開業醫師最常使用的廣效性抗生素cephalosporin是否可被使用於葡萄球菌之感染，該問題長久以來一直是令人關心的問題，傳統上一般實驗室通常是使用表現型(phenotyping)傳統方法亦即使用oxacillin紙錠來鑑定MRS (methicillin resistant Staphylococcus)，進一步以做為監測cephalosporin使用的適當性，也因為有的菌株並未被正確的鑑定出來而有一些爭議。
MRS誤鑑定為MSS (methicillin susceptible Staphylococcus)， 結果將會造成治療上的失敗，因而造成MRS菌株的擴散；相反地，當MSS誤鑑定為MRS時也會造成健康者被隔離而造成的一些不必要花費；因此，正確的鑑定出MRS是實驗室所必須的。
在本論文中，我們採用2004年的NCCLS規範，亦即建議使用cefoxitin及oxacillin來鑑定MRSA(methicillin resistant Staphylococcus aureus)以及MRCNS(methicillin resistant coagulase negative Staphylococcus)菌株。
另外，利用PCR測試兩種不同引子(primers)的 mecA 基因當作依據 (gold standard)；同時也測定clfA和nucA基因來鑑定S. aureus菌株之正確性，並且利用Eubacterial 16S rRNA及 Staphylococcal 16S rRNA 都具有之保留區段(conserved regions)的house-keeping gene來確保細菌DNA的抽取成功；實驗偵測六個相關性基因直接或間接以得知Cephalosporin類藥物是否可有效的使用於治療該菌株的感染。由實驗得知鑑定S. aureus若能同時使用Staph Latex Kit試驗及DNase試驗其靈敏度及有效性可由單獨使用Staph Latex Kit試驗(分別為99.2%，99.4%) 提昇到均為100%；鑑定MRSA或MRCNS時單獨使用cefoxitin 紙錠之靈敏度及特異性(分別為96.1%，100%)優於單獨使用oxacillin紙錠(分別為94.7%，97.8%)，若兩種紙錠同時使用則靈敏度更可提高至98.7%，特異性維持在97.8%，精確度提昇至98.2%.

Whether the widely-effective antibiotics can be used to cure the infection of Staphylococcus has been a long-concerned question. Traditionally, investigators in microbiology laboratories use oxacillin to inspect the fitness of using cephalosporin and general laboratories adopt traditional phenotyping to identify MRS (methicillin resistant Staphylococcus). But some strains are not correctly identified causing a little confusion.
When MRS is wrongly identified as MSS (methicillin susceptible Staphylococcus), it will lead to the failure of treatment. Furthermore, if there is no efficient infection control, it will cause the spread of MRS strains. When MSS is wrongly identified as MRS, it will cause money waste resulting from unnecessary isolation of healthy people. Therefore, it’s necessary to correctly identify MRS.
In this study, we adopt NCCLS guideline of 2004, with which cefoxitin and oxacillin are both used to identify MRSA (methicillin resistant Staphylococcus aureus) and MRCNS (methicillin resistant coagulase negative Staphylococcus) strains. Besides, Then compare with using PCR technique to identify mecA gene as gold standard by two different primer pairs. At the same time, detect clfA and nucA gene (also by PCR) to monitor the accuracy of S. aureus identification, and using the house-keeping gene in the conserved regions of Eubacterial 16S rRNA and Staphylococcal 16S rRNA to be a positive control. We can evaluate whether cephalosporin is a efficient treatment of S. aureus directly or indirectly by assaying these six related gene. In this report, bother Staph Latex kit and DNase test were evaluated for their sensitivity and specificity on the identification of S. aureus by using it separately or combined together. The results showed that the sensitivity and accuracy are 99.2% and 99.4% respectively and could be improved to 100% by combining two tests. For identifying of MRSA or MRCNS, both cefoxitin and oxacillin were tested. The results indicated that in cefoxitin alone, the sensitivity and specificity are 96.1% and 100% respectively and in oxacillin alone the sensitivity and specificity are 94.7% and 97.8% respectively. When two tests were combined, the sensitivity and specificity could be improved to 98.7% and 97.8% respectively and the accuracy could be increased to 98.2%.

