本文選題：斷奶仔豬 + 大腸桿菌耐藥性�。� 參考：《南京農(nóng)業(yè)大學》2015年碩士論文

【摘要】：畜牧業(yè)中,抗生素或用于預防和治療動物疾病,或添加到飼料中促進動物生長,為現(xiàn)代畜牧業(yè)的發(fā)展做出了巨大的貢獻,但是這也導致了動物腸道細菌嚴重的耐藥問題。銅不僅是動物細胞和細菌正常生長所必需,高水平銅也能抑制細菌生長,因此養(yǎng)豬生產(chǎn)中常在仔豬飼料中添加高銅以抗腹瀉、促生長,但是高銅也會誘導細菌的銅抗性。研究發(fā)現(xiàn)細菌的重金屬抗性和抗生素耐藥性之間存在多種形式的聯(lián)系,但是飼料高銅對于腸道大腸桿菌抗生素耐藥的形成是否存在協(xié)同作用?如果存在,其可能的機制是什么?這些問題迄今尚未明了,本論文擬圍繞上述兩個關鍵科學問題開展深入系統(tǒng)研究,試驗分為四個部分:一、斷奶前后仔豬和母豬糞便源大腸桿菌耐藥性和銅抗性的分析本試驗旨在比較斷奶前后仔豬和母豬大腸桿菌耐藥性和銅抗性的變化,并分析其可能原因,為尋求控制大腸桿菌耐藥性形成與傳播的營養(yǎng)措施提供依據(jù)。試驗中,采用美國臨床試驗室標準化委員會(CLSI)推薦的瓊脂稀釋法,對分離自仔豬及其哺乳母豬的224株大腸桿菌的7種抗生素耐藥性及銅抗性進行檢測。結果表明,224株大腸桿菌對7種抗生素均有不同程度的耐藥(耐藥率范圍28.13%～93.30%),70.09%的菌株表現(xiàn)出多重耐藥性(MDR)。分離自母豬與哺乳仔豬的大腸桿菌抗生素耐藥率相似度較高,僅有對氨芐西林和慶大霉素的耐藥率差異顯著。而斷奶后仔豬源大腸桿菌的耐藥率相對于母豬和哺乳仔豬變化明顯,耐受頭孢噻呋、氟苯尼考和氯霉素的菌株都極顯著增加(P0.01),耐受氨芐西林的菌株則顯著增加(P0.05)。相對于母豬和哺乳仔豬,分離自斷奶后仔豬的大腸桿菌對銅的抗性程度明顯增加(P0.01)。大腸桿菌對銅抗性增加時,對抗生素耐藥性也有所變化,兩者之間有一定的相關性。母豬腸道內(nèi)的耐藥菌及飼料中的抗生素添加劑對仔豬大腸桿菌耐藥性產(chǎn)生、維持與加劇發(fā)揮了重要作用,仔豬飼料中添加的高水平銅與大腸桿菌耐藥水平的變化存在一定的關系。二、飼料高銅對斷奶仔豬大腸桿菌恩諾沙星耐藥協(xié)同作用的研究本試驗旨在研究飼料高銅和恩諾沙星對腸道中大腸桿菌耐藥性和銅抗性水平的影響,并探討飼料高銅在腸道大腸桿菌耐藥性形成中的協(xié)同作用。試驗以腸道細菌抗生素耐藥水平較低的10頭巴馬香豬為對象,分為低銅組(4 mg/kg)和高銅組(125 mg/kg),每組5頭仔豬。試驗開始后的第15天分別在兩組日糧中加入亞治療劑量的恩諾沙星,連續(xù)加藥10天。第25天,停止加藥,繼續(xù)分別飼喂低銅、高銅無抗日糧。于試驗的0、14、19、24、34、48天收集仔豬新鮮糞便樣品,分離并鑒定大腸桿菌。采用CLSI推薦的瓊脂稀釋法,對分離株進行抗生素及銅敏感性的檢測。結果顯示:試驗過程中,因受飼料中銅和恩諾沙星的影響,兩組大腸桿菌分離數(shù)都不斷下降(P0.01)。低、高銅組隨飼料中恩諾沙星的添加和撤除,菌株耐藥性水平都先極顯著上升(P0.01)而后極顯著下降(P0.01)。高銅組菌株還表現(xiàn)為添加抗生素前恩諾沙星耐藥率極顯著高于同時期的低銅組(49.44%4.05%,P0.01);撤除抗生素后大腸桿菌耐藥率仍顯著高于同時期的低銅組(14.86%2.70%,P0.05)。高銅組因高水平銅在飼料中持續(xù)添加,菌株銅抗性顯著高于低銅組(P0.05),而低銅組菌株隨低銅日糧的持續(xù)飼喂銅抗性極顯著下降(P0.01)。大腸桿菌恩諾沙星耐藥性和銅抗性的變化上存在相關性,飼料添加恩諾沙星后,高銅組菌株銅抗性和耐藥性變化正相關(P0.01);低銅組在給藥的中、后期,菌株銅抗性和耐藥性由負相關轉變?yōu)檎嚓P(P0.01)。因此,飼料中添加高銅和恩諾沙星對大腸桿菌耐藥性和銅抗性形成影響明顯,高銅飼料對大腸桿菌耐藥性增加具有促進和維持作用。飼料高銅和恩諾沙星對大腸桿菌耐藥性形成可能具有協(xié)同作用。三、斷奶香豬大腸桿菌質(zhì)粒介導喹諾酮類耐藥基因、耐銅基因檢測及差異性分析本試驗旨在研究斷奶香豬大腸桿菌質(zhì)粒介導喹諾酮類耐藥基因、耐銅基因的攜帶情況,探討抗性基因與抗性表型的相關性、抗性基因間的相關性。本研究依據(jù)上一章的菌株MIC結果選取四種不同抗性表型菌株進行質(zhì)粒的提取,分別為高耐藥高耐銅菌株(恩諾沙星MIC32μg/mL,銅MIC≥24mM);高耐藥低耐銅菌株(恩諾沙星MIC32μg/mL,銅MIC24mM);高敏感高耐銅菌株(恩諾沙星MIC ≤0.125μg/mL,銅MIC≥24mM);高敏感低耐銅菌株(恩諾沙星≤0.125μg/mL,銅MIC24mM),共125株。PCR檢測質(zhì)粒介導的4種喹諾酮類耐藥基因(qnrS、qepA、oqxAB、aac(6')-lb-cr)和兩種耐銅基因(pcoA、pcoD)。結果表明,125株大腸桿菌中除qepA基因外,其余抗性基因檢出率均較高,oqxAB達到95.2%,其次是qnrS(92.0%),aac(6')-lb-cr檢出率為81.6%;pcoA和pcoD的檢出率分別為48.8%、66.4%。111株菌株攜帶3種或3種以上的抗性基因。通過表型和基因型相關性分析,發(fā)現(xiàn)抗性表型與抗性基因的檢出率有一定的相關性,表型耐藥菌株抗性基因檢出率較高。飼料中亞治療劑量恩諾沙星的添加導致aac(6')-lb-cr基因的檢出率增加(P0.01)。多數(shù)抗性基因的檢出率具有相關性,如oqxAB和qepA、aac(6')-lb-cr與qepA都呈極顯著正相關(P0.01);qepA和qnrS呈極顯著負相關(P0.01)。除qepA外,其他幾種喹諾酮類耐藥基因和抗銅基因間呈顯著正相關(P0.05)。綜上所述,大腸桿菌普遍攜帶多種質(zhì)粒介導抗性基因,菌株耐藥表型與耐藥基因攜帶情況并不完全一致。耐藥基因和抗銅基因間具有相關性,從基因水平上可以初步推測大腸桿菌質(zhì)粒介導耐銅基因和喹諾酮類耐藥基因間有同時水平傳播的可能性。四、恩諾沙星和銅對體外模型中大腸桿菌恩諾沙星敏感性的影響研究本試驗旨在體外研究銅對大腸桿菌恩諾沙星敏感性的影響和可能的機制。試驗選取分離自斷奶香豬糞便的兩株(M1,M2)敏感型大腸桿菌(恩諾沙星MIC=0.5μg/mL,銅MIC=20mM),分別于添加恩諾沙星(0.25μg/mL)、硫酸銅(10mM)、硫酸銅(10mM)和恩諾沙星(0.25μg/mL)的MH肉湯(恩諾沙星組、銅組、恩諾沙星加銅組)中培養(yǎng)并傳代,采用瓊脂稀釋法測定第3、6、9、12、15、18代菌株的恩諾沙星和銅MIC值,PCR檢測第0和18代菌株質(zhì)粒介導抗性基因的攜帶情況。結果表明:經(jīng)誘導后,兩株菌株恩諾沙星和銅MIC值都有所變化。兩株菌的恩諾沙星MIC值,在恩諾沙星組中都顯著提高8倍,產(chǎn)生耐藥性;恩諾沙星加銅組中,兩株菌株的恩諾沙星MIC值提高了4倍,處于耐藥中介范圍。兩株菌株的銅MIC值,在恩諾沙星組均有所上升,M2達28mM;而銅組和恩諾沙星加銅組中,銅MIC都升至24mM。對抗性基因檢測可知,三種不同的環(huán)境誘導后,都能從大腸桿菌中檢出質(zhì)粒上喹諾酮類耐藥基因qnrS,其他基因無變化。綜上所述,通過恩諾沙星和銅的誘導,大腸桿菌耐藥性和銅抗性有所增加。低濃度恩諾沙星對大腸桿菌MIC影響明顯,從體外證實了低濃度恩諾沙星能誘導大腸桿菌逐步產(chǎn)生耐藥性。而體外模型中,銅和恩諾沙星的協(xié)同作用不明顯,與體內(nèi)分離菌株試驗結果有所差異,需進一步研究探討。
