CN111254100A - Pseudomonas aeruginosa biofilm and preparation method and application thereof - Google Patents
Pseudomonas aeruginosa biofilm and preparation method and application thereof Download PDFInfo
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- CN111254100A CN111254100A CN202010234076.6A CN202010234076A CN111254100A CN 111254100 A CN111254100 A CN 111254100A CN 202010234076 A CN202010234076 A CN 202010234076A CN 111254100 A CN111254100 A CN 111254100A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention belongs to the technical field of macromolecules, and discloses a pseudomonas aeruginosa biomembrane and a preparation method and application thereof, wherein bacteria are inoculated on an agar plate, a single colony is selected and inoculated in 5mLLB culture solution, the bacteria solution is centrifuged after shaking, the supernatant is discarded, the bacteria precipitate is washed by sterile normal saline, and the bacteria suspension is adjusted by normal saline to be ready for use; 10ml of the prepared experimental strain liquid is added into a 50ml conical flask, 10 carriers are added, and the bacteria are adhered by shaking culture at the constant temperature of 37 ℃ and 110rpm for 20 hours. And (3) culturing the pseudomonas aeruginosa biofilm structure in vitro, adding a carrier into the adjusted concentration of the bacterial suspension to culture the biofilm structure, and then detecting and adjusting the bacterial concentration. The bacterial biofilm has stable structure and good repeatability. The prepared carrier is transplanted into the abdominal cavity of a mouse to cause pseudomonas aeruginosa biofilm infection, and then the pathogenic mechanism and the mild moxibustion intervention treatment effect of the pseudomonas aeruginosa biofilm infection are researched.
Description
Technical Field
The invention belongs to the technical field of macromolecules, and particularly relates to a pseudomonas aeruginosa biomembrane, and a preparation method and application thereof.
Background
The present invention relates to a method for treating chronic infectious diseases, such as chronic infectious bronchitis, cystic fibrosis, chronic urethral infection and the like, wherein the prior art commonly used in the industry is that bacterial Biofilm (BF) related infection is a disease which widely exists and relates to multidisciplinary science, BF means that bacteria are adsorbed on the surface of a solid substance (such as medical organic materials or the surface of body mucous membrane), polysaccharide-protein complexes such as polysaccharide matrix, fibrin, lipoprotein and the like are secreted, the bacteria are mutually adhered, the bacteria are wrapped in the biofilm, so that a compact membrane-like state is formed, three-dimensional aggregates of the bacteria are not known, the clinical infection related to the bacterial biofilm is increased gradually along with the wide use of biomedical materials, according to statistics, 65% of human bacterial infection is related to the biofilm, wherein Pseudomonas aeruginosa (Pseudomonas aeruginosa is one of the very common bacterial BF related infectious pathogenic bacteria) is one of the chronic intractable infectious pathogenic bacteria BF related to BF, many chronic infection which is difficult to treat, such as chronic panbronchiolitis, cystic fibrosis, chronic urethral infection, the chronic infectious disease is closely related to BF forming a plurality of antibiotic, the antibiotic is widely used for treating infectious disease, the antibiotic.
In summary, the problems of the prior art are as follows: the first western medicine antibiotic resistance phenomenon of pseudomonas aeruginosa biofilm infection has certain curative effect with the treatment of traditional Chinese medicine, but the treatment is difficult to completely cure, the economic burden is large, and dysbacteriosis, liver and kidney damage and the like can be caused.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a pseudomonas aeruginosa biomembrane and a preparation method and application thereof.
The invention is realized in such a way that the preparation method of the pseudomonas aeruginosa biomembrane comprises the following steps:
step one, bacteria are inoculated on an agar plate, a single colony is selected and inoculated in 5mLLB culture solution at 37 ℃ and 200rmin-1After shaking for 20h, 3000rmin of bacterial liquid is added-1Centrifuging for 10min, removing supernatant, washing bacterial precipitate with sterile normal saline for 2 times, and adjusting OD600 of bacterial suspension with normal saline to 0.05 bacterial liquid;
and step two, adding 10ml of the prepared experimental strain bacterial liquid into a 50ml conical flask, adding 10 carriers, and performing constant-temperature shaking culture at 37 ℃ and 110rpm for 20 hours to enable the bacteria to be adhered.
The invention also aims to provide the pseudomonas aeruginosa biomembrane prepared by the preparation method of the pseudomonas aeruginosa biomembrane.
The invention also aims to provide a medicament for treating chronic refractory diseases prepared from the pseudomonas aeruginosa biomembrane.
In summary, the advantages and positive effects of the invention are: and (3) culturing the pseudomonas aeruginosa biofilm structure in vitro, adding a carrier into the adjusted concentration of the bacterial suspension to culture the biofilm structure, and then detecting and adjusting the bacterial concentration. The bacterial biofilm has stable structure and good repeatability. The prepared carrier is transplanted into the abdominal cavity of a mouse to cause pseudomonas aeruginosa biofilm infection, and then the pathogenic mechanism and the mild moxibustion intervention treatment effect of the pseudomonas aeruginosa biofilm infection are researched. In recent years, people pay more and more attention to the clinical application of traditional Chinese medicine and criticize some pseudo-sciences. The curative effects of traditional Chinese medicine treatment such as moxibustion, acupuncture, cupping and massage are well accepted by people, and the traditional Chinese medicine treatment is popular with people all over the world after going out of China. The traditional Chinese medicine treatment has unique curative effect on chronic intractable diseases with poor curative effect of western medicine, the treatment cost is very low, the pain in the treatment process is small, the effect is fast, and the traditional Chinese medicine treatment is easy to accept by patients. A large number of clinical and experimental researches find that the moxibustion has the function of obviously improving the cellular immunity of the organism, the cellular immunity of the organism is often low in the infection process of the pseudomonas aeruginosa biomembrane, and the cellular immunity is obviously related to the prognosis. Intervention of such treatment regimens therefore provides new, more clinically significant therapeutic directions for the treatment of such diseases.
