEC 19.4cmol·kg-1。
The experiment was set up with four sets of treatments: control group (CK): planting 1 potato in each pot, covering 2-3 cm of soil on potato tubers, compacting and covering soil, and keeping the temperature at 15 +/-2 ℃; ② bacteriophage
Treatment (P1): 100mL of 10 concentration at optimal MOI was inoculated on a control basis6pfu·mL-1Bacteriophage of
③ bacteriophage
Treatment (P2): inoculation with 100mL of 10 concentration on the basis of the control group5pfu·mL-1Bacteriophage of
(iv) mixed "cocktail" treatment (P3 ═ 1/2P1+1/2P 2): inoculation with 50mL of 10 concentration on the basis of the control group6pfu·mL-1Bacteriophage of
And 50mL of 105pfu·mL-1Bacteriophage of
Sampling soil and potatoes on site after the potatoes grow for 90 days, and determining the background pollution concentration of the bacillus cereus in the soil polluted by a control group as follows: 4.1X 108cfu·g-1The tetracycline resistance gene tetW background contamination abundance is: 2.2X 109copies·g-1(ii) a The number of Bacillus cereus in the treatments inoculated with phages P1, P2, P3 decreased to: 4.6X 105cfu·g-1、1.8×105cfu·g-1、5.3×103cfu·g-1The abundance of the resistance gene tetW decreased to: 2.3X 106copies·g-1、1.2×106copies·g-1、2.4×104copies·g-1(ii) a Compared with the number of bacillus cereus in a control group (CK), the number of the three groups of treatments for inoculating the phage is respectively reduced by 1.9, 2.2 and 4.9 orders of magnitude, and the abundance of the resistance gene tetW is respectively reduced: 2.9, 3.1, 4.9 orders of magnitude. The determination of the number of bacillus cereus in the potato stem block comprises the following steps in four groups of treatments of CK, P1, P2 and P3: 8.6X 104cfu·g-1、1.3×103cfu·g-1、1.1×103cfu·g-1、1.2×102cfu·g-1The abundance of the resistance gene tetW decreased to: 4.3X 105copies·g-1、6.5×103copies·g-1、4.5×103copies·g-1、2.1×102 copies·g-1(ii) a The number of bacillus cereus in the stem block is respectively reduced by 1.7, 1.9 and 2.7 orders of magnitude compared with the control group, and the abundance of the resistance gene tetW in the stem block is respectively reduced: 1.8, 1.9, 2.4 orders of magnitude. Wherein the removal effect of the treatment of the mixed 'cocktail' therapy (P3) on the resistant pathogenic bacteria and the resistant genes is obviously higher than that of the treatment of the phage (P1/P2) alone.
The analysis finds that indexes of ecological diversity of soil environment microorganisms under four groups of treatments, namely CK, P1, P2 and P3, and the AWCD indexes are respectively as follows: 0.83 + -0.1, 0.79 + -0.4, 0.80 + -0.2, 0.86 + -0.2, inoculating bacteriophage alone
And
the diversity of the soil microorganisms is reduced to a certain extent, and the mixed 'cocktail' therapy (P3) can remarkably promote the diversity and stability (P is less than 0.05) of the functions of the repaired soil microorganisms, which indicates that the repairing technology has remarkable effect on repairing the pollution of resistant bacteria.
