CN1699559A - An imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection therefrom - Google Patents
An imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection therefrom Download PDFInfo
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- CN1699559A CN1699559A CNA2005100189230A CN200510018923A CN1699559A CN 1699559 A CN1699559 A CN 1699559A CN A2005100189230 A CNA2005100189230 A CN A2005100189230A CN 200510018923 A CN200510018923 A CN 200510018923A CN 1699559 A CN1699559 A CN 1699559A
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Abstract
Disclosed is an imine-resistant bacillus pyocyaneus bacteriophage and its use for treating infection, wherein a stain of broad-spectrum drug-resistant bacillus pyocyaneus bacteriophage phiA392, which can effectively kill drug-resistant bacillus pyocyaneus in vivo and in vitro, the bacteriophage can decompose multiple strains of clinically segregated imipenem-resistant bacillus pyocyaneus in vivo. The invention proves the superiority and feasibility of bacteriophage as a therapeutic substance in the clinical application to the treatment of bacillus pyocyaneus resistant affection, and provides a novel therapeutic means for clinical imipenem-resistant bacillus pyocyaneus affection.
Description
[technical field]
The present invention relates to a strain and separate phage with broad host range and the therapeutic application thereof that obtains
[background technology]
Infection is the disease that always perplexs human health for a long time.Though since microbiotic comes out, the infectious diseases that the look of many news in the past becomes is controlled effectively, but because chemical sproof appearance, make clinical infection control become a difficult problem again, particularly the appearance speed of clinical drug-resistant bacterium has surmounted antibiotic renewal speed greatly at present, and the mankind have been faced with antibiotic-free available condition.And the microbiotic that has self toxic side effect is big, and selectivity is not strong, and itself just can cause damage or cause superinfection body.Therefore be necessary to study new treatment means.
The phage treatment obtains people's attention in recent years gradually again as a kind of biological treatment means.As far back as this century two, the '30s, one of discoverer of phage Felix d ' herelle is with regard to the therapeutic studies of once being devoted to phage and obtain positively effect.Owing to antibiotic appearance, effectively solved the control problem of infectious diseases afterwards.Thereby the phage treatment has been limited in western countries gradually.But in the former Soviet Union area and areas such as the East European countries, France, the phage treatment is able to prolonged preservation and obtains certain effect as a kind of treatment means, and as in USSR (Union of Soviet Socialist Republics), phage preparation is used for the control that army suffers from diarrhoea for a long time in a large number and obtains unusual effect.In the East European countries (as Poland) and France, phage as a kind of means for the treatment of infection also by life-time service and there is finished product preparation to sell in market.
Principle that phage is used for the treatment of infectious diseases is based on the biological characteristics of himself and develops out.Phage has two kinds of growth patterns: lysogeny growth and cracking performance growth.After lysogeny growth is meant that promptly phage DNA enters host's mycetocyte, integrates with the DNA of host's mycetocyte, and divide, but do not cause the death of host bacterium along with the division of host bacterium.After cracking performance growth is meant that then phage enters host's mycetocyte, destroy the DNA of host bacterium, and be synthetic self the DNA of raw material, utilize the interior protein synthesis protein enclosure of host cell then, and then be assembled into a complete new phage.After the phage in the host cell reaches some amount, will cause the cracking of host bacterium, death.Bacterium around its phage that discharges can continue again to infect is realized multiplication effect, thereby realizes the purpose of treatment infectation of bacteria.In addition, phage preparation adsorption rate height, latent period are short, burst size (being the phage quantity that cracking discharged behind the single phage-infect bacterium) is big, (phage preparation is general only to have infringement to the host bacterium to high specificity, and harmless to human normal cell and normal microflora, can not cause superinfection).Therefore, treating infection with phage is a up-and-coming anti-infective therapy new way.Certainly, phage still has arguement at present to the mechanism of host bacterium infringement, but this feasibility that does not hinder phage preparation to be used for the treatment of.
