CN109706125A - Bacillus cereus bacteriophage composition and its application - Google Patents
Bacillus cereus bacteriophage composition and its application Download PDFInfo
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Abstract
Bacillus cereus bacteriophage composition and its application, including two plants of bacteriophages, respectively phage phi YSZBA1, deposit number are as follows: CCTCC M 2018517, classification naming areBacillus cereusphage φYSZBA1；Phage phi YSZBA2, deposit number are as follows: CCTCC M 2018518, classification naming areBacillus cereusphage φYSZBA2.The present invention is inoculated with phage mixture into contaminated soil-Vegetable System with mixing therapeuticcocktail of anti-retrovirals, the repair mode that orientation infects and resistance bacillus cereus pollution synergetic removes resistant gene in deactivation system, after reparation, resistance bacillus cereus and its resistant gene are significantly cut down, while can maintain ecological environment of soil functional diversity and stability.
Description
Technical field
The invention belongs to antibiotic resistance pathogenic bacteria contaminated soil remediation technical fields, and in particular to bacillus cereus
Bacteriophage composition and its application.
Background technique
In recent years due to medicine, the abuse of veterinary drug class antibiotic, sewage treatment plant and feces of livestock and poultry safe processing technique
Deficiency and environmental management missing, the agricultural land soil-Vegetable System in many national outskirts of a town, Chang Chengwei in China and world wide
Residual and growth antibiotic-resistant bacteria (Antibiotic Resistance Bacteria, ARB) and resistant gene
The high risk hot spot source of (Antibiotic Resistance Genes, ARGs) moves gene especially in a large amount of environment
Under element (plasmid, integron, transposons) horizontal transfer or the facilitation vertically transduceed, some antibiotic resistance pathogenic bacterias
Diffusive transport risk but will greatly increase.Bacillus cereus (Bacillus cereus) is a kind of rough surface, quality
It is soft, without pod membrane, produce gemma, facultative aerobic Gram-positive bacillus, belong to bacillus (Bacillus).It is primarily present in
In soil, sewage and animal intestinal tract, have stronger temperature tolerance (10~45 DEG C).Bacillus cereus is a kind of condition
Pathogenic bacteria, the meeting mass propagation in refrigerating improper and rotten food (rice, wheaten food etc.), while generation enterotoxin can be secreted
(vomiting type enterotoxin, diarrhea-type enterotoxin) can lead to nausea,vomiting,diarrhea, abdominal pain and wait illnesss, can cause eye when serious
The diseases such as portion's disease, bacteremia, meningitis.Such pathogenic bacteria mainly passes through the way such as contaminated soil, irrigation water, organic fertilizer
Diameter enters in Vegetable System, and then causes safely to human health potentially hazardous.Currently, being ground to such bacillus cereus
It is less to study carefully concern, also lacks effective biological risk management and control technology.Thus, carry out agriculture phagotherapy targeting inactivation soil-
The technological invention of antibiotic resistance pathogenic bacteria is very necessary and urgent in Vegetable System.
Bacteriophage (Bacteriaphage, Phage) is a kind of specificity predation living body host bacteria and the life survived
Object, it is widely distributed in soil, water, air or even humans and animals body surface or enteron aisle, it is estimated that its total amount reaches 1031~
1032It is a；Agriculture phagotherapy (Agriculture Phage Therapy) refers to through separation, screening, purifying and enrichment place
After the exclusive bacteriophage of main bacterium, the bacteriophage that high-titer, burst times are short, resistance is strong is filtered out, then to contaminated soil-
Different bacteriophages bacterium solution mixture is added in Vegetable System, orientation infects and inactivate the repair mode of pathogenic bacteria.Phagocytosis physical exercise therapy
The appearance of method (Phage Therapy) provides a kind of practicable biological prevention in order to solve the above problem.
Major defect of the existing technology is: the spore of bacillus cereus is easy to diffusive transport in the soil, attached
To on vegetables, and eat such mishandling food by mistake and its be easy to cause food poisoning, to human health and ecology peace
Extremely serious potential threat is brought entirely.Antibiotic treatment is mostly used greatly for such complications, antibiotic cause is excessively used
Bacillus cereus is set to generate drug resistance, however, there is no the waxes that a kind for the treatment of of effective measures carries antibiotic resistance at present
Sample packet bacillus.Existing pathogenic bacteria contamination phenomenon is to be treated and prevented using antibiotic, however frequent use resists mostly
Raw element causes to generate a large amount of resistance pathogenic bacteria in soil, so as to cause no effective risk management and control technology.