英文摘要
中文摘要
目次
圖目錄
表目錄
一、緒論
(一).細菌概述
(二).抗藥性
(三).菌株鑑定
二、文獻探討
(一).細菌一般鑑定
1.葡萄球菌具有Eubacterial 16S rRNA及Staphylococcal 16S rRNA
2.金黃色葡萄球菌具有coagulase，clumping factor及proteinA
3.金黃色葡萄球菌可利用latex agglutination test和clfA gene檢測
4.金黃色葡萄球菌可利用DNase test和nucA gene檢測
(二).利用抗生素紙錠鑑定菌株
1.細菌室長久以來一直以OX-1 µg紙錠鑑定MRSA
2.近年來有人嘗試利用ceftizoxime (ZOX)，moxalactam (MOX)或cefoxitin (FOX)紙錠鑑定MRSA
3.BORSA及heterogeneous MRSA菌株
4.NCCLS 2005年已正式提出建議
三、實驗目的與設計
(一).目的
(二).菌株
(三).實驗設計
四、材料與方法
(一). 實驗菌株
(二). 細菌室使用傳統方法(目的、原理、材料、方法、判讀)
1. Staph Latex Kit 試驗
2. DNase 試驗
3. FOX-30及OX-1紙錠試驗
(三). PCR使用方法
1. DNA萃取
2.引子設計
3. PCR實驗
五、實驗結果與分析
1. Eubacterial 16SrRNA與Staphylococcal 16S rRNA
2. clfA基因與Staph Latex Kit 試驗
3. nucA基因與DNase試驗
4. Staph Latex Kit與DNase試驗同時進行效果之評估
5. mecA基因兩組引子比較
6. FOX-30µg紙錠試驗與mecA基因
7. OX-1µg紙錠試驗與mecA基因
8. FOX-30µg與OX-1µg紙錠同時進行試驗之評估
9. 抗生素紙錠抑制環直徑結果分析
六、討論
1. 污染是實驗室最常見的困擾
2. 僅憑菌落外觀做鑑定常被誤導
3. clfA基因與Staph Latex Kit試劑的比較
4. nucA基因與DNase test的比較
5. clfA基因與nucA基因效果的比較
6. Staph Latex Kit與DNase試驗效果的比較
7. CNS菌株最好設法鑑定到"種"的層次
8. 同時使用FOX-30µg 及OX-1µg紙錠與單獨使用FOX-30µg或OX-1µg紙錠試驗之比較
9. MRSA菌株抗生素紙錠抑制環大小的比較
10.MRCNS菌株抗生素紙錠抑制環大小的比較
11.BORSA菌株討論
12.NCCLS 2004年及2005年guideline中已正式建議MRS菌株應將β-lactams類藥物報告無效或不予報告
13.培養溫度方面的選擇性
14.PCR準確性的探討
七、結論與建議
1.同時使用Staph Latex Kit及DNase test鑑定較為正確
2. NCCLS 2005年guideline中已對於OX-1µg 及FOX-30µg紙錠試驗建S.lugdunensis菌株應採用與S. aureus菌株相同的標準做判定
3.以往單獨使用OX-1µg紙錠鑑定MRS會有所缺失
4. FOX-30µg紙錠效果優於OX-1µg紙錠，同時使用正確性較高
5.不論MRSA或MRCNS的抗藥性都不容忽視
6.對BORSA菌株的建議
7.現有條件下之務實做法
附圖與表
參考文獻

Reference

1. Boutiba-Ben Boubaker, I., Ben Abbes, R., Ben Abdallah, H., Mamlouk, K., Mahjoubi, F., Kammoun, A., Hammami, A. & Ben Redjeb, S. (2004). Evaluation of a cefoxitin disk diffusion test for the routine detection of methicillin-resistant Staphylococcus aureus. Clin Microbiol Infect 10, 762-5.
2. Brakstad, O. G., Aasbakk, K. & Maeland, J. A. (1992). Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J Clin Microbiol 30, 1654-60.
3. Brown, D. F. & Walpole, E. (2001). Evaluation of the Mastalex latex agglutination test for methicillin resistance in Staphylococcus aureus grown on different screening media. J Antimicrob Chemother 47, 187-9.
4. Cauwelier, B., Gordts, B., Descheemaecker, P. & Van Landuyt, H. (2004). Evaluation of a disk diffusion method with cefoxitin (30 microg) for detection of methicillin-resistant Staphylococcus aureus. Eur J Clin Microbiol Infect Dis 23, 389-92.
