CN107619805B - Tetracycline-resistant Escherichia coli and application thereof - Google Patents
Tetracycline-resistant Escherichia coli and application thereof Download PDFInfo
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Abstract
The invention discloses application of tetracycline-resistant Escherichia coli, wherein a strain is preserved in China general microbiological culture Collection center (CGMCC) in 7-25.7-2017 with the preservation number of CGMCC (China general microbiological culture Collection center) of 14456. The invention also discloses application of the tetracycline-resistant Escherichia coli (Escherichia coli) in preparation of tetracycline-resistant products. The Escherichia coli (Escherichia coli) provided by the invention has stable drug resistance, and still shows drug resistance after drug sensitivity test for half a year, and the drug-resistant strain is stable and difficult to reverse.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to tetracycline-resistant Escherichia coli (Escherichia coli) and application thereof.
Background
Colibacillosis is a common disease of both human and animals, and the colibacillosis of animals is common for many reasons, and one of the reasons is that the clinical repeated use of the same antibiotic for many times easily causes the appearance of a drug-resistant strain, and the drug-resistant strain causes the antibiotic to lose antibacterial activity and lose effectiveness. Therefore, the acquisition of the drug-resistant strain is the key for researching drug-resistant mechanism and searching for alternative antibacterial drugs in laboratories, and is also a necessary material for effectively controlling the occurrence of colibacillosis in farms.
The current research shows that the drug-resistant strains are obtained mainly through clinical separation, environmental acquisition, induction of bacterial drug resistance by laboratory drugs and the like. The drug induction of the bacterial drug resistance is the most convenient way for obtaining drug-resistant strains in a laboratory, and the drug-resistant conditions of the bacteria can be found in the drug induction process, and the apparent bacteria are sensitive to the drugs, the drug-resistant process, the drug-resistant rule and the exploration of the drug-resistant mechanism. The quality control bacterial strain is induced by adopting a medicament with sub-bacteriostatic concentration in a laboratory, and the problems of high requirement, complex operation, long time consumption, easy pollution of the strain and the like often occur, so that the method is not easy to obtain the drug-resistant bacterial strain in a short time. How to obtain the drug-induced drug-resistant strain in the laboratory simply, effectively and with less time consumption is a great problem which troubles laboratory researchers at present.
Disclosure of Invention
In view of the above, the application provides a tetracycline-resistant Escherichia coli (Escherichia coli) and an application thereof, aiming at the problems that a sub-inhibitory concentration drug-induced bacterial drug resistance method has high requirements, is complex to operate, consumes long time, and is prone to causing pollution to strains.
In order to solve the technical problems, the application discloses tetracycline-resistant Escherichia coli (Escherichia coli), which is preserved in China general microbiological culture Collection center (CGMCC) at 25.7.2017 with the preservation number of CGMCC No. 14456.
Further, the morphological characteristics of the strain are as follows: the isolated strain can grow on a MacconyKa culture medium and is a red, convex, smooth and moist microcolony.
The invention also discloses a preparation method of the tetracycline-resistant Escherichia coli, which comprises the following steps:
step 1, isolated culture and morphological observation of bacteria: taking dead duck liver tissues, inoculating the dead duck liver tissues to a Mackanka agar plate culture medium in a sterile manner, and culturing for 18-24h at the constant temperature of 37 ℃ to observe the growth condition of bacteria; picking a pink single bacterial colony on a culture medium for gram stain microscopic examination, then hooking the same single bacterial colony, inoculating the single bacterial colony on a Macconkey agar plate culture medium for further separation and purification, inoculating the separated and purified bacteria on an agar slant, numbering A, and storing for later use;
step 2, tetracycline drug sensitivity test of the strain A: uniformly coating and inoculating the strain A on a nutrient agar culture medium, carrying out tetracycline drug sensitivity test according to a K-B paper diffusion method, and judging the standard according to the standard of the drug sensitivity paper diffusion method of American clinical laboratory standards institute (2010 version);
step 3, animal regression test of the strain A: inoculating the strain A into nutrient broth culture medium, and shake-culturing at 37 deg.CCulturing for 12 hr, and making the content of the bacterial liquid 1 × 10 by turbidimetry8cfu/mL; testing 10 healthy ducklings, and randomly dividing the ducklings into a test group and a control group, wherein each group comprises 5 ducklings; injecting 1mL of bacterial liquid into muscle of the ducklings of the test group, injecting 1mL of normal saline into muscle of the ducklings of the control group, observing for 1 week, and recording the morbidity and mortality of the ducklings;
step 4, tetracycline resistant strain induction test: performing tetracycline drug sensitivity test on the strain A, selecting bacteria at the edge of the inhibition zone of tetracycline drug sensitive paper sheet for the first time, performing tetracycline drug sensitivity test again, repeating the tetracycline drug sensitivity induction test for multiple times until a drug-resistant strain with diameter of the inhibition zone of tetracycline being 0mm is selected, and numbering the strain B3Namely, a tetracycline-resistant Escherichia coli (Escherichia coli) according to claim 1 is produced.
