臺灣博碩士論文加值系統

金黃色葡萄球菌為臨床上常見的革蘭氏陽性菌，其與葡萄球菌屬其他菌種在凝固&;#37238;等生化特性上有較大的差異。金黃色葡萄球菌以其金黃色菌落得名，然而近年澳洲研究發現了一群不產生金黃色葡萄球菌色素的品系，其菌落呈現白色。
現行葡萄球菌屬菌種鑑定多以生化反應與菌落型態做為判斷依據，然而生化反應往往會有例外，在實驗室難以作為鑑定依據，進行16S rRNA定序卻又太耗費人力物力，因此有文獻報導可使用dnaJ 基因 PCR-RFLP作為葡萄球菌屬檢體菌種鑑定、再確認的方法。
本實驗在進行臨床菌種再確認時發現了會產生不同樣式(pattern) dnaJ 基因 PCR-RFLP結果的金黃色葡萄球菌分離株，以此為契機開始研究dnaJ基因PCR-RFLP在金黃色葡萄球菌產生的兩種非典型樣式，其在臺大醫院金黃色葡萄球菌檢體中分別所佔的比例，以及菌株特性。
篩選出具非典型樣式的金黃色葡萄球菌後，進行了dnaJ基因的分析與序列比對，發現第一型非典型樣式的金黃色葡萄球菌的基因序列與前述不產生金黃色色素的金黃色葡萄球菌幾乎相同，而與一般金黃色葡萄球菌相差較多。而經由進一步的分子分型確認，發現第一型非典型樣式金黃色葡萄球菌為CC75，而第二型非典型樣式金黃色葡萄球菌則屬CC45。在脈衝式電泳及MALDI-TOF的實驗中也可明確區分典型樣式、第一型非典型樣式與第二型非典型樣式金黃色葡萄球菌。
第一型非典型樣式金黃色葡萄球菌在dnaJ基因的序列上與不產生金黃色葡萄球菌色素的金黃色葡萄球菌品系幾乎相同，而經由色素基因、色素萃取，也發現的確無色素表現。金黃色葡萄球菌色素為一毒力因子，在抵抗氧化壓力以及嗜中性球毒殺上被證實有功效，然而由本實驗發現缺乏金黃色葡萄球菌色素的第一型與正常產生色素的第二型非典型樣式金黃色葡萄球菌在臺大醫院均不少見，因此進行了毒力因子與生物膜生成等各方面實驗，分析是否具有較有利感染的因素使此二型非典型dnaJ 基因 PCR-RFLP樣式金黃色葡萄球菌能在金黃色葡萄球菌檢體中占有一席之地。
在毒力因子上有部分第二型非典型樣式金黃色葡萄球菌具有毒性休克症候群毒素基因(60.9%)較具有顯著不同，而生物膜生成實驗發現第一型非典型樣式金黃色葡萄球菌具有較佳的合成力，可能與其良好感染力相關。而在生化反應的部分則是藉由自動化鑑定儀器發現了第一型非典型樣式金黃色葡萄球菌尿素&;#37238;活性具有明顯較高的陽性率，並經生化管確認，然而進一步的生長速率分析初步排除尿素&;#37238;活性對於其生長之影響。

Staphylococcus aureus is a common Gram positive bacteria, and is different from other Staphylococcus species by biochemical activities such as coagulase. Staphylococcus aureus is reputed by its golden colony type, nevertheless, a lineage of non-pigmented S. aureus displaying white colonies had been found recently.
Currently, the major identification method for Staphylococcus species is by biochemical tests. But, biochemical activities usually have variation. Identification by 16S rRNA sequencing is more reliable, but it would cost more money and manpower. So, dnaJ gene PCR-RFLP would be a good choise for identification of Staphylococcus species.
In the beginning of this study, we found a S. aureus isolate with an atypical dnaJ gene PCR-RFLP pattern, further , we tried to study on 2 atypical S. aureus dnaJ gene PCR-RFLP patterns in NTU hospital, trying to find out its proportion and bacterial characteristics.