The main reason for defect generates has: in recent years, it is that middle antibiotic is anti-that academia, which gradually recognizes soils-vegetables system,
Property bacterium and resistant gene accumulation preservation " source " and " remittance ", and such novel resistance pathogenic bacteria and gene can pass through food
The transmitting effect of chain seriously threatens human health and ecological environment security；And existing research to the concern of bacillus cereus compared with
It is few, often ignore its potential pathogenic risk.It is several to the bacillus cereus removal technology of antibiotic resistance in Soil-Vegetable System
Do not report, thus, it needs to carry out specific aim reduction and eliminates antibiotic resistance bacillus cereus in soils-vegetables system
Accumulate the biological targeting inactivation technology R&D work of risk.
Summary of the invention
The technical issues of solution: the present invention is directed to above-mentioned prior art defect, provide a kind of bacteriophage composition and its
The application for inactivating bacillus cereus carries bacillus cereus and its related resistance of antibiotic resistance especially for removal
The bioremediation technology of gene.This method is by separation and purifies the screening using bacillus cereus as the bacteriophage of host bacteria
Out high sensitivity, two plants of bacteriophages that attacking ability is short compared with strong, pyrolysis time, with mixing therapeuticcocktail of anti-retrovirals to Polluted Soil
Phage mixture is inoculated in earth-Vegetable System, orientation infects and resistance bacillus cereus pollution synergetic is gone in deactivation system
Except the repair mode of resistant gene, after reparation, resistance bacillus cereus and its resistant gene are significantly cut down, while can
It is a kind of bioremediation technology for having both environment friendly to maintain ecological environment of soil functional diversity and stability.
Technical solution: a kind of bacteriophage composition, including two plants of bacteriophages, the bacteriophage protected on August 1st, 2018
It is hidden in China typical culture collection center, respectively bacteriophageDeposit number are as follows: CCTCC M 2018517,
Classification naming is Bacillus cereus phageBacteriophageDeposit number are as follows: CCTCC M
2018518, classification naming is Bacillus cereus phagePreservation address is Wuhan City, Hubei Province Wuchang Luo
Ka mountain, Wuhan University's China typical culture collection center.
Application of the above-mentioned bacteriophage composition in targeting inactivation soils-vegetables system in antibiotic resistance pathogenic bacteria.
Above-mentioned bacteriophage composition targets in inactivation soils-vegetables system in antibiotic resistance pathogenic bacteria product in preparation
Application.
The working principle of the invention is: 1, bacteriophage is a kind of specific " predation " host strain and the tiny organism survived
Body can be divided into cracking performance and two kinds of lysogenicity；2, virulent phage can identify host bacterial cells film during environmental transport and transfer
Surface receptor protein, tail portion will do it in specific adsorption to cell membrane, itself DNA is injected place by hollow tail portion by nucleic acid
In main bacterium body, execute invasion procedure, then phage DNA will using the intracorporal nucleic acid base of host to and energy matter, fastly
Speed completes itself nucleic acid replication and protein synthesis, and then assembles and be proliferated a large amount of progeny phages in bacterial body, and discharge
Cell wall lywallzyme causes host bacteria rupture dead, destroys bacterium internal structure, is finally completed the process of cracking release；3,
Therapeuticcocktail of anti-retrovirals is inoculated into progress depth inactivation pathogenic bacteria in contaminated soil after referring to the mixing of two or more bacteriophages；4,
Bacteriophage selected by phagotherapy is to be simulated to choose efficiently according to its contaminated soil environmental condition (temperature, pH etc.) in situ
Valence, lytic cycle be short, high-output stress-resistance bacteriophage is as preferred strain；Tracking " predation " can be targeted in the environment, and its is corresponding
Host bacteria, depth inactivate the resistance pathogenic bacteria in soils-vegetables system, while can prevent the secondary " anti-of pathogenic bacteria
Bullet "；5, phagocytosis body length is about equivalent to several hundred and upper one thousandths of bacterium at 20~200 μm, in the middle part of soils-vegetables system
Divide bacteriophage that can be transmitted in vegetables body with plant root osmosis and leaf table transpiration, synchronous tracking, which inactivates in vegetables, to be resisted
Property pathogenic bacteria, resistance control its Spreading and diffusion indirect its by food chain transmitting function influence human health；6, that selects bites
Thallus finally returns soil without any transformation, environmental-friendly, the ecological wind after applying to phagotherapy from soil
It is assessed danger, it is ensured that its microbial ecological functional diversity and stability.
