CN114085887A - Bionic microsphere-based pseudomonas aeruginosa drug resistance concentration detection method - Google Patents
Bionic microsphere-based pseudomonas aeruginosa drug resistance concentration detection method Download PDFInfo
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- CN114085887A CN114085887A CN202010860002.3A CN202010860002A CN114085887A CN 114085887 A CN114085887 A CN 114085887A CN 202010860002 A CN202010860002 A CN 202010860002A CN 114085887 A CN114085887 A CN 114085887A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
- C12N11/12—Cellulose or derivatives thereof
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
Abstract
The invention discloses a bionic microsphere-based pseudomonas aeruginosa drug resistance concentration detection method, which comprises the following steps: s1: sampling and activating thalli; s2: mixing the thallus with sodium alginate and sodium cellulose; s3: manufacturing pseudomonas aeruginosa microspheres; s4: placing the microspheres in a 96-well plate; s5: adding a culture medium containing antibiotics with specific concentration into a 96-well plate; s6: putting the 96-well plate into an incubator for 24 hours; and S7, the minimum antibiotic concentration corresponding to the hole without growth is the drug-resistant concentration for inhibiting the pseudomonas aeruginosa. The invention simulates the physiological state of the pseudomonas aeruginosa forming a biological membrane in vivo in vitro, and detects the drug resistance concentration of the pseudomonas aeruginosa in the state, thereby improving the accuracy of the evaluation of the drug resistance degree of the pseudomonas aeruginosa.
Description
Technical Field
The invention relates to the technical field of bacteria drug resistance detection, in particular to a method for detecting pseudomonas aeruginosa drug resistance concentration based on bionic microspheres.
Background
Pseudomonas aeruginosa is a common conditional pathogen in hospitals, belongs to gram-negative bacteria, and can cause serious infection in a plurality of tissues of a human body, such as respiratory tracts, urinary systems, digestive tracts and the like. Due to the abuse of antibiotics in recent years, pseudomonas aeruginosa clinically develops serious multiple drug resistance phenomena. One of the reasons why drug resistance is serious is that pseudomonas aeruginosa forms a biofilm in vivo, and the drug resistance of pseudomonas aeruginosa is enhanced by limiting the contact of antibiotics and thalli and regulating various metabolic pathways.
The existing pseudomonas aeruginosa drug sensitivity detection method adopts an M-H agar plate overnight culture method to judge the antibacterial concentration of bacteria. But the traditional method weakens the influence of the generation of the biomembrane on the drug resistance of the pseudomonas aeruginosa.
Disclosure of Invention
The invention aims to provide a method for detecting the drug resistance concentration of pseudomonas aeruginosa based on bionic microspheres, which provides culture conditions for producing biomembranes of pseudomonas aeruginosa in vitro, so that bacteria can be subjected to drug sensitive detection in a state of having the biomembranes, and the drug resistance degree of clinical pseudomonas aeruginosa strains can be more accurately evaluated.
In order to achieve the purpose, the invention provides the following technical scheme: a method for detecting the drug-resistant concentration of pseudomonas aeruginosa based on bionic microspheres comprises the following steps: activating the strain, mixing the thallus with sodium alginate cellulose, preparing pseudomonas aeruginosa microspheres, adding a culture medium containing antibiotics with specific concentration into a 96-well plate for culture, wherein the minimum antibiotic concentration corresponding to a non-growing well is the drug resistance concentration for inhibiting the pseudomonas aeruginosa. The method comprises the following steps:
s1: sampling and activating the strain. Pseudomonas aeruginosa samples were taken from a minus eighty refrigerator and inoculated into 5mL LB medium, and cultured overnight at 37 ℃ with a shaker.
S2: the thallus is mixed with sodium alginate cellulose. Centrifuging the activated strain in 1mL S1 in a 1.5mL centrifuge tube, removing the supernatant, resuspending the centrifuged thallus by 10mL of mixed solution of sodium alginate and sodium carboxymethylcellulose, and reversing the upside down for several times to mix evenly.
S3: and (4) manufacturing the pseudomonas aeruginosa microspheres. The mixture in S2 was aspirated by a 5mL syringe, and then dropped dropwise into an aqueous solution containing 2% calcium chloride. And fishing out the solidified microspheres for later use.
S4: the microspheres were placed in a 96-well plate. The sodium alginate microspheres prepared in S3 were added to a 96-well plate, one ball per well.
S5: media containing antibiotics at specific concentrations were added to 96-well plates. Adding antibiotic into the culture medium, adjusting the concentration to 128ug/mL, sequentially diluting the antibiotic concentration culture medium by half to obtain the concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 ug/mL, sequentially adding the gradient diluted culture medium into a 96-well plate, and sequentially adding 100 ul per well.
