食源性革兰氏阴性肠道病原菌PFGE分型和大肠杆菌耐药性研究

发布时间：2018-03-02 18:19

本文选题：食源性大肠杆菌　切入点：药敏性　出处：《西北农林科技大学》2009年博士论文　论文类型：学位论文

【摘要】：大肠杆菌、沙门氏菌和空肠弯曲杆菌是目前世界范围内报到最多、引起食源性疾病暴发事件最多的3种致病性革兰氏阴性肠道菌。对暴发病原菌的快速确定和溯源是防止疫情恶化、减少对人们身体和经济带来更大危害的关键。脉冲场电泳(PFGE)分子分型技术由于其高分辨力、实验室间的高度可重复性和易于分析并通过网络进行对比,被誉为病原菌分型的“黄金方法”。越来越严重的病原菌耐药问题,对临床上相关感染的治疗带来极大挑战,成为目前困扰医学的一大难题。肠道菌群因为密度比较大,更容易进行耐药基因的水平传递,作为粪便污染指示菌的大肠杆菌更是被认为是肠道致病菌的耐药基因库。对其耐药性现状、耐药性产生和发展、耐药性转移和相关影响因素的研究,对各种肠道病原菌的多重耐药性的控制、临床治疗具有重要意义。本研究对陕西西安和杨凌地区的食源性大肠杆菌进行分离、鉴定、毒力因子、整合子和药敏性研究,并分别利用6种限制性内切酶对大肠杆菌O157:H7、空肠弯曲杆菌和四个血清型的沙门氏菌进行了PFGE分子分型研究,所得主要结果如下: 1、陕西西安和杨凌地区各种原料肉中,大肠杆菌污染100%存在,其污染程度跟产品种类无关,跟产品来源有一定相关性。凉拌菜样品大肠杆菌分离率86.7%,调味品可能是抑制大肠杆菌在凉拌菜中繁殖的主要原因。在毒力因子检测中,粘附性毒素主要表达基因fimA基因阳性率为56.1%(273株)。通过对STXI、STXII、ST、LT等毒素表达基因的检测,发现产志贺样毒素大肠杆菌阳性率为0.62%,产肠毒素大肠杆菌阳性率是14.4%。 2、陕西食源性大肠杆菌对四环素的耐药率最高,占受试菌株的99.3%(551);耐受率均超过50%的抗生素依次还有链霉素(65.6%)、阿莫西林(61.3%)、萘啶酮酸(57.7%)、氨苄青霉素(55.1%),其余依次是环丙沙星(38.7%)、氯霉素(38.0%)、卡那霉素(34.6%)、庆大霉素(30.8%)、头孢哌酮(23.4%)、头孢西丁(12.4%);试验菌株对阿米卡星最为敏感,有8.8%耐药菌株产生。 3、575株受试菌中,发现2.1%(12)的菌株对12种抗生素全部敏感,其余563(97.9%)株大肠杆菌至少对一种抗生素有耐药性。多重耐药(≥3)率为62.3%(358),37.0%的株菌对7种以上抗生素耐受。多重耐药菌存在最多的是9重耐药菌株,有58株菌, 6重和10重耐药菌次之,均都有49株,其余依次是8(43)、3(35)、4(34)、7(31)、5(27)、11(25)、12(7)。表明多重耐药现象在陕西食源性大肠杆菌中比较严重。 4、大肠杆菌菌株来源不同则耐药性存在很大差异,鸡肉分离株的耐药性和多重耐药率明显高于其它分离株。304株鸡肉分离株100%的耐药,多重耐药(≥3)率为85.5%(260),以9重耐药菌株最多(56),其次是10(47)、8(40)、7(26)、11(25)、6(21)、4(14)、5(14)、3(11)、12(7)重耐药株。对7种以上抗生素耐受率达66.1%(201)。说明陕西鸡肉分离大肠杆菌的耐药性非常严重。 5、118株肉样品分离菌株中,49.1%的菌株携带有750～2000bp的I类整合子。其中鸡肉分离株的整合子率为55.1%。多重耐药菌株(≥3)中,整合子阳性率为59.1%,其中鸡肉多重耐药菌株的整合子阳性率为59.5%,其它肉多重耐药菌株整合子阳性率为55.6%。非多重耐药菌中,I类整合子检出率仅16.7%。在耐药重数≥7的菌株中,66.7%的菌株I类整合子呈阳性。整合子存在与大肠杆菌耐药性之间有一定的相关性。 6、58株携带整合子的菌株,携带约2.0kbI类整合子的菌最多,有29株,有19株菌在750bp左右有扩增带,在1.5kb左右有扩增带的菌12株,其中1株有750bp和1.5kb两条带,2株在750bp和2.0kb均有扩增。 7、在对大肠杆菌O157:H7单酶PFGE分型中,以SpeI效果最好,将43株试验菌株切割后,产生21～32条有效带,产生基因型22个。BlnI/SpeI/PacI3酶结合和XbaI/BlnI/NheI/SpiI/SpeI/PacI 6种酶结合分析做树状图,所得基因型相、束平均数、束菌株百分比、结与菌株比值和SID指数等5个参数完全相同,分别是26、4.4、52%、0.67和0.902。 8、对4个血清型的沙门氏菌S. Heidelberg、S.Kentucky、S.SaintPaul和S.Hadar进行6种酶PFGE分子分型,分型效果最好的酶各不相同,分别是PacI、SpeI、SpiI、NotI;最佳3酶组合分别依次是:SpeI/PacI/BlnI、SpeI/NotI/SfiI、SpeI/BlnI/SfiI和SpeI/NotI/SfiI。6种酶结合使用,血清型不同则产生的分型能力不同。3酶结合就可以达到很好的分型效果。 9、限制性内切酶BamHI是对空场弯曲杆菌PFGE分子分型效果最好的内切酶。对空肠弯曲杆菌,要获得更好的溯源和分型效果,PFGE应该与其它方法结合使用才能达到预期结果。 10、综合分析PFGE分子分型结果,发现与大肠杆菌和沙门氏菌分型结果相比较,PFGE对空肠弯曲杆菌的分型能力要差一些。