The utility model has the advantages that 1, targeting inactivates resistance pathogenic bacteria in contaminated soil and synchronous its related resistance genes of abatement are rich
Degree；2, bacteriophage therapeuticcocktail of anti-retrovirals cost prepare it is cheap, convenient for storage, convenient transportation, it is easy to use, it is accurate inactivate,
Broad spectrum activity is high, can prevent " rebound " after replying, is easy to spread；3, bacteriophage returns from soil in soil, to the micro- life of soil
Object ecological functions diversity and stability have active promoting function, environmental-friendly.This method is for antibiosis in China's agricultural land soil
The repair of plain resistance pathogenic bacteria and resistant gene contaminated site soil has been widely used prospect.
Detailed description of the invention
Fig. 1 is bacillus cereus bacteriophageDouble-layer plate plaque figure；
Fig. 2 is bacillus cereus bacteriophageDouble-layer plate plaque figure；
Fig. 3 is bacillus cereus bacteriophageTransmission electron microscope picture；
Fig. 4 is bacillus cereus bacteriophageTransmission electron microscope picture；
Fig. 5 is bacillus cereus bacteriophageGrowth curve；
Fig. 6 is bacillus cereus bacteriophageGrowth curve；
Fig. 7 is bacteriophageWithThe fungistatic effect figure of mixing " cocktail " pair；
Fig. 8 uses technical solution of the present invention, when planting potato on contaminated soil, to Nanjing crossbeam milk cow
Farm's excrement aheap in contaminated soil-Vegetable System pathogenic bacteria inactivating efficacy proof diagram；
Fig. 9 uses technical solution of the present invention, when planting carrot on contaminated soil, to Nanjing crossbeam milk cow
Farm's excrement aheap in contaminated soil-Vegetable System pathogenic bacteria inactivating efficacy proof diagram；
Figure 10 uses technical solution of the present invention, when planting romaine lettuce on contaminated soil, to Nanjing crossbeam milk cow
Farm's excrement aheap in contaminated soil-Vegetable System pathogenic bacteria inactivating efficacy proof diagram.
Specific embodiment
The following specific embodiments technical solution that the invention is not limited in any way, it is all to use equivalent replacement or wait
The mode technical solution obtained of effect transformation all falls within protection scope of the present invention.
The phage phi YSZBA1 is the bacteriophage of specificity " predation " bacillus cereus saved early period, and preservation is compiled
Number are as follows: CCTCC M 2018517, preservation date: on August 1st, 2018.BacteriophageThe head of visible ball-type and
One long-tail, head major diameter about 60nm, transverse diameter about 70nm, tail length about 220nm；Its plaque is in Clear & Transparent, neat in edge, halo-free
The round spot of ring, diameter about 1~2mm；Incubation period 40min, outbreak period 70min；Optimal multiplicity of infection (MOI) is 0.1.According to state
The 9th report of the border virus taxis committee, bacteriophageBelong to Stylovinidae (Siphoviridae
Bacteriophage)。
The phage phi YSZBA2 is the bacteriophage of specificity " predation " bacillus cereus saved early period, and preservation is compiled
Number are as follows: CCTCC M 2018518, preservation date: on August 1st, 2018.BacteriophageThe polyhedron of visible rule
The long-tail on head and contraction, head major diameter about 90nm, transverse diameter about 80nm, tail length about 190nm；Its plaque is in Clear & Transparent, edge
Neatly, without the round spot of halo, diameter about 2~3mm；Incubation period 20min, outbreak period 55min；Optimal multiplicity of infection (MOI) is
0.01.According to the 9th report of International Commission on Virus Classification, bacteriophageBelong to Stylovinidae
(Siphoviridae Bacteriophage)。
The resistant gene tetW, which refers to, carries related tetracycline resistance gene on the intracellular plasmid of bacillus cereus.