[Abstract]:In animal husbandry, antibiotics are used to prevent and treat animal diseases, or to be added to feed to promote animal growth, which has made a great contribution to the development of modern animal husbandry, but this also leads to the serious drug resistance of animal intestinal bacteria. Copper is not only necessary for the normal growth of animal cells and bacteria, but also the high level copper can also inhibit the bacteria. As a result, high copper is often added to piglet feed to resist diarrhea and growth, but high copper can also induce copper resistance of bacteria. What is the possible mechanism of the same effect? If it exists, what are the possible mechanisms? These questions have not been understood so far. This paper is intended to carry out a thorough and systematic study of the two key scientific problems mentioned above. The experiment is divided into four parts: first, the analysis of the resistance of Escherichia coli and copper resistance of piglets and sows before and after weaning is aimed at comparing the Aberdeen before and after weaning. The changes in resistance to Escherichia coli and copper resistance in pigs and sows and their possible causes were analyzed to provide a basis for the search for the nutritional measures to control the formation and transmission of resistance to Escherichia coli. In the experiment, the agar dilution method recommended by the American clinical laboratory standardization committee (CLSI) was used to separate 224 coliform rods from piglets and their lactation sows. The resistance of 7 kinds of antibiotics and copper resistance were detected. The results showed that 224 strains of Escherichia coli were resistant to 7 antibiotics in varying degrees (the range of resistance rate was 28.13% to 93.30%), and 70.09% of the strains showed multiple resistance (MDR). The similarity of antibiotic resistance of isolated bacilli from sow and suckling piglets was higher and only ampicin was used. The resistance rate of Xilin and gentamicin was significantly different, but the resistance rate of Escherichia coli in weaned piglets was significantly higher than that of sow and lactation piglets, and the strains resistant to ceftif, florfenicol and chloramphenicol were significantly increased (P0.01), and the strains tolerant ampicillin were significantly increased (P0.05). The resistance of Escherichia coli to copper increased significantly after weaning (P0.01). When the resistance of Escherichia coli to copper was increased, the resistance to antibiotics was also changed, and there was a certain correlation between the resistance of Escherichia coli and the antibiotic additives in the pig's intestines. The important role is that there is a certain relationship between the high level copper added in the piglet feed and the change of the resistance level of Escherichia coli. Two, the study of the synergistic effect of high copper on the resistance to enrofloxacin in weanling piglets was designed to study the effect of high copper and enrofloxacin on the resistance to Escherichia coli and the level of copper resistance in the intestinal tract. The synergistic effect of high copper on resistance formation of intestinal Escherichia coli was investigated. 