5. Cavassini, M., Wenger, A., Jaton, K., Blanc, D. S. & Bille, J. (1999). Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus. J Clin Microbiol 37, 1591-4.
6. Chambers, H. F. (1997). Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev 10, 781-91.
7. de la Maza, L.M., Pezzlo, M.T., and Baron, E.J. (1997). Aerobic gram-positive bacilli & actonomyces. : Color atlas of diagnostic microbiology. pp.46-55, Mosby, Inc., St.Louis USA
8. Felten, A., Grandry, B., Lagrange, P. H. & Casin, I. (2002). Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test. J Clin Microbiol 40, 2766-71.
9. Garbacz K, Piechowicz L, Wisniewska K and Galinski J (2002) [Characteristics of defective strains of methicillin resistance Staphylococcus aureus that do not produce coagulase or CF]. Med Dosw Mikrobiol 54:1-8.
10. Geha, D. J., Uhl, J. R., Gustaferro, C. A. & Persing, D. H. (1994). Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory. J Clin Microbiol 32, 1768-72.
11. Gerberding, J. L., Miick, C., Liu, H. H. & Chambers, H. F. (1991). Comparison of conventional susceptibility tests with direct detection of penicillin-binding protein 2a in borderline oxacillin-resistant strains of Staphylococcus aureus. Antimicrob Agents Chemother 35, 2574-9.
12. Greisen, K., Loeffelholz, M., Purohit, A. & Leong, D. (1994). PCR primers and probes for the 16S rRNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. J Clin Microbiol 32, 335-51.
13. Hussain, M., Haggar, A., Heilmann, C., Peters, G., Flock, J. I. & Herrmann, M. (2002). Insertional inactivation of Eap in Staphylococcus aureus strain Newman confers reduced staphylococcal binding to fibroblasts. Infect Immun 70, 2933-40.
14. Hussain, Z., Stoakes, L., Lannigan, R., Longo, S. & Nancekivell, B. (1998). Evaluation of screening and commercial methods for detection of methicillin resistance in coagulase-negative staphylococci. J Clin Microbiol 36, 273-4.
15. Isenberg,H.D (1995). Identification of commonly isolated aerobic gram-positive bacteria catalase-positive aerobic gram-positive cocci: Guide to regulatory requirements. A special supplement for users of the clinical microbiology procedures handbook. 1, pp.1.20.4 -7, American Society for Microbiology, Washington, D.C. USA
16. Lachica, R. V., Genigeorgis, C. & Hoeprich, P. D. (1971). Metachromatic agar-diffusion methods for detecting staphylococcal nuclease activity. Appl Microbiol 21, 585-7.
17. Louie, L., Matsumura, S. O., Choi, E., Louie, M. & Simor, A. E. (2000). Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus. J Clin Microbiol 38, 2170-3.
18. MacFaddin, J.F. (2000). Coagulase test, Deoxyribonuclease (DNase) and Thermonuclease (TNase ) tests: Biochemical tests for identification of medical bacteria. 3rd ed. pp.105-119, 136-159, Lippincott Williams & Wilkins, Inc., London British
19. Madison, B. M. & Baselski, V. S. (1983). Rapid identification of Staphylococcus aureus in blood cultures by thermonuclease testing. J Clin Microbiol 18, 722-4.
20. Maranan, M. C., Moreira, B., Boyle-Vavra, S. & Daum, R. S. (1997). Antimicrobial resistance in staphylococci. Epidemiology, molecular mechanisms, and clinical relevance. Infect Dis Clin North Am 11, 813-49.
21. Mariani, B. D., Martin, D. S., Levine, M. J., Booth, R. E., Jr. & Tuan, R. S. (1996). The Coventry Award. Polymerase chain reaction detection of bacterial infection in total knee arthroplasty. Clin Orthop Relat Res, 11-22.
22. Mason, W. J., Blevins, J. S., Beenken, K., Wibowo, N., Ojha, N. & Smeltzer, M. S. (2001). Multiplex PCR protocol for the diagnosis of staphylococcal infection. J Clin Microbiol 39, 3332-8.
23. McDevitt, D., Francois, P., Vaudaux, P. & Foster, T. J. (1994). Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol Microbiol 11, 237-48.
24. McDonald, L. C., Lauderdale, T. L., Shiau, Y. R., Chen, P. C., Lai, J. F., Wang, H. Y. & Ho, M. (2004). The status of antimicrobial resistance in Taiwan among Gram-positive pathogens: the Taiwan Surveillance of Antimicrobial Resistance (TSAR) programme, 2000. Int J Antimicrob Agents 23, 362-70.
