CN111378715A - Caenorhabditis elegans model for identifying bovine escherichia coli virulence - Google Patents
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Abstract
The invention discloses a caenorhabditis elegans model for identifying bovine escherichia coli virulence, which comprises the following steps: carrying out synchronization treatment on caenorhabditis elegans; culturing Escherichia coli to form thallus Porphyrae; placing the C.elegans subjected to the synchronization treatment in a culture medium containing escherichia coli lawn, namely a C.elegans model for identifying the bovine-derived escherichia coli virulence; the synchronization treatment comprises the steps of culturing for 40-60 hours after cracking, centrifuging and precipitating to obtain synchronized L4-phase nematodes; the escherichia coli is firstly placed in an LB solid culture medium for culturing for 18 h-24 h at 37 ℃, then placed in an LB broth for shaking for 18 h-24 h at 37 ℃, and finally placed in an SK culture medium and placed in a biochemical incubator at 20 ℃ for overnight drying to form lawn. The establishment of the nematode model is quicker, simpler and more convenient, saves time and cost, can use the caenorhabditis elegans pathogenic model to research the pathogenic mechanism of pathogenic bacteria, and further can be used for screening antibacterial drugs.
Description
Technical Field
The invention relates to the technical field of biological models, in particular to a caenorhabditis elegans model for identifying bovine escherichia coli virulence.
Background
Escherichia coli (Escherichia coli) is an important component of enterobacteriaceae, Escherichia, gram-negative brevibacterium, has the size of 0.5 × 1-3 mu m, is an important part of normal flora of intestinal tract, generally has no pathogenicity, but some special serotypes can cause severe diarrhea and septicemia of human and animals, and is generally called pathogenic Escherichia coli.
Caenorhabditis elegans (Caenorhabditis elegans), a small nematode that lives in soil, feeds on bacteria, as a typical model organism, and has several characteristics: the body is transparent, the structure is simple, and the observation is easy under a microscope; the life cycle is short and is divided into 4 stages: embryonic development stage, growth development stage, reproductive stage and late reproductive stage. Under the environment of 20 ℃, a life cycle can be completed within 3.5 days, and the test time can be greatly shortened; the gene sequence is known, and the origin of each cell is completely clear; the genetic sequence has 40% homology with mammals and the like. These characteristics make caenorhabditis elegans have very big simplicity and practicality with experimental animals such as mouse, rat, rabbit, more save time, energy and cost. At present, caenorhabditis elegans is widely applied to the researches of toxicology, disease models, pathogenic bacteria pathogenic models and the like. Caenorhabditis elegans is frequently reported for aging research, drug toxicology, drug screening, etc., and researchers have established over 50 nematode models including bacteria, fungi, and viruses. The bacteria include Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and the like. However, the pathogenic model of bovine Escherichia coli infected with caenorhabditis elegans is rarely reported in China. The test uses two test animals of mice and caenorhabditis elegans as contrast, and simultaneously infects 13 bovine escherichia coli separated in clinic to compare the influence of the escherichia coli on the two animals, so as to search a simpler, more convenient and faster method for researching the pathogenic mechanism of pathogenic bacteria, and provide scientific medication basis for clinical infection resistance.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a caenorhabditis elegans model for identifying the virulence of bovine escherichia coli, provide a simpler and faster method for researching the pathogenic mechanism of pathogenic bacteria for bovine escherichia coli infection, carry out drug rescue on the pathogenic model by using veterinary antibacterial drugs, and facilitate the prevention and treatment of bovine escherichia coli infection and the screening of drugs.
In order to solve the technical problems, the invention adopts the following technical scheme:
a caenorhabditis elegans model for identifying bovine escherichia coli virulence comprises the following steps:
(1) carrying out synchronization treatment on caenorhabditis elegans;
(2) culturing escherichia coli in an SK culture medium to form lawn;
(3) placing the C.elegans subjected to the synchronization treatment in a culture medium containing escherichia coli lawn, namely identifying a C.elegans model of bovine-derived escherichia coli virulence;
the synchronization treatment comprises the steps of culturing for 40-60 hours after cracking, centrifuging and precipitating to obtain synchronized L4-phase nematodes;
the escherichia coli is firstly placed in an LB solid culture medium for culturing for 18 h-24 h at 37 ℃, then is placed in an LB broth for shaking for 18 h-24 h in a shaking table at 37 ℃, and finally is placed in an SK culture medium and is placed in a biochemical incubator at 20 ℃ for overnight drying to form lawn.
Preferably, the cracking time of the synchronization treatment process is 4-5 min.
