饲料高铜对猪肠道大肠杆菌抗生素耐药协同作用的研究

发布时间：2018-06-20 11:47

本文选题：断奶仔猪 + 大肠杆菌耐药性　；参考：《南京农业大学》2015年硕士论文

【摘要】：畜牧业中,抗生素或用于预防和治疗动物疾病,或添加到饲料中促进动物生长,为现代畜牧业的发展做出了巨大的贡献,但是这也导致了动物肠道细菌严重的耐药问题。铜不仅是动物细胞和细菌正常生长所必需,高水平铜也能抑制细菌生长,因此养猪生产中常在仔猪饲料中添加高铜以抗腹泻、促生长,但是高铜也会诱导细菌的铜抗性。研究发现细菌的重金属抗性和抗生素耐药性之间存在多种形式的联系,但是饲料高铜对于肠道大肠杆菌抗生素耐药的形成是否存在协同作用?如果存在,其可能的机制是什么?这些问题迄今尚未明了,本论文拟围绕上述两个关键科学问题开展深入系统研究,试验分为四个部分:一、断奶前后仔猪和母猪粪便源大肠杆菌耐药性和铜抗性的分析本试验旨在比较断奶前后仔猪和母猪大肠杆菌耐药性和铜抗性的变化,并分析其可能原因,为寻求控制大肠杆菌耐药性形成与传播的营养措施提供依据。试验中,采用美国临床试验室标准化委员会(CLSI)推荐的琼脂稀释法,对分离自仔猪及其哺乳母猪的224株大肠杆菌的7种抗生素耐药性及铜抗性进行检测。结果表明,224株大肠杆菌对7种抗生素均有不同程度的耐药(耐药率范围28.13%～93.30%),70.09%的菌株表现出多重耐药性(MDR)。分离自母猪与哺乳仔猪的大肠杆菌抗生素耐药率相似度较高,仅有对氨苄西林和庆大霉素的耐药率差异显著。而断奶后仔猪源大肠杆菌的耐药率相对于母猪和哺乳仔猪变化明显,耐受头孢噻呋、氟苯尼考和氯霉素的菌株都极显著增加(P0.01),耐受氨苄西林的菌株则显著增加(P0.05)。相对于母猪和哺乳仔猪,分离自断奶后仔猪的大肠杆菌对铜的抗性程度明显增加(P0.01)。大肠杆菌对铜抗性增加时,对抗生素耐药性也有所变化,两者之间有一定的相关性。母猪肠道内的耐药菌及饲料中的抗生素添加剂对仔猪大肠杆菌耐药性产生、维持与加剧发挥了重要作用,仔猪饲料中添加的高水平铜与大肠杆菌耐药水平的变化存在一定的关系。二、饲料高铜对断奶仔猪大肠杆菌恩诺沙星耐药协同作用的研究本试验旨在研究饲料高铜和恩诺沙星对肠道中大肠杆菌耐药性和铜抗性水平的影响,并探讨饲料高铜在肠道大肠杆菌耐药性形成中的协同作用。试验以肠道细菌抗生素耐药水平较低的10头巴马香猪为对象,分为低铜组(4 mg/kg)和高铜组(125 mg/kg),每组5头仔猪。试验开始后的第15天分别在两组日粮中加入亚治疗剂量的恩诺沙星,连续加药10天。第25天,停止加药,继续分别饲喂低铜、高铜无抗日粮。于试验的0、14、19、24、34、48天收集仔猪新鲜粪便样品,分离并鉴定大肠杆菌。采用CLSI推荐的琼脂稀释法,对分离株进行抗生素及铜敏感性的检测。结果显示:试验过程中,因受饲料中铜和恩诺沙星的影响,两组大肠杆菌分离数都不断下降(P0.01)。低、高铜组随饲料中恩诺沙星的添加和撤除,菌株耐药性水平都先极显著上升(P0.01)而后极显著下降(P0.01)。高铜组菌株还表现为添加抗生素前恩诺沙星耐药率极显著高于同时期的低铜组(49.44%4.05%,P0.01);撤除抗生素后大肠杆菌耐药率仍显著高于同时期的低铜组(14.86%2.70%,P0.05)。高铜组因高水平铜在饲料中持续添加,菌株铜抗性显著高于低铜组(P0.05),而低铜组菌株随低铜日粮的持续饲喂铜抗性极显著下降(P0.01)。大肠杆菌恩诺沙星耐药性和铜抗性的变化上存在相关性,饲料添加恩诺沙星后,高铜组菌株铜抗性和耐药性变化正相关(P0.01);低铜组在给药的中、后期,菌株铜抗性和耐药性由负相关转变为正相关(P0.01)。因此,饲料中添加高铜和恩诺沙星对大肠杆菌耐药性和铜抗性形成影响明显,高铜饲料对大肠杆菌耐药性增加具有促进和维持作用。