S6: the 96-well plate was placed in an incubator for 24 hours. The incubator temperature was set at 37 ℃.
S7: the minimum antibiotic concentration corresponding to the wells that did not grow was the concentration that inhibited the resistance of the strain of Pseudomonas aeruginosa.
The drug for drug resistance detection is carbenicillin, meropenem, imipenem, aztreonam, levofloxacin, ciprofloxacin, tetracycline or chloramphenicol.
The concentration of sodium alginate in the mixed solution of sodium alginate and sodium carboxymethylcellulose in the S2 is 2%, and the concentration of sodium carboxymethylcellulose is 1%.
The culture medium in the S5 is a DMEM culture medium containing 5% fetal bovine serum.
The invention has the advantages that:
the invention has the advantages of the technical aspects:
1. the invention simulates the growth environment of the pseudomonas aeruginosa in vivo generating the biofilm state, and carries out drug resistance detection under the culture condition.
2. The invention can accurately obtain the minimum inhibitory concentration of the strain under the growth of the biofilm state.
3. High flux, and can detect multiple antibiotics at one time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
Fig. 1 is a picture of a bionic microsphere.
FIG. 2 is a schematic diagram of a bionic microsphere-based Pseudomonas aeruginosa drug resistance concentration detection method; 1. a 96-well plate; 2. sodium alginate microspheres containing pseudomonas aeruginosa; 3. antibiotic concentration; 4. antibiotic name: carbenicillin (CAR); meropenem (MEM); imipenem (IPM); aztreonam (ATM); levofloxacin (LVX); ciprofloxacin (CIP); tetracycline (TET); chloramphenicol (CHL).
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The method for detecting the drug-resistant concentration of pseudomonas aeruginosa according to the embodiment of the invention is specifically described below.
Example 1
A method for detecting the drug-resistant concentration of pseudomonas aeruginosa based on bionic microspheres comprises the following steps:
s1: sampling and activating the strain. The pseudomonas aeruginosa sample strain PAO1 is taken from a minus eighty refrigerator and inoculated in 5mL of LB culture medium, and is cultured by a shaker at 37 ℃ overnight.
S2: the thallus is mixed with sodium alginate cellulose. Centrifuging the activated strain in 1mL S1 in a 1.5mL centrifuge tube, removing the supernatant, resuspending the centrifuged thallus by 10mL of mixed solution of sodium alginate and sodium carboxymethylcellulose, and reversing the upside down for several times to mix evenly.
S3: and (4) manufacturing the pseudomonas aeruginosa microspheres. The mixture in S2 was aspirated by a 5mL syringe, and then dropped dropwise into an aqueous solution containing 2% calcium chloride. And fishing out the solidified microspheres for later use. As shown in fig. 1.
S4: the microspheres were placed in a 96-well plate. The sodium alginate microspheres prepared in S3 were added to a 96-well plate, one ball per well.
S5: media containing antibiotics at specific concentrations were added to 96-well plates. Adding antibiotic into the culture medium, adjusting the concentration to 128ug/mL, sequentially diluting the antibiotic concentration culture medium by half to obtain the concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 ug/mL, sequentially adding the gradient diluted culture medium into a 96-well plate, and sequentially adding 100 ul per well. As shown in fig. 2.
S6: the 96-well plate was placed in an incubator for 24 hours. The incubator temperature was set at 37 ℃.
S7: the minimum antibiotic concentration corresponding to the wells that did not grow was the concentration that inhibited the resistance of the strain of Pseudomonas aeruginosa. The invention provides a bionic microsphere-based pseudomonas aeruginosa drug resistance concentration detection method, the test results of a detection strain PAO1 are as follows:
Claims (6)
1. a method for detecting the drug-resistant concentration of pseudomonas aeruginosa based on bionic microspheres is characterized by comprising the following steps: the method simulates the physiological state of the pseudomonas aeruginosa forming a biological membrane in vivo in vitro, and detects the drug-resistant concentration of the pseudomonas aeruginosa in the state.
2. The bionic microsphere-based pseudomonas aeruginosa drug resistance concentration detection method according to claim 1, wherein: the drug for drug resistance detection is carbenicillin, meropenem, imipenem, aztreonam, levofloxacin, ciprofloxacin, tetracycline or chloramphenicol.
