CN114752542B - Method for cultivating mycoplasma synoviae biofilm, application and screening method thereof - Google Patents
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Abstract
The invention relates to the technical field of pathogen separation identification and pathogen culture, in particular to a method for culturing mycoplasma synoviae biofilm, and application and a screening method thereof. The method for cultivating the mycoplasma synoviae biofilm comprises the following steps: diluting chicken synovial bursa mycoplasma grown to logarithmic growth phase to (0.9-1.5) × 10 6 CFU/mL, carrying out first cultivation, growing to logarithmic growth phase, removing suspended thalli, and preparing the mycoplasma synoviae biofilm; the liquid culture medium is an improved Frey liquid culture medium, and the judgment standard of the growth to logarithmic phase is that the improved Frey liquid culture medium changes from red to yellow; the incubation temperature for the first incubation was 30-37 ℃. The method can be used for culturing the mycoplasma synoviae biofilm in vitro, so that the data measured in the biological experiment can be more accurate.
Description
Technical Field
The invention relates to the technical field of pathogen separation identification and pathogen culture, in particular to a method for cultivating mycoplasma synoviae (MRM) biofilm, and an application and a screening method thereof.
Background
Infection with Mycoplasma Synoviae (MS) can cause infectious synovitis in some birds, such as chickens, ducks, geese, turkeys, etc., which is characterized by synovial cyst membrane and tendon sheath synovitis with joint exudation. MS infection can also cause respiratory diseases, but is primarily subclinical and thus not readily observable, and can cause airsacculitis if complicated with newcastle disease or infectious bronchitis. In addition, MS infection can also cause abnormalities at the top of the eggshell. Therefore, MS infected chickens often show symptoms of lameness, joint swelling, respiratory inflammation and the like, and further have the consequences of egg laying reduction, growth and development retardation, feed conversion rate reduction and the like.
At present, the prevention and treatment of MS infection mainly depends on antibacterial drugs, but due to unreasonable use of the antibacterial drugs, the sensitivity of the antibacterial drugs to MS is gradually reduced, and at the moment, the requirement on the screening accuracy of the antibacterial drugs is further improved. However, no study has been made on the growth of live chickens for their presence of Mycoplasma synoviae biofilm. However, currently, for the in vitro antibacterial activity screening of mycoplasma synoviae of antibacterial drugs, the mycoplasma synoviae is generally prepared by simply suspension culture, and a growth form of mycoplasma synoviae biofilm is not formed. The cultured mycoplasma synoviae is generally considered to be the mycoplasma synoviae existing in the environment of live chickens, so that the antibacterial activity result of the screened medicine has a great risk of difference from the practical application, and the theoretical dosage can not guide the practical application.
Therefore, a method for cultivating mycoplasma synoviae biofilm, and an application and a screening method thereof are needed to be provided, so that the antibacterial activity result of the screened drug is closer to the practical application.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a culture method of mycoplasma synoviae biofilm, application and a screening method thereof, and the culture method of the mycoplasma synoviae biofilm, the application and the screening method thereof can enable the antibacterial activity result of the screened medicine to be closer to the practical application.
The invention conception of the invention is as follows: the mycoplasma synoviae biofilm is cultivated and prepared by increasing the cultivation stage, selecting proper initial cultivation concentration and selecting a specific range of cultivation temperature.
The first aspect of the invention provides a method for cultivating mycoplasma synoviae biofilm, which comprises the following steps:
a method for culturing mycoplasma synoviae biofilm is characterized by comprising the following steps: diluting chicken synovial bursa branch antigen grown to logarithmic growth phase to (0.9-1.5) × 10 with liquid culture medium 6 CFU/mL, carrying out first cultivation, growing to logarithmic growth phase, removing suspended thalli, and preparing the mycoplasma synoviae biofilm;
the liquid culture medium is an improved Frey's liquid culture medium, and the judgment standard of the growth period to the logarithmic phase is that the improved Frey's liquid culture medium changes from red to yellow;
the incubation temperature for the first incubation was 30-37 ℃.
Preferably, the modified Frey's liquid medium is prepared according to the method for preparing a modified Frey's liquid medium described in "preparation of medium for examination" section three parts of pharmacopoeia of the people's republic of china 2015. The color change principle of the modified Frey's liquid culture medium is that mycoplasma decomposes glucose in the culture medium, so that the pH of the modified Frey's liquid culture medium is reduced from 7.7 to 6.5-6.9, phenol red in the culture medium is changed from red (pH = 7.7) to yellow (pH = 6.5-6.9), and the judgment of growth to logarithmic growth phase is realized.
Compared with the prior art, the method for culturing the mycoplasma synoviae biofilm has the following beneficial effects: currently, mycoplasma synoviae used in vitro biological experiments is usually obtained by isolation culture, subculture and purification culture of joint inflammatory exudates, and a growth form of mycoplasma synoviae biofilm is not formed. The mycoplasma synoviae biofilm is prepared by increasing the cultivation stage, selecting a proper initial cultivation concentration and selecting a cultivation temperature within a specific range, so that data measured in a biological experiment (such as antibacterial activity of a medicament on the mycoplasma synoviae) can be more accurate.