The potting soil is the pathogenic bacteria that same concentrations are added in the native soil of acquisition, is inoculated in pedotheque
Bacillus cereus, until final concentration of 107Cfu/g is dispensed into flowerpot (upper diameter × lower diameter × height: 26.8cm by 10kg/ basin
× 19.78 cm × 28cm) in.
Embodiment 1:
1. bacillus cereus bacteriophage isolates and purifies
Nanjing crossbeam cattle farm excrement accumulation pond ambient contamination soil is picked up from for examination pedotheque.Soil is basic
Physicochemical property: the grains of sand 23.8%, earth grain 45.4%, clay 31.8%, pH 7.7, full nitrogen 1.7gkg-1, water-soluble nitrogen 1.7g
kg-1, full phosphorus 1.3gkg-1, full potassium 17.5gkg-1, CEC 19.4cmolkg-1。
Fresh soil samples 10g is taken, is added in 100mL sterile water, 30 DEG C, 250rpm shaken cultivation 5h, 10 000rpm
It is centrifuged 5min, supernatant takes 9mL filtrate and 1mL to grow into the bacillus cereus suspension of logarithmic phase through 0.22 μm of filter membrane degerming
3 × LB liquid medium of 40mL is added, adds calcium chloride solid to solution final concentration 1mmolL-1, 30 DEG C, the training of 250rpm shaking table
12h is supported, 10 000rpm of gained culture solution is centrifuged 5min, then through 0.22 μm of filter membrane degerming, i.e. acquisition bacteriophage stoste；Using double
Layer flat band method screening bacteriophage simultaneously purifies, and takes the bacillus cereus suspension of above-mentioned 100 μ L of filtrate and 100 μ L to mix, room temperature is quiet
20min is set, the 0.75% semisolid LB agar medium of 5mL is added, tiling is poured on LB solid plate after mixing, 30 DEG C of trainings
10~12h is supported, observes plaque, (phagocytosis spot diameter is about 1-2mm in Fig. 1, and phagocytosis spot diameter is about in Fig. 2 for plaque to appear
For 2-3mm), the transparent plaque of the single edge clear of picking is into the LB liquid containing host strain, and 30 DEG C, 250rpm culture
8h；10 000rpm are centrifuged 5min, and 0.22 μm of filter membrane degerming obtains two plants of bacteriophages, is respectively designated as), it is stored in SM buffer, 4 DEG C of low-temperature storages.
2. the Microbiological Characteristics of bacillus cereus bacteriophage are identified
To bacteriophageCarry out Electronic Speculum observation.By prophage drop on copper mesh, use
After pH 7.0,2% phosphotungstic acid negative staining 90s, extra dye liquor is sucked with filter paper, it is aobvious in Hitachi H-7650 type transmitted electron after dry
Its form of micro- microscopic observation.BacteriophageThe long-tail of the head sum of visible ball-type, head major diameter about 60nm, transverse diameter is about
70nm, tail length about 220nm；According to the 9th report of International Commission on Virus Classification, bacteriophageBelong to long-tail phagocytosis
Body section (Siphoviridae Bacteriophage)；BacteriophageThe polyhedron head of visible rule and contraction
Long-tail, head major diameter about 90nm, transverse diameter about 80nm, tail length about 190nm；Its plaque is in Clear & Transparent, neat in edge, without halo
Round spot, diameter about 2~3mm；According to the 9th report of International Commission on Virus Classification, bacteriophageBelong to long-tail
Phagaceae (Siphoviridae Bacteriophage).
Infection multiplicity (Multiplicity of Infection, MOI) is also referred to as phage titre, refers to infection
Prophge and host strain quantity ratio.Two plants of bacteriophages are obtained based on aforesaid operations, 100 μ L of bacteriophage filtrate is taken, presses
Infection multiplicity is respectively 100: 1,10: 1,1: 1,1: 100,1: 1 000,1: 10 000 addition 100 μ L of logarithmic phase host bacteria suspension.
30 DEG C of shaking table shaken cultivation 5h.Each group phage titre (table 1) is measured with double-layer agar technique.BacteriophageBest sense
Dye plural number is 0.1, bacteriophageOptimal multiplicity of infection is 0.01.
