Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/51773
標題: Analysis of the Toxin Types, Antibiograms and Integron Genes for Pathogens Escherichia coli Isolates from Human and Porcine Origins.
人類及豬隻來源病原性大腸桿菌之毒素型,抗生素耐性及其抗藥基因integron之分析
作者: 軒轅照秀
Chao-Hsiu, Hsuan Yuan
關鍵字: integron;大腸桿菌;E. coli;毒素型;抗藥性
出版社: 食品科學系
摘要: 
中文摘要
抗生素已普遍應用於治療由細菌感染之疾病,例如病原性大腸桿菌引起的食物中毒、出血性結腸炎、旅行者下痢等疾病。在藥物的篩選壓力下,病原菌藉由質體轉移、轉位子 (transposons) 等模式而產生多重抗藥性,且將抗藥性基因在人類與動物間轉移,造成臨床上疾病醫療日益困難。
Integron是細菌獲得新的抗藥性基因的重要機制之一,依其重組酶基因 (int) 序列之不同,分為四種類型 (classⅠ、Ⅱ、Ⅲ、Ⅳ),其中以classⅠ integron在臨床菌株最為常見,因而本研究主要探討classⅠintegron與大腸桿菌抗藥性之關係。
本研究首先針對大腸桿菌malate hydrogenase之mdh基因,及各病原型致病相關之基因,實驗以鑑定分型的方式自台灣地區人類與豬隻檢體所分離之158株大腸桿菌進行實驗。使用引子組Emdh 1/2 (mdh) , LT I 1/2 (ltI), LTI 51/31 (ltI) , ST II 1/2 (stII) , ST Ia 1/2 (stIa), SLT I 5/3 (sltI) , SLT II 5/3 (sltII), SK1/2 (eae) , INV5/3 (inv) , BFP1/2 (bfp) 等共10組,進行聚合酶連鎖反應 (PCR)。結果顯示,人類來源之79株大腸桿菌有57株 (72%) 為ETEC,其中為LTI毒素型菌株有31株 (39%),另有2株 (3%) 具STII毒素,而STIa毒素則有9株 (11%),3株 (4%) 菌株同時具LTI及STII毒素,12株 (15%) 菌株具LTI與STIa 2種毒素。動物分離之大腸桿菌中,則有46株 (58%) 為ETEC毒素型,其中有9株 (11%) 為LTI毒素型之菌株,9株 (11%) 具STII毒素,以及5株 (6%) 具STIa毒素。2株 (3%) 同時具有LTI及STII、16株 (20%) 同時具有STIa與STII毒素、5株 (6%) 則同時具有此3種毒素型。人類來源之病源性大腸桿菌以ETEC-LTI毒素型為主 (20%);豬來源菌株則以ETEC-STIa-STII毒素型為主 (10%),另有1株 (1%) 為STEC。
並進一步,分析人類與豬隻來源之大腸桿菌抗生素圖譜,以明瞭菌株產生抗藥性之情況。結果顯示兩者來源之大腸桿菌菌株皆屬多重抗藥性類型,以豬隻來源菌所產生之抗藥性較為嚴重,如Fluoroquinolones類。人類來源菌株以AmCfKGS3SxtETeCb為主要抗藥性類型;豬隻則以AmGmNaETeOtMyNAprSpt為主。菌株皆對第一線抗生素、動物常用藥物及第一線頭孢子菌素cephalothin具高度抗藥性,所有菌株對Erythromycin、Lincomycin皆具抗性。對第二、三代頭胞子菌素cefmetazole、ceftazidime較不具抗性 (各佔3%、5%),對fluoroquinolone類抗生素如norfloxacin、ofloxacin、ciprofloacin,已有22%、23%、30%之抗性,另外,豬來源菌株對於禁用藥物仍有高抗藥性比例。
針對病原性大腸桿菌之class I integron的PCR偵測,採用integron結構中之保守區域,設計5’-CS/3’-CS 與Int1F/3’-CS引子組。經實驗結果得知,有106 (佔67%) 株可增幅3種片段分別為1380、1500與2500 bp之integron PCR產物,其中以佔61%之2500 bp integron最多,7株 (6%) 同時擁有2種integron片段。經分析顯示1380 bp之integron攜帶sat-1 基因,抗streptothricin;1500 bp之integron為攜帶aadA基因,抗streptomycin-spectinomycin;2500 bp 之integron則攜帶抗藥性基因 ”dfrA12-orfF-aadA2”,抗streptomycin-spectinomycin與trimethoprim。另以Int1F/Int1R 引子組檢測int基因,進行再確認,發現有134株 (佔85%) 具int基因。另外,就病原性大腸桿菌抗藥性與integron之關係而言,與integron抗藥性相關之抗生素,於抗生素圖譜上可見較高比例之抗藥性表現。

Abstract
Pathogens Escherichia coli which have several virulent factors like heat-labile toxin (LT), heat-stable toxin (ST), shiga-like toxin (SLT) and attaching mechanism (eae) is one of the most important microorganisms causing foodborne illness worldwide. When human was infected with pathogens E. coli, it is potentially fatal and may be associated with serious complication symptoms such as hemolytic-uremic syndrome (HUS), porcine diarrhoea. The curing of these infections are traditionally treated with antibiotics. However, in the past few years, many E. coli strains isolated from human and animals also have seen the development of antibiotic resistance, and multidrug resistance in human and veterinary medicine. For pathogenic bacteria strains, their drug resistance may be obtained by mutation or the transfer of plasmid or transposon. Recently, for Gram negative bacteria, integrons (class I, II, III and IV) which contain the antibiotic resistant genes have be reported. Of them, class I integron is most important. The integrons are mobile DNA elements with a specific structure which acquires or exchanges the antibiotic resistance genes. In recent years, we have collected hundreds of E. coli strains from both the human and animal origins. This study was focused on the analyze of the toxin types, antibiotic susceptibility pattern, and integron of each E. coli strain.