After the atypical pattern S. aureus had been selected out, we did the sequence blast and found out that atypical pattern 1 S. aureus is very similar to non-pigmented S. aureus mentioned above. Then we did some molecular typing, and found that atypical pattern 1 S. aureus isolates belongs to clonal complex 75, and atypical pattern 2 S. aureus isolates belongs to clonal complex 45. All of these three pattern could be separated by Pulsed-field gel electrophoresis and MALDI-TOF.
Atypical pattern 1 S. aureus shows similarity in dnaJ gene sequence with the lineage of non-pigmented S. aureus. By pigment gene detection and the extraction of pigment, we found that atypical pattern 1 S. aureus truly was unable to produce pigment. The golden pigment is recognized as one of important virulence factor of S. aureus, which plays a great role in reducing the oxidative stress damage and resisting netrophil phagocytosis. Though lacking of this pigment, atypical pattern 1 S. aureus still had its stand in isolates in NTU hospital. Thus this study tried to examine virulence factors and biofilm activity on 2 atypical pattern S. aureus for studying its advantage during infection.
The toxin shock syndrome toxin gene was found in many atypical pattern 2 S. aureus (60.9%), and so did it in biofilm activity in atypical pattern 1 S. aureus. These results might be an explanation for the well ability of infection. We then used Vitek 2 automated system to check the biochemical activities of 2 atypical pattern S. aureus, and found a dramatic high proportion on urease activity in atypical pattern 1 S. aureus. This result was confirmed by tube biochemical test, but after testing growth curve, we found it might not enhance the growth activity even in urea broth.

目錄
致謝………………………………………………….........i
中文摘要…………………………………………….....ii
英文摘要………………………………………………...iii
目錄……………………………………………………...iv
圖目錄. …………………………………………………..v
表目錄…………………………………………………...vi
第一章 緒論………………………………………….… 1
第二章 研究目的…………………………………….….4
第三章 實驗材料及方法………………………………..5
第四章 實驗結果………………………………………27
第五章 討論…………………………………….……...36
第六章 實驗結果附圖…………………………………43
第七章 實驗結果附表…………………………………54
第八章 參考文獻………………………………………71

1.Lowy FD: Staphylococcus aureus infections. The New England journal of medicine 1998, 339(8):520-532.
2.Pinho MG, de Lencastre H, Tomasz A: An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug-resistant staphylococci. P Natl Acad Sci USA 2001, 98(19):10886-10891.
3.Pantosti A, Venditti M: What is MRSA? Eur Respir J 2009, 34(5):1190-1196.
4.Katayama Y, Ito T, Hiramatsu K: A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob Agents Ch 2000, 44(6):1549-1555.
5.Hartman BJ, Tomasz A: Low-Affinity Penicillin-Binding Protein Associated with Beta-Lactam Resistance in Staphylococcus-Aureus. J Bacteriol 1984, 158(2):513-516.
6.Deleo FR, Chambers HF: Reemergence of antibiotic-resistant Staphylococcus aureus in the genomics era. J Clin Invest 2009, 119(9):2464-2474.
7.Hanssen AM, Sollid JUE: SCCmec in staphylococci: genes on the move. Fems Immunol Med Mic 2006, 46(1):8-20.
8.Chambers HF, Deleo FR: Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009, 7(9):629-641.
9.Baird-Parker A: Classification and identification of staphylococci and their resistance to physical agents. The staphylococci John Wiley &; Sons, Inc, New York 1972:1-20.
10.Willis A, Jacobs S, Goodburn GM: Pigment production, enzymatic activity and antibiotic sensitivity of staphylococci: subdivision of the pathogenic group. The Journal of pathology and bacteriology 1964, 87(1):157-167.
11.Marshall JH, Rodwell ES: Int Symp Carotenoids Abstr Commun 1972: 56-57.
12.Marshall JH, Wilmoth GJ: Pigments of Staphylococcus aureus, a series of triterpenoid carotenoids. J Bacteriol 1981, 147(3):900-913.
