亚抑菌浓度抗生素对野生黑颈长尾雉源大肠杆菌耐药基因水平传播效率的影响

发布时间：2018-05-01 09:32

本文选题：亚抑菌浓度 + 抗生素　；参考：《广西师范大学》2016年硕士论文

【摘要】：近年来,随着抗生素使用量的增加,细菌的耐药性逐渐增强、获得耐药性的速度逐渐加快,甚至出现了能够耐受大多数抗生素的“超级细菌”。因此人们更多地关心抗生素的杀菌效率,但对亚抑菌浓度抗生素对细菌耐药性传播效率的影响少有关注。本文通过对野生黑颈长尾雉源大肠杆菌耐药性、携带的可移动遗传元件及几种常用的抗生素在亚抑菌浓度条件下对耐药基因传播效率的影响进行研究,了解野生状态下大肠杆菌的耐药性,并对该野生雉类的人工驯养回放保护性措施可能引起的耐药性传播风险及亚抑菌浓度抗生素对耐药基因水平传播效率的影响的评价提供资料。采集广西百色市田林县野生黑颈长尾雉自然分布区野生黑颈长尾雉新鲜粪便,并利用显色培养基分离筛选大肠杆菌；以苹果酸脱氢酶基因(mdh)特异性引物对所分离大肠杆菌进行鉴定,并对其16S rRNA进行扩增验证；对所分离的大肠杆菌肠杆菌基因间保守重复序列进行PCR (ERIC-PCR)分析剔除重叠菌株；利用Kirby-Bauer法(K-B纸片法)对所获得的不同ERIC指纹型大肠杆菌菌株的耐药性进行检测；经聚合酶链反应(PCR)检测所获得的ERIC指纹型大肠杆菌菌株所携带的可移动遗传元件；以原位杂交方法确定其中一株大肠杆菌的四环素耐药基因类型及存在位置；通过在含有不同浓度抗生素的培养基中混合培养大肠杆菌和产酸克雷伯氏菌的方式检测亚抑菌浓度抗生素对大肠杆菌耐药基因传播效率的影响。结果如下：(1)本研究利用大肠杆菌显色培养基从5份粪便样品中分离到100株大肠杆菌,mdh基因检测并经16S rRNA验证,排除其中2株非大肠杆菌。可见该显色培养基筛选大肠杆菌的准确率可达98%。(2)对98株大肠杆菌进行ERIC-PCR检测,共获得17个ERIC指纹型。当聚类重新标定距离为15时,可被分为4个类群。表明本研究中采集的野生黑颈长尾雉粪便内大肠杆菌种群结构较复杂。(3)药敏实验结果显示p内酰胺类及四环素类抗生素对所分离大肠杆菌抑制效果较差,其中青霉素G对所分离大肠杆菌无抑制作用,头孢曲松耐药株占11.76%,所有菌株对四环素均不敏感；相反,喹诺酮类、脂肽类及氨基糖苷类抗生素具有很好的抑菌效果,喹诺酮类抗生素环丙沙星和氧氟沙星的抑菌率分别高达88.24%和76.47%,多粘菌素B的抑菌也达到82.35%。(4)17个ERIC指纹型大肠杆菌中耐受4种抗生素的菌株占35.30%(6/17),耐受3种的占29.40%(5/17),耐受2种及以下的仅占35.30%(6/17)。所有菌株均对至少一种抗生素具有抗性。若不考虑革兰氏阴性菌对青霉素G天然耐受这一因素,则在所有耐药菌株中,未出现可以同时耐受3类及以上抗生素的“超级细菌”。(5)17个ERIC指纹型大肠杆菌中均检测到F质粒遗传标记traA与整合子遗传标记intl,并检出5种大小不同的1类整合子可变区但不含耐药基因盒的片段；未检出traF、 int Ⅱ、intⅢ及Int-Tn遗传标记。表明菌株携带的可移动遗传元件种类虽然较单一,但仍然具有传播与获得耐药基因的风险。(6)15号大肠杆菌E15所含强力霉素耐药基因为tetB。该基因存在于质粒上但其并非F质粒,共培养时此基因可转移至产酸克雷伯氏菌突变株TR-M30-1。在培养基中共培养时,传播效率与硫酸卡那霉素和氨苄青霉素的亚抑菌浓度呈正相关(P0.05),与磺胺二甲基嘧啶、强力霉素及铜离子浓度无显著性相关；在模拟自然条件中,传播效率与上述药品浓度间均无显著相关性。
[Abstract]:In recent years, with the increase of the use of antibiotics, the drug resistance of bacteria has gradually increased, the speed of obtaining drug resistance is accelerating gradually, and even the "superbacteria" which can tolerate most antibiotics. Therefore, people are more concerned about the bactericidal efficiency of antibiotics, but the effect of antibiotics on the transmission efficiency of antimicrobial resistance to subbacteria concentration antibiotics In this paper, the effects of the resistance of the wild black necked Pheasant on Escherichia coli, the transportable genetic components and several commonly used antibiotics on the transmission efficiency of the resistant genes under the subinhibitory concentration were studied in order to understand the resistance of Escherichia coli in the wild state and to replay the artificial domestication of the wild pheasant. The effects of protective measures on the risk of transmission of drug resistance and the effect of subinhibitory concentration of antibiotics on the transmission efficiency of drug resistance genes were evaluated. The fresh feces of wild black necked Pheasant in the wild black necked Pheasant in Tianlin County, Baise, Guangxi, were collected and Escherichia coli was isolated and screened by coloring medium; The MDH specific primers were identified for the isolated Escherichia coli, and the 16S rRNA was amplified and verified. PCR (ERIC-PCR) analysis of the isolated sequence of isolated Escherichia coli gene was used to eliminate the overlapped strains, and the different ERIC fingerprints obtained by the Kirby-Bauer method (K-B paper method) were used. Detection of resistance to Enterobacteriaceae; detection