- 學位論文
台灣環境水體中腸道致病菌暨金黃色葡萄球菌之環境調查及檢測方法學之研究
Investigation and Detection Method Development of Intestinal Pathogenic Bacteria and Staphylococcus Aureus in Aquatic Environment of Taiwan
摘要
腸道細菌普遍的存在世界上各個地方，其中有些腸道細菌會造成人類生病，因此，檢測環境水體中腸道致病菌是必要的。本研究針對台灣環境水體中沙門氏桿菌、致病性大腸桿菌、李斯特菌、曲狀桿菌和金黃色葡萄球菌做檢測。在本研究中主要利用聚合酶連鎖反應(PCR)檢測環境中沙門氏桿菌存在，並利用多位點序列分析(MLST)分析環境中沙門氏桿菌的血清型別。研究結果顯示沙門氏桿菌 在朴子溪的檢出率為4.9%、全國河川8.5%、全國水庫中無檢出。利用MLST鑑定分離的12株沙門氏桿菌菌株，其血清型為S. Mbandaka(5)、S. Oranienburg(2)、 S. Derby(1)、S. Newport(1)、S. Stanley(1)、S. Typhimurium(1)、S. Livingstone(1)。本研究針對致病性大腸桿菌中常見的EPEC、ETEC、EHEC做調查，包含朴子溪、全國河川、全國水庫、南北溫泉，EPEC在朴子溪的檢出率為28%、全國水庫1.7%、全國河川17%、南北溫泉2.5%，ETEC在朴子溪的檢出率為13.4%、全國水庫1.7%、全國河川17%、南北溫泉2.5%，EHEC在朴子溪的檢出率為1.2%、全國水庫無檢出、全國河川2.1%、南北溫泉1.2%。在本實驗中利用液態前增殖和2次的選擇性固態培養基分離出1株EPEC和5株ETEC。在沙門氏桿菌和致病性大腸桿菌的實驗中也發現其具有季節性的變化。在李斯特菌方面，本研究調查台灣環境水體中李斯特菌的存在情形，Listeria spp.的prs基因在朴子溪的檢出率為19.4%、全國水庫無檢出、全國河川16.7%，本實驗使用目標基因lmo1030和iap分別檢測L. monocytogenes和L. innocua，其在朴子溪的檢出率各為1.3%和18.1%。曲狀桿菌方面，本研究利用曲狀桿菌的16S rRNA基因做檢測，在朴子溪和全國水庫河川皆無檢出。在金黃色葡萄球菌方面也對於台灣環境水體中，朴子溪和全國水庫河川調查，利用目標基因nuc檢測環境中是否存在Staphylococcus aureus，進一步再利用mecA基因來看是否為Methicillin-Resistant Staphylococcus aureus(MRSA)，其檢出率方面在朴子溪為5.56%、全國河川和全國水庫皆無檢出。本研究也檢測環境水體中是否攜帶ampc、 blaTEM、cmlA、mecA、strA、sul1、tetA、 tetB基因，在朴子溪的檢出率分別為sul1 47.6(78)、cmlA 34.1(56)、tetA 32.3(53)、blaTEM 26.8(44)、tetB 4.3(7)、strA 0.6(1)、ampc和mecA皆無檢出，全國水庫為blaTEM 10.3(6)、tetB 6.9(4)、cmlA 5.1(3)、strA 1.7(1)、sul1 1.7(1)、ampc、mecA和tetA皆無檢出，全國河川為cmlA 19.1(9)、blaTEM 14.9(7)、tetB 6.4(3)、tetA 4.3(2)、sul1 4.3(2)、strA 2.1(1)、ampc和mecA皆無檢出，在本實驗中也發現抗藥性基因的檢出會隨著季節變化而發生改變。 關鍵字：沙門氏桿菌、多位點序列分析、致病性大腸桿菌、李斯特菌、曲狀桿菌、金黃色葡萄球菌、抗藥性
並列摘要
Intestinal pathogenic bacteria are ubiquitous in the world, some of intestinal pathogenic bacteria can cause diseases, and therefore, the detection of intestinal pathogenic bacteria in aquatic environment is important. In this study, we detection Salmonella, Diarrheagenic E. coli, Listeria, Campylobacter and Staphylococcus aureus in aquatic environment of Taiwan. We detection Salmonella by polymerase chain reaction (PCR) and identified the serotypes of Salmonella by multilocus sequence typing (MLST). The result of Salmonella show that Puzih river (4.9%), Nationwide rivers (8.5%), and Nationwide reservoir (none). The twelve Salmonella isolate from culture method were identified by MLST, that are S. Mbandaka (5), S. Oranienburg (2), S. Derby (1), S. Newport (1), S. Stanley (1), S. Typhimurium (1) and S. Livingstone (1). In Diarrheagenic E. coli, we detection Enteropathogenic E.coli (EPEC), Enterotoxigenic E.coli (ETEC) and Enterohemorrhagic E. coli (EHEC), the sampling locations include Puzih river, North and south of the hot spring area Nationwide rivers, and Nationwide reservoir. The detection rate of EPEC is Puzih river (28%), North and south of the hot spring area (2.5%), Nationwide rivers (17%), and Nationwide reservoir (1.7%). The detection rate of ETEC is Puzih river (13.4%), North and south of the hot spring area (2.5%), Nationwide rivers (17%), and Nationwide reservoir (1.7%). The detection rate of EHEC is Puzih river (1.2%), North and south of the hot spring area (1.2%), Nationwide rivers (2.1%), and Nationwide reservoir (none). Finally, we use a liquid pre-enrichment and two selective solid medium isolated one EPEC and five ETEC strains. In addition, we also found that Salmonella and Diarrheagenic E. coli are seasonal. In Listeria, the prs gene carried by Listeria spp. The detection rate of Listeria spp. is Puzih river (19.4%), Nationwide rivers (16.7%), and Nationwide reservoir (none). Moreover, this study also use lmo1030 and iap genes to detection L. monocytogenes and L. innocua, the detection rate is L. monocytogenes (1.3%), and L. innocua (18.1%) in Puzih river. In Campylobacter, we use 16S rRNA gene of Campylobacter to detection, but the result showed no detectable in Puzih river, Nationwide rivers, and Nationwide reservoir. In Staphylococcus aureus, we use target gene nuc to detection Staphylococcus aureus in aquatic environment. Furthermore, use mecA gene to identification Methicillin-Resistant Staphylococcus aureus (MRSA), The detection rate of MRSA is Puzih river (5.56%), Nationwide rivers (none), and Nationwide reservoir (none). In addition, this study also detection the presence of drug resistance gene. The result showed that sul1 47.6% (78), cmlA 34.1% (56), tetA 32.3% (53),blaTEM 26.8% (44), tetB 4.3% (7), strA 0.6% (1), ampc (none), and mecA (none) in Puzih river, blaTEM 10.3%(6), tetB 6.9%(4), cmlA 5.1%(3), strA 1.7%(1), sul1 1.7%(1), ampc (none), mecA (none), and tetA (none) in Nationwide reservoir, cmlA 19.1%(9), blaTEM 14.9%(7), tetB 6.4%(3), tetA 4.3%(2), sul1 4.3%(2), strA 2.1%(1), ampc (none),and mecA (none). In addition, we also found that the presence of drug resistance gene is seasonal. Key words：Salmonella, Diarrheagenic E. coli, Listeria, Campylobacter, Staphylococcus aureus, multilocus sequence typing (MLST), and drug resistance gene.