3. The bionic microsphere-based pseudomonas aeruginosa drug resistance concentration detection method according to claim 1, wherein: the drug resistance concentration detection specifically comprises the following steps: activating the strain, mixing the thallus with sodium alginate cellulose, preparing pseudomonas aeruginosa microspheres, adding a culture medium containing antibiotics with specific concentration into a 96-well plate for culture, wherein the minimum antibiotic concentration corresponding to a non-growing well is the drug resistance concentration for inhibiting the pseudomonas aeruginosa.
4. The bionic microsphere-based pseudomonas aeruginosa drug resistance concentration detection method according to claim 1, wherein: the method comprises the following steps:
s1: sampling and activating strains; taking a pseudomonas aeruginosa sample from a minus eighty refrigerator, inoculating the pseudomonas aeruginosa sample into 5mL of LB culture medium, and carrying out shaking table overnight culture at 37 ℃;
s2: mixing the thallus with sodium alginate cellulose; centrifuging 1mL of activated strain in S1 in a 1.5mL centrifuge tube, removing supernatant, resuspending the centrifuged thallus by 10mL of mixed solution of sodium alginate and sodium carboxymethylcellulose, and turning upside down for several times to mix uniformly;
s3: manufacturing pseudomonas aeruginosa microspheres; the mixture in S2 was aspirated by a 5mL syringe, and then dropped dropwise into an aqueous solution containing 2% calcium chloride. Fishing out the solidified microspheres for later use;
s4: placing the microspheres in a 96-well plate; adding the sodium alginate microspheres prepared in the step S3 into a 96-hole plate, wherein each hole is filled with one microsphere;
s5: adding a culture medium containing antibiotics with specific concentration into a 96-well plate; adding antibiotics into the culture medium, adjusting the concentration to be 128ug/mL, sequentially diluting the culture medium with the antibiotic concentration by half to be 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 mug/mL, sequentially adding the culture medium with the gradient dilution into a 96-well plate, wherein each well is 100 mug/mL;
s6: putting the 96-well plate into an incubator for 24 hours; the incubator temperature is set to 37 ℃;
s7: the minimum antibiotic concentration corresponding to the wells that did not grow was the concentration that inhibited the resistance of the strain of Pseudomonas aeruginosa.
5. The method for detecting the drug-resistant concentration of pseudomonas aeruginosa according to claim 4, wherein the concentration of sodium alginate in the mixed solution of sodium alginate and sodium carboxymethylcellulose in S2 is 2%, and the concentration of sodium carboxymethylcellulose is 1%.
6. The method for detecting the drug-resistant concentration of pseudomonas aeruginosa according to claim 4, wherein the culture medium component in the S5 is DMEM culture medium containing 5% fetal bovine serum.
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Citations (5)
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|---|---|---|---|---|
| US20070244059A1 (en) * | 2003-07-28 | 2007-10-18 | Karaolis David K | Method For Attentuating Virulence Of Microbial Pathogens And For Inhibiting Microbial Biofilm Formation |
| CN103509781A (en) * | 2012-06-29 | 2014-01-15 | 中国科学院大连化学物理研究所 | Research model of bacterial bio-film |
| CN105255851A (en) * | 2015-10-10 | 2016-01-20 | 深圳爱生再生医学科技有限公司 | Carboxymethyl cellulose/sodium alginate stem cell micro capsule and preparation and cultivation method thereof |
| CN106967779A (en) * | 2017-05-04 | 2017-07-21 | 廊坊恒益生物技术有限公司 | Suitable for the examination culture medium and preparation method of extensive tolerant Pseudomonas aeruginosa |
| 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
- 2020-08-25 CN CN202010860002.3A patent/CN114085887A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070244059A1 (en) * | 2003-07-28 | 2007-10-18 | Karaolis David K | Method For Attentuating Virulence Of Microbial Pathogens And For Inhibiting Microbial Biofilm Formation |
| CN103509781A (en) * | 2012-06-29 | 2014-01-15 | 中国科学院大连化学物理研究所 | Research model of bacterial bio-film |
| CN105255851A (en) * | 2015-10-10 | 2016-01-20 | 深圳爱生再生医学科技有限公司 | Carboxymethyl cellulose/sodium alginate stem cell micro capsule and preparation and cultivation method thereof |
| 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 |
| CN106967779A (en) * | 2017-05-04 | 2017-07-21 | 廊坊恒益生物技术有限公司 | Suitable for the examination culture medium and preparation method of extensive tolerant Pseudomonas aeruginosa |
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| BAO CAO等: "Antibiotic penetration and bacterial killing in a Pseudomonas aeruginosa biofilm model", J ANTIMICROB CHEMOTHER, vol. 70, no. 7, pages 2058 * |
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