Further preferably, the chicken synovial bursa mycoplasma grown to logarithmic growth phase is diluted to the concentration of 1 × 10 by using a liquid culture medium 6 CFU/mL。
Preferably, the mycoplasma synoviae is prepared by isolated culture, subculture and purified culture.
Preferably, the mycoplasma synoviae is prepared from arthritic exudates of poultry by isolated culture, subculture and purified culture.
Preferably, the isolated culture comprises the following steps: inoculating the arthritic exudates of the poultry to a liquid culture medium, and carrying out second cultivation to logarithmic phase to obtain a bacteria liquid which is subjected to separation cultivation.
Preferably, the environment of the second cultivation is a sealed environment and the temperature is 35-38 ℃; further preferably, the temperature of the second incubation is 37 ℃.
Preferably, the second incubation cultivation vessel comprises at least one of a penicillin bottle, a culture dish, a 96-well plate, a 12-well plate and a conical flask.
Preferably, the purpose of said subculture is to obtain a culture capable of stable subculture; further preferably, the subculture is performed 2 to 10 times; still further preferably, the number of times of subculture is 3 to 5; still more preferably, the subculture is performed 3 times.
Preferably, the subculture comprises the steps of: separating thallus from the bacteria liquid after separation culture, inoculating 10-50% of thallus into a new liquid culture medium for culture, and growing to logarithmic phase.
Preferably, the purification culture comprises the following steps: diluting the bacteria liquid which is subjected to subculture and grows to the logarithmic growth phase to be 800-1500 times, taking 10-100 mu L of the bacteria liquid to be in a solid culture medium, performing third culture, taking the bacteria liquid culture medium of a single colony to perform second culture after the egg-fried bacteria liquid appears, and preparing the mycoplasma synoviae when the bacteria liquid grows to the logarithmic growth phase; further preferably, the dilution factor is 900-1100; even more preferably, the dilution factor is 1000-fold.
Preferably, the solid culture medium is prepared by applying agar powder to the liquid culture medium; further preferably, the agar powder is applied in an amount of (0.5-1.5) wt% of the liquid medium; still more preferably, the agar powder is applied in an amount of 0.9 to 1.1wt% of the liquid medium.
Preferably, the culturing conditions for the third culturing are 30-40 ℃ and 4-8% CO 2 The volume percentage concentration and the incubation time are 3-7 days; further preferably, the CO is 2 The volume percentage concentration is 5%.
Preferably, the mycoplasma synoviae is further subjected to freeze-drying preservation and resuscitation, wherein the freeze-drying preservation temperature is 0-80 ℃; further preferably, the temperature of freeze-drying preservation is-50 to-80 ℃; still more preferably, the temperature for freeze-drying preservation is from-70 to-80 ℃.
Preferably, the freeze-drying preservation uses freeze-drying liquid, and the freeze-drying liquid is prepared by mixing skimmed milk powder, cane sugar and water and sterilizing at high temperature and high pressure.
Preferably, the freeze-drying preservation and recovery are performed before the chicken synovial bursa mycoplasma growing to the logarithmic growth phase is diluted by a liquid culture medium, after the subculture and purification culture, the freeze-drying preservation is performed firstly, and then the recovery is performed.
Preferably, the incubation environment of the first incubation is a sealed environment.
Preferably, in the cultivation method, the cultivation temperature of the first cultivation is selected to be 30-31 ℃ when the maximum biofilm size is required, and the cultivation temperature of the first cultivation is selected to be 32-34 ℃ when the maximum biofilm biomass is required; still more preferably, in the cultivation method, the cultivation temperature for the first cultivation is selected to be 30 ℃ when the maximum biofilm size is required and 33 ℃ when the maximum biofilm biomass is required. In the invention, the first cultivation is used for cultivating the mycoplasma synoviae biofilm, the second cultivation is used for separating and culturing the mycoplasma synoviae, and the third cultivation is used for purifying and culturing.
Preferably, the first incubated cultivation vessel comprises at least one of a 12-well plate, a 96-well plate, a penicillin bottle, a petri dish, and a conical flask.
Preferably, the step of removing suspended cells comprises: the suspension in the medium was washed with buffer.
Preferably, in the step of removing suspended bacteria, the buffer is a phosphate buffer; further preferably, the phosphate buffer has a pH of 6.5 to 7.5 and a concentration of 0.05 to 0.15M.
Preferably, the mycoplasma synoviae biofilm is also subjected to staining observation, and the staining method for the staining observation is Congo red.