[summary of the invention]
The inventor is through extensive studies, so that a kind of biological means for the treatment of clinical imipenem-resistant charrin's disease to be provided.The result separates to obtain a strain wide spectrum drug-resistant pseudomonas aeruginosa phage phi A392.It can be effectively in vivo, the killing in vitro drug-resistant pseudomonas aeruginosa.The inventor has done further research based on the purpose that a kind of novel anti-infection treatment means is provided, the clinical separation imipenem-resistant of these many strains of phagocytosis physical efficiency cracking in vivo and in vitro Pseudomonas aeruginosa.
Thereby, the object of the present invention is to provide a strain phage with broad host range, it can be in vivo, externally kill clinical isolating imipenem-resistant Pseudomonas aeruginosa effectively.
Another object of the present invention is to have confirmed superiority and the feasibility of phage as a kind of therapeutic substance applying clinical anti Bacillus pyocyaneu Flugge treatment of infection, and a kind of new treatment means of clinical imipenem-resistant charrin's disease is provided.
Imipenem-resistant pyocinophages φ A392 of the present invention on May 16th, 2005 in (Chinese Wuhan City, Chinese representative microbial preservation center, Wuhan University, budapest treaty depositary institution) preservation, and receive preservation registration number CCTCC NO:M205045.
It has anti-infectious effect in the body the present invention.And the host range of broad arranged, experiment that can cracking 75% is with clinical separation Resistant strain.Phage of the present invention is applied to treatment of infection as anti-infective material,, particularly use phage phi A392 (CCTCC NO:M205045) that separation screening obtains and confirmed its validity in experimentation on animals is used for preparing treatment clinical drug-resistant charrin's disease as a kind of therapeutic substance biotechnological formulation for but clinical treatment resistant organism problem provides a kind of selection approach.
According to the present invention, imipenem-resistant phage phi A392 can form bigger bright plaque on culture dish, and 22 strains in the 30 collected strain imipenem-resistant Pseudomonas aeruginosa clinical separation strains there is splitting action, cleavage rate is 75%, and its institute's energy cracked imipenem-resistant clinical separation strain is as follows: IMR-Pa 91,15,9915,9472,9986,9747,9484,8095,8145,505,9431,7827,1014,1092, A392, A399,1423,1505,1612,1182,1578,2024.Adopt phage of the present invention to be used for the bacteremic treatment of mouse, can cure whole mouse when (multiple of infection, infection multiplicity) in MOI 〉=0.01.Even under the situation that postpones treatment in 3 hours (this moment, tangible systemic infection symptom such as debility, frizzle, the back of a bow, purulent exudate gathering etc. near the eyes appearred in mouse) still have 40% curative effect.Body is interior, experiment in vitro proves that all phage of the present invention is a kind of effective anti-infective material, has the potential value of clinical application.
It is as follows to separate the process that obtains phage of the present invention: get the preceding sewage 1L of Tongji University's Hospital Sewage Treatment center processing, add NaCl58g, the centrifugal 10min of 10,000 * g.Get supernatant, add PEG-8000 to final concentration be 10% (w/v), put after 4 ℃ of refrigerator overnight at 4 ℃ the centrifugal 20min of 10,000 * g.With the resuspended precipitation of 5ml SM Buffer.The extracting of equal-volume chloroform once.Sewage after 300 μ l handle mixes with the imipenem-resistant Pseudomonas aeruginosa φ A392 of 200 μ l incubated overnight, hatches 20min for 37 ℃, is 50 ℃ of mixings with 3ml fusing top-agar, paves ware, is inverted in 37 ℃ of incubated overnight after the cooling.On culture dish, find several bright plaques that differ in size next day.Take off wherein maximum plaque with the tip head, be dissolved in the 2mlLB nutrient solution, add the imipenem-resistant Pseudomonas aeruginosa φ A392 of incubated overnight in 1: 100 ratio in this nutrient solution, 37 ℃, 250 rotational oscillations shook 4.5～5 hours.12000 left the heart 5 minutes, got 4 ℃ of preservations of 80% supernatant.Get this supernatant and cultivate altogether on solid medium with φ A392 bacterium again, with the big plaque of tip picking.3 times so repeatedly, can obtain uniform plaque.Take out plaque, carry out a small amount of amplification as stated above, obtain liquid phage amplification liquid.The host bacterium of incubated overnight was diluted by 1: 100, continue to be cultured to early stage logarithmic phase.Add aforementioned concentrated phage 10 μ l, continue to cultivate 5h, add the chloroform of 1/10 volume, continue jolting after 10 minutes, add DNase I and RNase A respectively and be 1 μ g/ml to final concentration, room temperature was placed 30 minutes, the centrifugal bacterial debris of removing.The PEG/NaCl that adds 1/6 volume, 4 ℃ are spent the night.Next day, 4 ℃, the centrifugal 20min of 12,000 * g.With the resuspended precipitation of 1ml SM Buffer.Add 1/6 volume PEG/NaCl and precipitate once more, hatch 1h on ice, 4 ℃, the centrifugal 20min of 12,000 * g.With the resuspended precipitation of 200 μ l SM Buffer, promptly obtain the phage of increasing, can reach required titre through amplification repeatedly.
