本文選題：食源性大腸桿菌　切入點：藥敏性　出處：《西北農(nóng)林科技大學(xué)》2009年博士論文　論文類型：學(xué)位論文

【摘要】：大腸桿菌、沙門氏菌和空腸彎曲桿菌是目前世界范圍內(nèi)報到最多、引起食源性疾病暴發(fā)事件最多的3種致病性革蘭氏陰性腸道菌。對暴發(fā)病原菌的快速確定和溯源是防止疫情惡化、減少對人們身體和經(jīng)濟(jì)帶來更大危害的關(guān)鍵。脈沖場電泳(PFGE)分子分型技術(shù)由于其高分辨力、實驗室間的高度可重復(fù)性和易于分析并通過網(wǎng)絡(luò)進(jìn)行對比,被譽為病原菌分型的“黃金方法”。越來越嚴(yán)重的病原菌耐藥問題,對臨床上相關(guān)感染的治療帶來極大挑戰(zhàn),成為目前困擾醫(yī)學(xué)的一大難題。腸道菌群因為密度比較大,更容易進(jìn)行耐藥基因的水平傳遞,作為糞便污染指示菌的大腸桿菌更是被認(rèn)為是腸道致病菌的耐藥基因庫。對其耐藥性現(xiàn)狀、耐藥性產(chǎn)生和發(fā)展、耐藥性轉(zhuǎn)移和相關(guān)影響因素的研究,對各種腸道病原菌的多重耐藥性的控制、臨床治療具有重要意義。本研究對陜西西安和楊凌地區(qū)的食源性大腸桿菌進(jìn)行分離、鑒定、毒力因子、整合子和藥敏性研究,并分別利用6種限制性內(nèi)切酶對大腸桿菌O157:H7、空腸彎曲桿菌和四個血清型的沙門氏菌進(jìn)行了PFGE分子分型研究,所得主要結(jié)果如下: 1、陜西西安和楊凌地區(qū)各種原料肉中,大腸桿菌污染100%存在,其污染程度跟產(chǎn)品種類無關(guān),跟產(chǎn)品來源有一定相關(guān)性。涼拌菜樣品大腸桿菌分離率86.7%,調(diào)味品可能是抑制大腸桿菌在涼拌菜中繁殖的主要原因。在毒力因子檢測中,粘附性毒素主要表達(dá)基因fimA基因陽性率為56.1%(273株)。通過對STXI、STXII、ST、LT等毒素表達(dá)基因的檢測,發(fā)現(xiàn)產(chǎn)志賀樣毒素大腸桿菌陽性率為0.62%,產(chǎn)腸毒素大腸桿菌陽性率是14.4%。 2、陜西食源性大腸桿菌對四環(huán)素的耐藥率最高,占受試菌株的99.3%(551);耐受率均超過50%的抗生素依次還有鏈霉素(65.6%)、阿莫西林(61.3%)、萘啶酮酸(57.7%)、氨芐青霉素(55.1%),其余依次是環(huán)丙沙星(38.7%)、氯霉素(38.0%)、卡那霉素(34.6%)、慶大霉素(30.8%)、頭孢哌酮(23.4%)、頭孢西丁(12.4%);試驗菌株對阿米卡星最為敏感,有8.8%耐藥菌株產(chǎn)生。 3、575株受試菌中,發(fā)現(xiàn)2.1%(12)的菌株對12種抗生素全部敏感,其余563(97.9%)株大腸桿菌至少對一種抗生素有耐藥性。多重耐藥(≥3)率為62.3%(358),37.0%的株菌對7種以上抗生素耐受。多重耐藥菌存在最多的是9重耐藥菌株,有58株菌, 6重和10重耐藥菌次之,均都有49株,其余依次是8(43)、3(35)、4(34)、7(31)、5(27)、11(25)、12(7)。表明多重耐藥現(xiàn)象在陜西食源性大腸桿菌中比較嚴(yán)重。 4、大腸桿菌菌株來源不同則耐藥性存在很大差異,雞肉分離株的耐藥性和多重耐藥率明顯高于其它分離株。304株雞肉分離株100%的耐藥,多重耐藥(≥3)率為85.5%(260),以9重耐藥菌株最多(56),其次是10(47)、8(40)、7(26)、11(25)、6(21)、4(14)、5(14)、3(11)、12(7)重耐藥株。對7種以上抗生素耐受率達(dá)66.1%(201)。說明陜西雞肉分離大腸桿菌的耐藥性非常嚴(yán)重。 5、118株肉樣品分離菌株中,49.1%的菌株攜帶有750～2000bp的I類整合子。其中雞肉分離株的整合子率為55.1%。多重耐藥菌株(≥3)中,整合子陽性率為59.1%,其中雞肉多重耐藥菌株的整合子陽性率為59.5%,其它肉多重耐藥菌株整合子陽性率為55.6%。非多重耐藥菌中,I類整合子檢出率僅16.7%。在耐藥重數(shù)≥7的菌株中,66.7%的菌株I類整合子呈陽性。整合子存在與大腸桿菌耐藥性之間有一定的相關(guān)性。 6、58株攜帶整合子的菌株,攜帶約2.0kbI類整合子的菌最多,有29株,有19株菌在750bp左右有擴(kuò)增帶,在1.5kb左右有擴(kuò)增帶的菌12株,其中1株有750bp和1.5kb兩條帶,2株在750bp和2.0kb均有擴(kuò)增。 7、在對大腸桿菌O157:H7單酶PFGE分型中,以SpeI效果最好,將43株試驗菌株切割后,產(chǎn)生21～32條有效帶,產(chǎn)生基因型22個。BlnI/SpeI/PacI3酶結(jié)合和XbaI/BlnI/NheI/SpiI/SpeI/PacI 6種酶結(jié)合分析做樹狀圖,所得基因型相、束平均數(shù)、束菌株百分比、結(jié)與菌株比值和SID指數(shù)等5個參數(shù)完全相同,分別是26、4.4、52%、0.67和0.902。 8、對4個血清型的沙門氏菌S. Heidelberg、S.Kentucky、S.SaintPaul和S.Hadar進(jìn)行6種酶PFGE分子分型,分型效果最好的酶各不相同,分別是PacI、SpeI、SpiI、NotI;最佳3酶組合分別依次是:SpeI/PacI/BlnI、SpeI/NotI/SfiI、SpeI/BlnI/SfiI和SpeI/NotI/SfiI。6種酶結(jié)合使用,血清型不同則產(chǎn)生的分型能力不同。3酶結(jié)合就可以達(dá)到很好的分型效果。 9、限制性內(nèi)切酶BamHI是對空場彎曲桿菌PFGE分子分型效果最好的內(nèi)切酶。對空腸彎曲桿菌,要獲得更好的溯源和分型效果,PFGE應(yīng)該與其它方法結(jié)合使用才能達(dá)到預(yù)期結(jié)果。 10、綜合分析PFGE分子分型結(jié)果,發(fā)現(xiàn)與大腸桿菌和沙門氏菌分型結(jié)果相比較,PFGE對空腸彎曲桿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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R378

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 王宏;牛源大腸桿菌耐藥性及耐藥機制研究[D];東北農(nóng)業(yè)大學(xué);2011年

2 李州;圈養(yǎng)野生動物大腸桿菌耐藥性流行病學(xué)研究[D];福建農(nóng)林大學(xué);2012年

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本文編號：1557640