Preferably, the dyeing method comprises the following steps:
(1) cleaning: the mycoplasma synoviae biofilm is cleaned in a buffer solution and naturally dried;
(2) fixing: fixing the washed mycoplasma synoviae biofilm for 5-20min, and naturally drying;
(3) dyeing: placing the fixed mycoplasma synoviae biofilm into Congo red staining solution for staining for 10-20 min;
(4) washing: and (3) putting the dyed mycoplasma synoviae biofilm into distilled water for cleaning and naturally drying.
Preferably, in the step (1) of the staining method, the buffer solution is phosphate buffer solution, and the concentration of the phosphate buffer solution is 0.05-0.5mol/L, pH and is 6.5-7.5; further preferably, the concentration of the phosphate buffer solution is 0.1mol/L, pH of 7.
Preferably, in the step (2) of the dyeing method, the reagent used for the immobilization is methanol; further preferably, the methanol is anhydrous methanol.
Preferably, in the step (2) of the dyeing method, the fixed time is 5-15 min; further preferably, the fixed time is 9-11 min.
Preferably, in the step (3) of the dyeing method, the congo red dyeing solution contains (0.5-1.5)% by mass of congo red; more preferably, the mass percentage of the congo red in the congo red dyeing solution is 0.9-1.1%.
Preferably, in the step (3) of the dyeing method, the dyeing time is 13-17 min; further preferably, the dyeing time is 15 min.
A second aspect of the present invention provides a drug screening method comprising the steps of: the mycoplasma synoviae biofilm prepared by the cultivation method of the medicine is used for carrying out an antibacterial activity test.
Preferably, the medicament comprises at least one of enrofloxacin, doxycycline, tylosin and tiamulin.
The third aspect of the invention provides an application of the mycoplasma synoviae biofilm prepared by any one of the cultivation methods in drug screening.
Compared with the prior art, the invention has the following beneficial effects:
(1) currently, mycoplasma synoviae used in vitro biological experiments is usually obtained by isolation culture, subculture and purification culture of joint inflammatory exudates, and a growth form of mycoplasma synoviae biofilm is not formed. The method can be used for culturing the mycoplasma synoviae biofilm in vitro, so that data (such as antibacterial activity of the medicine on the mycoplasma synoviae) measured in a biological experiment can be more accurate.
(2) At present, the color of the biofilm after crystal violet staining is obvious, but in the test process, the crystal violet staining is found to be deviated due to the state of the staining solution and the staining method, and can not reach a uniform color. Congo red dyeing is based on the combination with a polysaccharide component. The Congo red is generally stained by adding Congo red to a solid medium, and the depth of Congo red staining varies according to the content of polysaccharide components in colonies growing on the solid medium, and the color gradually changes from red to black as the content of polysaccharide components in the colonies increases ("Modification of the colony red method to detection bio-resistant microorganism by Staphylococcus epidermidis", DOI: 10.1016/j.diagnostic microbio.2012.11.014). Currently, there is no relevant literature showing direct biofilm staining using congo red. In the invention, Congo red directly dyes the mycoplasma synoviae biofilm of chicken, so that the biofilm is stable and uniform in dyeing effect. Moreover, the Congo red staining solution is easy to prepare and can be prepared for use.
(3) The biomass varied very significantly with temperature change (p < 0.01), with a higher biomass at a cultivation temperature of 33 ℃ and a larger Biofilm (BF) size at a cultivation temperature of 30 ℃.
Drawings
FIG. 1 is a microscopic view of a single colony of the MS isolate in step (2) of example 1;
FIG. 2 is a scanning electron micrograph of Mycoplasma synoviae biofilm in step (3) of example 1;
FIG. 3 is a microscopic picture of crystal violet staining of MS biofilm growth at different culture temperatures;
FIG. 4 shows the result of MS biofilm staining;
FIG. 5 is a graph comparing the change in drug sensitivity before and after growth of MS biofilms.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
The antibacterial agents used in the present invention: enrofloxacin sodium (active ingredient content 92.1%, lot number: DK06-2011022, Guangdong Huachen pharmaceutical Co., Ltd.), doxycycline hydrochloride (active ingredient content 92.3%, lot number: A202012066, Guangdong Wenzhong Dahua nong Biotech Co., Ltd.), tylosin tartrate (active ingredient content 80.45%, lot number: 01105010008, Shandong Lukang Sheerle pharmaceutical Co., Ltd.), and tiamulin fumarate (active ingredient content 80.48%, lot number: 402070010771, Guangdong Wenzhong Dahua nong Biotech Co., Ltd.).
The preparation method of the improved Frey's liquid culture medium comprises the following steps: the preparation was carried out according to the preparation method of modified Frey's liquid medium described in "preparation of culture Medium for examination" section three of pharmacopoeia of the people's republic of China 2015 edition. After the preparation is finished, the product is stored in a refrigerator at 4 ℃ and is used within 1 month.
The preparation method of the solid culture medium comprises the following steps: adding 1wt% of agar powder on the basis of the improved Frey's liquid culture medium, cooling to 60 ℃ after autoclaving, and pouring the culture medium into a disposable sterile culture dish in an ultra-clean workbench. After the agar is cooled and solidified, the agar is packaged, sealed and placed in a refrigerator at 4 ℃ for storage and is used within 1 month.