Substratum of the present invention has following composition: LB substratum: 1%tryptone, 0.5%Yeast extract and 1%NaCl.Solid agar: 1%tryptone, 0.5%Yeastextract and 1%NaCl and 1.5%Agar.Top-agar: 1%tryptone, 0.5%Yeastextract and 1%NaCl and 0.7%Agarose.SM Buffer is by 0.1M NaCl, 0.01MMgSO
47H
2O, 0.05M Tris-HCl (pH7.5) and 0.01%gelatin (gelatin) are formulated.PEG/NaCl is formulated by 20%PEG-8000 and 2.5MNaCl.
Phage strain φ A392 of the present invention (CCTCC NO:M205045) has following microbial characteristic:
1, morphological characteristic:
Observe the phage particle (Fig. 1) of phospho-wolframic acid negative staining under electron microscope, this phage is the head construction of regular polygon, and diameter 60nm has tailing axle, long 116nm.
2, cultivate characteristic
Phage strain of the present invention can form bigger bright plaque on the solid medium of indicator φ A392, plaque diameter 1～2mm does not have halo on every side.
3, biological characteristics:
Phage strain of the present invention can be adsorbed on the host bacterium φ A392 fast, and the adsorption rate in 5 minutes reaches 96% (as Fig. 2), and the adsorption rate constant K is 1.28 * 10
-8Ml/min.
The one step growth of this phage such as Fig. 3, therefrom be 20～25 minutes the latent period of this phage-infect host bacterium as can be seen, average burst size is 125.Latent period is short, and burst size is big, illustrates that this phage has very strong cracking effect.
Phage splitting spectrum of the present invention is not single-minded, and it can form bright plaque on the culture dish of 22 strains in the clinical isolating imipenem-resistant Pseudomonas aeruginosa of 30 strains, and the wide rate of biting reaches 75%.And it does not have splitting action to other Pseudomonas such as intestinal bacteria, streptococcus aureus.
The inventor is verified, separates the phage strain φ A392 that obtains and can treat the infection of imipenem-resistant Pseudomonas aeruginosa effectively in experimentation on animals, and do not have toxic side effect.Because the bacterium beyond it does not belong to Pseudomonas aeruginosa has splitting action, therefore also flora imbalance can not take place.
[description of drawings]
Fig. 1: the electromicroscopic photograph of φ A392, this phage is the head construction of regular polygon, and diameter 60nm has tailing axle mechanism.The long 116nm of tailing axle.
Fig. 2: the absorption curve of phage phi A392, the adsorption rate in 5 minutes reach 96% (as Fig. 2), and the adsorption rate constant K is 1.28 * 10
-8Ml/min.
Fig. 3: the one step growth of phage phi A392, be 20～25 minutes the latent period of this phage-infect host bacterium, average burst size is 125.Latent period is short, and burst size is big, illustrates that this phage has very strong cracking effect.
Fig. 4: the screening of phage with broad host range: with Pseudomonas aeruginosa φ 9747 is that the host bacterium is made uniform lawn, divides 20 lattices at the back of culture dish then, drips phage in lattice, and the phage with cracking performance then can form bright plaque.