The measurement of 1 optimal multiplicity of infection of table
Measure bacteriophage one step growth curve.By optimal multiplicity of infection, 500 μ L bacteriophages and 500 μ L host bacteria suspensions are taken
It is added in 9mL LB liquid medium, 37 DEG C, 150rpm shaken cultivation, every 10min sampling, centrifugal filtration, and uses double-layer plate
Method measures phage titre.BacteriophageIncubation period 40min, outbreak period 70min (Fig. 5)；BacteriophageIncubation period 20min, outbreak period 55min (Fig. 6).
Embodiment 2:
Single bacteriophage and combinations thereof is verified in water phase to the fungistatic effect of bacillus cereus.
4 groups of test process are set.Control group (processing 1): the bacillus cereus (tetW) of 100 μ L logarithmic phases is taken to be added
In 100mL LB liquid medium, according to best MOI value, it is inoculated with 100 μ LIt is inoculated with single bacteriophage
To the fungistatic effect (processing 2) of host strain: on the basis of control group, according to best MOI value, being inoculated with 100 μ L
Oscillation mixes；It is inoculated with single bacteriophage(processing 3): on the basis of control group, according to best MOI value, inoculation
100 μ L bacteriophagesOscillation mixes；It is inoculated with 1/2 bacteriophageWith 1/2 bacteriophage(processing
4): on the basis of control group, according to best MOI value, being inoculated with 50 μ LWith 50 μ LEvery group is handled
After oscillation mixes, 37 DEG C, 150rpm shaken cultivation is primary every 2h sampling, and carries out bacterium to bacillus cereus (tetW)
It counts, fungistatic effect figure is drawn according to bacillus cereus quantity.According to Fig. 7 result it is found that after cultivating 12h in water phase, four
The quantity that group handles lower bacillus cereus is respectively as follows: 3.2 × 1010cfu·g-1、 1.8×108cfu·g-1、7.6×
107cfu·g-1、4.6×106cfu·g-1, the related tetracycline resistance gene tetW abundance carried is respectively as follows: 1.6 ×
1011copies·g-1、8.3×108copies·g-1、3.3×108copies·g-1、2.6×107copies·g-1.Individually
Or combined inoculation bacteriophage significantly inhibits effect (p < 0.05) to bacillus cereus and its resistant gene tetW.Inoculation is bitten
Three groups of processing of thallus bacillus cereus quantity compared with CK has dropped 2.1,2.6,3.9 orders of magnitude, and tetW has dropped
2.4,2.9,3.9 orders of magnitude.Bacteriophage processing (processing 4) is mixed to the removal effect of pathogenic bacteria and resistant gene the most
Obviously (P < 0.05).Be carry out phagotherapy in contaminated soil-Vegetable System application provide effectively theoretical foundation and
Technical support.
Embodiment 3:
Nanjing crossbeam cattle farm excrement accumulation pond ambient contamination soil is picked up from for examination potting soil.Long-term cropping
For potato (Solanum tuberosum), Jiangsu Province Agriculture Science Institute.Physiochemical properties of soil: the grains of sand 23.8%, earth grain
45.4%, clay 31.8%, pH 7.7, full nitrogen 1.7gkg-1, water-soluble nitrogen 1.7gkg-1, full phosphorus 1.3gkg-1, full potassium
17.5g·kg-1, CEC 19.4cmolkg-1。
Four groups of processing are arranged in experiment altogether: 1. control group (CK): every basin plants 1 potato, potato tubers 2-4cm, on
2~3cm of earthing, compacting earthing, 15 ± 2 DEG C of room temperature；2. bacteriophageHandle (P1): on the basis of the control group according to
Best MOI inoculation 100mL concentration is 106pfu·mL-1Bacteriophage3. bacteriophageIt handles (P2):
Inoculation 100mL concentration is 10 on the basis of the control group5pfu·mL-1Bacteriophage4. mixing " cocktail " processing
(P3=1/2P1+1/2P2): being inoculated with 50mL concentration on the basis of the control group is 106pfu·mL-1BacteriophageWith
50mL concentration is 105pfu·mL-1BacteriophageSoil and potato are carried out after potato growth the 90th day
Spot sampling measures bacillus cereus background contamination concentration in control group contaminated soil are as follows: 4.1 × 108cfu·g-1, Fourth Ring
Plain resistant gene tetW background contamination abundance are as follows: 2.2 × 109copies·g-1；It is inoculated with wax in the processing of bacteriophage P1, P2, P3
Sample bacillus quantity drops to respectively: 4.6 × 105cfu·g-1、1.8×105cfu·g-1、5.3×103cfu·g-1, resistance
Gene tetW abundance drops to respectively: 2.3 × 106copies·g-1、1.2×106copies·g-1、2.4×104copies·
g-1；Three groups of processing of inoculation bacteriophage have dropped 1.9,2.2,4.9 compared with bacillus cereus quantity in control group (CK) respectively