The preparation of the freeze-drying solution of the invention: adding skimmed milk powder and sucrose into distilled water, mixing, dissolving, and autoclaving. Cooling, sealing, storing in 4 deg.C refrigerator, and using within 1 month.
The preparation of the dyeing liquid of the invention: the crystal violet staining solution of 1wt% was a commercial finished staining solution. When preparing the 1wt% Congo red staining solution, firstly weighing 1g of Congo red, adding the Congo red into a beaker, adding 100mL of distilled water or 0.1M phosphate buffer (pH7.0) and ultrasonically mixing the Congo red and the phosphate buffer, pouring the mixture into a brown bottle after the preparation is finished, and storing the mixture at normal temperature for use within 1 month.
The standard strain of MS (Mycoplasma synoviae) in the present invention is WVU1853, and was awarded by Gansu university of agriculture.
PCR amplification in the inventionProgram-increasing reference TaKaRa Taq TM The instructions of Version 2.0 plus dye (TaKaRa Bio Inc) were set up as follows: firstly, pre-denaturation is carried out for 2min at 94 ℃; ② melting at 94 ℃ for 30s, annealing at 52 ℃ for 30s and extending at 72 ℃ for 32s, and 35 cycles in total; extension at 72 deg.C for 5 min. The primer sequence is as follows: upstream primer 5'-GGCCATTGCTCCTRCTGTTAT-3' (SEQ ID NO: 1); downstream primer 5'-AGTAACCGATCCGCTTAATGC-3' (SEQ ID NO: 2) (primer synthesized by Biotechnology engineering (Shanghai) Co., Ltd.). The PCR reaction system was prepared in 50. mu.L: TaKaRa Taq TM Version 2.0 plus dye 25. mu.L, upstream primer (10. mu.M) 1.5. mu.L, downstream primer (10. mu.M) 1.5. mu. L, ddH 2 O17. mu.L, and 5. mu.L of a bacterial solution.
Example 1
The separation of the mycoplasma synoviae and the cultivation of the mycoplasma synoviae biofilm comprise the following steps:
(1) and (3) MS separation culture: 148 sick chickens suspected of being MS infected and found in different areas were killed by exsanguination from the neck, amputated from the femoral joint, sterilized with 75% alcohol and labeled, and samples such as arthritic exudates were collected in a clean bench and inoculated into penicillin bottles containing 3mL of modified Frey's broth. And covering a bottle stopper after inoculation is finished, simultaneously setting a blank control group, sealing a penicillin bottle by using a sealing film, culturing at 37 ℃, and immediately performing subculture when the color of the culture medium is changed from red to yellow or orange yellow to obtain a culture capable of being subjected to stable subculture.
(2) And (3) MS purification: the MS bacterial liquid growing to logarithmic phase is diluted by 1000 times. 50. mu.L of the diluted cell suspension was uniformly applied to a solid medium with a coating rod. After the operation, the plate was sealed with a sealing film at 37 ℃ with 5% CO 2 After egg-shaped colonies grow on the solid culture medium, selecting a single colony to an improved Frey liquid culture medium for culture (third culture), subculturing the culture medium again when the culture medium turns yellow from red, continuously carrying out subculturing for 3 times to obtain an MS bacterial liquid growing to a logarithmic growth phase, and identifying, typing and naming the extracted purified bacterial strains to finally obtain 29 MS isolates.
(3) Culturing chicken mycoplasma synoviae biofilm: diluting MS bacterial liquid growing to logarithmic phase to 10 degree by using modified Frey's liquid culture medium 6 CFU/mL is ready for use. Placing sterilized 20mm round cover glass into 12-well plate, adding 1mL, and diluting to 10% 6 Preparing a culture system by using CFU/mL bacterial liquid, meanwhile, using a blank culture medium as a negative control, sealing, culturing at 37 ℃ (first culture), and taking out a cover glass when the culture medium turns yellow from red to obtain the mycoplasma synoviae biofilm.
In the invention, the specific steps of identifying and typing are as follows: in the test, the separated vlhA (variable lipoprotein haemagglutinin A) genes of 29 MS isolates are subjected to PCR amplification, and amplification products are subjected to sequencing and splicing and then are compared on GenBank according to gene sequences corresponding to GenBank accession numbers to determine the genotypes of the amplification products.
In the present invention, the names, the separation region, the separation year, the genotype, the GenBank accession number corresponding to the names of 29 MSs are shown in Table 1 below for the details of MS isolates and the results of typing.