25. Michael, J., Hibbert, R. & Plewes, J. (1996). Providing intensive care. Criticisms of situation in Birmingham are unsubstantiated. Bmj 313, 111.
26. Moriyasu, I., Igari, J., Yamane, N., Oguri, T., Takahashi, A., Tosaka, M., Takemori, K., Toyoshima, S. & Minamide, W. (1994). [Multi-center evaluation of Showa ceftizoxime disk susceptibility test to discriminate between the strains of methicillin-resistant Staphylococcus aureus (MRSA) and those susceptible (MSSA)]. Rinsho Byori 42, 271-7.
27. National Committee for Clinical Laboratory Standard.2004. Performance standards for antimicrobial disk susceptibility testing,14th ed. Approved standard M2-A8. National Committee for Clinical Laboratory Standards, Wayne,Pa.
28. National Committee for Clinical Laboratory Standard.2005. Performance standards for antimicrobial disk susceptibility testing,15th ed. Approved standard M2-A8. National Committee for Clinical Laboratory Standards, Wayne,Pa.
29. Noskin, G. A., Siddiqui, F., Stosor, V., Hacek, D. & Peterson, L. R. (1999). In vitro activities of linezolid against important gram-positive bacterial pathogens including vancomycin-resistant enterococci. Antimicrob Agents Chemother 43, 2059-62.
30. Resende, C. A. & Figueiredo, A. M. (1997). Discrimination of methicillin-resistant Staphylococcus aureus from borderline-resistant and susceptible isolates by different methods. J Med Microbiol 46, 145-9.
31. Sakoulas, G., Gold, H. S., Venkataraman, L., DeGirolami, P. C., Eliopoulos, G. M. & Qian, Q. (2001). Methicillin-resistant Staphylococcus aureus: comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains. J Clin Microbiol 39, 3946-51.
32. Strommenger, B., Kettlitz, C., Werner, G. & Witte, W. (2003). Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol 41, 4089-94.
33. Swenson, J. M., Spargo, J., Tenover, F. C. & Ferraro, M. J. (2001a). Optimal inoculation methods and quality control for the NCCLS oxacillin agar screen test for detection of oxacillin resistance in Staphylococcus aureus. J Clin Microbiol 39, 3781-4.
34. Swenson, J. M., Williams, P. P., Killgore, G., O'Hara, C. M. & Tenover, F. C. (2001b). Performance of eight methods, including two new rapid methods, for detection of oxacillin resistance in a challenge set of Staphylococcus aureus organisms. J Clin Microbiol 39, 3785-8.
35. Tokue, Y., Shoji, S., Satoh, K., Watanabe, A. & Motomiya, M. (1992). Comparison of a polymerase chain reaction assay and a conventional microbiologic method for detection of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 36, 6-9.
36. Urbaskova, P., Melter, O., Mackova, B., Jakubu, V. & Wunschova, M. (2004). [Detection of MRSA in a group of 752 strains of S. aureus using a cefoxitin disk]. Epidemiol Mikrobiol Imunol 53, 62-5.
37. van Griethuysen, A., Pouw, M., van Leeuwen, N., Heck, M., Willemse, P., Buiting, A. & Kluytmans, J. (1999). Rapid slide latex agglutination test for detection of methicillin resistance in Staphylococcus aureus. J Clin Microbiol 37, 2789-92.
38. von Eiff, C., Reinert, R. R., Kresken, M., Brauers, J., Hafner, D. & Peters, G. (2000). Nationwide German multicenter study on prevalence of antibiotic resistance in staphylococcal bloodstream isolates and comparative in vitro activities of quinupristin-dalfopristin. J Clin Microbiol 38, 2819-23.
39. Warren, D. K., Liao, R. S., Merz, L. R., Eveland, M. & Dunne, W. M., Jr. (2004). Detection of methicillin-resistant Staphylococcus aureus directly from nasal swab specimens by a real-time PCR assay. J Clin Microbiol 42, 5578-81.
40. York, M. K., Gibbs, L., Chehab, F. & Brooks, G. F. (1996). Comparison of PCR detection of mecA with standard susceptibility testing methods to determine methicillin resistance in coagulase-negative staphylococci. J Clin Microbiol 34, 249-53.