本文關(guān)鍵詞： 產(chǎn)志賀毒素大腸埃希菌 分子流行病學(xué) 血清分型 耐藥性　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景產(chǎn)志賀毒素大腸埃希菌(Shiga toxin-producing Escherichia coli,STEC)是一類能夠產(chǎn)1種或多種志賀毒素(Shigatoxin,STX)大腸埃希菌。是能引起一系列人畜共患病的重要食源性病原菌。反芻動物是引起人類發(fā)病的STEC主要宿主。自1982年0157:H7血清型STEC在美國首次被確認為食物中毒新型致病菌以來,前后發(fā)現(xiàn)了超過400種血清型的STEC致病菌株,血清型的復(fù)雜性嚴重威脅著人類的健康和生命。目前,細菌耐藥已成為全球性的公共衛(wèi)生難題,STEC菌株的耐藥現(xiàn)狀在我國也較為嚴峻,因此,及時監(jiān)測和比較不同來源的STEC的耐藥性并分析其分子流行病學(xué)特征,有助于分析菌株親緣關(guān)系,掌握其變異規(guī)律,并為臨床用藥提供一定指導(dǎo)意見。研究目的1.通過收集不同來源的STEC菌株,了解STEC的檢出及流行情況,獲得菌株實驗室基礎(chǔ)數(shù)據(jù),探索全面分析不同來源STEC的技術(shù)路線,為其長期監(jiān)測奠定基礎(chǔ);2.了解STEC的藥物敏感性及多重耐藥性現(xiàn)狀,為指導(dǎo)臨床用藥提供參考。3.分析STEC的分子分型結(jié)果,探究不同來源的菌株間的親緣關(guān)系,并比較分子分型與表型結(jié)果間的聯(lián)系。研究方法1.樣本來源:(1)采集山東省濟寧微山縣及煙臺萊州市農(nóng)村散養(yǎng)家禽家畜新鮮糞便樣本;(2)依托于山東省食品安全風(fēng)險監(jiān)測工作,收集食品源大腸埃希菌;(3)依托山東省食源性疾病主動監(jiān)測工作,收集食源性患者源大腸埃希菌。2.STEC菌株分離:(1)對于動物糞便樣本,分別采用免疫磁珠捕獲法及ECC顯色平板培養(yǎng)法初篩O157:H7型及非0157:H7型STEC可疑菌落,飛行質(zhì)譜鑒定可疑菌落菌種,以PCR擴增法檢測毒力基因,攜帶一種或兩種STX毒力基因的大腸埃希菌鑒定為STEC;(2)采用飛行質(zhì)譜鑒定收集到的食品源及食源性疾病患者源大腸埃希菌的菌種,通過PCR擴增法檢測毒力基因,攜帶毒力基因的大腸埃希菌鑒定為STEC。3.生化特征分析:采用DBI-09致瀉大腸埃希菌干制生化鑒定試劑盒對分離到的STEC進行一系列生化特征的鑒定和分析。4.血清型:通過特異性引物PCR擴增測序法結(jié)合血清玻板凝集法確認STEC菌株的血清型。5.藥物敏感性試驗:微量肉湯稀釋法對7類、16種常用抗生素進行藥敏試驗,得到最低抑菌濃度(MIC)值,分析其藥物敏感性。6.分子分型:對分離到的STEC菌株進行PFGE分型及MLST分型。結(jié)果1.從1022份動物糞便樣本中共分離出26株STEC;從157株食品源大腸埃希菌中檢測出10株致瀉性大腸埃希菌,未檢測出STEC陽性菌株;從726株食源性疾病患者源大腸埃希菌中檢測出3株STEC;另獲得6株2007-2012年分離到的歷史動物源STEC。2.全部35株STEC中,23株只攜帶STX2基因,2株只攜帶STX1基因;9株同時攜帶STX2與黏附基因eae;1株同時攜帶STX1、STX2及eae三種基因3.35株STEC菌株中有8株鑒定為0157:H7血清型菌株,占22.86%。27株非0157:H7型STEC菌株血清型分布分散,無優(yōu)勢血清型。10株食品源致瀉性大腸埃希菌的血清型各不相同。4.35株STEC有不同程度的耐藥性,對磺胺異惡唑的耐藥率最高(65.71%),其次為四環(huán)素(60.00%)、復(fù)方新諾明(57.14%)和萘啶酸(54.29%)�？傮w上對β-內(nèi)酰胺類抗生素耐藥率呈現(xiàn)較低水平,對磺胺類抗生素耐藥率呈現(xiàn)較高水平。10株食品源致瀉大腸埃希菌對磺胺異惡唑耐藥率最高(90.00%),對其他抗生素耐藥率較低。35株STEC中共24株多重耐藥菌株,占68.57%;10株食品源致瀉大腸埃希菌中2株為多重耐藥菌,占 20.00%。5.35株STEC與10株食品源致瀉大腸埃希菌分子分型結(jié)果較為復(fù)雜,全部45株菌株分屬30個基因型別,36個PFGE基因指紋圖譜,同源性在57.6%-100%之間,反映了一定基因多態(tài)性。SD025和SD028基因型別的菌株最多,均為4株(8.89%);其次為SD011、SD021和SD023,均為3株(6.67%)。全部45株菌株共有24種MLST型別。其中35株STEC有14種MLST型別,ST11型別的菌株最多,包含8株菌(22.86%)。其次為ST5133和ST540,均包含4株菌(11.43%)。10株食品中分離的致瀉性大腸埃希菌的MLST型別結(jié)果各不相同。結(jié)論1.STEC菌株血清型分布分散,無優(yōu)勢血清型,表型結(jié)果反映了一定的菌株變異性。2.STEC菌株對β-內(nèi)酰胺類抗生素耐藥率呈現(xiàn)較低水平,對磺胺類抗生素耐藥率呈現(xiàn)較高水平;3.分子分型結(jié)果呈多態(tài)性和復(fù)雜性,STEC菌株變異性較高,不同來源STEC菌株間未形成明顯的親緣聚集性�？傮w上PFGE的分型能力高于MLST,MLST分型結(jié)果與血清型相關(guān)性較高。
[Abstract]:The research background of Shiga toxin producing Escherichia coli (Shiga toxin-producing Escherichia coli, STEC) is a kind of can produce 1 or more kinds of Shiga toxin (Shigatoxin, STX) of Escherichia coli. Can cause a series of important foodborne pathogens of zoonosis. Ruminant animal STEC is caused by the main host disease in humans. Since 1982 0157:H7 serotype STEC in America was first recognized as a new food poisoning pathogen, before and after