The order of magnitude, resistant gene tetW abundance have dropped respectively: 2.9,3.1,4.9 orders of magnitude.Measure waxy bud in potato haulm block
The quantity of spore bacillus is respectively as follows: 8.6 × 10 in tetra- groups of processing of CK, P1, P2, P34cfu·g-1、1.3×103cfu·g-1、
1.1×103cfu·g-1、1.2×102cfu·g-1, resistant gene tetW abundance drops to respectively: 4.3 × 105copies·g-1、6.5×103copies·g-1、4.5×103copies·g-1、2.1×102 copies·g-1；Waxy gemma bar in stem block
Bacterium number amount has dropped 1.7,1.9,2.7 orders of magnitude compared with control group respectively, and resistant gene tetW abundance has dropped respectively in stem block:
1.8,1.9,2.4 orders of magnitude.Wherein mixing therapeuticcocktail of anti-retrovirals (P3) handles lower antagonism pathogenic bacteria and resistant gene
Removal effect is significantly higher than individually addition bacteriophage (P1/P2).
Analysis finds soil environment microbial ecological diversity index under tetra- groups of processing of CK, P1, P2, P3, AWCD index point
Not are as follows: 0.83 ± 0.1,0.79 ± 0.4,0.80 ± 0.2,0.86 ± 0.2, individually it is inoculated with bacteriophageWithProcessing, there is a degree of reduction in diversity of soil microorganism, and mixing therapeuticcocktail of anti-retrovirals (P3) can be shown
Edaphon functional diversity and stability (p < 0.05) after promoting to repair are write, illustrates that the recovery technique is thin to resistance is repaired
The pollution of bacterium has remarkable result.
Embodiment 4:
Nanjing crossbeam cattle farm excrement accumulation pond ambient contamination soil is picked up from for examination potting soil.Planting vegetable
For six cun of carrot Seoul (Daucus L.), Beijing Seminis Vegetable Seeds Inc., middle peasant Tentium.Physiochemical properties of soil: the grains of sand
23.8%, earth grain 45.4%, clay 31.8%, pH 7.7, full nitrogen 1.7gkg-1, water-soluble nitrogen 1.7gkg-1, full phosphorus
1.3g·kg-1, full potassium 17.5gkg-1, CEC 19.4cmolkg-1。
Four groups of processing are arranged in experiment altogether: 1. control group (CK): every basin plant 1 carrot (on seed earthing 0.5~
1cm, 20 ± 2 DEG C of room temperature)；2. bacteriophageProcessing (P1): 100mL concentration is individually inoculated on the basis of control group
It is 106pfu·mL-1Bacteriophage3. bacteriophageIt handles (P2): on the basis of the control group individually
Being inoculated with 100mL concentration is 105pfu·mL-1Bacteriophage4. bacteriophageProcessing (P2): it is compareing
It is 10 that 100mL concentration is inoculated on the basis of group5pfu·mL-1Bacteriophage4. mixing " cocktail " handles (P3=1/
2 P1+1/2P2): being inoculated with 50mL concentration on the basis of the control group is 106pfu·mL-1BacteriophageIt is dense with 50mL
Degree is 105pfu·mL-1BacteriophageScene is carried out to soil and carrot after Carrot the 70th day to adopt
Sample measures bacillus cereus background contamination concentration in control group contaminated soil are as follows: 1.7 × 108cfu·g-1, tetracyclin resistance
Gene tetW background contamination abundance are as follows: 8.3 × 109copies·g-1；It is inoculated with waxy gemma in the processing of bacteriophage P1, P2, P3
Bacillus quantity drops to respectively: 8.6 × 105cfu·g-1、3.4×105cfu·g-1、4.2×103cfu·g-1, resistant gene
TetW abundance drops to respectively: 4.3 × 106copies·g-1、1.7×106copies·g-1、2.1×104copies·g-1；
Three groups of processing of inoculation bacteriophage have dropped 2.3,2.8,4.7 quantity compared with bacillus cereus quantity in control group (CK) respectively
Grade, resistant gene tetW abundance have dropped respectively: 2.4,2.1,4.6 orders of magnitude.Measure bacillus cereus in carrot block
Quantity be respectively as follows: 6.4 × 10 in tetra- groups of processing of CK, P1, P2, P34cfu·g-1、3.2×103cfu·g-1、1.6×
103cfu·g-1、1.1×102cfu·g-1, resistant gene tetW abundance drops to respectively: 7.2 × 105copies·g-1、1.6
×104copies·g-1、8.2×103copies·g-1、2.5×102 copies·g-1, waxy gemma bar in Carrot Roots block
Bacterium number amount has dropped 1.3,1.5,2.5 orders of magnitude compared with control group respectively, and resistant gene tetW abundance has dropped respectively in stem block:
1.6,1.9,2.2 orders of magnitude.Wherein mixing therapeuticcocktail of anti-retrovirals (P3) handles lower antagonism pathogenic bacteria and resistant gene
Removal effect is significantly higher than individually addition bacteriophage (P1/P2).