of removable genetic elements carried by ERIC fingerprinted Escherichia coli strain by polymerase chain reaction (PCR); in situ hybridization method to determine the type and location of the tetracycline resistance gene of one of the Escherichia coli strains; through the cultivation of antibiotics in different concentrations The effects of the mixed culture of Escherichia coli and Klebsiella producing bacteria on the transmission efficiency of Escherichia coli resistance genes were detected. The results were as follows: (1) 100 strains of Escherichia coli were isolated from 5 fecal samples by coliform coliform medium, and the MDH gene was detected and verified by 16S rRNA. 2 strains of non Escherichia coli were found. The accuracy of screening Escherichia coli in the color culture medium could reach 98%. (2) and 98 strains of Escherichia coli were detected by ERIC-PCR. A total of 17 ERIC fingerprints were obtained. When the cluster re calibration distance was 15, it could be divided into 4 groups. The population structure of the feces of the wild black necked Pheasant was collected in this study. (3) the results of drug sensitivity test showed that the inhibitory effect of P lactam and tetracycline antibiotics on the isolated Escherichia coli was poor, of which penicillin G had no inhibitory effect on the isolated Escherichia coli, ceftriaxone resistant strains accounted for 11.76%, all strains were not sensitive to tetracycline; on the contrary, quinolones, lipopeptides and aminoglycoside antibiotics With good bacteriostasis, the bacteriostasis rates of ciprofloxacin and ofloxacin were 88.24% and 76.47% respectively. The Bacteriostasis of polymyxin B was 82.35%. (4) 17 ERIC fingerprinting Escherichia coli, 35.30% (6/17), 29.40% (5/17) tolerant and 35.30% (6), and 35.30% (6) tolerated. /17). All strains were resistant to at least one antibiotic. If the natural tolerance of Gram-negative bacteria to penicillin G was not considered, the "superbacteria" that could tolerate 3 or more antibiotics were not found in all resistant strains. (5) the F plasmid genetic marker traA was detected in 17 ERIC fingerprint Escherichia coli. Integron genetic markers Intl, and detection of 5 different 1 types of integron variable regions but not resistant gene boxes; traF, int II, int III and Int-Tn genetic markers were not detected. It showed that the types of mobile genetic elements carried by the strain still had the risk of spreading and obtaining resistance genes. (6) 15 E. coli. The gene of doxycycline resistance in E15 is tetB., which exists on plasmids but not F plasmids. In co culture, this gene can be transferred to Klebsiella producing mutant TR-M30-1. in culture medium. The transmission efficiency is positively correlated with the inhibitory concentration of kanamycin sulfate and ampicillin sulphate (P0.05), and sulfonamide two methyl azoxurin. There was no significant correlation between the concentration of pyridine, doxycycline and copper ion. There was no significant correlation between the transmission efficiency and the concentration of these drugs in simulated natural conditions.

【学位授予单位】：广西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R378

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