Determining of Fig. 5 imipenem-resistant Pseudomonas aeruginosa φ A392 infecting mouse minimum lethal dose, 3 * 10
7-1 * 10
8The bacterium liquid of CFU all can cause whole dead mouses, and 1 * 10
7-2 * 10
7The bacterium liquid of CFU can not cause whole dead mouses.Therefore, 3 * 10
7CFU is the minimum lethal dose that causes dead mouse.
Phasic property is analyzed during Fig. 6 minimum lethal dose accurate, and in 10 mouse, 90% mouse is all dead in 6～14 hours through abdominal injection for the bacterium liquid of MLD.
Fig. 7 various dose phage result of treatment, phage dosage whole mouse under the situation of MOI 〉=0.01 all can survive and finally recovery from illness.
Fig. 8 phage postpones result of treatment, and under situation about postponing in 1 hour, all mouse all can avoid death.And postponing treatment more than 1 hour the time since this moment mouse state relatively poor, tangible infection symptoms such as frizzle, the back of a bow, exudate gathering etc. have near the eyes appearred, the effect of phage descends to some extent, but still has statistical significance (P＜0.01).
[embodiment]
The present invention will be by being described in more detail the present invention by the following example.Following examples only are illustrative, should be pointed out that the present invention is not subjected to the restriction of these embodiment.
Embodiment 1:
Get the Hospital Sewage Treatment center sewage 1L of Tongji University, add NaCl58g, the centrifugal 10min of 10,000 * g.Get supernatant, add PEG-8000 to final concentration be 10% (w/v), put after 4 ℃ of refrigerator overnight at 4 ℃ the centrifugal 20min of 10,000 * g.With the resuspended precipitation of 5ml SM Buffer.The extracting of equal-volume chloroform once.Sewage after 300 μ l handle mixes with the host bacterium of 200 μ l incubated overnight, hatches 20min for 37 ℃, is 50 ℃ of mixings with 3ml fusing top-agar, paves ware, is inverted in 37 ℃ of incubated overnight after the cooling.Take off wherein maximum plaque with the tip head, be dissolved in the 2mlLB nutrient solution, add the imipenem-resistant Pseudomonas aeruginosa φ A392 of incubated overnight in 1: 100 ratio in this nutrient solution, 37 ℃, 250 rotational oscillations shook 4.5～5 hours.12000 left the heart 5 minutes, got 4 ℃ of preservations of 80% supernatant.Get this supernatant and cultivate altogether on solid medium with φ A392 bacterium again, with the big plaque of tip picking.3 times so repeatedly, can obtain uniform plaque.Get this plaque by the amplification of preceding method liquid, obtain concentrating phagocytosis body fluid.
Embodiment 2:
The host bacterium φ A392 of incubated overnight was diluted by 1: 100, continue to be cultured to early stage logarithmic phase.Add the concentrated phage 10 μ l among the embodiment 1, continue to cultivate 5h, add the chloroform of 1/10 volume, continue jolting after 10 minutes, add DNase I and RNase A respectively and be 1 μ g/ml to final concentration
[3], room temperature was placed 30 minutes, the centrifugal bacterial debris of removing.The PEG/NaCl that adds 1/6 volume, 4 ℃ are spent the night.Next day, 4 ℃, the centrifugal 20min of 12,000 * g.With the resuspended precipitation of 1ml SM Buffer.Add 1/6 volume PEG/NaCl and precipitate once more, hatch 1h on ice, 4 ℃, the centrifugal 20min of 12,000 * g.With the resuspended precipitation of 200 μ l SM Buffer, promptly obtain the phage of increasing, can reach required titre through amplification repeatedly.
Embodiment 3:
Imipenem-resistant Pseudomonas aeruginosa with 30 strains dilution is made uniform lawn on the LB substratum.Divide 16-20 grid at this substratum back side and mark, dropping phage phi A392 liquid in corresponding grid then treats that being inverted in 37 ℃ behind the droplet drying hatches 12-16h, observations (Fig. 4).As seen form plaque on φ A392 22 bacterial strains therein, specific as follows: IMR-Pa 91,15,9915,9472,9986,9747,9484,8095,8145,505,9431,7827,1014,1092, A392, A399,1423,1505,1612,1182,1578,2024.
