CN102199576A - Method for separating and screening phage for degrading Pseudomonas aeruginosa biofilm - Google Patents
Method for separating and screening phage for degrading Pseudomonas aeruginosa biofilm Download PDFInfo
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Abstract
The invention relates to a phage for degrading a Pseudomonas aeruginosa biofilm. The phage is characterized in that: the phage is named C12, the collection number is CGMCC No. 4249, the collection unit is China General Microbiological Culture Collection Center located at No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing City, and the collection date is October 26, 2010. The invention provides a method for amplifying, separating and preserving the phage for degrading Pseudomonas aeruginosa. The capacity of degrading the Pseudomonas aeruginosa biofilm by the phage is also determined, finally the pure phage C12 with high titer and high biofilm degradation capacity is obtained, and the degradation amount of the pure phage C12 reaches 85.3 percent; the phage can degrade biofilms and crack viable bacteria in the biofilms; the clinical application of the phage can be realized; treatment problems of medicine resistance and biofilms of the Pseudomonas aeruginosa are solved; and the method is a feasible biological method.
Description
Technical field
The invention belongs to biological technical field, relate to separation and the screening method of a kind of phage, separation and the screening method of especially a kind of Pseudomonas aeruginosa biofilm load phage of degrading.
Background technology
Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a conditioned pathogen, extensively is present in the various environment, and be one of important Gram-negative bacteria that causes nosocomial infection.This bacterium has stronger adaptability, can form biofilm load at various biologies and abiotic surface, comprises medicine equipment or tissue, is difficult to remove.Discover that 80% charrin disease is caused by biofilm load, common infection comprises fire victim's infection, urinary tract infections, catheter infections, otitis media and eyes corneal infection etc.
For a long time, microbiotic is widely used in the treatment of bacteriosis, causes endurance strain constantly to occur, and brings great difficulty to clinical treatment.Be easy to clinically be separated to and have multidrug resistant or general resistant Pseudomonas Aeruginosa bacterial strain, the infection that is caused by Pseudomonas aeruginosa can not effectively be treated and control to the microbiotic that causes using at present.
Phage (Bacteriophage or phage) be can bacterial infection etc. the virus of microorganism, cracking host bacterium specifically, whether resistance is irrelevant with the host bacterium simultaneously.On the other hand, some phage a kind of enzyme of can encoding, the exocellular polysaccharide polymkeric substance (EPS) of the host bacterium of can degrading, thus destroy the biofilm load that pathogenic bacterium form.These characteristics make phage can be used for controlling the infection of host bacterium.Therefore, from environmental sample, separate phage, detect the influence that it forms biofilm load, thereby filter out the phage of effective cracking Pseudomonas aeruginosa and its biofilm load of degrading, finally reach the purpose of treatment and control charrin disease.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art part, separation and the screening method of a kind of Pseudomonas aeruginosa biofilm load phage of degrading are provided, the present invention can access the height of tiring, pure phage that degradation biological tunicle ability is strong, and this phage can degradation biological tunicle and cracking viable bacteria wherein.
The objective of the invention is to be achieved through the following technical solutions:
A kind of degraded Pseudomonas aeruginosa biofilm load phage, name is called: C12, deposit number: CGMCC No.4249, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is: on October 26th, 2010, the classification name of suggestion: Pseudomonas aeruginosa double-stranded DNA phage.
Separation and the screening method of a kind of Pseudomonas aeruginosa biofilm load phage of degrading, the step of method is:
(1) from environment, gathers mud sample or sewage sample;
(2) amplification phage: incubated overnight host bacterium in the test tube, the host bacterium is a Pseudomonas aeruginosa, with 1% inoculum size enlarged culturing to logarithmic phase, add mud sample or sewage mixed culture after, centrifugal, collect supernatant liquor, membrane filtration degerming;
(3) separate single plaque: it is mixed with the Pseudomonas aeruginosa that is in logarithmic phase to get supernatant liquor, fall double-layer plate with 0.6% semisolid medium mixing again, cultivate 2-8h for 20-40 ℃, the picking plaque dilutes in the LB liquid nutrient medium, get each dilute sample respectively with the bacteria suspension mixing, be poured on rapidly on the 1.5% bottom-layer agar substratum again with behind the 0.6% semi-solid LB substratum mixing, observe behind 37 ℃ of cultivation 4h, repeat this step until forming single plaque;
(4) phage preserves: after obtaining single plaque,, use the membrane filtration degerming, add aseptic glycerine the phage amplification cultivation, and mixing ,-70 ℃ of preservations are standby;
(5) phage titer is measured: the Pseudomonas aeruginosa of phage diluent and logarithmic phase mixes, and double-layer plate is observed plaque behind 37 ℃ of cultivation 4～6h, calculates phage titer;
(6) Pseudomonas aeruginosa biofilm load: get the Pseudomonas aeruginosa overnight culture in Tissue Culture Plate, then add 1-10 μ L phage to each hole respectively, add the LB liquid nutrient medium of 1/3 volume, mixing, 37 ℃ leave standstill cultivation, with the amount of Pseudomonas aeruginosa clinical strains biofilm load in Tissue Culture Plate of Viola crystallina analytical method survey;
(7) determining of phage increment: after cultivating 24h,, after the filtration sterilization, tiring of phage measured, and calculated the increment of phage according to tiring of initial phage with liquid sucking-off gently;
(8) mensuration of biofilm load degradation amount: get 24 porocyte culture plates, the LB liquid nutrient medium that adds 100 μ L Pseudomonas aeruginosa overnight culture, 5 μ L phages, 1mL 1/3 volume respectively, mixing leaves standstill cultivation for 37 ℃, the amount of measuring space Pseudomonas aeruginosa biofilm load, calculate the degradation amount of biofilm load, select the highest phage of degradation amount.