The invention also discloses application of the tetracycline-resistant Escherichia coli (Escherichia coli) in preparation of tetracycline-resistant products.
The invention also discloses a traditional Chinese medicine for inhibiting the tetracycline-resistant Escherichia coli, wherein the traditional Chinese medicine is one of scutellaria baicalensis, rheum officinale, folium isatidis, radix isatidis or fructus forsythiae.
Furthermore, the minimum inhibitory concentration of the forsythia suspense to tetracycline-resistant Escherichia coli (Escherichia coli) is 0.125g/mL, the scutellaria baicalensis is 0.5g/mL, and the rheum officinale is 0.25 g/mL.
Compared with the prior art, the application can obtain the following technical effects:
1) the method for acquiring the escherichia coli drug-resistant strain provided by the invention is strong in pertinence and clear in target.
2) The method provided by the invention is established on the principle that the original sub-bacteriostatic concentration medicament induces the medicament resistance of bacteria, and is simpler, more convenient and quicker than the former method, short in time consumption and obvious in effect.
3) The Escherichia coli B3 strain provided by the invention is inoculated in common nutrient broth for enrichment culture, and animal regression test is carried out on ducklings, so that good pathogenic capability is shown.
4) The escherichia coli B3 strain provided by the invention has stable drug resistance, and still shows drug resistance after drug sensitivity test for half a year, which indicates that the drug resistant strain is stable and difficult to reverse.
Of course, it is not necessary for any one product to achieve all of the above-described technical effects simultaneously.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a diagram of tetracycline-sensitive (right) and drug-resistant (left) E.coli strains of the present application;
FIG. 2 shows the results of in vitro bacteriostatic tests of Scutellaria baicalensis Georgi against tetracycline-resistant strains of the present application, wherein a is a photograph taken from the front side and b is a photograph taken from the back side;
FIG. 3 shows the results of in vitro inhibition of the tetracycline-resistant strains of rheum officinale of the present invention against tetracycline-resistant strains of the present application;
FIG. 4 shows the results of in vitro bacteriostatic tests of Isatis root of the present invention on tetracycline-resistant strains of the present application;
FIG. 5 shows the results of in vitro bacteriostatic tests of forsythia suspense according to the present invention against tetracycline-resistant strains of the present application;
FIG. 6 shows the results of in vitro bacteriostatic tests of the folium Isatidis of the present invention on tetracycline-resistant strains of the present application.
Detailed Description
Embodiments of the present application will be described in detail with reference to the drawings and examples, so that how to implement technical means to solve technical problems and achieve technical effects of the present application can be fully understood and implemented.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
15 healthy ducklings of 10 days old are purchased from the duck fry market in Rongchang district, Chongqing, and are randomly divided into 1 control group, 1 sensitive bacteria group and 1 drug-resistant bacteria group, and 3 test groups are provided, wherein each group comprises 5 ducklings.
The killed duck comes from a certain duck farm in Rongchang district in Chongqing city. The duck farm can be used for feeding about 500 laced meat ducks with age of 30 days and weight of about 1.5 kg. In a certain day, the duck is suddenly hot, the drinking water of the duck is gradually reduced, the sanitary conditions of a duck farm are gradually poor, and the individual duck only has the symptoms of reduced appetite, mental retardation and white and thin feces discharge. The treatment is mainly carried out by combining penicillin, oxytetracycline, florfenicol and other medicines with traditional Chinese medicines for clearing heat and drying dampness, such as Chinese pulsatilla root, rhubarb, scutellaria baicalensis, golden cypress and the like, drinking water and mixing materials, the effect is not ideal, 7-8 dead duck livers are dead every day, and the duck livers dead of diseases are collected for laboratory diagnosis.
Tetracycline drug sensitive test paper (30 μ g/tablet) available from Hangzhou Bing and microbiological reagents, Inc.; nutrient broth, nutrient agar, Macconyya agar medium, 20 enterobacteriaceae bacteria biochemical micro-reaction tubes were purchased from Hangzhou Bing and microbial reagents GmbH.
