中藥對臨床型奶牛乳房炎源大腸桿菌的體外抑制及耐藥消除研究
發(fā)布時間：2019-05-09 19:01
【摘要】：奶牛乳房炎是奶牛乳腺組織受到內(nèi)、外界環(huán)境中的刺激因素如物理、化學(xué)、微生物等因素刺激所產(chǎn)生的一種炎性反應(yīng),是奶牛生產(chǎn)中的常見病和多發(fā)病,而多種病原微生物混合感染是引起奶牛乳房炎發(fā)病的主要原因,嚴(yán)重制約了當(dāng)前奶牛業(yè)的發(fā)展。奶牛場乳房炎的防控與治療目前主要依賴抗菌藥物,但由于長期和廣泛使用抗菌藥物,使得細(xì)菌耐藥性增強(qiáng)現(xiàn)象普遍發(fā)生,導(dǎo)致乳汁中抗菌藥物殘留超標(biāo)、環(huán)境污染嚴(yán)重及乳制品安全受到威脅等問題日益突出,引起了全社會的廣泛關(guān)注。我國傳統(tǒng)中草藥具有天然性、多功能性、基本無耐藥性、不易在畜禽及其產(chǎn)品中殘留、毒副作用小和不污染環(huán)境等優(yōu)點(diǎn),在奶牛乳房炎防治中的應(yīng)用越來越受到重視。其中,中草藥黃連、黃芩及其復(fù)方制劑具有清熱解毒、抑菌、抗病毒等作用,在臨床上的研究和應(yīng)用已成為熱點(diǎn)。本研究收集陜西省部分地區(qū)奶牛場患臨床型乳房炎奶牛乳樣共95份,采用革蘭氏染色鏡檢、生理生化試驗(yàn)、分子生物學(xué)特性檢測等方法對其進(jìn)行大腸桿菌的分離和鑒定;采用二倍稀釋法對分離得到的大腸桿菌進(jìn)行四組中藥(黃連、黃芩、黃連黃芩(1:1)水煎劑及雙黃連凍干粉針劑)體外抑菌試驗(yàn);采用掃描電鏡及酶標(biāo)板法檢測抑菌效果較好的黃芩和雙黃連對細(xì)菌胞壁及細(xì)菌外觀微形態(tài)的影響;進(jìn)一步檢測了所分離大腸桿菌對常用14種抗菌藥物的耐藥情況以及四組中藥對大腸桿菌耐藥性的消除作用,通過RT-PCR方法對比了四組中藥對大腸桿菌所含耐藥基因的影響,結(jié)果如下:本研究共分離得到大腸桿菌15株。四組中藥對所分離15株大腸桿菌均有一定程度的體外抑制作用,藥物體外MIC50測定結(jié)果顯示,雙黃連在所選中藥中抑菌作用最強(qiáng),其MIC50為3.90 mg/mL;其次是黃芩,其MIC50為7.81 mg/mL;然后是黃連和黃芩與黃連的組合,其MIC50為15.625 mg/mL。發(fā)射場電子掃描顯微鏡觀察大腸桿菌胞體表面微觀形態(tài)顯示隨著中藥作用時間的延長,大腸桿菌胞體表面完整性破壞越發(fā)嚴(yán)重,說明中藥黃芩和雙黃連均對大腸桿菌有破壞作用;中藥培養(yǎng)的大腸桿菌上清液中AKP隨著中藥作用時間的增加含量為上升趨勢,表明中藥黃芩和雙黃連可增加大腸桿菌細(xì)胞壁的通透性,提示黃芩和雙黃連主要通過對大腸桿菌細(xì)胞壁的破壞及菌體的分解破壞發(fā)揮抑制作用。15株大腸桿菌分離株對現(xiàn)階段臨床上普遍使用的大部分抗菌藥物均表現(xiàn)出不同程度的耐藥,其中對林可霉素、紅霉素、青霉素的耐藥率高達(dá)100%,而對頭孢西丁、頭孢噻肟的耐藥率為0%。經(jīng)中藥培養(yǎng)后各組菌株敏感性普遍增強(qiáng),其中以黃芩的作用最為顯著;對單種抗菌藥物(如萬古霉素)耐藥率降低最多的是黃芩(53%),其次是雙黃連(40%)。15株大腸桿菌檢測出β-內(nèi)酰胺酶(Extended spectrumβ-lactamase,ESBL)基因型兩種blaTEM和blaSHV,檢出率分別為40%和13.3%;檢測出質(zhì)粒介導(dǎo)的喹諾酮耐藥(Plasmid-mediated quinolone resistance,PMQR)基因型三種qnrB,qnrD和qnrS檢出率均為13.3%。經(jīng)中藥作用后,耐藥基因均呈現(xiàn)不同的減弱或消除,以黃芩對5種耐藥基因的消除效果最好,黃芩黃連(1:1)次之。以上試驗(yàn)結(jié)果表明,黃連、黃芩、雙黃連對臨床型奶牛乳房炎乳源大腸桿菌具有一定的體外抑菌作用,黃芩、雙黃連主要通過破壞大腸桿菌細(xì)胞壁完整性及分解破壞菌體發(fā)揮抑菌作用。藥敏試驗(yàn)和分子生物學(xué)檢測表明,中藥黃連、黃芩、雙黃連對大腸桿菌的耐藥性和耐藥基因均有不同程度的減弱或消除作用。
[Abstract]:The mastitis of the milk cow is an inflammatory reaction caused by the stimulation factors such as physical, chemical and micro-organisms in the dairy cow's breast tissue, which is a common disease and a disease in the production of the dairy cow, The mixed infection of a plurality of pathogenic microorganisms is the main cause of the occurrence of the mastitis of the dairy cow, and the development of the current dairy cattle industry is seriously restricted. The prevention and control and treatment of the mastitis of the dairy cattle field are mainly dependent on the antibacterial drugs, The problem of serious environmental pollution and the threat of the safety of dairy products has become more and more prominent, which has attracted wide attention from the whole society. The traditional Chinese herbal medicine has the advantages of natural nature, multi-function, basically no drug resistance, no residue in the livestock and poultry and the products thereof, small toxic and side effect, no environmental pollution and the like, and the application of the traditional Chinese herbal medicine in the prevention and treatment of the mastitis of the dairy cow is more and more important. The Chinese herbal medicine coptis, the Chinese medicinal preparation and the compound preparation have the effects of clearing away heat and toxic materials, inhibiting bacteria and resisting virus, and has become a hot spot in clinical research and application. In this study,95 samples of dairy cows with clinical mastitis were collected in the dairy cattle farm in some parts of Shaanxi, and the isolation and identification of E. coli were carried out by the methods of Gram staining, physiological and biochemical test and molecular biological characteristic test. in vitro antibacterial test of four groups of traditional Chinese medicine (coptis root, yellow root, coptis chinensis (1:1), and Shuanghuanglian freeze-dried powder injection) are carried out by adopting a double dilution method; The effects of yellow and double coptis on the micromorphology of the bacterial cell wall and the bacterial appearance are detected by using the scanning electron microscope and the enzyme labeling method, the drug resistance of the isolated E. coli to the common 14 antibacterial drugs and the elimination effect of the four groups of the traditional Chinese medicine on the drug resistance of the Escherichia coli are further detected, The effect of four