And, the measuring method of the degradation amount of described biofilm load is as follows: take out Tissue Culture Plate, water washes repeatedly, remove and stick to biofilm load surface free thalline, 1% Viola crystallina-3% glacial acetic acid aqueous solution dyeing the 15min of 1mL, water washes repeatedly removes unnecessary dye liquor, and the ethanol of 500 μ L 95% washes Viola crystallina, collect the ethanol washing lotion, measure its OD540 value.
Advantage of the present invention and positively effect are:
The invention provides amplification, separation, the store method of the phage of a kind of Pseudomonas aeruginosa of degrading, also carried out the mensuration of degraded Pseudomonas aeruginosa biofilm load ability at phage, height, pure phage C12 that degradation biological tunicle ability is strong finally obtain tiring, its degradation amount reaches 85.3%, this phage can degradation biological tunicle and cracking viable bacteria wherein, can realize phage application clinically, overcoming the treatment difficult problem of the Pseudomonas Aeruginosa property of medicine and biofilm load, is a kind of feasible biological method.
Embodiment
The invention will be further described below by specific embodiment, and following examples are descriptive, is not determinate, can not limit protection scope of the present invention with this.
Need to prove:
The phage of taking sample separation to obtain from various environment, therefore the ability difference of its degraded Pseudomonas aeruginosa biofilm load adopts the Viola crystallina analytical method to calculate the degradation amount of biofilm load, thereby selects the strong phage of degradation capability.
The source of phage is earth, mud or sewage, the requirement of no region.
Technical scheme:
A is collected specimens from different environment;
The B phage of increasing: incubated overnight host bacterium in the test tube, the host bacterium is: Pseudomonas aeruginosa, with 1% inoculum size enlarged culturing to logarithmic phase, after adding 5g mud sample (or 5mL sewage) mixed culture 48h, most of thalline of centrifugal removal and sample impurity, collect supernatant liquor, filter membrane (0.22 μ m) filtration sterilization;
C separates single plaque: get the mixed 10min of Pseudomonas aeruginosa that 100 μ L supernatant liquors and 200 μ L are in logarithmic phase, fall double-layer plate with semi-solid LB substratum (60 ℃) mixing of 5mL 0.6% again, cultivate 4h for 37 ℃, picking 2 ring plaques dilute in 1mL LB liquid nutrient medium, get each dilute sample 100 μ L respectively with 200 μ L bacteria suspension mixing 5min, be poured on rapidly on the 1.5% bottom-layer agar substratum again with behind 5mL 0.6% semi-solid LB substratum (60 ℃) mixing, observe behind 37 ℃ of cultivation 4h, repeat this step until forming single plaque;
The D phage preserves: after obtaining single plaque,,, add the aseptic glycerine of 30% (v/v) with 0.22 μ m membrane filtration degerming with the phage amplification cultivation, and mixing ,-70 ℃ of preservations are standby;
The E phage titer is measured: get 10
-6, 10
-7, 10
-8The Pseudomonas aeruginosa of each 100 μ L of phage diluent and 200 μ L logarithmic phases mixes, and double-layer plate is observed plaque behind 37 ℃ of cultivation 4～6h, calculates phage titer;
Determining of F phage increment: after cultivating 24h,, note not destroying biofilm load with liquid sucking-off gently.After the filtration sterilization, tiring of phage measured, and calculated the increment of phage according to tiring of initial phage.