In order to identify the toxin types of 158 E. coli isolates from human and animal origins in Taiwan, the malate dehydrogenase (mdh) gene and several toxin related genes were investigated. The gene was amplified by primer set , i.e. Emdh 1/2, LT I 1/2, LTI 51/31, ST II 1/2, ST Ia 1/2, SLT I 5/3, SLT II 5/3, SK1/2, INV5/3, BFP1/2. For examples, 57 strains (72%) of 79 E. coli human isolates were identified as ETEC; included 31 strains (39%) were LTI toxin, 2 strains (3%) were STII toxin, 9 strains were STIa toxin, 3 strains (4%) were LTI and STII toxins, and 12 strains (15%) were LTI and STIa toxins. From the porcine isolates, i.e., 46 strains (58%) were identified as ETEC; included 9 strains (11%) were of LTI toxin, 9 strains (11%) were STII toxin, 5 strains (6%) were STIa toxin, 2 strains (3%) were LTI and STII toxins, 16 strains (20%) of STII, STIa toxins, and 5 strains (6%) both have the STII, LTI, STIa. One (1%) of 158 isolates was STEC-SLTII toxins.The major type from E. coli human isolates origin in Taiwan is ETEC-LTI type (20%), and the major type from E. coli porcine isolates is ETEC-STIa-STII (10%).
Furthermore, the use of the antibiograms to realize the drugs resistant of E. coli isolates from human and porcine origins. Results demonstrated that both human and porcine origins isolated E. coli strains resistant to multi-drugs, especially from porcine origin (ie., Fluoroquinolones). The major antibiogram type from human isolated is AmCfKGS3SxtETeCb, and the major antibiogram from porcine isolated is AmGmNaETeOtMyNAprSpt. All strains resisted to the first line antibiotics, i.e., cephalothin, erythromycin, and lincomucin. All strains had less resistant to the second and third generation antibiotics, such as cefmetazole (3%) and ceftazidime (5%). Strains had already resistance to fluoroquinolone-like drugs, ie., norfloxacin (22%), ofloxacin (23%), and ciprofloxacin (30%). In addition, E. coli from porcine isolates had still resisted to the banned drugs at a high rate.
In the detection of integron genes for pathogens E. coli isolates, the conserved primers 5'-CS/3'-CS and Int1F/3'-CS were used. The amplification results were 1380 bp,1500 bp, and 2500 bp. 106 strains (67%) had the three amplicons. 61% strains were amplified the 2500 bp products, and 6% strains were amplified two integron products. DNA sequencing analysis showed that the 1380 bp integron PCR product carried the sat-1 gene which resist to streptothricin, 1500 bp integron PCR product carried the aadA gene which responsible to the streptomycin-spectinomycin, and the 2500 bp integron PCR product was carried the dfrA12-orfF-aadA2 gene which resist to the streptomycin-spectinomycin and trimethoprim. Besides, we detected the int gene using Int1F/Int1R primers to confirm the present of integron. Data showed that 134 strains (85%) have the int gene. In addition, the integron related strains were highly resistant to antibiotic drugs.
Multiplex resistance was often found to erythromycin, lincomycin, and sulfisoxazole. These isolates resistant to fluoroquinolones, and the third-cephalothin, i.e. 106 of the 158 (67%) E. coli isolates were found to carry class 1 integrons. The amplified integron fragments are 1380 bp,1500 bp, and 2500 bp. Class 1 integrase gene were also detected, i.e. 85% (134/158) of E. coli isolates. Integrons were strongly associated with multiple-antibiotic-resistant strains, and integron-positive strains, it is demonstrating a greater predilection for antibiotic resistance than integron-native strains.
The data would be useful for developing the monitoring and prevention systems for these E. coli pathogens. In addition, the DNA sequences of integron could be used as the development of biochips, specifilly for the detection of integron related drug-resistant genes in the food pathogens.
URI: http://hdl.handle.net/11455/51773
Appears in Collections:食品暨應用生物科技學系

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