groups of traditional Chinese medicine on the resistance gene contained in E. coli was compared by RT-PCR. The results were as follows:15 strains of E. coli were isolated. The four groups of traditional Chinese medicines have a certain degree of in-vitro inhibition on the isolated 15 strains of E. coli. The results of the determination of the in vitro MIC50 of the drug show that the inhibitory effect of the Shuanghuanglian in the selected Chinese medicine is the strongest, the MIC50 of the two coptis is 3.90 mg/ mL, the second is the yellow root, the MIC50 of the traditional Chinese medicine is 7.81 mg/ mL, and then is a combination of the coptis and the Chinese goldthread and the Chinese goldthread. Its MIC50 is 15.625 mg/ mL. The micromorphology of the surface of E. coli cell was observed by the electron-scanning microscope of the launch site, and the surface integrity of the E. coli cell was more and more serious with the extension of the time of the traditional Chinese medicine. The content of AKP in the supernatant of E. coli cultured by traditional Chinese medicine is the rising trend with the increasing of the action time of the traditional Chinese medicine, indicating that the traditional Chinese medicine yellow and double coptis can increase the permeability of the cell wall of the Escherichia coli, The results showed that Huang-Huang and Shuanghuanglian had an inhibitory effect on the destruction of the cell wall of E. coli and the decomposition and destruction of the cells. The 15 strains of E. coli isolates showed different resistance to most of the antibacterial drugs commonly used at the present stage, including lincomycin and erythromycin. The resistance rate of the penicillin is as high as 100%, and the resistance rate to cefoxitin and cefixime is 0%. the sensitivity of each group of strains after the culture of the traditional Chinese medicine is generally enhanced, and the effect of the yellow-root is the most significant; the drug resistance rate of a single antibacterial agent (such as vancomycin) is reduced to a maximum of yellow-root (53%), followed by double coptis (40%), and 15 strains of Escherichia coli are used for detecting an extended spectrum-lactamase, The detection rate was 40% and 13.3%, respectively. The detection rate of three qnrB, qnrD and qnrS was 13.3%. After the action of the traditional Chinese medicine, the drug-resistant gene has different attenuation or elimination, and the effect of the yellow rice on the five drug-resistant genes is the best, and the Huanglian Coptis (1:1) is the second. The results of the above test show that Coptis chinensis, Huang-Huang and Shuanghuanglian have a certain in-vitro antibacterial effect on the clinical-type dairy cow mastitis milk-source E. coli. The drug sensitivity test and molecular biology test show that the drug resistance and the drug resistance gene of the Chinese medicine coptis root, the yellow root and the Shuanghuanglian have different degrees of attenuation or elimination.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.23
本文編號：2473005
[Abstract]:The mastitis of the milk cow is an inflammatory reaction caused by the stimulation factors such as physical, chemical and micro-organisms in the dairy cow's breast tissue, which is a common disease and a disease in the production of the dairy cow, The mixed infection of a plurality of pathogenic microorganisms is the main cause of the occurrence of the mastitis of the dairy cow, and the development of the current dairy cattle industry is seriously restricted. The prevention and control and treatment of the mastitis of the dairy cattle field are mainly dependent on the antibacterial drugs, The problem of serious environmental pollution and the threat of the safety of dairy products has become more and more prominent, which has attracted wide attention from the whole society. The traditional Chinese herbal medicine has the advantages of natural nature, multi-function, basically no drug resistance, no residue in the livestock and poultry and the products thereof, small toxic and side effect, no environmental pollution and the like, and the application of the traditional Chinese herbal medicine in the prevention and treatment of the mastitis of the dairy cow is more and more important. The Chinese herbal medicine coptis, the Chinese