The mensuration of G biofilm load degradation amount: the inoculation Pseudomonas aeruginosa is in the LB of 1/3 volume liquid nutrient medium, and 18h is cultivated in 37 ℃ of concussions.Get 100 μ L bacterium liquid respectively, add in the 24 porocyte culture plates, then add 1mL 1/3LB liquid nutrient medium, 37 ℃ leave standstill cultivation 48h, obtain sophisticated Pseudomonas aeruginosa biofilm load.Discard bacterium liquid, wash Tissue Culture Plate repeatedly, remain in the free thalline on biofilm load surface with removal with 0.9% stroke-physiological saline solution.Get 100 μ L phage diluents (tiring after measured) respectively and add in the 24 porocyte culture plates, each experiment do 3 parallel, control group is not except that adding the phage, all the other operation stepss are all identical.In 24 porocyte culture plates, add the LB liquid nutrient medium of 1mL 1/3 volume at last respectively, behind 37 ℃ of cultivation 24h,, note not destroying biofilm load liquid sucking-off gently.Measure the titre of phage according to the double-layer plate method, and utilize the amount of Viola crystallina assay biofilm load this moment.The method of measuring the biofilm load amount is as follows: the Tissue Culture Plate water washed repeatedly, removes and stick to biofilm load surface free thalline, and the 1% Viola crystallina-3% glacial acetic acid aqueous solution dyeing 15min of 1mL, water washes repeatedly removes unnecessary dye liquor; The ethanol of 500 μ L 95% washes Viola crystallina, collects the ethanol washing lotion, measures its OD
540Value, the degradation amount of calculating biofilm load.
Degradation amount (%)=(A
0-A
n) * 100%/A
0
A
0---the ethanol washing lotion OD540 value of parallel control pore membrane;
A
n---the ethanol washing lotion OD540 value of experiment pore membrane;
The method of calculation of degradation amount: it is the parallel control hole that 3 holes are all arranged on every 24 orifice plate, does not add phage, and the biofilm load of generation is not degraded, and the film of degrading with the part that adds the phage generation compares.
Embodiment 1:
The degraded Pseudomonas aeruginosa biofilm load phage name that present embodiment obtains is called: C12, deposit number: CGMCCNo.4249, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is: on October 26th, 2010.When this preserves phage the host bacterium is also given preservation simultaneously.
(1) collected specimens from Taida district, Tianjin Tai Feng park earth;
(2) amplification phage: 37 ℃, under the 120rpm condition, incubated overnight host bacterium in the test tube, with 1% inoculum size in 250mL triangular flask enlarged culturing to logarithmic phase (OD600 about 0.5), add 5g mud sample (or 5mL sewage), behind the mixed culture 48h, the centrifugal 10min of 8000g, remove most of thalline and sample impurity, collect supernatant liquor, with 0.22 μ m membrane filtration degerming.
(3) separate single plaque: get 100 μ L supernatant liquors in centrifuge tube, add 200 μ L and be in the Pseudomonas aeruginosa mixing of logarithmic phase, behind the 10min, semi-solid LB substratum (60 ℃) mixing with mixture and 5mL 0.6%, fall double-layer plate, cultivate 4h for 37 ℃, encircle in 1mL LB liquid nutrient medium at phagocytosis macular area picking 2 with transfering loop, after the dilution, get each dilute sample 100 μ L respectively with 200 μ L bacteria suspension mixing 5min, pour into rapidly on the 1.5% bottom-layer agar substratum again with behind 5mL 0.6% semi-solid LB substratum (60 ℃) mixing, observe behind 37 ℃ of cultivation 4h, repeat this step until forming single plaque; Filter out C1, C10, C12 totally 3 strain phages.
(4) phage preserves: the single plaque of each phage of picking, with each phage amplification cultivation,, add the aseptic glycerine of 30% (v/v) with 0.22 μ m membrane filtration degerming, and mixing ,-70 ℃ of preservations are standby.
(5) phage titer is measured: phage uses physiological saline with 10 times of gradient serial dilutions, until 10
-8, get 10
-6, 10
-7, 10
-8The Pseudomonas aeruginosa of each 100 μ L of diluent and 200 μ L logarithmic phases mixes, and double-layer plate is observed plaque behind 37 ℃ of cultivation 4～6h, calculates phage titer, and is as shown in the table.
| The phage numbering | (pfu/ml) tires |
| C1 | 2.1×10 9 |
| C10 | 3.6×10 8 |
| C12 | 6.6×10 9 |
(6) Pseudomonas aeruginosa biofilm load: (that adopt in the present embodiment is TJC729 to get 100 μ L Pseudomonas aeruginosas respectively, indicate the source of bacterium, from Tianjin Children's hospital, with phage preservation simultaneously) overnight culture is in 24 porocyte culture plates, then add 5 μ L phages to each hole respectively, add 1mL 1/3LB liquid nutrient medium, mixing, 37 ℃ leave standstill cultivation, every 24h, with the amount of Pseudomonas aeruginosa clinical strains biofilm load in Tissue Culture Plate of Viola crystallina analytical method survey;
(7) determining of phage increment: after cultivating 24h,, note not destroying biofilm load with liquid sucking-off gently.After the filtration sterilization, tiring of phage measured, and calculated the increment of phage according to tiring of initial phage.