Example 4:
soil for the test pot culture was collected from contaminated soil around a beam dairy farm excrement accumulation pond in Nanjing City of Jiangsu province. The vegetable is carrot Hecheng six inches (Daucus L.) and vegetable seeds of Tianteng, Beijing Zhongnong. Basic physicochemical properties of soil: 23.8 percent of sand grains, 45.4 percent of soil grains, 31.8 percent of clay grains, 7.7 percent of pH and 1.7 g/kg of total nitrogen-11.7 g.kg of water-soluble nitrogen-11.3 g.kg of total phosphorus-117.5 g/kg of total potassium-1，CEC 19.4cmol·kg-1。
The experiment was set up with four sets of treatments: control group (CK): planting 1 carrot in each pot (covering the seeds with 0.5-1 cm of soil at the room temperature of 20 +/-2 ℃); ② bacteriophage
Treatment (P1): 100mL of 10 concentration was inoculated alone on a control basis6pfu·mL-1Bacteriophage of
③ bacteriophage
Treatment (P2): 100mL of 10 concentration was inoculated alone on a control basis5pfu·mL-1Bacteriophage of
Bacteriophage
Treatment (P2): inoculation with 100mL of 10 concentration on the basis of the control group5pfu·mL-1Bacteriophage of
(iv) mixed "cocktail" treatment (P3 ═ 1/2P1+1/2P 2): inoculation with 50mL of 10 concentration on the basis of the control group6pfu·mL-1Bacteriophage of
And 50mL of 105pfu·mL-1Bacteriophage of
Sampling the soil and the carrots on site after the carrots grow for 70 days, and determining the background pollution concentration of the bacillus cereus in the polluted soil of the control group as follows: 1.7X 108cfu·g-1The tetracycline resistance gene tetW background contamination abundance is: 8.3X 109copies·g-1(ii) a The number of Bacillus cereus in the treatments inoculated with phages P1, P2, P3 decreased to: 8.6X 105cfu·g-1、3.4×105cfu·g-1、4.2×103cfu·g-1The abundance of the resistance gene tetW decreased to: 4.3X 106copies·g-1、1.7×106copies·g-1、2.1×104copies·g-1(ii) a Compared with the number of bacillus cereus in a control group (CK), the number of the three groups of treatments for inoculating the phage is respectively reduced by 2.3, 2.8 and 4.7 orders of magnitude, and the abundance of the resistance gene tetW is respectively reduced: 2.4, 2.1, 4.6 orders of magnitude. The number of bacillus cereus in the carrot blocks is determined in four groups of treatments of CK, P1, P2 and P3 as follows: 6.4X 104cfu·g-1、3.2×103cfu·g-1、1.6×103cfu·g-1、1.1×102cfu·g-1The abundance of the resistance gene tetW decreased to: 7.2X 105copies·g-1、1.6×104copies·g-1、8.2×103copies·g-1、2.5×102 copies·g-1The number of bacillus cereus in the carrot root block is respectively reduced by 1.3, 1.5 and 2.5 orders of magnitude compared with the control group, and the abundance of the resistance gene tetW in the stem block is respectively reduced: 1.6, 1.9, 2.2 orders of magnitude. Wherein the removal effect of the treatment of the mixed 'cocktail' therapy (P3) on the resistant pathogenic bacteria and the resistant genes is obviously higher than that of the treatment of the phage (P1/P2) alone.
The analysis finds that indexes of ecological diversity of soil environment microorganisms under four groups of treatments, namely CK, P1, P2 and P3, and the AWCD indexes are respectively as follows: 0.75 + -0.2, 0.71 + -0.3, 0.72 + -0.2, 0.79 + -0.2, inoculating bacteriophage alone
And
the diversity of the soil microorganisms is reduced to a certain extent, and the mixed 'cocktail' therapy (P3) can remarkably promote the diversity and stability (P is less than 0.05) of the functions of the repaired soil microorganisms, which indicates that the repairing technology has remarkable effect on repairing the pollution of resistant bacteria.