medicinal preparation and the compound preparation have the effects of clearing away heat and toxic materials, inhibiting bacteria and resisting virus, and has become a hot spot in clinical research and application. In this study,95 samples of dairy cows with clinical mastitis were collected in the dairy cattle farm in some parts of Shaanxi, and the isolation and identification of E. coli were carried out by the methods of Gram staining, physiological and biochemical test and molecular biological characteristic test. in vitro antibacterial test of four groups of traditional Chinese medicine (coptis root, yellow root, coptis chinensis (1:1), and Shuanghuanglian freeze-dried powder injection) are carried out by adopting a double dilution method; The effects of yellow and double coptis on the micromorphology of the bacterial cell wall and the bacterial appearance are detected by using the scanning electron microscope and the enzyme labeling method, the drug resistance of the isolated E. coli to the common 14 antibacterial drugs and the elimination effect of the four groups of the traditional Chinese medicine on the drug resistance of the Escherichia coli are further detected, The effect of four groups of traditional Chinese medicine on the resistance gene contained in E. coli was compared by RT-PCR. The results were as follows:15 strains of E. coli were isolated. The four groups of traditional Chinese medicines have a certain degree of in-vitro inhibition on the isolated 15 strains of E. coli. The results of the determination of the in vitro MIC50 of the drug show that the inhibitory effect of the Shuanghuanglian in the selected Chinese medicine is the strongest, the MIC50 of the two coptis is 3.90 mg/ mL, the second is the yellow root, the MIC50 of the traditional Chinese medicine is 7.81 mg/ mL, and then is a combination of the coptis and the Chinese goldthread and the Chinese goldthread. Its MIC50 is 15.625 mg/ mL. The micromorphology of the surface of E. coli cell was observed by the electron-scanning microscope of the launch site, and the surface integrity of the E. coli cell was more and more serious with the extension of the time of the traditional Chinese medicine. The content of AKP in the supernatant of E. coli cultured by traditional Chinese medicine is the rising trend with the increasing of the action time of the traditional Chinese medicine, indicating that the traditional Chinese medicine yellow and double coptis can increase the permeability of the cell wall of the Escherichia coli, The results showed that Huang-Huang and Shuanghuanglian had an inhibitory effect on the destruction of the cell wall of E. coli and the decomposition and destruction of the cells. The 15 strains of E. coli isolates showed different resistance to most of the antibacterial drugs commonly used at the present stage, including lincomycin and erythromycin. The resistance rate of the penicillin is as high as 100%, and the resistance rate to cefoxitin and cefixime is 0%. the sensitivity of each group of strains after the culture of the traditional Chinese medicine is generally enhanced, and the effect of the yellow-root is the most significant; the drug resistance rate of a single antibacterial agent (such as vancomycin) is reduced to a maximum of yellow-root (53%), followed by double coptis (40%), and 15 strains of Escherichia coli are used for detecting an extended spectrum-lactamase, The detection rate was 40% and 13.3%, respectively. The detection rate of three qnrB, qnrD and qnrS was 13.3%. After the action of the traditional Chinese medicine, the drug-resistant gene has different attenuation or elimination, and the effect of the yellow rice on the five drug-resistant genes is the best, and the Huanglian Coptis (1:1) is the second. The results of the above test show that Coptis chinensis, Huang-Huang and Shuanghuanglian have a certain in-vitro antibacterial effect on the clinical-type dairy cow mastitis milk-source E. coli. The drug sensitivity test and molecular biology test show that the drug resistance and the drug resistance gene of the Chinese medicine coptis root, the yellow root and the Shuanghuanglian have different degrees of attenuation or elimination.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.23
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