Analysis finds soil environment microbial ecological diversity index under tetra- groups of processing of CK, P1, P2, P3, AWCD index point
Not are as follows: 0.75 ± 0.2,0.71 ± 0.3,0.72 ± 0.2,0.79 ± 0.2, individually it is inoculated with bacteriophageWithProcessing, there is a degree of reduction in diversity of soil microorganism, and mixing therapeuticcocktail of anti-retrovirals (P3) can be shown
Edaphon functional diversity and stability (p < 0.05) after promoting to repair are write, illustrates that the recovery technique is thin to resistance is repaired
The pollution of bacterium has remarkable result.
Embodiment 5:
Nanjing crossbeam cattle farm excrement accumulation pond ambient contamination soil is picked up from for examination potting soil.Planting vegetable
For the Italian annual romaine lettuce of resistance to bolting (Lactuca sativa L), Hebei golden hair Zhong Ye Co., Ltd.Soil physics and chemistry substantially
Matter: the grains of sand 23.8%, earth grain 45.4%, clay 31.8%, pH 7.7, full nitrogen 1.7gkg-1, water-soluble nitrogen 1.7gkg-1,
Full phosphorus 1.3gkg-1, full potassium 17.5gkg-1, CEC 19.4cmolkg-1。
Four groups of processing are arranged in experiment altogether: 1. control group (CK): every basin plant 1 romaine lettuce (on seed 0.5~1cm of earthing,
18 ± 2 DEG C of room temperature)；2. bacteriophageHandle (P1): being individually inoculated with 100mL concentration on the basis of control group is
106pfu·mL-1Bacteriophage3. bacteriophageIt handles (P2): individually inoculation on the basis of the control group
100mL concentration is 105pfu·mL-1Bacteriophage4. bacteriophageIt handles (P2): in control group base
It is 10 that 100mL concentration is inoculated on plinth5pfu·mL-1Bacteriophage4. mixing " cocktail " handles (P3=1/2 P1
+ 1/2P2): being inoculated with 50mL concentration on the basis of the control group is 106pfu·mL-1BacteriophageIt is with 50mL concentration
105pfu·mL-1BacteriophageSpot sampling, measurement are carried out to soil and romaine lettuce after Growth of Lettuce the 60th day
Bacillus cereus background contamination concentration in control group contaminated soil are as follows: 6.1 × 108cfu·g-1, tetracycline resistance gene tetW
Background contamination abundance are as follows: 3.6 × 109copies·g-1；It is inoculated with bacillus cereus quantity in the processing of bacteriophage P1, P2, P3
Drop to respectively: 3.8 × 106cfu·g-1、1.1×106cfu·g-1、1.5×104cfu·g-1, resistant gene tetW abundance point
Do not drop to: 1.6 × 107copies·g-1、15.5×106copies·g-1、7.7×104copies·g-1；It is inoculated with bacteriophage
Three groups of processing have dropped 2.2,2.5,4.4 orders of magnitude, resistance base respectively compared with bacillus cereus quantity in control group (CK)
Because tetW abundance has dropped respectively:
2.2,2.8,4.7 orders of magnitude.The quantity of bacillus cereus in romaine lettuce blade is measured at tetra- groups of CK, P1, P2, P3
It is respectively as follows: 8.7 × 10 in processing4cfu·g-1、6.6×103cfu·g-1、2.3×103cfu·g-1、1.6×102cfu·g-1,
Resistant gene tetW abundance drops to respectively: 7.8 × 105copies·g-1、3.3×104copies·g-1、1.9×
103copies·g-1、8.2×102copies·g-1, bacillus cereus quantity declines respectively compared with control group in Carrot Roots block
1.2,1.6,2.7 orders of magnitude, resistant gene tetW abundance has dropped respectively in blade: 1.4,1.6,2.9 orders of magnitude.