TABLE 1 MS isolate details and typing results
The bacterial count of the MS bacterial liquid grown to the logarithmic phase in the step (2) of example 1 was calculated: diluting the bacterial liquid growing to logarithmic phase for 5 times according to 10 times gradient, sucking 10 μ L of the final 2 gradient bacterial liquid, dripping onto solid culture medium, sealing, and adding 5% CO at 37 deg.C 2 Until colonies visible to the naked eye grow. The counting gradient is selected according to the colony density grown from the last 2 gradients, the colony density cannot be too large or too small, and each group of experiments is repeated for 3 times. The amount of bacteria calculated by this method was CFU (Colony forming units), and the number of MS in the bacteria solution grown to the logarithmic phase in step (2) of example 1 was 10 in terms of CFU/mL 8 Of the order of CFU/mL.
FIG. 1 is a microscopic view (40-fold magnification) of a single colony of the MS isolate in step (2) of example 1; the colony formed by the separated MS isolate on a solid medium can be observed in a typical 'poached egg' shape under a microscope.
FIG. 2 is a scanning electron micrograph of Mycoplasma synoviae biofilm in step (3) of example 1; from this it is clear that the Biofilm (BF) typically has a complex structure of Extracellular Polymers (EPS) encapsulating bacteria. The whole structure of the MS biofilm can be seen from A of FIG. 2, and the MS biofilm is closely arranged and overlapped to form a certain thickness; FIG. 2B is a photograph taken at a magnification of the upper right notch of the entire Biofilm (BF) in FIG. 2A, in which the channel structure of the Biofilm (BF) can be seen.
Comparative example 1
Isolation and cultivation of Mycoplasma synoviae I
Comparative example 1 differs from example 1 in that: comparative example 1 did not have step (3).
Comparative example 2
Isolation and cultivation of common mycoplasma synoviae II
Comparative example 2 differs from example 1 in that: comparative example 2 step (3) the procedure was the same as in example step (2).
The initial concentration of mycoplasma synoviae is not (0.9-1.5) × 10 in the breeding process 6 CFU/mL (initial concentration of 10) 5 CFU/mL order of magnitude or less), still only common mycoplasma synoviae were obtained.
Example 2
Separation of mycoplasma synoviae and cultivation of mycoplasma synoviae biofilm
Example 2 differs from example 1 in that: example 2 was also freeze-dried for storage; the method specifically comprises the following steps:
(1) and (3) MS separation culture: 148 sick chickens suspected of being MS infected and found in different areas were killed by exsanguination from the neck, amputated from the femoral joint, sterilized with 75% alcohol and labeled, and samples such as arthritic exudates were collected in a clean bench and inoculated into penicillin bottles containing 3mL of modified Frey's broth. And covering a bottle stopper after inoculation is finished, simultaneously setting a blank control group, sealing a penicillin bottle by using a sealing film, culturing at 37 ℃, and immediately performing subculture when the color of the culture medium is changed from red to yellow or orange yellow to obtain a culture capable of being subjected to stable subculture.
(2) And (3) MS purification: the MS bacterial liquid growing to logarithmic phase is diluted by 1000 times. 50. mu.L of the diluted cell suspension was uniformly applied to a solid medium with a coating rod. After the operation, the plate was sealed with a sealing film at 37 ℃ with 5% CO 2 Culturing under the condition of (1), after a fried egg-shaped strain grows on a solid culture medium, selecting a single colony to an improved Frey's liquid culture medium for culturing (third culturing), subculturing the culture medium again when the culture medium turns yellow from red, continuously carrying out subculturing for 3 times to obtain an MS bacterial liquid growing to a logarithmic phase, and naming, identifying and typing 29 extracted MSs.
(3) Freeze-drying preservation of MS: and adding the purified MS bacterial liquid which grows to the logarithmic phase into the freeze-dried liquid according to the proportion of 1:1, uniformly mixing, and pre-freezing the mixture. Taking out the frozen sample, immediately freezing and drying for 24h by using a vacuum freeze dryer, taking out the sample after the operation is finished, sealing well, and freezing and storing in a refrigerator at the temperature of minus 80 ℃.
(4) And (3) recovery of MS: adding 3mL of MS liquid culture medium into a penicillin bottle, adding a proper amount of MS freeze-dried powder stored in a refrigerator at the temperature of-80 ℃, and simultaneously setting a blank control group. After the operation is finished, a bottle stopper is covered and sealed by a sealing film, the culture is carried out at the temperature of 37 ℃, when the culture medium is observed to change from red to yellow, the MS is shown to grow to the logarithmic phase, and the MS bacterial liquid growing to the logarithmic phase is prepared.
(5) Culturing chicken mycoplasma synoviae biofilm: diluting MS bacterial liquid growing to logarithmic phase to 10 degree by using improved Frey's liquid culture medium 6 CFU/mL is ready for use. The sterilized 20mm round cover glass is placed in a 12-well plate, and then 1mL of the cover glass is added to dilute the cover glass to 10 6 CFU/mL bacterial solution, and culturingAnd (3) simultaneously using a blank culture medium as a negative control, sealing, culturing at 37 ℃ (first culture), and taking out a cover glass when the culture medium turns yellow from red to prepare the mycoplasma synoviae biofilm.