| The phage numbering | Increment (doubly) |
| C1 | 2.5×10 6 |
| C10 | 3.9×10 2 |
| C12 | 1.7×10 7 |
(8) phage degraded Pseudomonas aeruginosa biofilm load: the Viola crystallina analytical method is measured the degradation amount of biofilm load, concrete grammar is as follows: take out Tissue Culture Plate, water washes repeatedly, remove the Pseudomonas aeruginosa that sticks to the biofilm load surface, 1% Viola crystallina-3% glacial acetic acid aqueous solution dyeing 15min with 1mL, then water washes repeatedly, remove unnecessary dye liquor, dissolve with ethanol bonded Viola crystallina with 500 μ L 95%, collect the ethanol washing lotion, measure its OD540 value, calculate the degradation amount of biofilm load, select the highest phage of degradation amount, be degraded Pseudomonas aeruginosa biofilm load phage C12.
| The phage numbering | Degradation amount (%) |
| C1 | 70.3 |
| C10 | 39.4 |
| C12 | 85.3 |
Embodiment 2:
Gather seashore, Bohai Sea Gulf muddy water sample; All the other steps are with embodiment 1.
Embodiment 3:
Gather pig methane-generating pit mud sample; All the other steps are with embodiment 1.
Claims (3)
1. degraded Pseudomonas aeruginosa biofilm load phage, it is characterized in that: name is called: C12, deposit number: CGMCCNo.4249, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is: on October 26th, 2010.
2. a separation and screening method of Pseudomonas aeruginosa biofilm load phage of degrading, it is characterized in that: the step of method is:
(1) from environment, gathers mud sample or sewage sample;
(2) amplification phage: incubated overnight host bacterium in the test tube, the host bacterium is a Pseudomonas aeruginosa, with 1% inoculum size enlarged culturing to logarithmic phase, add mud sample or sewage mixed culture after, centrifugal, collect supernatant liquor, membrane filtration degerming;
(3) separate single plaque: it is mixed with the Pseudomonas aeruginosa that is in logarithmic phase to get supernatant liquor, fall double-layer plate with 0.6% semisolid medium mixing again, cultivate 2-8h for 20-40 ℃, the picking plaque dilutes in the LB liquid nutrient medium, get each dilute sample respectively with the bacteria suspension mixing, be poured on rapidly on the 1.5% bottom-layer agar substratum again with behind the 0.6% semi-solid LB substratum mixing, observe behind 37 ℃ of cultivation 4h, repeat this step until forming single plaque;
(4) phage preserves: after obtaining single plaque,, use the membrane filtration degerming, add aseptic glycerine the phage amplification cultivation, and mixing ,-70 ℃ of preservations are standby;
(5) phage titer is measured: the Pseudomonas aeruginosa of phage diluent and logarithmic phase mixes, and double-layer plate is observed plaque behind 37 ℃ of cultivation 4～6h, calculates phage titer;
(6) Pseudomonas aeruginosa biofilm load: get the Pseudomonas aeruginosa overnight culture in Tissue Culture Plate, then add 1-10 μ L phage to each hole respectively, add the LB liquid nutrient medium of 1/3 volume, mixing, 37 ℃ leave standstill cultivation, with the amount of Pseudomonas aeruginosa clinical strains biofilm load in Tissue Culture Plate of Viola crystallina analytical method survey;
(7) determining of phage increment: after cultivating 24h,, after the filtration sterilization, tiring of phage measured, and calculated the increment of phage according to tiring of initial phage with liquid sucking-off gently;
(8) mensuration of biofilm load degradation amount: get 24 porocyte culture plates, the LB liquid nutrient medium that adds 100 μ L Pseudomonas aeruginosa overnight culture, 5 μ L phages, 1mL 1/3 volume respectively, mixing leaves standstill cultivation for 37 ℃, the amount of measuring space Pseudomonas aeruginosa biofilm load, calculate the degradation amount of biofilm load, select the highest phage of degradation amount.
3. separation and the screening method of degraded Pseudomonas aeruginosa biofilm load phage according to claim 2, it is characterized in that: the measuring method of the degradation amount of biofilm load is as follows in the described step (8): take out Tissue Culture Plate, water washes repeatedly, remove and stick to biofilm load surface free thalline, 1% Viola crystallina-3% glacial acetic acid aqueous solution dyeing the 15min of 1mL, water washes repeatedly removes unnecessary dye liquor, the ethanol of 500 μ L 95% washes Viola crystallina, collect the ethanol washing lotion, measure its OD540 value.
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