宰杀肉鸡中肺炎克雷伯菌耐药基因的检测和传播分析

发布时间：2018-04-11 10:09

  本文选题：肺炎克雷伯菌 + β-内酰胺酶　； 参考：《山东大学》2015年硕士论文

【摘要】：抗生素被认为是二十一世纪最伟大的医学发现,对细菌、病毒、寄生虫等具有抑制和杀灭作用。在畜牧养殖业中,它可以防治动物疫病、促进畜禽生长。然而大量使用甚至滥用抗生素,会使动物肠道微生物的生存环境遭到破坏,导致动物机体抵御外界的能力下降,造成疾病继发频发。养殖产品的抗生素残留,将直接影响到食品安全,通过从各条食物链源源不断侵入人体,诱导人类出现原发耐药性,威胁整个人类的健康。近年来,越来越多的多重耐药肺炎克雷伯菌随着抗生素尤其是由p-内酰胺类和喹诺酮类等广谱抗生素的大量使用而出现。肺炎克雷伯菌是典型的条件致病菌,可经过人或动物的呼吸道进入肺内引发病理性突变,其耐药机制主要是产生p-内酰胺酶、喹诺酮类等耐药基因。在分子水平上检测耐药基因型并对耐药基因的传播进行相关研究,以便采取科学有效的防控措施显得尤为重要。目前,对肺炎克雷伯菌耐药性的研究多以医院临床分离菌株为主,畜牧业养殖中产生耐药性菌株的研究较少。本文从山东地区某肉禽养殖厂的种鸡、商品鸡的身体表面以及屠宰场的污水中采集样品,分离纯化出75株肺炎克雷伯菌作为实验菌株,琼脂纸片药敏试验显示肺炎克雷伯菌对临床常用的头孢菌素类氨苄西林(AMP)的耐药率已经达到了100%,对头孢他啶(CAZ)、卡那霉素(KAN)、氯霉素(CHL)、环丙沙星(CIP)也具有很强耐药性,耐药率在80%以上。以75株肺炎克雷伯菌DNA为模板进行PCR扩增检测,共检出产p-内酰胺酶耐药基因菌株72株,其中有66株blaSHV呈阳性,55株blaTEM呈阳性,有60株blaCTX-M呈阳性；对blaSHV和blaCTX-M阳性菌株进行基因分型检测,以SHV-11、SHV-1型和CTX-M-1为主；此外还检测了细菌质粒介导的喹诺酮类耐药基因,发现有68株耐药基因阳性菌株,其中有4株qnrA呈阳性,56株qnrB呈阳性,23株qnrS呈阳性,检出qepA为阳性的菌株共有2株,aac(6')-1b-cr的阳性菌株共31株,共有66株菌株同时携带β-内酰胺酶与喹诺酮类耐药基因。根据整合子基因序列intI1、intI2 和intI3设计引物,对75株肺炎克雷伯菌进行整合子检测,结果显示有70株Ⅰ型整合子检测呈阳性,有9株含有Ⅱ型整合子,没有发现Ⅲ型整合子菌株。检测中共发现3种基因盒阵列,分别为dfrA17-aadA5、dfrA12-orfF-aadA2和dfrAl-sat2-aadA1,这其中出现频率最大的基因盒是dfrA12-orfF-aadA2,它的大小为1989bp,共出现19次。本文通过对肺炎克雷伯菌耐药性及耐药基因的分布情况进行初步研究,了解耐药性产生的原因和耐药基因转移机制,以此提高人们对抗菌药物在养殖业中滥用情况的认识,同时加强养殖场周边环境的监测,为预防和控制肺炎克雷伯菌的潜在传播和发展,减缓细菌耐药提供科学依据。
[Abstract]:Antibiotics are considered the greatest medical discoveries of the 21 century, inhibiting and killing bacteria, viruses, parasites, etc.In animal husbandry, it can prevent and control animal disease and promote the growth of livestock and poultry.However, the extensive use and even abuse of antibiotics will destroy the living environment of animal intestinal microbes, reduce the ability of animal body to resist the outside world, and lead to the frequent occurrence of diseases.Antibiotic residues in aquaculture products will directly affect food safety, through the continuous invasion of the human body from the various food chains, leading to the emergence of primary drug resistance, threatening the health of the whole human.In recent years, more and more multidrug resistant Klebsiella pneumoniae have emerged with the extensive use of antibiotics, such as p- lactams and quinolones.Klebsiella pneumoniae (Klebsiella pneumoniae) is a typical conditional pathogen, which can cause pathological mutation through the respiratory tract of human or animal. The drug resistance mechanism of Klebsiella pneumoniae is mainly the production of plactamases, quinolones and other drug resistant genes.It is very important to detect drug resistance genotypes at molecular level and to study the transmission of drug resistance genes in order to take scientific and effective prevention and control measures.At present, the drug resistance of Klebsiella pneumoniae is mainly studied by clinical isolates in hospitals.In this paper, 75 strains of Klebsiella pneumoniae were isolated and purified from the body surface of broiler, commercial chicken and the sewage of slaughterhouse in a meat and poultry breeding factory in Shandong province, and 75 strains of Klebsiella pneumoniae were isolated and purified as experimental strains.Agar disk drug sensitivity test showed that Klebsiella pneumoniae was resistant to the commonly used clinical cephalosporins ampicillin (AMP) and had a high resistance to ceftazidime, kanamycin, chloramphenicol, ciprofloxacin and CIPs, and also to ceftazidime, kanamycin, chloramphenicol and ciprofloxacin.The drug resistance rate was more than 80%.Using 75 strains of Klebsiella pneumoniae DNA as template, 72 strains of plactamase-resistant gene were detected by PCR amplification. Among them, 66 strains were blaSHV positive, 55 strains were blaTEM positive, and 60 strains were blaCTX-M positive.The genotypes of blaSHV and blaCTX-M positive strains were mainly SHV-11, SHV-1 and CTX-M-1, and the quinolone resistance genes mediated by bacterial plasmids were also detected, and 68 positive strains were found.Among them, there were 4 qnrA positive strains, 56 qnrB positive strains and 23 qnrS positive strains. There were 31 positive strains with qepA positive, 66 strains carrying 尾 -lactamases and quinolones resistance genes.75 strains of Klebsiella pneumoniae were tested for integron with primers designed according to the sequence of integron gene intI1tI2 and intI3. The results showed that 70 strains of type 鈪，

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