Wherein mixing therapeuticcocktail of anti-retrovirals (P3) handles lower antagonism pathogenic bacteria and the removal effect of resistant gene is significantly higher than individually
It adds bacteriophage (P1/P2).
Analysis finds soil environment microbial ecological diversity index under tetra- groups of processing of CK, P1, P2, P3, AWCD index point
Not are as follows: 0.65 ± 0.2,0.61 ± 0.3,0.62 ± 0.2,0.68 ± 0.2, individually it is inoculated with bacteriophageWithProcessing, there is a degree of reduction in diversity of soil microorganism, and mixing therapeuticcocktail of anti-retrovirals (P3) can be shown
Edaphon functional diversity and stability (p < 0.05) after promoting to repair are write, illustrates that the recovery technique is thin to resistance is repaired
The pollution of bacterium has remarkable result.
Illustrate the technology using multiple resistance pathogenic bacteria in the synchronous inactivation soils-vegetables system of multivalence type phagotherapy
Have the advantages that broad spectrum activity is high, ecological risk is low, environmental-friendly, is that a kind of multiple pathogenic bacterium with applications well prospect is dirty
Contaminate soil restoring technology.
Claims (3)
1. a kind of bacteriophage composition, it is characterised in that including two plants of bacteriophages, the bacteriophage was protected on August 1st, 2018
It is hidden in China typical culture collection center, respectively phage phi YSZBA1, deposit number are as follows: CCTCC M 2018517, point
Class is named asBacillus cereusphage φYSZBA1；Phage phi YSZBA2, deposit number are as follows: CCTCC M
2018518, classification naming isBacillus cereus phage φYSZBA2。
2. the answering in antibiotic resistance pathogenic bacteria in targeting inactivation soil environment of bacteriophage composition described in claim 1
With.
3. bacteriophage composition described in claim 1 targets antibiotic resistance in inactivation soils-vegetables system in preparation and causes a disease carefully
Application in bacterium product.
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| CN110205306A (en) * | 2019-06-10 | 2019-09-06 | 武汉轻工大学 | Bacillus cereus bacteriophage, bacteriophage composition and inhibiting-bacteria preparation |
| CN114480299A (en) * | 2020-10-27 | 2022-05-13 | 暨南大学 | Bacillus cereus bacteriophage and application thereof |
| CN118497149A (en) * | 2024-07-18 | 2024-08-16 | 中国科学院南京土壤研究所 | Phage composition and application thereof in strengthening soil carbon fixation |
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| CN108531461A (en) * | 2018-01-04 | 2018-09-14 | 海南师范大学 | One plant of bacillus cereus bacteriophage and its application |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110205306A (en) * | 2019-06-10 | 2019-09-06 | 武汉轻工大学 | Bacillus cereus bacteriophage, bacteriophage composition and inhibiting-bacteria preparation |
| CN110205306B (en) * | 2019-06-10 | 2023-03-03 | 武汉轻工大学 | Bacillus cereus bacteriophage, bacteriophage composition and bacteriostatic preparation |
| CN114480299A (en) * | 2020-10-27 | 2022-05-13 | 暨南大学 | Bacillus cereus bacteriophage and application thereof |
| CN114480299B (en) * | 2020-10-27 | 2023-08-29 | 暨南大学 | Bacillus cereus bacteriophage and application thereof |
| CN118497149A (en) * | 2024-07-18 | 2024-08-16 | 中国科学院南京土壤研究所 | Phage composition and application thereof in strengthening soil carbon fixation |
| CN118497149B (en) * | 2024-07-18 | 2024-09-20 | 中国科学院南京土壤研究所 | Phage composition and application thereof in strengthening soil carbon fixation |
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