Example 1 isolation and identification of bacteria
First, separation of bacteria
(I) isolated culture and morphological observation of bacteria
Taking dead duck liver tissues, inoculating the dead duck liver tissues to a Mackanka agar plate culture medium in a sterile manner, and culturing for 18-24h at the constant temperature of 37 ℃ to observe the growth condition of bacteria; picking single pink colony on the culture medium for gram stain microscopic examination, then hooking the same single colony to inoculate on a Macconkey agar plate culture medium for further separation and purification, re-inoculating the separated and purified bacteria on an agar slant with the number of A, and storing for later use.
(II) Strain A Tetracycline drug sensitivity test
Strain A was spread evenly on nutrient agar medium and subjected to tetracycline drug sensitivity test according to K-B paper diffusion method (sand, Song Sha, animal microbiology Laboratory course [ M ]. Chongqing: university of southwestern publisher, 2011.), the standard was determined with reference to the American society for Clinical Laboratory Standards drug sensitive paper diffusion method standard (2010 edition) (CLSI (Clinical and Laboratory Standards Institute), Methods for analytical Dilution and Disk characterization of Infreguency analysis or diagnostic Bacteria-Document M45-A2.Wayne, PA: Clinical and Laboratory Standards Institute; 2010.).
(III) Strain A animal regression test
Inoculating the strain A into nutrient broth culture medium, shaking at 37 deg.C, culturing for 12 hr, and turbidifying the bacterial liquid with Maifang to make the content of bacterial liquid 1 × 108cfu/mL (0.5 McLeod concentration). Testing 10 healthy ducklings, and randomly dividing the ducklings into a test group and a control group, wherein each group comprises 5 ducklings; injecting bacterial liquid into muscle of the ducklings of the test group by 1 mL/one, injecting physiological saline into muscle of the ducklings of the control group by 1 mL/one, observing for 1 week and recording the morbidity and mortality of the ducklings.
(IV) Tetracycline drug resistant Strain Induction test
Performing tetracycline drug sensitivity test on the strain A, selecting bacteria at the edge of the inhibition zone of tetracycline drug sensitive paper sheet for the first time, performing tetracycline drug sensitivity test again, repeating the tetracycline drug sensitivity induction test for multiple times until a drug-resistant strain with diameter of the inhibition zone of tetracycline being 0mm is selected, and numbering the strain B3The results of the tetracycline drug sensitive test paper induction are shown in Table 1 and FIG. 1.
TABLE 1 results of tetracycline sensitivity test of isolates
As can be seen from the table 1, the separating bacteria has high sensitivity to tetracycline, and gradually loses sensitivity along with repeated and multiple actions of the tetracycline, so that drug resistance is finally generated, and the diameter of a inhibition zone is 0 mm. After half a year, performing tetracycline drug sensitivity test again by using drug-resistant bacteria, and keeping drug resistance; indicating that the drug resistance of the drug-resistant strain is difficult to reverse.
II, identification
(I) Biochemical assay
Respectively inoculating the purified separated bacteria into different enterobacteriaceae bacteria biochemical identification micro-reaction tubes, culturing for 24-48h under the environment condition of 37 ℃, observing and recording results, wherein the biochemical identification results are as follows:
morphological characteristics of the cells:
the isolated strain can grow on a MacconyKa culture medium and is a red, convex, smooth and moist microcolony. Experiments show that the isolate is duck escherichia coli.
The physiological and biochemical characteristics of the thallus are shown in a table 2:
TABLE 2 Biochemical identification of isolated strains
Note: "+", positive; "-", negative
As is clear from the results of biochemical identification in Table 2, there was no difference in metabolism between the sensitive bacterium A and the drug-resistant bacterium B3.
(II) 16S rDNA assay
Extracting drug-resistant bacteria B3The total DNA of (1) is used as a template, the bacterial 16S rDNA universal primer is used for carrying out PCR amplification to obtain an amplification product with the length of about 1.4kb, the amplification product is recovered and sequenced, and the determined sequence is shown as SEQ ID No. 1.
Drug-resistant bacteria B based on Gen-Bank sequence homology comparison3The homology with Escherichia coli CAU2558(GenBank: MF428963.1) was 99%, and the bacterium was preliminarily determined to be Escherichia bacterium (E.
Based on the characteristics, the strain is resistant to drug-resistant bacteria B3Identified as Escherichia coli. The proposed classification was named E.coli; the strain is preserved in China general microbiological culture Collection center (CGMCC 9501 for short, the address: No.1 Xilu-Beijing university of Tokyo facing Yang district No. 3, Microbiol research institute of Chinese academy of sciences, postal code 100101) in 25.7.7.2017, and the preservation number is CGMCC No. 14456.