STEC was found more than 400 pathogenic strains of serotypes, the serotype complexity is a serious threat to human health and life. At present, bacterial resistance has become a public health problem worldwide, the status of resistance strains in STEC our country is also more severe, so the resistance of STEC timely monitoring and comparing the different sources and to analyze the molecular epidemiological characteristic, is helpful to the analysis of genetic relationship, grasp the variation of, And provide some guidance for clinical use. Objective: 1. through STEC isolates collected from different sources, understand the detection and prevalence of STEC was obtained, laboratory data, a comprehensive analysis of the technical route to explore different sources of STEC, lay the foundation for its long-term monitoring; 2. to understand the status of drug sensitivity of STEC and multiple drug resistance, to provide the results of STEC type molecular reference.3. analysis for clinical medication, explore the relationship between the strains of different sources, and to compare the molecular typing and phenotype of the relation between the result. The method of 1. sample sources: (1) collected in rural area of Shandong Province, Weishan in Jining county and Yantai Laizhou City backyard poultry and livestock (fresh stool samples; 2) based on the Shandong province food safety risk monitoring, collecting food source Escherichia coli; (3) based on the work of active surveillance of foodborne diseases in Shandong Province, collected from patients with foodborne E. The separation of E.coli.2.STEC strains: (1) for animal fecal samples, respectively using immunomagnetic capture method and ECC color plate culture method for screening O157:H7 and non 0157:H7 type STEC suspicious colonies, MALDI-TOF-MS suspicious colony strains, detection of virulence genes by PCR amplification, identification of Escherichia coli carrying one or two STX virulence gene STEC; (2) the source of foodborne diseases and food source Escherichia coli MALDI-TOF-MS collected strains, detection of virulence gene was amplified by PCR, carrying the identification of Escherichia coli virulence genes analysis of STEC.3. biochemical characteristics: identification and analysis of.4. serotypes by DBI-09 lapactic E. coli for biochemical identification kit of isolated STEC was a series of biochemical characteristics by specific primer PCR amplification sequencing method combined with serum agglutination method confirmed serotype.5. strain STEC Drug sensitivity test: broth dilution method of 7 kinds of 16 kinds of antibiotics, drug sensitive test, get the minimum inhibitory concentration (MIC) values, analyze the drug sensitivity of.6. molecular typing: STEC to the strains of PFGE and MLST classification. Results 1. from 1022 animal fecal samples 26 strains of STEC were isolated from 157 strains of food; Escherichia coli detected in 10 strains of pathogenic Escherichia coli, did not detect STEC positive strains; 3 strains were identified as STEC from 726 strains of Escherichia coli were the source of foodborne diseases; the other 6 strains isolated from 2007-2012 years history of animal STEC.2. 