沙氏桿菌及大腸桿菌之調查及其抑制作用之研究

Investigation of Salmonella spp. and Escherichia coli and research on their inhibition

丁秋煌(Ting Chiu-Huang)

指導教授 ： 吳弘毅

國立屏東科技大學/獸醫學院/獸醫學系所/博士(2019年)

https://doi.org/10.6346/NPUST202000386

摘要

本研究旨在調查沙氏桿菌（Salmonella spp.）和大腸桿菌（E. coli）及其抑制作用之探討。為了解以動物副產品作為飼料添加劑而被污染的細菌菌群。從2012年9月到2014年3月，自進口的貨櫃中採集了206個樣品。對所有菌落形成進行單位計數，並表示為每克菌落形成單位(colony-forming units per gram- CFU/g)。結果顯示，最低CFU/g為零，最高為189×105 CFU/g。選擇單一菌落進行革蘭氏染色，以1,000倍油鏡觀察細菌，總共對245個菌落進行了染色。結果大部分為球菌，其中G +佔57.5％。以特定引子對相應細菌核酸(DNA)進行聚合酶連鎖反應(PCR)，以觀察受污染飼料添加劑的細菌群基因。以九種藥物來測試不同菌株進行抗菌敏感性試驗。其中第6號藥物阿莫西林對細菌編號第442菌株產生直徑近2cm的抑制圈，評定為非常敏感。第442菌株對4號藥物沒有抑制作用，評定為不敏感。沙氏桿菌會藉由空氣或受污染的食物廣泛傳播。致病性大腸桿菌常見的有6種，佔所有大腸桿菌病原體的4-8％。研究中，使用傳統的聚合酶鏈反應（PCR）調查了台灣西部的沙氏桿菌和大腸桿菌感染犬隻而導致腹瀉的感染率。並以流行病學分析被感染犬的流行率與年齡，季節，區域和品種之間的相關性。自2015年1月至2017年1月，從動物醫院檢查240隻腹瀉犬，收集其糞便樣品，取0.1g萃取其DNA，以特定引子對應細菌DNA進行PCR。結果沙氏桿菌和大腸桿菌感染率分別為19.2％（46/240）和3.3％（8/240）。此研究提昇臨床獸醫診斷疾病的能力及預防和控制的關鍵信息。粘土礦物是自然界中的重要物質。為醫療上防制,於飼料中添加元素，諸如豬和家禽飼料中，添加鋅和銅。這項研究旨在體外觀察負載銅的蒙脫土（Cu-MMT）對沙氏桿菌的殺菌作用。實驗分為兩種對沙氏桿菌的影響方法。首先，使用銅離子(Cu2+)與細菌混合的作用試驗。第二，載銅的蒙脫土直接與細菌混合作用試驗。結果顯示，銅離子(Cu2+)與細菌混合的作用試驗，於24小時的最佳殺菌濃度為0.1 毫克/公升，直接與沙氏桿菌混合的載銅蒙脫土，於24小時最佳殺菌濃度為0.05毫克/公升。將沙氏桿菌與銅離子或載銅蒙脫土混合後，在電子顯微鏡下清楚地觀察到對細菌的影響。許多細菌被載銅蒙脫土吸附、細菌表面顯現凹凸不均勻、細胞壁破裂、細胞膜破裂、細胞出現氣泡以及由於銅離子液體的滲透壓導致細胞壁與細胞質分離等現象
。經調查進口動物副產品作為飼料添加劑，其污染菌之衛生條件確有差異，因此在進行添加時必需確定滅菌完成，否則容易造成動物胃腸道疾病，若因此而使用抗生素作防制，則又產生抗藥性及消費者的衛生安全問題，所以載銅的蒙脫土即為另一種防制的選擇。