10 Bama pigs with low antibiotic resistance of intestinal bacteria were divided into low copper group (4 mg/kg) and high copper group (125 mg/kg), and 5 piglets in each group. The fifteenth days after the trial were added to the two groups of diet to add subtherapeutic dose, respectively. Enrofloxacin, continuous addition of medicine for 10 days, twenty-fifth days, stopped feeding, continued feeding low copper, high copper and no Anti Japanese food. Collected fresh feces samples from piglets on 0,14,19,24,34,48 days, isolated and identified Escherichia coli. The agar dilution method recommended by CLSI was used to detect the antimicrobial and copper sensitivity of the isolates. The results showed: test process In the influence of copper and enrofloxacin in the feed, the number of Escherichia coli in the two groups decreased continuously (P0.01). The resistance level of the high copper group increased significantly (P0.01) with the addition and removal of enrofloxacin in the feed (P0.01). The high copper group also showed the resistance rate of Enrofloxacin before the antibiotic. The resistance rate of Escherichia coli was significantly higher than that of low copper group at the same time (14.86%2.70%, P0.05) after the removal of antibiotics (14.86%2.70%, P0.05). The copper resistance of the high copper group was significantly higher than that of the low copper group (P0.05), while the low copper group continued to feed copper resistance with low copper diet. There was a significant decrease in sex (P0.01). There was a correlation between the resistance of enrofloxacin and copper resistance in Escherichia coli. After the feed added enrofloxacin, the copper resistance and resistance changes in the high copper group were positively correlated (P0.01); the copper resistance and resistance of the low copper group were positively correlated with the negative correlation (P0.01) in the later period of the administration of the low copper group. Therefore, the feed was in the feed. The effect of high copper and enrofloxacin on resistance to Escherichia coli and formation of copper resistance is obvious. High copper feed can promote and maintain the resistance to Escherichia coli. Fodder high copper and enrofloxacin may have synergistic effect on the formation of Escherichia coli resistance. Three, the plasmid mediated quinolone resistance in weanling pig Escherichia coli, The test and difference analysis of copper resistant genes were designed to study the quinolone resistant gene of Escherichia coli in weaned pigs, the carrying situation of copper resistant genes, the correlation of resistance genes and resistant phenotype, and the correlation between resistance genes. This study selected four different resistant phenotypic strains according to the results of the strain MIC in the last chapter. High resistance and high copper resistant strains (enrofloxacin MIC32 g/mL, copper MIC > 24mM), high resistance and low copper resistant strain (enrofloxacin MIC32 g/mL, copper MIC24mM), high sensitive and high copper resistant strains (enrofloxacin MIC < 0.125 g/mL, copper MIC > 24mM), high sensitive and low copper resistant strains (enrofloxacin < 0.125 micron g/mL, copper), were 125. Strain.PCR detected 4 quinolone resistant genes (qnrS, qepA, oqxAB, AAC (6') -lb-cr) and two kinds of copper resistant genes (pcoA, pcoD). The results showed that in 125 strains of Escherichia coli, the detection rate of the other resistant genes was higher, oqxAB reached 95.2%, followed by qnrS (92%), and the detection rate was 81.6%. The rate of 66.4%.111 strain was 48.8%. 