台灣環境水體病原性大腸桿菌與沙門氏桿菌調查暨沙門氏桿菌抗藥性及整合子定量分析

Investigation of Enteropathogenic Escherichia coli and Salmonella spp. in Aquatic Environments of Taiwan and the Quantitative Analysis of Antibiotic Resistance Genes and Class 1 Integron in Salmonella spp

沈宗佑(Tzung-Yu Shen)

指導教授 ： 許昺慕

國立中正大學/理學院/地球與環境科學系應用地球物理與環境科學研究所/碩士(2013年)

摘要

沙門氏桿菌為一在各地相當普及的腸道細菌，具有多種血清型別，且對特定血清型別會有相對應的特定宿主，可藉此追蹤環境中汙染來源。而本研究多數的沙門氏桿菌具有一種以上抗藥性，此現象已成為一個公共健康的主要議題。在本研究主要利用聚合酶連鎖反應(PCR)檢測環境中沙門氏桿菌存在，並利用脈衝式膠體電泳(PFGE)、多位點序列分析(MLST)分析環境中沙門氏桿菌的血清型別。此外，本研究利用抗藥性基因PCR和Disk Diffusion 抗生素紙錠測試檢測環境中沙門氏桿菌的抗藥性存在情形。在培養法所培養出的13株沙門氏桿菌菌株當中，利用PFGE及MLST我們共檢測出了多種血清型別，主要血清型別包括了S. Typhimurium、S. Bareilly、S. Albany、S. Newport 等等，而MLST利用7段管家基因(aroC, dnaN, hemD, hisD, purE, sucA, thrA)即可分出11種血清型別並得到與PFGE類似的血清預測結果。本研究所檢測沙門氏桿菌菌株對以下幾種抗生素具有明顯抗藥性，磺胺類抗生素(41.7%)、氯黴素(33.3%)、青黴素(33.3%)、四環素(41.7%)、奈定酸(16.7%)、鏈黴素(25.0%)、健大黴素(25.0)、阿莫西林克拉(25.0%)、卡納黴素(8.3%)以及環丙沙星(8.3%)。而大部分皆對一種以上的抗生素具有抗藥性，從13株多重抗藥性菌株中檢測出不同的抗藥性組合型態。另外，本研究發現沙門氏桿菌菌株內帶有第一類整合子時，其多數具有多重抗藥性。研究結果顯示了在台灣不同沙門氏桿菌血清皆具有嚴重的多重抗藥性，所以建議需要持續監測在環境中抗藥性趨勢變化，此外，本研究並利用即時聚合酶連鎖反應去定量養豬廢水廠中每個處理單元的抗藥性基因存在量，並比較每個單元之去除效率。本研究中利用即時聚合酶連鎖反應定量養豬廢水廠中抗藥性基因的存在量，並評估廢水廠中的處理程序對於抗藥性基因的去除效率，在致病性大腸桿菌的方面，本研究利用即時聚合酶連鎖反應搭配增殖培養法，回推出在環境中致病性大腸桿菌的存在量。

關鍵字

多位點序列分析 ； 即時聚合酶連鎖反應 ； 紙錠測試 ； 抗藥性 ； 沙門氏桿菌 ； 致病性大腸桿菌

並列摘要

Salmonella is a common enteric pathogen with a worldwide distribution that comprises a large number of serotypes which characterized by corresponding to different hosts specificities. Most of Salmonella strains have great varieties of antibiotics resistance and it becomes a major public health concern. In this study, we detected the existence of Salmonella in Puzih river, Kaupin river, reservoir, and wastewater treatment plants by polymerase chain reaction (PCR) and identified the serotypes of Salmonella by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In addition, we used PCR and disk diffusion method to evaluate the antibiotic resistance and the resistant genes of Salmonella. The thirteen Salmonella isolates from culture method were further identified as S. Typhimurium, S. Bareilly, S. Albany, S. Newport by serological identification, PFGE and MLST. We also used seven house-keeping genes (aroC, dnaN, hemD, hisD, purE, sucA, thrA) to analyze the serotypes of Salmonella isolates. The results show that the predictions of 11 Salmonella serotypes by PFGE and MLST are with similar out of the total 13 colonies. For antibiotic resistance study Salmonella show that the highest percentage of resistance in the following antimicrobial agents: sulfadiazine (41.7%), chloramphenicol (33.3%), ampicillin (33.3%), tetracycline (41.7%), nalidixic acid (16.7%), streptomycin (25.0%), gentamicin (25.0%), amoxicillin-clavulanic acid (25.0%), kanamycin (8.3%)and ciprofloxacin (8.3%). Most of thirteen Salmonella isolates had more than one resistence gene and with different combinations of target antibiotics. Furthermore, we also found that the presence of the class 1 integron were often emergent with multi-drug resistance in Salmonella isolates. The results showed the serious situation of multi-drug resistant about different serotypes of Salmonella in Taiwan. We suggest keep monitoring the changes of antobiotics resistance trends of Salmonella in the environment continuously. In addition to monitoring the environmental water, we also quantified the resistance genes in several wastewater treatment plants by real-time PCR, and compared the removel percentage among different treatment units. In the enteropathogenic Escherichia coli, we used real-time PCR with broth method to quantity the amount of enteropathogenic Escherichia coli in environmental samples.

並列關鍵字

enteropathogenic Escherichia coli ； MLST ； real-time PCR ； antibiotic resistance ； Salmonella ； disk diffusion

參考文獻

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