關鍵字：蒙脫土，大腸桿菌，電子顯微鏡，聚合酶連鎖反應，沙氏桿菌，藥敏試驗。

關鍵字

蒙脫土 ； 大腸桿菌 ； 電子顯微鏡 ； 聚合酶連鎖反應 ； 沙氏桿菌 ； 藥敏試驗

並列摘要

Abstract
Student ID: P9916004
Title of Thesis: Investigation of Salmonella spp. and Escherichia coli and research on their inhibition
Total Page: 85
Name of Institute: National Pingtung University of Science and Technology.
Graduate Date: July, 2020 Degree Conferred: Ph.D.
Name of Student: Chiu-Huang Ting Adviser: Hung-Yi Wu
The Contents of Abstract in this thesis:
This study aims to research on Investigation of Salmonella spp. and Escherichia coli (E. coli) and research on their inhibition. The bacterial flora of contaminated animal by-products as feedstuff additives. A total of 206 samples were taken from the imported container from September 2012 to March 2014. The colony-forming units were counted and expressed as CFU/g (colony-forming units per gram) of all samples. The result showed the lowest CFU/g is zero and the highest is 18,900,000 CFU/g. A single colony was selected for Gram staining. Gram-stained bacteria were viewed with a brightfield microscope at 1,000× magnification with oil immersion. A total of 245 colonies were stained, and cocci or bacilli, gram-negative (G-) or gram-positive (G+) were recorded respectively. The results were mostly cocci with G+ accounting for 57.5% of it. The corresponding PCR is performed by the extracted DNA with the specific primer to observe the bacterial flora gene of contaminated feedstuff additives from different sources (country). 450 different strains were selected from all dishes and stored in frozen 30% glycerol. An antimicrobial disk sensitivity test was performed. Nine kinds of drugs were used to test the antimicrobial sensitivity of different strains. Drug No. 6 Amoxicillin with a diameter suppression ring nearly 2 cm on bacterium strain No. 442 was judged to be extremely sensitive. Drugs No. 4 on No. 442 bacterium non-inhibition which is judged insensitive as it was an herbal essential oil. This study has provided the clinical veterinarian the crucial information for the prevention and control of domestic animal digestive tract diseases. Salmonella spp. can be widely spread by air or contaminated food. There are 6 common pathogenic E. coli that accounts for 4-8% of all E. coli pathogens. Among them, E. coli O157 is the most widely known one. In this study, we investigated the prevalence of diarrhea pathogens in pet dogs referred to as Salmonella spp. and E. coli in western Taiwan using conventional polymerase chain reaction (PCR). The related analysis between the prevalence rates and the epidemiological data of pet dogs were correlated with the age, season, area and breed. Fecal samples were collected from 240 dogs which have the symptom of diarrhea examined by the veterinary hospitals from January 2015 to January 2017. The PCR sensitivity of total DNAs extracted from 0.1g fecal samples ranged from 10 fg to 100 ng. The prevalence of Salmonella spp. and E. coli infections were 19.2% (46/240) and 3.3% (8/240) respectively. The results showed that both diseases Salmonella spp. and E. coli have the highest occurrence in spring and summer, and the highest proportions might occur in puppies, suburbs dogs. Since both diseases are zoonosis, the more human being is exposed to it, the higher its occurrence will be. This study has provided the clinical veterinarian the advanced ability of both diagnosis diseases and crucial information for the prevention and control of Salmonella spp. and E. coli diseases in a dog with diarrhea in Taiwan and neighborhood countries. Clay minerals are important substances in nature. Montmorillonite is believed to enhance the absorption and antibacterial properties of many products in biological preparations. Due to the addition of elements in compound feed and medical treatment, livestock such as pigs and poultry also need additional zinc and copper in their diets. Another purpose of this study is aimed to observe the bactericidal activity of Cu2+-loaded montmorillonite (Cu-MMT) against Salmonella spp. in vitro. The experiments were divided into two methods of effect on Salmonella spp.. First, using Cu-MMT to precipitate the Cu2+ ion supernatant mixed with bacteria. The second, Cu-MMT directly mixed with bacteria. The results indicated that the optimal bactericidal concentration of the Cu2+ ion supernatant precipitated by Cu-MMT was 0.1 mg/l for 24 hours, and the optimal sterilization concentration of Cu-MMT direct mixed with Salmonella was 0.05 mg/l for 24 hours also. When Salmonella spp. is mixed with Cu2+ ion or Cu-MMT, the effects on cell types can be clearly discerned by an electron microscope. Under the Scanning electron microscope (SEM) or Transmission electron microscope (TEM) showing many bacteria are adsorbed by Cu-MMT. The appearances of the bacteria are performed unevenly, the cell wall was ruptured, the cell membrane was broken, the cells appear as bubbles, and the osmotic pressure of Cu+2 ion liquid caused the cell wall to separate from the cytoplasm. Imported animal by-products are used as feed additives. According to the investigation, the sanitary conditions of the contaminated bacteria are different depending on the type of additive, batch number, importing country and other factors. Therefore, the sterilization must be completed when adding; otherwise it will easily cause animal gastrointestinal diseases. The use of antibiotics and other bacteriostatic agents for control will cause drug resistance and health with safety issues for human consumers, so copper-loaded montmorillonite is another control option.
Keywords: Cu-Montmorillonite, E. coli, electron microscope, PCR assays, Salmonella, susceptibility test

並列關鍵字

Cu-Montmorillonite ； E. coli ； electron microscope ； PCR assays ； Salmonella ； susceptibility test

參考文獻

1.2 References

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