Example 3
The influence of temperature on the growth of MS biofilm specifically comprises the following steps:
(1) biofilm (BF) culture at different temperatures: the 96-well plate is added with modified Frey's liquid culture medium to be diluted to 10 6 CFU/mL standard strain WVU185 bacterial solution, and simultaneously using blank culture medium to establish a control group, respectively culturing at 25 deg.C, 30 deg.C, 33 deg.C, 37 deg.C, and 40 deg.C, taking out when the culture medium turns from red to yellow, and repeating each group of experiments for 3 times.
(2) Biofilm (BF) biomass count: the BF-grown 96-well plate was taken out, the medium was aspirated in a clean bench and washed 2 to 3 times with sterile 0.1M phosphate buffer (pH 7.0) to remove residual medium and suspended bacteria. Adding 200 μ L fresh culture medium (modified Frey's liquid culture medium) after washing, repeatedly blowing and beating MS dispersed in BF with micropipette, diluting the dispersed bacterial liquid with modified Frey's liquid culture medium for 4 times in 10-fold gradient, sucking 10 μ L of each gradient and dropping onto solid culture medium, sealing, and adding 5% CO at 37 deg.C 2 Culturing for 3-7 days until macroscopic colonies grow. The gradient of counts was selected based on the density of colonies grown from the 4 gradients, and each set of experiments was repeated 3 times.
(3) And (3) data analysis: experimental data statistical analysis was performed on the data using the One-Way ANOVA method using SPSS 22.0 and the effect of different culture temperatures on MS biofilm growth is shown in table 2 below.
TABLE 2 Effect of different culture temperatures on MS biofilm growth
No growth means that the culture medium does not change from red to yellow over a longer period of time when the biofilm is cultured.
The results obtained in Table 2 were statistically analyzed to show that the biomass varied very significantly (p < 0.01) with temperature changes, with the highest biomass at 33 ℃ and the biomass being close to but inferior to the results at 33 ℃ at 30 ℃ and 37 ℃ and with no results at 25 ℃ and 40 ℃.
FIG. 3 is also a photograph which shows the growth of MS biofilm at different culture temperatures, and FIG. 3 is a microscope photograph (40 times magnification) of the crystal violet staining; it is apparent from 33 ℃ in FIG. 3 that BF was significantly denser at a culture temperature of 33 ℃ and the difference in the overall amount of BF was not large between 30 ℃ (30 ℃ in FIG. 3 and 37 ℃ (37 ℃ in FIG. 3), but the size of BF was larger at a culture temperature of 30 ℃.
Example 4
A method of staining biofilms, comprising the steps of:
(1) cleaning: the round coverslip on which Biofilm (BF) growth was completed in step (3) of example 1 was taken out, washed from the culture medium and suspended cells in 0.1M phosphate buffer (pH = 7.0), and then dried.
(2) Fixing: and (3) putting the cleaned round cover glass into absolute methanol for fixing for 10min, and taking out and drying.
(3) Dyeing: and (3) putting the fixed round cover glass into 1wt% of crystal violet staining solution or 1wt% of Congo red staining solution respectively for staining for 15 min.
(4) Washing: after dyeing, the cloth is put into distilled water to be washed off with loose color gently, and finally the cloth is dried.
(5) After completion of BF staining, the round cover glass was placed under an optical microscope for observation (with the face on which BF was grown facing upward).
Fig. 4 shows the MS biofilm staining results (magnification of 100 times), and the stained Biofilm (BF) in a lump or in a dotted state was observed under an optical microscope, and the crystal violet stain (a) in fig. 4) was darker than the congo red stain (B) in fig. 4).
At present, the color of the biofilm after crystal violet staining is obvious, but in the test process, the crystal violet staining is found to be deviated due to the state of the staining solution and the staining method, and can not reach a uniform color. Congo red dyeing is based on the combination with a polysaccharide component. Currently, Congo red is generally stained by adding Congo red into a solid medium, the depth of Congo red staining varies according to the content of polysaccharide components in colonies grown on the solid medium, and the color gradually changes from red to black as the content of polysaccharide components in the colonies increases ("Modification of the Congo red marker method to detect biological microorganism production by bacterial microorganisms", DOI: 10.1016/j.diagnostically microorganism 2012.11.014), and no relevant literature shows that the biofilm is directly stained by using Congo red. In the invention, Congo red directly dyes the mycoplasma synoviae biofilm of chicken stably, and the dyeing effect is uniform. Moreover, the Congo red staining solution is easy to prepare and can be prepared for use.
