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以PMA-qPCR進行空氣中金黃色葡萄球菌之採樣技術評估

Assessment of Bioaerosols Sampling Techniques for Viable Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus by Propidium Monoazide Quantitative PCR

作者:陳琬婷
畢業學校:慈濟大學
出版單位:慈濟大學
核准日期:2012-10-04
類型:Electronic Thesis or Dissertation
權限:Copyright information available at source archive--Tzu Chi University....

中文摘要

金黃色葡萄球菌(Staphyloccocus aureus; S. aureus) 是一種常引起食物中毒和傷口感染的細菌,近年由於抗生素的濫用,導致抗甲氧西林金黃色葡萄球菌(methicillin-resistant S. aureus; MRSA)的出現。因此定量環境中具有活性的S. aureus或是MRSA便成為重要的公共衛生議題。有別於傳統的培養法(Culture based methods),即時定量聚合酶鏈鎖反應(Real-time quantitative polymerase chain reaction; Real-time PCR)具備快速且高敏感度的優點,具有定量生物氣膠的潛力。但由於Real-time PCR並無法辨別微生物的活性,因此,近年來有研究將 Propidium Monoazide (PMA)核酸染劑與Real-time PCR結合,藉由辨別細菌細胞膜的完整度達到區分其活性的目的。因此,本研究的目的是以PMA結合Real-time PCR (PMA-qPCR)後,應用在偵測環境空氣中具有活性的S. aureus以及MRSA上。
本研究所建立的PMA標準方法是以濃度為10 μg/ml的PMA搭配500 W的鹵素光源照射7分鐘。在死活百分比相關性測試的部分,PMA-qPCR與培養法均可用來辨別S. aureus的死活。而由PMA-qPCR應用於生物氣膠樣本的保存測試測驗中則可發現PMA與樣本DNA鍵結之後,其鍵結力之穩定性至少可持續28天。後續環境偵測部分,內科加護病房(medical intensive care unit; MICU)以及重訓室偵測到的空氣中總S. aureus濃度範圍皆為105 copies/m3,該濃度中分別有2.61E+03 copies/m3及9.04E+03 copies/m3為具有活性的S. aureus,且經後續檢驗後有超過一半的機率為活的MRSA。而培養法於MICU和重訓室採集到的空氣中S. aureus濃度則分別為109 CFU/m3和296 CFU/m3,其中有19 %及9 %為MRSA。
研究中使用PMA-qPCR偵測到空氣中活的S. aureus及MRSA的濃度比使用培養法測到的濃度高10 ~ 400倍,由此可知,無論是S. aureus或是MRSA均有可能在空氣中呈現具有活性但無法培養(viable but non-culturable; VBNC)的狀態。此結果說明我們若單純以培養法進行感染控制監測的手段,則可能會明顯低估空氣中S. aureus以及MRSA所帶來的風險。本研究透過PMA-qPCR可快速得知空氣中S. aureus的濃度及活性,搭配Methicillin前處理更可有效縮短鑑定空氣中具有活性MRSA的時間,此方法相當值得發展與並應用於實際空氣偵測中。

英文摘要

Staphylococcus aureus (S. aureus) are bacteria which are commonly found in human epidermis and environment may cause food poisoning and wound infections. Because antibiotics have been abused in recent years, the emergence of methicillin-resistant Staphylococcus aureus (methicillin-resistant S. aureus; MRSA) have attracted public attention. Therefore, how to quantify viable S.aureus or MRSA in indoor air has become an important public health issue. Traditional bioaerosols detection methods are based on culture methods, in comparison with culture methods, real-time quantitative polymerase chain reaction (Real-time PCR) which is rapid, and with highly sensitivity and specificity has been proved to quantify bioaerosols. However, real-time PCR can not distinguish whether the bacteria are viable or not is the major shortcoming. In recent years, the Propidium Monoazide (PMA), a nucleic acid dye which could combine with real-time PCR to determine the viability of bacteria by identifying the integrity of bacterial cell membrane are able to address the viability of bacteria. Therefore, the purpose of this study is to establish the method by using PMA combined with real-time PCR (PMA-qPCR) to detect S. aureus in indoor air. In addition, the PMA-qPCR method will be subjected to apply in the medical environment.
The optimal method for PMA-qPCR was staining sample with 10 g/ml of PMA and then exposed to 500 W halogen light for 7 min. Based on the viability test, PMA-qPCR has the potential to distinguish whether S. aureus are viable or not. Moreover, the stability of PMA binding DNA samples were evaluated when stored at refrigerated temperature. Once PMA was bound with DNA, the stability of the DNA bindings was stable up to 28 days. For environmental monitoring, the S. aureus concentration was 105 copies/m3 in medical intensive care unit (MICU) and gym air, which containing 2.61E +03 copies/m3 and 9.04E +03 copies/m3 viable S. aureus. In the sampling sites, nearly half of the viable S. aureus was viable MRSA. The S. aureus concentration was 109 CFU/m3 and 296 CFU/m3 in MICU and gym air by culture based methods, respectively. There was 19 % and 9 % of total bacteria was identified for MRSA in MICU and gym, respectively.
In this study, the concentration of viable S.aureus and MRSA in air detected by PMA-qPCR method was 10 to 400 times higher than those detected by using culture method. Therefore, S. aureus and MRSA are likely to present in air with viable but non-culturable (VBNC) stage in the environment. The results of this study indicated that culture methods may significantly underestimate the concentration of S. aureus and MRSA. PMA-qPCR maybe used to quantify the concentration of viable S. aureus in air. PMA-qPCR combined with Methicillin pretreatment may shorten time for identification of MRSA in air. Therefore, PMA-qPCR is a potential methd to be applied for bioaerosol sampling.


召集人 - 吳佩芝

指導教授 - 曾俊傑

審核委員 - 張凱誌


 

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