Preferably, the centrifugal rotation speed in the synchronization treatment process is 2500-3500 r/min, and the centrifugal time is 1-3 min.
Preferably, the lysed lysate is placed in a shaker at 20 ℃ and 150r/min for 20h with shaking.
Preferably, the culture of the synchronization treatment process is to transfer L1 stage nematodes at the bottom of the centrifugal tube into a clean NGM plate, and place the plate in a biochemical incubator at 20 ℃ for 40-60 hours to obtain synchronized L4 stage nematodes.
Preferably, the escherichia coli is the viscera or the feces of the cattle dead of diseases, the single colony of the escherichia coli is subjected to gram staining and microscopic examination, and is inoculated on a Mackanka culture medium, cultured for 18-24 h at 37 ℃, further purified and inoculated in nutrient broth for secondary culture.
Preferably, the DNA of the Escherichia coli needs to be extracted for PCR reaction.
Preferably, the sequence of the PCR reaction primer is shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
Preferably, the PCR reaction system is 2 × ES Taq MasterMix 25 muL, the upstream and downstream primers are 2 muL respectively, the template is 4 muL, and ddH2O is supplemented to 50 muL, the reaction program is pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30S, annealing at 52 ℃ for 30S, extension at 72 ℃ for 1min, 35 cycles, and final extension at 72 ℃ for 10min
The invention also provides a method for using the caenorhabditis elegans pathogenic model for identifying bovine escherichia coli virulence to establish a bovine escherichia coli virulence-caenorhabditis elegans pathogenic model in drug screening.
The invention has the beneficial effects that:
the establishment of the nematode model is quicker, simpler and more convenient, saves time and cost, can use the caenorhabditis elegans pathogenic model to research the pathogenic mechanism of pathogenic bacteria, and further screens antibacterial drugs, thereby providing theoretical basis for the anti-infection treatment and the medication of clinical bovine colibacillosis.
Drawings
FIG. 1 shows the survival rate of C.elegans infected with E.coli of bovine origin, which is (number of surviving nematodes/number of busworms) × 100%.
Figure 2 is EC3 nematode intestinal bacteria counts.
Figure 3 is EC10 nematode intestinal bacteria counts.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited to the scope of the examples. These examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. In addition, various modifications may occur to those skilled in the art upon reading the present disclosure, and such equivalent variations are within the scope of the present invention as defined in the appended claims. 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.
1. Materials and methods
1.1 materials
1.1.1 preparation methods of main reagents and instruments MacconKai agar, eosin methylene blue agar, LB agar and nutrient broth from Beijing land bridge technology GmbH, bacterial DNA extraction kit, 2 × ES Taq Mastermix, DNA Marker from Beijing kang century Biotechnology GmbH, serum detection kit from Botanhuan biopharmaceutical industry GmbH, micro biochemical reaction tube from Qingdao Haibo biotechnology GmbH, agarose gel from Spanish GENE, PCR instrument from Bio-Rad, gel imaging analyzer from Alpha Inotech, peristaltic pump from fixed application Pump Inc. NGM medium, SK medium, M9 buffer, NOR lysate, S base solution and other references such as Abay [ TEJ L, ABAY A.A connected To-like electrophoresis for expression of microorganism reagent ] and instruments [ 11J, 9] prepared by biological assay (playback J, 9) and so on.
1.1.2 samples derived from Escherichia coli are separated from cattle diseased material collected in 2017-Guangxi Nanning, Guilin, Liuzhou and other areas, and the cattle mostly have symptoms of pulmonary hemorrhage and diarrhea. Coli OP50 was gifted by the university of duck molecular genetics and microbiology laboratories, usa. Pathogenic E.coli O157 was maintained by the bacterial institute of veterinary research, Guangxi.
1.1.3 test animals 70 SPF-level Kunming mice with 4 weeks old, half male and half female, and 18-22 g of body weight were purchased from the center of laboratory animals of Guangxi medical university. N2 wild type C.elegans was offered by the university of Duke molecular genetics and microbiology laboratories, USA.
1.2 methods
1.2.1 isolation and identification of bacteria isolated from cattle disease material collected in Guangxi Nanning, Guilin, Liuzhou, etc. were inoculated into eosin methylene blue solid medium, and 13 colonies with metallic luster of black-purple bands were isolated, which are suspected to be escherichia coli. Respectively selecting single bacterial colonies for gram staining, performing microscopic examination, inoculating on a MacconKa culture medium, culturing at 37 ℃ for 18-24 h, further purifying, and inoculating in nutrient broth for re-culturing. And taking the bacteria liquid cultured for 18h for trace biochemical identification of sucrose, maltose, lactose and the like.
