臺灣博碩士論文加值系統

金黃色葡萄球菌感染（Staphylococcus aureus）是臨床重要議題，它除了會引發嚴重敗血症、感染性心內膜炎、壞死性肺炎以及軟組織感染外，亦可以在10-20%無症狀的健康人皮膚上發現。持續性的無症狀移生宿主較之於間歇性移生者更有可能演變成侵入性感染。一般的金黃色葡萄球菌(MSSA)可藉由獲得一個移動型基因片段，staphylococcal cassette chromosome mec (SCCmec)，所攜帶的mecA基因而合成額外的青黴素接合蛋白(PBP2a)而變成抗甲氧苯青黴素金黃色葡萄球菌(MRSA)。目前SCCmec可至少分為十一個分型。過去MRSA多屬於院內感染常見菌株，不過近二十年來亦可由許多無就醫紀錄的感染病患分離出來，由於這些菌株所攜帶的SCCmec在基因分型多攜帶SCCmecIV或SCCmecV，又稱之為社區型抗甲氧苯青黴素金黃色葡萄球菌(CA-MRSA)。台灣的CA-MRSA在多位點序列分型（MLST）中大多為攜帶SCCmecV(5C2&5)的第59型並約可在1-5%無症狀成人身上分離出來。多位點序列分型（MLST）為常用分析群聚的分子生物分型方式，其原理為分析七個結構基因部分定序結果來分型。由於抗生素的長期使用會造成移生菌叢的改變甚至誘發抗藥性，因此我們一開始想藉由觀察使用抗結核藥物（利福平）病患移生的金黃色葡萄球菌，以了解在接受治療後是不是對利福平產生抗藥性，亦或直接由他處獲得已產生利福平抗藥性的菌株。我們藉由持續追蹤兩百位在亞東醫院服用利福平治療結核菌的患者鼻腔帶菌，發現有5%病人一開始有鼻腔帶菌，其中1%為MRSA(均為ST59-SCCmecIV)。在治療開始後病人身上移生的金黃色葡萄球菌就消失了，然而在治療過程中發現卻有三位病人在服藥期間發生新的移生，其中兩位持續帶菌的病人所分離的菌株對利福平有抗藥性，這兩位持續帶菌的菌株有一位是MSSA(ST7)，一位是MRSA(ST45-SCCmecV[5C2&5])，這兩位持續帶菌者的菌株的分子分型前後各自相同。而在服藥結束兩個月後總共有十三位病人鼻腔有金黃色葡萄球菌移生，除了一位持續帶菌者的菌株外，其餘菌株對於利福平仍維持感受性。在後續十三位帶菌者中，有兩位在一開始篩檢時也有帶菌，不過前後菌株的分子分型是不一樣的。另外抗利福平菌株的抗藥基因突變點在MSSA與MRSA是不同的。第一部分的研究發現在利福平使用下，原本帶有的移生菌會被帶有抗藥性的菌株所取代，原生的菌株似乎不會在治療期間自己發展出新的抗藥基因。藉由MLST分析發現這MRSA不是過去常見的社區型(ST59)而是過去在台灣以及亞洲地區很少被分離出來的ST45。這個新引入的分型過去在歐洲已成為主要院感以及社區型的主要菌株。這個分型有一個重要特色，它對苯唑西林為低程度抗藥(最低抑菌濃度介於0.5-8μg/ml)，因此過去可能被誤判成MSSA。不過在台灣苯唑西林低程度抗藥的MRSA(borderline oxacillin-resistant S. aureus; BORSA)過去以MRSA-ST59為主，有關MRSA-ST45的資料並不是很清楚，於是我們利用急診金黃色葡萄球菌菌血症資料庫，找尋苯唑西林最低抑菌濃度介於0.5-4μg/ml的MRSA菌株做MLST分型分析，同時分析十五歲以上病人的臨床表現。總共分析了六十五位BORSA菌血症病人，其總死亡率為24.6%，較MRSA菌血症低許多(38.5%)。這些BORSA感染者的共病除了較少的洗腎病患外與感染MRSA菌血症患者十分接近，不過臨床表現卻與MRSA菌血症表現不同；患者以下呼吸道感染為主，且BORSA菌血症的三十天死亡率與敗血性休克以及肺部感染有關。MLST分型結果發現過去這些低程度抗藥的菌株多為ST59，然而在最近五年卻逐漸被ST45所取代。這些ST45的抗菌譜完全不同於ST59，對环丙氟哌酸100%抗藥。由於ST45在最近研究發現已經在長照機構的移生菌中發現，因此第三部分則是分析中南部安養機構的抗甲氧西林金黃色葡萄球菌，結果已發現ST45是中南部第二多的移生菌，僅次於ST59，至於會不會進一步取代ST59則需要後續監測。藉由我們的研究發現，ST45為這幾年新發生在台灣的抗甲氧西林金黃色葡萄球菌，它獨特的抗藥性以及臨床表現值得持續追蹤研究。

Staphylococcus aureus is an important human pathogen causing severe infections including severe sepsis, infective endocarditis, necrotizing pneumonia and soft tissue infection. It could colonize in 10-20% asymptomatic healthy adults. Patients persistently colonized with S. aureus are more likely to develop subsequent infections compared with non-colonizers and intermittent carriers. Methicillin-resistant S. aureus could emerge from methicillin-susceptible S. aureus (MSSA) progenitor strains after acquired the ability to produce additional penicillin-binding protein (PBP2a) encoded by mecA gene on a genetic mobile element calledstaphylcoccal cassette chromosome (SCCmec). Currently SCCmec could be clarified into 11 types by analyzing their structure. In recent two decades, MRSA could be isolated form patients without health-care facilities exposure and the community-acquired MRSA (CA-MRSA) clones usually carry SCCmecIVorSCCmecV. In Taiwan, the most commonly encountered CA-MRSA