Example 5
Drug screening method 1: in example 1, 29 MS isolates and MS standard strains (WVU 1853) were subjected to MIC (minimum inhibitory concentration) and MBIC (minimum biofilm inhibitory concentration) measurements (it should be noted that MIC and MBIC measured in this test were both measured in vitro):
(1) culturing MS biofilm: adding 200 mu L of modified Frey's liquid culture medium into 1-11 wells of a 96-well plate respectively to dilute the solution to 10 6 CFU/mL of the bacterial liquid of 29 MS isolates and the bacterial liquid of an MS standard strain (WVU 1853) prepared in the step (2) of example 1, meanwhile, the 12 th hole is subjected to negative control by using a blank culture medium, the culture is carried out under the condition of 33 ℃ after sealing, the subsequent tests are carried out when the culture medium turns yellow from red, and each group of tests is repeated for 3 times;
(2) determination of Minimum Biofilm Inhibitory Concentration (MBIC): and (3) placing the BF-grown 96-well plate into a super clean bench, sucking out the culture medium, and then gently washing the plate for 2-3 times by using 0.1M phosphate buffer (pH = 7.0) for later use. Another 96-well plate is taken, the antibacterial drug is diluted to a proper concentration in 1-10 holes by 2-time gradient of a fresh culture medium (an improved Frey liquid culture medium), 220 mu L of the antibacterial drug is prepared in each hole, and the enrofloxacin is diluted to the concentration range of 64-0.125 mu g/mL; doxycycline and tylosin are diluted to the concentration range of 6.4-0.0125 mug/mL; tiamulin is diluted to the concentration range of 0.64-0.00125 mu g/mL. Adding the diluted 1-10-hole antibacterial agent into 1-10 holes of the washed 96-hole plate correspondingly, adding 200 μ L of the antibacterial agent into each hole, adding 200 μ L of fresh culture medium (modified Frey's liquid culture medium) into the 11 th hole and the 12 th hole respectively to serve as a positive control and a negative control, culturing at 37 ℃ after sealing, and repeating each group of experiments for 3 times. Observing color change every day after the operation is finished, recording the result when the positive control group changes from red to yellow, and when the color of the hole site is not changed after the test group exceeds 2 days, determining the drug concentration of the last hole site which does not change color as the MBIC value of the antibacterial drug, and recording the data in the following table 3;
(3) determination of Minimum Inhibitory Concentration (MIC): adding 100 μ L of antibacterial liquid 2 times diluted with fresh culture medium (modified Frey's liquid culture medium) into 1-10 wells of 96-well plate, and diluting to 10 μ L 6 CFU/mL of the culture of 29 MS isolates, MS standards (WVU 1853) prepared in step (2) of comparative example 1 (same as step (2) of example 1), 200. mu.L of the culture at 11 th well having the same turbidity as that of the test group was used as a positive control, 200. mu.L of the blank medium at 12 th well was used as a negative control, the culture was sealed and incubated at 37 ℃ and each test was repeated 3 times. According to the method, the enrofloxacin is diluted to the concentration range of 32-0.0625 mug/mL; diluting doxycycline and tylosin to a concentration range of 3.2-0.00625 mug/mL; tiamulin is diluted to the concentration range of 0.32-0.000625 mug/mL; the amount of MS was finally diluted to 5X 10 5 CFU/mL. And observing color change every day after the operation is finished, recording the result when the color of the hole site of the positive control group is changed from red to yellow, and recording the data in the following table 3, wherein the drug concentration of the last hole site which does not change the color is the MIC value of the antibacterial drug when the color of the hole site of the test group does not change for more than 2 days.
TABLE 3 Change in drug sensitivity before and after growth of MS biofilm
In Table 3, the units of MIC and MBIC are both μ g/mL.
FIG. 5 is a graph comparing the change in drug sensitivity before and after the growth of MS biofilm (ratio of MBIC to MIC), and for enrofloxacin (A) of FIG. 5, 63.3% (19/30) of the strains had an increase in the ratio of MBIC to MIC of more than 2-fold and more after biofilm formation, with an average increase of 1.83-fold; for doxycycline (B) of fig. 5, 63.3% (19/30) of the strains had an increase in the ratio of MBIC to MIC after biofilm formation of more than 2-fold and more, with an average increase of 1.79-fold; for tylosin (C) of fig. 5, 70% (21/30) of the strains showed an increase in the ratio of MBIC to MIC after biofilm formation of more than 2-fold and more, with an average increase of 4.24-fold; for tiamulin (D) of fig. 5, 80% (24/30) of the strains showed an increase in the ratio of MBIC to MIC after biofilm formation of more than 2-fold and more, with an average increase of 2.7-fold.
Example 6
Drug screening method 2: correlation analysis of MBIC and MS biofilm biomass
(1) MS biofilm culture prior to biomass determination: the 96-well plate is added with a diluted solution of 200. mu.L to 10 with modified Frey's liquid medium 6 CFU/mL of the culture of 29 MS isolates prepared in the step (2) of example 1 and the culture of MS standard strain (WVU 1853) were used as negative controls in a blank medium, and after sealing, the culture was incubated at 37 ℃ until the medium turned yellow from red, and the subsequent tests were repeated 3 times.