1.2.2 extraction of bacterial DNA the viscera or feces of cattle died of illness (feces are diluted with sterile PBS buffer solution in advance) are collected by a sterile inoculating loop, inoculated into a Macconyk solid culture medium, cultured for 18 h-24 h at 37 ℃, selected to fall into a nutrient broth, cultured for 18 h-24 h at 37 ℃, and extracted according to the steps of a bacterial DNA extraction kit.
1.2.3PCR (polymerase chain reaction) with extracted DNA as template, 16S rRNA gene fragment was amplified with the primer sequence of 27F:5 '-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1) and 1492R: 5'-GGCTACCTTGTTACGACTT-3' (SEQ ID NO: 2) and the amplified fragment size of 1400 bp. as 2 × ES TaqMasterMix 25. mu.L, 2. mu.L each of upstream and downstream primers, 4. mu.L template and ddH2O to 50. mu.L. the reaction procedure was pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30S, annealing at 52 ℃ for 30S, extension at 72 ℃ for 1min, 35 cycles, final extension at 72 ℃ for 10 min. the amplified PCR product was electrophoresed on 1% agarose gel and sent to Guangzhou Tianyihui Mingyuan for sequencing.
1.2.4 identification of Escherichia coli serotype O is identified by a slide agglutination method according to the steps of an Escherichia coli serotype detection kit.
1.2.5 mice pathogenicity test 70 Kunming mice are randomly divided into 14 groups, each group comprises 5 mice, the mice of the test group are injected with 3.0 × 109CFU/mL bacterial diluent in the abdominal cavity, the mice of the control group are injected with 0.3mL sterile physiological saline in the abdominal cavity, the 14 groups of mice are separately raised, the mice are freely fed and drunk, the death time and the number of the mice are observed, the mice with symptoms and death in the pathogenicity test are subjected to autopsy, the lesion parts are aseptically collected, and the pathogenic bacteria are separated.
1.2.6 nematode pathogenicity test
1.2.6.1 Simultaneous treatment of caenorhabditis elegans, selecting an NGM plate which is large in adult nematodes and worm eggs and free of mould pollution, repeatedly washing the NGM plate with M9 buffer solution to wash the nematodes and the worm eggs from OP50 lawn, collecting the washed NGM plate into a sterilized 15mL centrifuge tube, adding M9 buffer solution to a constant volume of 10mL, centrifuging the mixture at 3000r/min for 1min, discarding the supernatant, adding 1mL nematode lysate, performing vortex oscillation for 4min (the lysis time is not too long so as to avoid killing the worm eggs), centrifuging the mixture at 3000r/min for 1min, and discarding the supernatant. Adding M9 buffer solution to 10mL, centrifuging at 3000r/min for 1min, and washing repeatedly for 3 times to remove the lysate. And finally adding the S basic solution to a constant volume of 10mL, and placing the mixture in a shaking table at 20 ℃ and 150r/min for shaking for 20 h. Centrifuging at 3000r/min for 1min, discarding supernatant, transferring L1 stage nematode at the bottom of the centrifuge tube into a clean NGM plate, and placing in a biochemical incubator at 20 ℃ for 40h to obtain synchronized L4 stage nematode for later use. Three regions of 13 strains of escherichia coli are streaked on an LB solid culture medium, after the escherichia coli is cultured for 18 h-24 h at 37 ℃, single colonies are picked to be placed into 5mL of LB broth, and the escherichia coli is shaken for 18 h-24 h in a shaking table at 37 ℃. And then 300 mu L of bacterial liquid is taken to be put into an SK culture medium, the bottom of a sterilization test tube is uniformly coated to ensure that the bacterial liquid is completely paved in the culture medium, and the culture medium is placed in a biochemical incubator at the temperature of 20 ℃ for overnight drying to form bacterial lawn for later use.
1.2.6.2 Escherichia coli infection elegans L4 phase nematodes were collected by a needle into SK culture medium with Escherichia coli lawn, each plate containing 20 worms and each group was repeated 3 times. Coli OP50 was used as a negative control.