Example 2B3Bacterial animal regression test
B is to be3Inoculating the strain in nutrient broth culture medium, shaking at 37 deg.C, culturing for 12 hr, and making the content of the strain to be 1 × 108cfu/mL (0.5 McLeod concentration). 10 healthy ducklings are divided into 2 groups, namely a test group and a control group, and each group comprises 5 ducklings; duckling muscle injection for test groupAnd (3) inoculating 1mLl bacterial liquid per duck, injecting 1mL of normal saline into the muscle of the ducklings of the control group, observing for 1 week, and recording the morbidity and mortality of the ducklings.
Sensitive bacteria group A and drug-resistant bacteria group B3After the tested ducks of the group are attacked by poison for 24 hours, the tested ducks are manifested as depressed spirit, anorexia, unstable standing and thin excrement, and die after 36 hours and all die after 96 hours. The duckling died of illness is subjected to autopsy, the cellulosic pericarditis and the liver are swollen, and a layer of film-shaped cellulosic exudates is covered on the surface of the liver; the liver tissue is inoculated on the MacconKa agar medium, and typical Escherichia coli colonies can be seen. The 2 control groups 10 ducklings are normal in diet, mental state and growth and development, no pathological change is seen in a autopsy, and no colony growth is seen when the liver tissues are inoculated on the Mackanka agar medium.
The results of animal regression experiments show that: the test duckling has typical colibacillosis clinical symptoms and pathological change characteristics; meanwhile, sensitive bacteria and drug-resistant bacteria have very strong toxicity, the lethality rate reaches 100 percent, and the toxicity of the drug-resistant strains which are successfully induced is not changed.
The pathological tissues of clinical suspected colibacillosis can be used for distinguishing duck colibacillosis, riemerella anatipestifer and pasteurella multocida by inoculating the pathological tissues to a distinguishing culture medium, microscopic examination and biochemical test. Only the Escherichia coli can grow on the MacConkey culture medium and is red, convex, smooth and moist microcolonies; the isolated bacterium was Escherichia coli as indicated by a red small rod under a microscope.
The mechanism of action of tetracycline is known to be via the hydrophilic pores of the outer membrane of bacterial cells, enter cells from an energy-dependent transfer system on the inner membrane, specifically bind to the ribosomal 30s subunit, prevent aminoacyl RNA binding, and thereby inhibit peptide chain elongation and protein synthesis. In addition, the permeability of bacterial cell membrane can be changed, so that important components in cells leak out, and the replication of DNA is rapidly inhibited. At present, the known tetracycline-resistant genes of escherichia coli respectively comprise six genes of tetA, tetB, tetC, tetD, tetK and tetL, and sensitive bacteria and drug-resistant bacteria have no change in aspects of morphology, physiological and biochemical characteristics and virulence, so that one of possible reasons for the generation of drug-resistant strains is presumed to be that the bacteria generate actively-discharged proteins.
Example 3 in vitro inhibition of Tetracycline-resistant B3 bacteria by traditional Chinese medicines
Preparing a traditional Chinese medicine solution for test: taking 4g of each of five traditional Chinese medicine extracts (each g of the extracts is equal to 10g of crude drug) of radix scutellariae, rheum officinale, folium isatidis, radix isatidis and fructus forsythiae, respectively putting the extracts into different beakers, adding 20ml of distilled water, stirring to dissolve, filtering, and sterilizing the filtrate in a penicillin bottle by reduced pressure steam (115 ℃ for 20min) for later use. The concentration of the drug solution at this time was 2 g/ml.
(II) preparing a bacterial liquid: recovering and inoculating the glycerol preserved strain B3 at the temperature of-20 ℃ to a common agar culture medium, culturing for 24 hours at the temperature of 37 ℃, selecting a typical bacterial colony, inoculating to a common broth culture medium, and culturing for 18 to 24 hours at the temperature of 37 ℃ for later use.
(III) an oxford cup method traditional Chinese medicine bacteriostasis test: the cultured bacterial liquid is subjected to Maifand turbidimetry to reach 0.5 Maifand concentration, namely 1.5 multiplied by 108CFU/ml (if it exceeds 0.5 McLee's concentration, diluted with nutrient broth or normal saline), dipping sterilized cotton swab with appropriate amount of bacteria to be tested, and applying bacteria to the surface of plate culture medium by streak method. After the bacterial liquid is absorbed by the agar, a sterilized Oxford cup (with the inner diameter of 6.0mm +/-0.1 mm, the height of 10.0mm +/-0.1 mm and the outer diameter of 7.8mm +/-0.1 mm) is lightly placed on the culture medium by using a pair of tweezers in an aseptic operation, the Oxford cup is lightly pressed to ensure that no gap exists between the bottom of the cup and the agar, 240ul of liquid medicine is added into the Oxford cup, the three repeated positions are arranged, the culture is carried out for 24 hours at 37 ℃, and the result is observed.