Embodiment 4:
1, get the bacterial cultures of 100ul overnight growth, insert in the Erlenmeyer flask of 10ml substratum, 37 ℃ after shaking culture 2.5-3 hour, through the microscopy counting, cell can reach 10
8More than/the ml,, be mixed with 5*10 with the substratum dilution
7/ ml.
2, the day before yesterday is measured phage titer in experiment, is mixed with 5*10 by this dilution
9PFU/ml.
3, with said two devices balance in 37 degree water-baths, press MOI=10 1ml phagocytosis body fluid is added the 9ml bacterial suspension, mix, put back to insulation or vibration insulation in the water-bath.
4, press certain hour (1-5min) sampling at interval, dilute 1000 times, be placed in the ice, stop absorption.
5, get dilute sample 1ml, the centrifugal 5min of centrifugal 3min or 4000rpm in the Ep pipe, the cell of sedimentation bacterium and absorption phage.
6, after suitably dilution (10-100 doubly),, cultivate the back record by the free bacteriophage in the double-deck agar method mensuration supernatant liquor.
7, with the logarithm of free bacteriophage percentage ratio function construction, should obtain a straight line, ask K value (1.28 * 10 from the collinear slope as the time
-8Ml/min).
Embodiment 5:
1, dilution phagocytosis body fluid is to 5*10
7PFU/ml.
2, bacterium liquid is diluted to 5*10
7CFU/ml.
3, get 20 Ep pipes, every pipe adds 900ulLB liquid.
4, get 20 Ep pipes, every pipe adds 200ul bacterium liquid.
5, get 20 test tubes, every pipe adds the top layer glue of 3ml fusing, and insulation is at 50 degree.
6, with above-mentioned phagocytosis body fluid, bacterium liquid is put 37 degree incubations.
7, get 0.1ml phagocytosis body fluid and add in the bacterial suspension of 0.9ml, adsorbed 5 minutes.
8, get the 0.1ml mixed solution and add to 9.9ml ice TSBM liquid, respectively get after mixing in 1ml to Ep8, the pipe, centrifugal 5 minutes of 4 ℃ of 8000g are with the resuspended precipitation of 1mlLB.
9, get the resuspended liquid of 0.1ml and add to 9.9mlLB liquid, 37 ℃ of shaking culture, got the common nutrient solution of 0.1ml respectively at the 0th, 5,10,20,30,40,50,60,70,80,90 minute during this time and mix, pave ware after 5 minutes, place 37 ℃ of cultivations respectively with 200ul bacterium liquid.Make curve (Fig. 3) by the plaque number-time of the premises, can this phage latent period be 20～25min, burst size is 125.
Embodiment 6:
The φ A392 bacterium of setting up the systemic infection model overnight incubation in 100ml LB, continues to be cultured to early stage logarithmic phase (OD by 1: 100 dilution proportion
600Value is about 0.5), 4 ℃, centrifugal 5 minutes of 8000g abandons supernatant, will precipitate with the equal-volume sterile saline resuspended, recentrifuge, resolution of precipitate is in 5ml physiological saline.With the φ A392 of physiological saline serial dilution through abdominal cavity (intraperitoneal, i.p) be injected in 11 groups of mouse, cause one group of (if no special instructions, being 5/group) all dead minimum dose of mouse be minimum lethal dose (minimal lethal dose, MLD).After the bacterium liquid of serial dilution is injected in mouse, 3 * 10
7-1 * 10
8The bacterium liquid of CFU all can cause whole dead mouses, and 1 * 10
7-2 * 10
7The bacterium liquid of CFU can not cause whole dead mouses (as Fig. 5).Therefore, 3 * 10
7CFU is the minimum lethal dose that causes dead mouse.In 10 mouse, 90% mouse is in 6～14 hours dead (as Fig. 6) all through abdominal injection for the bacterium liquid of MLD.