1.2.6.3 in vivo Escherichia coli count and identification of nematodes two strains of Escherichia coli (EC3 and EC10) with strong toxicity were selected to infect C.elegans, every 24h, 10 infected nematodes were picked with a picking needle into a 1.5mLEP tube into which 1mL of M9 buffer had been added, centrifuged at 3000r/min for 1min, washed repeatedly 3 times to remove bacteria on the surface of the nematodes, then 400 mg of sterilized quartz sand was added, vortexed for several minutes to grind the nematodes. Diluting and coating the supernatant, counting, determining the number of bacteria in each nematode, purifying and culturing the obtained bacteria, extracting DNA, PCR, sequencing, detecting O serotype, and determining whether the nematode is the original pathogenic bacteria. TABLE 1 determination of the pathogenicity of E.coli to nematodes
2. Results
2.1 results of separation and identification, molecular biology identification and serotype detection of Escherichia coli
The results of the staining microscopy show that 13 strains of bacteria are gram-negative bacilli, the thalli are red, round at two ends and in a short rod shape, the results of the biochemical tests show that the separated 13 strains of escherichia coli sucrose, maltose, peptone water, MR and VP tests are negative, the results of the lactose, mannitol, glucose, nitrate and trisaccharide iron tests are positive, and the results are consistent with the results measured by the escherichia coli standard strains. 16S rRNA sequencing and BLAST comparison on GenBank prove that the homology with the sequence of the Escherichia coli reaches over 99 percent, and the 13 strains are all the Escherichia coli. Serologically identified, the O serotypes of the Escherichia coli are O127, O126, O44 and the like, wherein the O126 is the dominant serotype (4/13), and the 13 strains of the Escherichia coli are numbered as EC 1-EC 13.
TABLE 213O serotypes of E.coli
2.2 mouse pathogenicity test
The results of pathogenicity tests on mice are shown in table 3, 11 of 13 escherichia coli strains have strong pathogenicity on the mice, the lethality rate is 40% -100%, the mice mainly take pulmonary hemorrhage and diarrhea as the main, and 2 escherichia coli strains have no lethality on the mice and mainly show lassitude and anorexia.
Results of the pathogenicity test of Table 313 strains of Escherichia coli on mice
2.3 nematode pathogenicity test
As can be seen from the pathogenic model of Escherichia coli-caenorhabditis elegans, EC3 and EC10 in 13 strains of Escherichia coli have the strongest pathogenicity to nematodes, the longest survival time of the nematodes infecting the 2 strains of Escherichia coli is 7d and 9d respectively, but half of the death time is 3d, and the survival rate of the nematodes on the third day is reduced to 50%. Judging that the nematode has strong pathogenicity. In addition, EC5 and EC13 are the weakest pathogenicity to nematodes, the longest survival time of the nematodes infecting these 2 strains of Escherichia coli is 11d and 10d respectively, and the half lethal time is 6d and 5d respectively. It is judged to be weak to the nematode. After other 9 strains of escherichia coli infect nematodes, the longest survival time is 7 d-10 d, and the half lethal time is 3.1 d-4.5 d. It is judged to be moderately pathogenic to nematodes.
In order to determine the pathogenicity of escherichia coli to nematodes more quickly, the 13 strains of escherichia coli are divided into three standards of weak pathogenicity to nematodes, medium pathogenicity to nematodes and strong pathogenicity to nematodes by combining with nematode survival test analysis, and the three standards are shown in table 2.
As can be seen from Table 2, the pathogenicity of E.coli against C.elegans began to differ significantly from day 2 onwards: the lethality of the strain with weak pathogenicity to the nematode is less than or equal to 5 percent; the lethality of strains with moderate pathogenicity to nematodes is 5-20%; the lethality of the strain with strong pathogenicity to the nematode is more than or equal to 20 percent. On day 3, the lethality of the strains with weak pathogenicity to the nematodes is 5-10%; the lethality of strains with moderate pathogenicity to nematodes is 10-48%; the lethality of the strain with strong pathogenicity to the nematode is more than or equal to 50 percent. On the 4 th day, the lethality of the strain with weak pathogenicity to the nematode is 20 to 30 percent; the lethality of strains with moderate pathogenicity to nematodes is 30-70%; the lethality of the strain with strong pathogenicity to the nematode is more than or equal to 85 percent. As can be seen from the above, the difference of pathogenicity of the nematode caused by Escherichia coli is mainly reflected in 2 d-4 d.
The mortality rate of escherichia coli with strong pathogenicity of the mouse to the nematode is the highest, and the mortality rate of escherichia coli with weak pathogenicity of the mouse to the nematode is the lowest. Thus, the pathogenicity of the Escherichia coli to the mouse can obtain the same result in vivo. The survival rate of the nematodes infected by the O157 positive control is reduced fastest, and the mortality rate is most obvious; most of the Escherichia coli have higher lethality, and the nematode survival rate is obviously reduced compared with that of the positive control SM 022.