13.Pelz A, Wieland KP, Putzbach K, Hentschel P, Albert K, Gotz F: Structure and biosynthesis of staphyloxanthin from Staphylococcus aureus. The Journal of biological chemistry 2005, 280(37):32493-32498.
14.Liu GY, Essex A, Buchanan JT, Datta V, Hoffman HM, Bastian JF, Fierer J, Nizet V: Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. The Journal of experimental medicine 2005, 202(2):209-215.
15.Holt DC, Holden MTG, Tong SYC, Castillo-Ramirez S, Clarke L, Quail MA, Currie BJ, Parkhill J, Bentley SD, Feil EJ et al: A Very Early-Branching Staphylococcus aureus Lineage Lacking the Carotenoid Pigment Staphyloxanthin. Genome Biol Evol 2011, 3:881-895.
16.Tong SY, Sharma-Kuinkel BK, Thaden JT, Whitney AR, Yang SJ, Mishra NN, Rude T, Lilliebridge RA, Selim MA, Ahn SH et al: Virulence of endemic nonpigmented northern Australian Staphylococcus aureus clone (clonal complex 75, S. argenteus) is not augmented by staphyloxanthin. The Journal of infectious diseases 2013, 208(3):520-527.
17.Weisburg WG, Barns SM, Pelletier DA, Lane DJ: 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991, 173(2):697-703.
18.Hauschild T, Stepanovic S: Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism analysis of dnaJ gene. Journal of clinical microbiology 2008, 46(12):3875-3879.
19.Ito T, Katayama Y, Asada K, Mori N, Tsutsumimoto K, Tiensasitorn C, Hiramatsu K: Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2001, 45(5):1323-1336.
20.Ito T, Ma XX, Takeuchi F, Okuma K, Yuzawa H, Hiramatsu K: Novel type v staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob Agents Ch 2004, 48(7):2637-2651.
21.Hanssen AM, Kjeldsen G, Sollid JUE: Local variants of staphylococcal cassette chromosome mec in sporadic methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci: Evidence of horizontal gene transfer? Antimicrob Agents Ch 2004, 48(1):285-296.
22.Kobayashi N, Urasawa S, Uehara N, Watanabe N: Distribution of insertion sequence-like element IS1272 and its position relative to methicillin resistance genes in clinically important staphylococci. Antimicrob Agents Ch 1999, 43(11):2780-2782.
23.Brakstad OG, Aasbakk K, Maeland JA: Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. Journal of clinical microbiology 1992, 30(7):1654-1660.
24.Bod&;#233;n MK, Flock J: Fibrinogen-binding protein/clumping factor from Staphylococcus aureus. Infection and immunity 1989, 57(8):2358-2363.
25.Chung M, De Lencastre H, Matthews P, Tomasz A, Adamsson I, De Sousa MA, Camou T, Cocuzza C, Corso A, Couto I et al: Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: Comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb Drug Resist 2000, 6(3):189-198.
26.Harmsen D, Claus H, Witte W, Rothg&;#228;nger J, Claus H, Turnwald D, Vogel U: Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. Journal of clinical microbiology 2003, 41(12):5442-5448.
27.Maiden MCJ: Multilocus sequence typing of bacteria. Annu Rev Microbiol 2006, 60:561-588.
28.Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG: Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones ofStaphylococcus aureus. Journal of clinical microbiology 2000, 38(3):1008-1015.
29.Bannerman TL, Hancock GA, Tenover FC, Miller JM: Pulsed-Field Gel-Electrophoresis as a Replacement for Bacteriophage-Typing of Staphylococcus aureus. Journal of clinical microbiology 1995, 33(3):551-555.
30.Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B: Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel-Electrophoresis - Criteria for Bacterial Strain Typing. Journal of clinical microbiology 1995, 33(9):2233-2239.
31.Morikawa K, Maruyama A, Inose Y, Higashide M, Hayashi H, Ohta T: Overexpression of sigma factor, sigma(B), urges Staphylococcus aureus to thicken the cell wall and to resist beta-lactams. Biochem Bioph Res Co 2001, 288(2):385-389.