[Abstract]:Escherichia coli, Salmonella and Campylobacter jejuni is currently in the world report most foodborne disease outbreaks caused by up to 3 kinds of pathogenic gram negative enteric bacteria. The rapid identification and traceability of pathogenic bacteria to prevent epidemic outbreaks of deterioration, reduce the key to bring greater harm to human body and economy. Pulsed field gel electrophoresis (PFGE) because of its high resolution molecular technology, inter laboratory height can be easily repeated and compared and analyzed through the network, known as the pathogen type "gold" method. The drug resistance of pathogenic bacteria is becoming more and more serious, which brings a tremendous challenge of clinical treatment of infection, a medical problem at present. The intestinal flora because of higher density, more resistance and horizontal gene transfer, as fecal contamination indicator bacteria of Escherichia coli is considered intestinal pathogenic The resistance gene pool of bacteria. The drug resistance status, drug resistance and development, factors of resistance transfer and related control, multiple drug resistance to various enteric pathogenic bacteria, has important significance in clinical treatment. This study was separated on food borne Escherichia coli in Shaanxi Xi'an and Yangling area identification, virulence factor. Research on Integration and drug sensitivity, and using 6 kinds of restriction endonuclease O157:H7 of Escherichia coli, Salmonella, Campylobacter jejuni and four serotypes of genotyping of PFGE molecules, the main results are as follows:
1, all kinds of meat raw materials in Shaanxi Xi'an and Yangling area, there are 100% Escherichia coli contamination, contamination of products with independent, have a certain correlation with the source of the products. The cold food samples of Escherichia coli was 86.7%, spices may inhibit Escherichia coli in the main reason in salad propagation. In the detection of virulence factors. Adhesion toxin positive expression rate of fimA gene was 56.1% (273 strains). Based on STXI, STXII, ST, LT toxin gene expression detection, found that the positive rate of Shiga toxin producing Escherichia coli was 0.62%, the positive rate of enterotoxigenic Escherichia coli is 14.4%.