2.4 count and identification of nematode intestinal bacteria
The bacteria obtained by dilution coating are subjected to 16S sequencing and O serotype identification, and the result shows that the bacteria are escherichia coli, and the serotype is consistent with that of an infectious bacterium. As can be seen from FIGS. 2 and 3, the number of E.coli in the nematode body was positively correlated with the infection time, and the longer the infection time was, the larger the number of E.coli. The number of escherichia coli from 1d to 2d is not obviously increased, and the survival rate of the nematodes is slightly reduced but is not obvious when the figure 1 is combined, and the death rate is 0-25%; the number of colibacillus in the nematode bodies of 3 rd to 4 th days begins to increase, and the death rate is 20-85%. The number of colibacillus in the 3 d-6 d nematode body is continuously increased and propagated, and the death rate reaches the highest.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
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<120> caenorhabditis elegans model for identifying bovine escherichia coli virulence
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Claims (10)
1. A caenorhabditis elegans model for identifying virulence of bovine Escherichia coli, which is characterized by comprising the following steps:
(1) carrying out synchronization treatment on caenorhabditis elegans;
(2) culturing escherichia coli in an SK culture medium to form lawn;
(3) placing the C.elegans which is subjected to the synchronization treatment in a culture medium containing escherichia coli lawn; namely a caenorhabditis elegans model for identifying the virulence of bovine escherichia coli;
the synchronization treatment comprises the steps of culturing for 40-60 hours after cracking, centrifuging and precipitating to obtain synchronized L4-phase nematodes;
the escherichia coli is firstly placed in an LB solid culture medium for culturing for 18 h-24 h at 37 ℃, then placed in an LB broth for shaking for 18 h-24 h at 37 ℃, and finally placed in an SK culture medium and placed in a biochemical incubator at 20 ℃ for overnight drying to form lawn.
2. The caenorhabditis elegans model for identifying bovine-derived escherichia coli virulence as claimed in claim 1, wherein the disruption time of the synchronization process is 4-5 min.
3. The caenorhabditis elegans model for identifying bovine-derived escherichia coli virulence as recited in claim 1, wherein the centrifugation speed of the synchronization process is 2500-3500 r/min, and the centrifugation time is 1-3 min.
4. The caenorhabditis elegans model for identifying virulence of bovine Escherichia coli as claimed in claim 2, wherein the lysed lysate is first placed in a shaker at 20 ℃ and 150r/min and shaken for 20 h.
5. The caenorhabditis elegans model for identifying bovine escherichia coli virulence as claimed in claim 1, wherein the culture of the synchronization treatment process is to transfer L1 phase nematodes at the bottom of the centrifuge tube into a clean NGM plate, and place the plate in a biochemical incubator at 20 ℃ for 40-60 h to obtain synchronized L4 phase nematodes.
6. The caenorhabditis elegans model for identifying bovine-derived escherichia coli virulence as claimed in claim 1, wherein escherichia coli is collected from viscera or feces of cattle died of illness, and single escherichia coli colony is gram-stained, microscopic, inoculated on MacconyKa medium, cultured at 37 ℃ for 18-24 h, further purified, and inoculated in nutrient broth for re-culture.
7. The caenorhabditis elegans model for identifying virulence of bovine Escherichia coli as claimed in claim 6, wherein said Escherichia coli DNA is extracted for PCR reaction.
8. The caenorhabditis elegans model for identifying bovine escherichia coli virulence of claim 7, wherein the PCR reaction primer sequence is set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
9. The caenorhabditis elegans model for identifying bovine-derived escherichia coli virulence as claimed in claim 7, wherein the PCR reaction system comprises 25 μ L of 2 × ES Taq MasterMix, 2 μ L of each of upstream and downstream primers, 4 μ L of template, and up to 50 μ L of ddH2O, and the reaction procedures comprise pre-denaturation at 95 ℃ for 5min, denaturation at 94 ℃ for 30S, annealing at 52 ℃ for 30S, elongation at 72 ℃ for 1min, 35 cycles, and final elongation at 72 ℃ for 10 min.
10. The use of the caenorhabditis elegans pathogenic model of bovine origin escherichia coli virulence, which is established by the caenorhabditis elegans model for identifying bovine origin escherichia coli virulence according to claim 1, in drug screening.
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| CN112575055A (en) * | 2020-12-21 | 2021-03-30 | 广西壮族自治区兽医研究所 | Method for rapidly detecting virulence of bovine-derived Shigella by using defective caenorhabditis elegans |
| WO2023236496A1 (en) * | 2022-06-09 | 2023-12-14 | 上海大学 | Method for counting number of intrauterinely developed embryos of caenorhabditis elegans |
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