Example 5:
soil for the test pot culture was collected from contaminated soil around a beam dairy farm excrement accumulation pond in Nanjing City of Jiangsu province. The vegetable is Italy bolting-resistant lettuce (Lactuca sativa L) all year round, and Hebei Jinfa breed ltd. Basic physicochemical properties of soil: 23.8 percent of sand grains, 45.4 percent of soil grains, 31.8 percent of clay grains, 7.7 percent of pH and 1.7 g/kg of total nitrogen-11.7 g.kg of water-soluble nitrogen-11.3 g.kg of total phosphorus-117.5 g/kg of total potassium-1，CEC 19.4cmol·kg-1。
The experiment was set up with four sets of treatments: control group (CK): planting 1 lettuce in each pot (covering the seeds with 0.5-1 cm of soil at the room temperature of 18 +/-2 ℃); ② bacteriophage
Treatment (P1): 100mL of 10 concentration was inoculated alone on a control basis6pfu·mL-1Bacteriophage of
③ bacteriophage
Treatment (P2): 100mL of 10 concentration was inoculated alone on a control basis5pfu·mL-1Bacteriophage of
Bacteriophage
Treatment (P2): inoculation with 100mL of 10 concentration on the basis of the control group5pfu·mL-1Bacteriophage of
(iv) mixed "cocktail" treatment (P3 ═ 1/2P1+1/2P 2): inoculation with 50mL of 10 concentration on the basis of the control group6pfu·mL-1Bacteriophage of
And 50mL of 105pfu·mL-1Bacteriophage of
Sampling soil and lettuce on site after the lettuce grows for 60 days, and determining background pollution concentration of the bacillus cereus in the polluted soil of a control group as follows: 6.1X 108cfu·g-1The tetracycline resistance gene tetW background contamination abundance is: 3.6X 109copies·g-1(ii) a The number of Bacillus cereus in the treatments inoculated with phages P1, P2, P3 decreased to: 3.8X 106cfu·g-1、1.1×106cfu·g-1、1.5×104cfu·g-1The abundance of the resistance gene tetW decreased to: 1.6X 107copies·g-1、15.5×106copies·g-1、7.7×104copies·g-1(ii) a Three groups of treatments inoculated with phage compared to Bacillus cereus in Control (CK)The bacterial number is respectively reduced by 2.2, 2.5 and 4.4 orders of magnitude, and the abundance of the resistance gene tetW is respectively reduced:
2.2, 2.8, 4.7 orders of magnitude. The number of bacillus cereus in the lettuce leaves is determined in four groups of treatments of CK, P1, P2 and P3 as follows: 8.7X 104cfu·g-1、6.6×103cfu·g-1、2.3×103cfu·g-1、1.6×102cfu·g-1The abundance of the resistance gene tetW decreased to: 7.8X 105copies·g-1、3.3×104copies·g-1、1.9×103copies·g-1、8.2×102copies·g-1The number of bacillus cereus in the carrot root block is respectively reduced by 1.2, 1.6 and 2.7 orders of magnitude compared with the control group, and the abundance of the resistance gene tetW in the leaf is respectively reduced: 1.4, 1.6, 2.9 orders of magnitude. Wherein the removal effect of the treatment of the mixed 'cocktail' therapy (P3) on the resistant pathogenic bacteria and the resistant genes is obviously higher than that of the treatment of the phage (P1/P2) alone.
The analysis finds that indexes of ecological diversity of soil environment microorganisms under four groups of treatments, namely CK, P1, P2 and P3, and the AWCD indexes are respectively as follows: 0.65 + -0.2, 0.61 + -0.3, 0.62 + -0.2, 0.68 + -0.2, inoculating bacteriophage alone
And
the diversity of the soil microorganisms is reduced to a certain extent, and the mixed 'cocktail' therapy (P3) can remarkably promote the diversity and stability (P is less than 0.05) of the functions of the repaired soil microorganisms, which indicates that the repairing technology has remarkable effect on repairing the pollution of resistant bacteria.
The technology for synchronously inactivating multiple resistant pathogenic bacteria in a soil-vegetable system by using the multivalent phage therapy has the advantages of high broad spectrum, low ecological risk and environmental friendliness, and is a composite pathogenic bacteria contaminated soil remediation technology with good application prospect.
Claims (3)
1. A phage composition comprising two phage, said phage being deposited at the chinese type culture collection on 2018, 8, 1, respectively, phage Φ YSZBA1, with the collection numbers: CCTCC M2018517, classified and named asBacillus cereusphase phi YSZBA 1; phage Φ YSZBA2, accession number: CCTCC M2018518, classified and named asBacillus cereus phage φYSZBA2。
2. Use of the phage composition of claim 1 for targeted inactivation of tetracycline resistant bacillus cereus in a soil environment.
3. Use of the phage composition of claim 1 for the preparation of a tetracycline resistant bacillus cereus product for targeted inactivation of a soil-vegetable system.
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Non-Patent Citations (2)
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| Complete genome sequence of bacteriophage Deep-Purple, a novel member of the family Siphoviridae infecting Bacillus cereus;Louise Hock等;《Arch Virol.》;20180511;第163卷(第9期);2555-2559 * |
| Genomic Sequence of Temperate Phage 250 Isolated from Emetic B. cereus and Cloning of Putative Endolysin;Young-Duck Lee等;《Food Science and Biotechnology》;20101231;第19卷(第16期);1643-1648 * |
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