Embodiment 7:
Different bacteriophages dosage is to curative effects: get 9 groups of mouse, inject through a ventrolateral compartment with the bacterium liquid of MLD dosage, through the phage preparation (LB dilution) of opposite side abdominal injection various dose, wherein one group of i.p injects the LB nutrient solution in contrast immediately.The state of health of observation mouse is more than 20 days.As Fig. 7, phage dosage whole mouse under the situation of MOI 〉=0.01 all can survive and finally recovery from illness.There is significant significant difference (P＜0.01) in the mouse survival rate under MOI=0 and MOI 〉=0.01 situation.
Embodiment 8:
Postpone the result of treatment monitoring: get 7 groups of mouse, all the bacterium liquid inductance with MLD dosage dyes, and respectively at injection back the 0th, 20,40,60, phagocytosis body fluid (MOI=200) the i.p injection with the higher titre of 500 μ l in 180,360 minutes, observation is more than 20 days.Every group of mouse all is provided with corresponding control group (being the LB nutrient solution of i.p injection equivalent).As Fig. 8, under situation about postponing in 1 hour, all mouse all can avoid death.And postponing treatment more than 1 hour the time since this moment mouse state relatively poor, tangible infection symptoms such as frizzle, the back of a bow, exudate gathering etc. have near the eyes appearred, the effect of phage descends to some extent, but still has statistical significance (P＜0.01).
Claims (4)
1, strain imipenem-resistant pyocinophages φ A392 (CCTCCNO:M205045), it has anti-infectious effect in the body.
2, by the described imipenem-resistant pyocinophages of claim 1 φ A392 (CCTCC NO:M205045), it is characterized in that having the host range of broad, experiment that can cracking 75% is with clinical separation Resistant strain.
3, a kind of body φ A392 (CCTCC NO:M205045) phage with broad host range is used for the therapeutic use of resistance charrin's disease, it is characterized in that the treatment that phage is applied to infect as anti-infective material, but provide the another kind selection approach for clinical treatment resistant organism problem.
4,, it is characterized in that using separation, screening phage phi A392 (CCTCCNO:M205045) that obtain and confirmed its validity in experimentation on animals are used for preparing treatment clinical drug-resistant charrin's disease as a kind of therapeutic substance biotechnological formulation by the described therapeutic use of claim 3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104140957A (en) * | 2013-05-07 | 2014-11-12 | 兰州大学 | Cleavable multiple-drug resistant pseudomonas aeruginosa bacteriophage and application thereof in infection treatment |
| CN108070572A (en) * | 2018-01-25 | 2018-05-25 | 青岛诺安百特生物技术有限公司 | A kind of width fragmentation pattern pyocinophages and its disinfection application |
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| JPH0617291B2 (en) * | 1986-07-14 | 1994-03-09 | 日本たばこ産業株式会社 | Soil disease control method for solanaceous plants |
| CN1216712A (en) * | 1998-10-09 | 1999-05-19 | 卢杲 | Traditional Chinese medicine for treating cancer |
| DK1458856T3 (en) * | 2001-12-13 | 2012-07-09 | Nestle Sa | ISOLATED SUBJECTS AND THEIR USE IN FOOD OR PETS FEED PRODUCTS |
| EP1663265B1 (en) * | 2003-07-23 | 2015-09-23 | Biocontrol Limited | Bacteriophage-containing therapeutic agents |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104140957A (en) * | 2013-05-07 | 2014-11-12 | 兰州大学 | Cleavable multiple-drug resistant pseudomonas aeruginosa bacteriophage and application thereof in infection treatment |
| CN104140957B (en) * | 2013-05-07 | 2017-04-26 | 兰州大学 | Cleavable multiple-drug resistant pseudomonas aeruginosa bacteriophage and application thereof in infection treatment |
| CN108070572A (en) * | 2018-01-25 | 2018-05-25 | 青岛诺安百特生物技术有限公司 | A kind of width fragmentation pattern pyocinophages and its disinfection application |
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