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FIG. 1 is a flow chart of a method for preparing a Pseudomonas aeruginosa biofilm according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
As shown in fig. 1, the preparation method of the pseudomonas aeruginosa biofilm provided by the embodiment of the invention comprises the following steps:
s101: inoculating bacteria on agar plate, selecting single colony, inoculating in 5mLLB culture solution at 37 deg.C, 200rmin-1After shaking for 20h, 3000rmin of bacterial liquid is added-1Centrifuging for 10min, removing supernatant, washing the bacterial precipitate with sterile normal saline for 2 times, adjusting OD600 of bacterial suspension with normal saline to 0.05 bacterial liquid for use, and obtaining the bacteria which are the planktonic bacteria of P.aeruginosa;
s102: 10ml of the prepared experimental strain liquid is added into a 50ml conical flask, then 10 carriers are added, and the bacteria are adhered by shaking culture at the constant temperature of 37 ℃ and 110rpm for 20 h.
Smearing the smear after the preparation is finished in the step S102, and dyeing and observing the condition of the pseudomonas aeruginosa biomembrane; under the anesthesia of an abdominal cavity with 1% sodium pentobarbital of 0.1ml, a left lower abdomen is locally disinfected and then enters the abdominal cavity layer by layer through an incision of about 8mm, a carrier adhered with a biofilm is lightly washed in sterile physiological saline to remove planktonic bacteria and then is implanted into the abdominal cavity, and the incision is sutured by silk threads. After the model is successfully made, the moxibustion group is used for mild moxibustion treatment on the incision part; the moxa stick is vertically suspended on the skin 10cm away from the skin, and the distance between the moxa stick and the skin is adjusted up and down to the extent that the rat does not struggle, and the moxibustion is performed for 15min once a day. After 1d, 3d and 7d of infection, the abdominal infection condition and pathological changes of the mice can be detected, and the effect of the intervention treatment is observed compared with a control group.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. The preparation method of the pseudomonas aeruginosa biomembrane is characterized by comprising the following steps:
step one, bacteria are inoculated on an agar plate, a single colony is selected and inoculated in 5mLLB culture solution at 37 ℃ and 200rmin-1After shaking for 20h, 3000rmin of bacterial liquid is added-1Centrifuging for 10min, discarding supernatant, and precipitating bacteria with sterile physiological mediumWashing with saline for 2 times, and adjusting OD600 of the bacterial suspension to 0.05 with physiological saline to obtain bacterial liquid for later use;
and step two, adding 10ml of the prepared experimental strain bacterial liquid into a 50ml conical flask, adding 10 carriers, and performing constant-temperature shaking culture at 37 ℃ and 110rpm for 20 hours to enable the bacteria to be adhered.
2. The pseudomonas aeruginosa biofilm prepared by the preparation method of the pseudomonas aeruginosa biofilm according to the claim 1.
3. A medicament for treating chronic refractory diseases, which is prepared from the pseudomonas aeruginosa biomembrane of claim 2.
4. A treatment of infection by the pseudomonas aeruginosa peritoneal indwelling catheter of claim 2.
5. A control of chronic bacterial biofilm infection of the abdominal cavity of claim 2.
6. A use regimen of mild moxibustion for abdominal disorders as defined in claim 2.
7. A prophylactic and therapeutic treatment of infection by an intracorporeal indwelling catheter of claim 2.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2702141A2 (en) * | 2011-04-25 | 2014-03-05 | California Institute of Technology | Methods and system for interfering with viability of bacteria and related compounds and compositions |
| CN104789508A (en) * | 2015-04-29 | 2015-07-22 | 广东省微生物研究所 | Culture medium for promoting bacterial biofilm formation and culture method |
| CN106572662A (en) * | 2014-06-27 | 2017-04-19 | 凯恩生物科技有限公司 | Antimicrobial-antibiofilm compositions and methods of use thereof for personal care products |
| CN107488653A (en) * | 2016-06-12 | 2017-12-19 | 中国科学院大连化学物理研究所 | A kind of bacterium micro-capsule with biomembrane and its application |
-
2020
- 2020-03-27 CN CN202010234076.6A patent/CN111254100A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2702141A2 (en) * | 2011-04-25 | 2014-03-05 | California Institute of Technology | Methods and system for interfering with viability of bacteria and related compounds and compositions |
| CN106572662A (en) * | 2014-06-27 | 2017-04-19 | 凯恩生物科技有限公司 | Antimicrobial-antibiofilm compositions and methods of use thereof for personal care products |
| CN104789508A (en) * | 2015-04-29 | 2015-07-22 | 广东省微生物研究所 | Culture medium for promoting bacterial biofilm formation and culture method |
| CN107488653A (en) * | 2016-06-12 | 2017-12-19 | 中国科学院大连化学物理研究所 | A kind of bacterium micro-capsule with biomembrane and its application |
Non-Patent Citations (2)
| Title |
|---|
| 冯礼尚: "铜绿假单胞菌生物膜形成和发育分子机制研究进展", 《生物化工》 * |
| 陈华等: "细菌生物膜形成过程成分及结构的研究进展", 《黑龙江科学》 * |
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