(2) Biomass was determined by plate counting: diluting the bacterial liquid grown to logarithmic phase in the step (1) for 5 times according to 10-fold gradient, sucking 10 mu L of the bacterial liquid of the last 2 gradients, dripping the bacterial liquid onto a solid culture medium, sealing, and then carrying out 5% CO treatment at 37 DEG C 2 Until colonies visible to the naked eye grow. The counting gradient is selected according to the density of the colonies growing from the last 2 gradients, the colony density cannot be too high or too low, and each group of experiments is repeated for 3 times. The amount of bacteria calculated by this method is the Colony Forming Units (CFU), unitThe biomass measurement data is reported in Table 4 below as CFU/mL.
(3) The experimental data were statistically analyzed using SPSS 22.0 and the correlation between MBIC and MS biofilm biomass in example 5 was examined using Pearson correlation analysis, the results of which are shown in table 5 below.
TABLE 4 results of biomass measurement
The biomass of the MS isolate BF determined was mostly 10 6 ~10 7 Few clinical strains of MS BF have a biomass of 10 5 The biomass of BF of different MS clinical isolates has great difference which can be up to 2 orders of magnitude different.
TABLE 5 Pearson correlation analysis of MBIC with BF Biomass
Correlation between MBIC and biomass was examined in the software SPSS 22 using Pearson correlation analysis and the results are summarized in table 5. There is a significantly low negative correlation between MBIC and BF biomass for enrofloxacin (r <0, 0.3 ≦ r | <0.5, P < 0.05), and there was no correlation between MBIC and BF biomass for the other 3 antibacterials.
Example 7
Example 7 differs from example 1 in that: in the step (3), the MS bacterial liquid growing to the logarithmic growth phase is diluted to the concentration of 0.9 multiplied by 10 by using an improved Frey's liquid culture medium 6 CFU/mL. Example 7 also enables the development of mycoplasma synoviae biofilms.
Example 8
Example 8 differs from example 1 in that: in the step (3), the MS bacterial liquid growing to the logarithmic growth phase is diluted to the concentration of 1.5 multiplied by 10 by using an improved Frey's liquid culture medium 6 CFU/mL. Example 8 can also be carried outAnd (5) breeding the mycoplasma synoviae biofilm.
SEQUENCE LISTING
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<120> cultivation method of mycoplasma synoviae biofilm, application and screening method thereof
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Claims (8)
1. A method for culturing mycoplasma synoviae biofilm is characterized by comprising the following steps: diluting chicken synovial bursa branch antigen grown to logarithmic growth phase to (0.9-1.5) × 10 with liquid culture medium 6 CFU/mL, carrying out first cultivation, growing to logarithmic growth phase, removing suspended thalli, and preparing the mycoplasma synoviae biofilm; the first cultivation environment is a sealed environment;
the liquid culture medium is an improved Frey's liquid culture medium, and the judgment standard of the growth period to the logarithmic phase is that the improved Frey's liquid culture medium changes from red to yellow;
the cultivation temperature of the first cultivation is 30-37 ℃;
the mycoplasma synoviae biofilm is also subjected to dyeing observation, and Congo red is used as a dyeing method for the dyeing observation.
2. The method of claim 1, wherein the Mycoplasma synoviae is produced by isolation culture, subculture, and purification culture.
3. The culture method according to claim 1, wherein the mycoplasma synoviae is further subjected to freeze-drying preservation and resuscitation, and the temperature of freeze-drying preservation is 0-80 ℃.
4. Method of incubation according to claim 1, characterized in that the incubation temperature of the first incubation is 32-34 ℃.
5. The method of culturing according to claim 1, wherein the step of removing suspended bacteria comprises: and (3) washing suspended bacteria in the culture medium by using a buffer solution, wherein the buffer solution is a phosphate buffer solution, and the pH value of the phosphate buffer solution is 6.5-7.5, and the concentration of the phosphate buffer solution is 0.05-0.15M.
6. A cultivation method as claimed in claim 1, characterised in that the staining method comprises the following steps:
(1) cleaning: the mycoplasma synoviae biofilm is cleaned in a buffer solution and naturally dried;
(2) fixing: fixing the washed mycoplasma synoviae biofilm for 5-20min, and naturally drying;
(3) dyeing: placing the fixed mycoplasma synoviae biofilm into Congo red staining solution for staining for 10-20 min;
(4) washing: and (3) putting the dyed mycoplasma synoviae biofilm into distilled water for cleaning and naturally drying.
7. A drug screening method, characterized in that it comprises the following steps: performing an antibacterial activity test on the mycoplasma synoviae biofilm prepared by the cultivation method according to any one of claims 1-6 by using a medicament; the medicine is at least one of enrofloxacin, doxycycline, tylosin and tiamulin.
8. The use of said mycoplasma synoviae biofilm prepared by the method of any one of claims 1-6 in drug screening; the medicine is at least one of enrofloxacin, doxycycline, tylosin and tiamulin.
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