2, Shaanxi food borne Escherichia coli resistance to tetracycline was the highest, accounting for 99.3% of the tested strains (551); tolerance rate of more than 50% of the antibiotics in turn and streptomycin (65.6%), amoxicillin (61.3%), nalidixic acid (57.7%), ampicillin (55.1%), followed by chloramphenicol, ciprofloxacin (38.7%) (38%), kanamycin (34.6%), gentamicin (30.8%), cefoperazone (23.4%), cefoxitin (12.4%); the test strain was most sensitive to Amikacin, has produced 8.8% resistant strains.
3575 isolates, 2.1% (12) strains were sensitive to 12 kinds of antibiotics, the remaining 563 (97.9%) strains of Escherichia coli to at least one antibiotic resistance. Multidrug resistance (more than 3) rate was 62.3% (358), 37% strains were more than 7 kinds of antibiotics for multi drug resistant bacteria tolerance. There is the largest 9 resistant strains, 58 strains of bacteria, 6 fold and 10 fold resistant strains of all 49 strains, followed by 8 (43), 3 (35), 4 (34), 7 (31), 5 (27), 11 (25), 12 (7). The results indicated that the phenomenon of multiple drug resistance in Shaanxi food borne Escherichia coli is more serious.
4 strains of Escherichia coli from different sources, there is a big difference, drug resistance, drug resistance of chicken strains and multi drug resistance rate was higher than that of other isolates of.304 resistant isolates of 100% strains of chicken, multi drug resistance (more than 3) rate was 85.5% (260), with 9 resistant strains (56), followed by up to 10 (47), 8 (40), 7 (26), 11 (25), 6 (21), 4 (14), 5 (14), 3 (11), 12 (7) heavy resistance strains. Over 7 kinds of antibiotic resistance rate of 66.1% (201). The drug resistance of Escherichia coli in Shaanxi the chicken is very serious.
5118 strains of meat samples were isolated, 49.1% strains carrying 750 ~ 2000bp I integron. The integron chicken isolates was 55.1%. multi drug resistant strains (more than 3), the positive rate of integron was 59.1%, the positive rate of integron in multi drug resistant strains of chicken was 59.5%, the positive rate of the whole other meat zygotic multi drug resistant strains of multidrug-resistant bacteria 55.6%., I integron detection rate of only 16.7%. in number more than 7 resistant strains, 66.7% strains of integron class I integron positive. There is a certain relationship between drug resistance of Escherichia coli.
6,58 strains carrying integron strains carried most of the 2.0kbI class integrons, including 29 strains, 19 strains had amplification bands at about 750bp, and 12 strains had amplification bands around 1.5kb, 1 of them had two bands of 750bp and 1.5kb, and 2 strains were amplified in 750bp and 2.0kb.
In 7, the Escherichia coli O157:H7 single enzyme PFGE type, the SpeI is the best, will cut 43 test strains, 21~32 valid bands, produce genotype analysis tree diagram combined with 22.BlnI/SpeI/PacI3 and XbaI/BlnI/NheI/SpiI/SpeI/PacI 6 kinds of enzyme bound enzyme gene, income type, the average number of beam, beam strain the percentage of nodes and strain ratio and SID index of the 5 parameters are exactly the same, are 26,4.4,52%, 0.67 and 0.902.
8, Salmonella S. Heidelberg, of the 4 serotypes of S.Kentucky, S.SaintPaul and S.Hadar are 6 kinds of enzyme PFGE molecular typing, typing the best enzyme is different, namely PacI, SpeI, SpiI, NotI; 3 best enzyme combination respectively is: SpeI/PacI/BlnI, SpeI/NotI/ SfiI, with the use of SpeI/BlnI/SfiI and SpeI/NotI/SfiI.6 enzymes, can achieve good effect with typing typing ability of different serotypes of.3 enzyme is produced.
9, the restriction enzyme BamHI is open to Campylobacter PFGE molecular typing the best enzyme. For Campylobacter jejuni, to obtain better traceability and classification effect, PFGE should be combined with other methods used to achieve the desired results.
10, by analyzing the results of PFGE molecular typing, it was found that the typing ability of PFGE to Campylobacter jejuni was a little worse than that of Escherichia coli and Salmonella.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R378

【引证文献】