3 or more resistance genes were carried by the strain of 66.4%.111 strain. Through the analysis of phenotypic and genotype correlation, it was found that the resistance phenotype was related to the detection rate of resistance genes, and the detection rate of resistance genes of phenotypic resistant strains was higher. The addition of enrofloxacin in feed Central Asian therapeutic agents led to the AAC (6') -lb-cr gene. The detection rate increased (P0.01). The detection rates of most resistant genes were correlated, such as oqxAB and qepA, AAC (6') -lb-cr and qepA had very significant positive correlation (P0.01); qepA and qnrS showed significant negative correlation (P0.01). Except qepA, there was a significant positive correlation between several other quinolones resistance genes and copper resistant genes. A variety of plasmid mediated resistance genes are all carried, and the resistance phenotype of the strain is not identical with that of the resistant gene. The correlation between the resistance gene and the copper resistance gene is related. The possibility of simultaneous horizontal transmission between the coliform plasmid mediated copper resistant gene and the quinolone resistant gene can be preliminarily deduced from the gene level. Four, enosand The effect of star and copper on the sensitivity of enrofloxacin in vitro model in vitro study on the effect and possible mechanism of copper on the sensitivity of enrofloxacin to Escherichia coli in vitro. Two strains of Escherichia coli (M1, M2) sensitive Escherichia coli (enrofloxacin MIC=0.5 g/mL, copper MIC=20mM) isolated from the feces of weaned pigs were selected, respectively. G/mL, copper sulphate (10mM), copper sulfate (10mM) and enrofloxacin (0.25 g/mL) MH broth (enrofloxacin group, copper group, enrofloxacin plus copper group) were cultured and passaged, and agar dilution method was used to determine the value of enrofloxacin and copper MIC in the 3,6,9,12,15,18 generation strain, and PCR was used to detect the plasmid mediated resistance gene of the zeroth and eighteenth generation strain. The results showed that the values of enrofloxacin and copper MIC in the two strains were all changed after induction. The MIC value of enrofloxacin in the two strains of enrofloxacin was significantly increased by 8 times in the enrofloxacin group and in the enrofloxacin group, the MIC value of enrofloxacin was increased by 4 times, and the copper MIC of the two strains. Value, in the enrofloxacin group all increased, M2 reached 28mM, and copper and enrofloxacin and copper groups were promoted to 24mM. antagonistic genes, and three different environments could detect the quinolone resistance gene qnrS of plasmid from Escherichia coli, and the other bases were not changed. In summary, enrofloxacin and copper were induced. Resistance to Escherichia coli and copper resistance increased. Low concentration enrofloxacin had an obvious effect on Escherichia coli MIC. It proved that low concentration enrofloxacin could induce resistance to Escherichia coli in vitro. In vitro model, the synergistic effect of copper and enrofloxacin was not obvious. One step of research.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S828.5

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