The result of the bacteriostatic experiment of the traditional Chinese medicine is taken as the standard for judging the sensitivity according to the diameter of the bacteriostatic circle. The diameter of the bacteriostatic zone: not less than 20 is high-sensitive, 10-19mm is medium-sensitive, and <10mm is non-sensitive. The results are shown in Table 3 and FIGS. 2-6.
TABLE 3 diameter of inhibition zone of five Chinese medicinal solutions to B3 bacteria
The results show that the Chinese herbal medicines of the baical skullcap root, the weeping forsythia and the rhubarb have good bacteriostatic effect on tetracycline-resistant Escherichia coli B3 bacteria, and the bacteriostatic effect of the Chinese herbal medicines of the weeping forsythia and the rhubarb is most obvious.
(IV) minimum inhibitory concentration of traditional Chinese medicine to tetracycline-resistant B3 bacteria
Preparing a decoction of a crude drug to be detected with a concentration of 1g/ml, determining the minimum inhibitory concentration by a 96-well plate method, under an aseptic environment, taking 100 mu l of broth after bacteria detection, respectively adding the broth into 1-10 rows of the 96-well plate, respectively, adding 100 mu l of liquid medicine into the 1 st row, diluting the broth to the 8 th row (the final volume of each hole is 100 mu l) in a successive multiple ratio, and discarding the redundant liquid after diluting the 8 th row. Adding 10 mul of bacterial liquid to be detected into the 1 st to 9 th holes, wherein the final volume of each hole is 110 mul; column 9 is a positive control, column 10 is a broth blank, and column 11 is a drug blank. The preservative film covers the surface, and the results are shown in Table 4 after culturing for 18-24 hours in an incubator at 37 ℃.
TABLE 4 minimum inhibitory concentration of aqueous extracts of Chinese herbs on tetracycline-resistant E.coli B3
Note: "+" indicates a bacterial growth and "-" indicates a sterile growth.
The invention shows that the Chinese herbal medicines of the scutellaria baicalensis, the forsythia suspense and the rhubarb have good bacteriostatic effect on tetracycline-resistant escherichia coli B3 bacteria, and the bacteriostatic effect of the traditional Chinese medicines of the forsythia suspense and the rhubarb is most obvious; the minimum inhibitory concentration of fructus forsythiae to B3 strain in 5 Chinese medicines is 0.125g/mL, the baical skullcap root is 0.5g/mL, and the rhubarb is 0.25g/mL, and the fructus forsythiae serving as a traditional antibacterial medicine has the advantages of small toxic and side effects of 4, wide source, low price, less occurrence of drug resistance and strong inhibitory effect on 3 pathogenic bacteria.
Modern medicine considers that the disease caused by the same pathogenic bacteria is treated by repeatedly applying the same antibiotic for many times, and drug resistance is easy to generate; the effect of adopting antibiotics to treat diseases is very little no matter in clinical practice of human medicine or veterinarian, so that the search for new effective antibacterial drugs becomes a new hotspot of the current medical drug research; the Chinese herbal medicine has the advantages of low toxicity, small side effect, no drug resistance and the like, and Chinese herbal medicine resources are rich, the variety is complete, and the processing is convenient. Therefore, the research on the bacteriostatic effect and bacteriostatic mechanism of the Chinese herbal medicine on the drug-resistant bacteria has positive clinical significance, the drug paper sheet method adopted by the test for preparing the drug-resistant strain provides a more convenient and effective way for obtaining the drug-resistant strain in a laboratory, the complexity of clinical sample collection is reduced, and the drug-resistant strain has stable drug-resistant property and is suitable for experimental research.
As used in the specification and claims, certain terms are used to refer to particular components or methods. As one skilled in the art will appreciate, different regions may refer to a component by different names. The present specification and claims do not intend to distinguish between components that differ in name but not in name. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.
It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (2)
1. Tetracycline-resistant Escherichia coli (A)Escherichia coli) The strain is characterized in that the strain is preserved in the China general microbiological culture Collection center in 2017, 7 and 25 months with the preservation number of CGMCC No. 14456;
the morphological characteristics of the strain are as follows: the isolated strain can grow on a MacconyKa culture medium and is a red, convex, smooth and moist microcolony.
2. The tetracycline-resistant Escherichia coli (C) of claim 1 (C:)Escherichia coli) The application of the strain in preparing tetracycline-resistant products.
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