32.Iandolo J: Genetic analysis of extracellular toxins of Staphylococcus aureus. Annual Reviews in Microbiology 1989, 43(1):375-402.
33.van Wamel WJ, Rooijakkers SH, Ruyken M, van Kessel KP, van Strijp JA: The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on β-hemolysin-converting bacteriophages. J Bacteriol 2006, 188(4):1310-1315.
34.Peacock SJ, Moore CE, Justice A, Kantzanou M, Story L, Mackie K, O''Neill G, Day NP: Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infection and immunity 2002, 70(9):4987-4996.
35.Fitzpatrick F, Humphreys H, O''Gara J: The genetics of staphylococcal biofilm formation—will a greater understanding of pathogenesis lead to better management of device&;#8208;related infection? Clinical microbiology and infection 2005, 11(12):967-973.
36.Freeman D, Falkiner F, Keane C: New method for detecting slime production by coagulase negative staphylococci. Journal of clinical pathology 1989, 42(8):872-874.
37.Frank KL, Patel R: Poly-N-acetylglucosamine is not a major component of the extracellular matrix in biofilms formed by icaADBC-positive Staphylococcus lugdunensis isolates. Infection and immunity 2007, 75(10):4728-4742.
38.Test SU: Further studies on urease production by Proteus and related organisms. 1944.
39.Kuroda M, Yamashita A, Hirakawa H, Kumano M, Morikawa K, Higashide M, Maruyama A, Inose Y, Matoba K, Toh H: Whole genome sequence of Staphylococcus saprophyticus reveals the pathogenesis of uncomplicated urinary tract infection. P Natl Acad Sci USA 2005, 102(37):13272-13277.
40.Christensen WB: Urea decomposition as a means of differentiating Proteus and paracolon cultures from each other and from Salmonella and Shigella types. J Bacteriol 1946, 52(4):461.
41.Ho P-L, Chow K-H, Lo P-Y, Lee K-F, Lai EL: Changes in the epidemiology of methicillin-resistant Staphylococcus aureus associated with spread of the ST45 lineage in Hong Kong. Diagnostic microbiology and infectious disease 2009, 64(2):131-137.
42.Ho P-L: Carriage of methicillin-resistant Staphylococcus aureus, ceftazidime-resistant Gram-negative bacilli, and vancomycin-resistant enterococci before and after intensive care unit admission. Critical care medicine 2003, 31(4):1175-1182.
43.Ghebremedhin B, K&;#246;nig W, K&;#246;nig B: Heterogeneity of methicillin-resistant Staphylococcus aureus strains at a German university hospital during a 1-year period. European Journal of Clinical Microbiology and Infectious Diseases 2005, 24(6):388-398.
44.Denis O, Deplano A, Nonhoff C, De Ryck R, De Mendon&;#231;a R, Rottiers S, Vanhoof R, Struelens MJ: National surveillance of methicillin-resistant Staphylococcus aureus in Belgian hospitals indicates rapid diversification of epidemic clones. Antimicrob Agents Ch 2004, 48(9):3625-3629.
45.Gillaspy AF, Patti JM, Pratt Jr FL, Iandolo JJ, Smeltzer MS: The Staphylococcus aureus collagen adhesin-encoding gene cna is within a discrete genetic element. Gene 1997, 196(1):239-248.
46.Cramton SE, Gerke C, Schnell NF, Nichols WW, G&;#246;tz F: The intercellular adhesion (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infection and immunity 1999, 67(10):5427-5433.
47.Krakow JS, Fronk E: Azotobacter vinelandii Ribonucleic Acid Polymerase. Journal of Biological Chemistry 1969, 244(21):5988-5993.
48.Costerton J, Stewart PS, Greenberg E: Bacterial biofilms: a common cause of persistent infections. Science 1999, 284(5418):1318-1322.
49.Donlan RM, Costerton JW: Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical microbiology reviews 2002, 15(2):167-193.