饲料高铜和恩诺沙星对大肠杆菌耐药性形成可能具有协同作用。三、断奶香猪大肠杆菌质粒介导喹诺酮类耐药基因、耐铜基因检测及差异性分析本试验旨在研究断奶香猪大肠杆菌质粒介导喹诺酮类耐药基因、耐铜基因的携带情况,探讨抗性基因与抗性表型的相关性、抗性基因间的相关性。本研究依据上一章的菌株MIC结果选取四种不同抗性表型菌株进行质粒的提取,分别为高耐药高耐铜菌株(恩诺沙星MIC32μg/mL,铜MIC≥24mM);高耐药低耐铜菌株(恩诺沙星MIC32μg/mL,铜MIC24mM);高敏感高耐铜菌株(恩诺沙星MIC ≤0.125μg/mL,铜MIC≥24mM);高敏感低耐铜菌株(恩诺沙星≤0.125μg/mL,铜MIC24mM),共125株。PCR检测质粒介导的4种喹诺酮类耐药基因(qnrS、qepA、oqxAB、aac(6')-lb-cr)和两种耐铜基因(pcoA、pcoD)。结果表明,125株大肠杆菌中除qepA基因外,其余抗性基因检出率均较高,oqxAB达到95.2%,其次是qnrS(92.0%),aac(6')-lb-cr检出率为81.6%;pcoA和pcoD的检出率分别为48.8%、66.4%。111株菌株携带3种或3种以上的抗性基因。通过表型和基因型相关性分析,发现抗性表型与抗性基因的检出率有一定的相关性,表型耐药菌株抗性基因检出率较高。饲料中亚治疗剂量恩诺沙星的添加导致aac(6')-lb-cr基因的检出率增加(P0.01)。多数抗性基因的检出率具有相关性,如oqxAB和qepA、aac(6')-lb-cr与qepA都呈极显著正相关(P0.01);qepA和qnrS呈极显著负相关(P0.01)。除qepA外,其他几种喹诺酮类耐药基因和抗铜基因间呈显著正相关(P0.05)。综上所述,大肠杆菌普遍携带多种质粒介导抗性基因,菌株耐药表型与耐药基因携带情况并不完全一致。耐药基因和抗铜基因间具有相关性,从基因水平上可以初步推测大肠杆菌质粒介导耐铜基因和喹诺酮类耐药基因间有同时水平传播的可能性。四、恩诺沙星和铜对体外模型中大肠杆菌恩诺沙星敏感性的影响研究本试验旨在体外研究铜对大肠杆菌恩诺沙星敏感性的影响和可能的机制。试验选取分离自断奶香猪粪便的两株(M1,M2)敏感型大肠杆菌(恩诺沙星MIC=0.5μg/mL,铜MIC=20mM),分别于添加恩诺沙星(0.25μg/mL)、硫酸铜(10mM)、硫酸铜(10mM)和恩诺沙星(0.25μg/mL)的MH肉汤(恩诺沙星组、铜组、恩诺沙星加铜组)中培养并传代,采用琼脂稀释法测定第3、6、9、12、15、18代菌株的恩诺沙星和铜MIC值,PCR检测第0和18代菌株质粒介导抗性基因的携带情况。结果表明:经诱导后,两株菌株恩诺沙星和铜MIC值都有所变化。两株菌的恩诺沙星MIC值,在恩诺沙星组中都显著提高8倍,产生耐药性;恩诺沙星加铜组中,两株菌株的恩诺沙星MIC值提高了4倍,处于耐药中介范围。两株菌株的铜MIC值,在恩诺沙星组均有所上升,M2达28mM;而铜组和恩诺沙星加铜组中,铜MIC都升至24mM。对抗性基因检测可知,三种不同的环境诱导后,都能从大肠杆菌中检出质粒上喹诺酮类耐药基因qnrS,其他基因无变化。综上所述,通过恩诺沙星和铜的诱导,大肠杆菌耐药性和铜抗性有所增加。低浓度恩诺沙星对大肠杆菌MIC影响明显,从体外证实了低浓度恩诺沙星能诱导大肠杆菌逐步产生耐药性。而体外模型中,铜和恩诺沙星的协同作用不明显,与体内分离菌株试验结果有所差异,需进一步研究探讨。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828.5

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