35 STEC strains, 23 strains carried STX2 gene, 2 strains carried STX1 gene; 9 strains carrying STX2 and adhesion gene EAE; 1 strains carrying STX1, STX2 and EAE three genes of 3.35 STEC strains in 8 strains were identified as serotype 0157:H7 strains,.27 strains accounted for 22.86% of non 0157:H7 S TEC strain serotype distribution, no serotype of diarrheagenic Escherichia coli isolated from the dominant serotype.10 strain food source in different.4.35 STEC strains have different degrees of resistance, resistance to sulfamethoxazole was the highest (65.71%), followed by tetracycline (60%), SMZ-TMP (57.14%) and naphthalene nalidixic acid (54.29%). The overall effect of beta lactam antibiotic resistance rate showed low levels of sulfa antibiotic resistance rate showed higher levels of.10 were the food source of diarrheogenic Escherichia coli on sulfamethoxazole resistance rate was the highest (90%), to other antibiotics were low.35 strain STEC of the 24 strains multi drug resistant strains, accounting for 68.57%; 10 strains of food source of diarrheogenic Escherichia coli 2 strains of multidrug-resistant bacteria, accounting for 20.00%.5.35 STEC strains and 10 strains of food source diarrheogenic Escherichia coli molecular typing results is more complex, all 45 strains belong to 30 gene type 36 PFGE gene Fingerprint, homology between 57.6%-100%, reflects a certain degree of genetic polymorphism of.SD025 and SD028 genotypes of the strains were 4 strains (up to 8.89%); followed by SD011, SD021 and SD023, were 3 strains (6.67%). All the 45 strains there were 24 genotypes of MLST. 35 of them were STEC 14 kinds of MLST type, ST11 type was most, contains 8 strains (22.86%), followed by ST5133 and ST540, including 4 strains (11.43%) type MLST results of diarrheagenic Escherichia coli isolated from.10 strains in food are different. Conclusion 1.STEC strains of serotype distribution, serum no advantage type phenotype results reflect the variability of certain strains of.2.STEC strain of beta lactam antibiotic resistance rate showed low levels of sulfa antibiotic resistance rate showed a high level of 3.; molecular typing results showed the polymorphism and complexity of STEC strain variability is high, STEC strains from different sources were not On the whole, the typing ability of PFGE was higher than that of MLST, and the correlation between the MLST typing and serotype was higher.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R446.5

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