strain is multilocus sequencing type 59 (MLST type ST59) carrying SCCmecV (5C2&5) and could be found in 1-5% asymptomatic adults. The MLST typing is one of the commonly used methods for determine genetic relatednessby analyzing seven house keeping genes.Prolonged exposure to antibiotics could result in changing bacterial colonization and emergence of drug resistant strains. In our first study, we observed the epidemiology of S. aureus nasal colonization in patients receiving long-term rifampin for tuberculosis. The results would clarify whether de novo resistance to rifampin developed during treatment or colonized with additional resistant strains. First, we prospectively collect nasal swab from TB patients taking rifampin for S. aureus carriage screening from March 2009 to March 2011. A total of 200 patients were enrolled during the study period with 152 patients completed follow-up during treatment and 114 patients had been followed 2 months after discontinuing treatment for tuberculosis. Ten patients (5%) had nasal colonization and two of them had MRSA (1%) at enrollment. Two of the ten patients were recolonized with unrelated MSSA strains two months after drug discontinuation. Of the 30 S. aureus isolates from the 23 patients with S. aureus colonization during surveillance, seven were MRSA with two ST59-SCCmecIV-RIF-S (two patients) and five ST45-SCCmecV(5C2&5)-RIF-R (two patients). The results of rpoB gene sequencing of RIF-R S. aureus revealed different point mutation sites between MSSA and MRSA isolates. We found that the RIF-R S aureus nasal isolates from patients receiving rifampin-containing regimens might acquire from extrinsic sources rather than develop during rifampin exposure. The ST45-SCCmecV(5C2&5) clone is an epidemic clone that was rarely reported in Asia before and disseminated in recent years in Taiwan. One of its characters is that the minimum inhibitory concentrations (MICs) of oxacillin fall into the rage of 0.5-8 μg/ml and wound be misidentified as MSSA using oxacillin disk diffusion method. In Taiwan, the MRSA isolates with low oxacillin MICs (borderline oxacillin-resistant S. aureus; BORSA) belonged to ST59 in previous reports. We investigate clinical presentations of bacteremia caused by BORSA and performed MLST typing for these isolates. We enrolled patients >15 year-old who admitted to emergency department in a tertiary hospital with BORSA bacteremia (oxacillin MICs: 0.5-4 μg/ml) during 2001-2015. Clinical presentations of BORSA, MSSA and MRSA bacteremia were compared. A total of 65 BORSA bacteremia patients were identified with in hospital mortality of 24.6%, significantly lower then MRSA (38.5%, p=0.03). The underlying conditions were similar between BORSA and MRSA patients except less dialysis of the former (16.9% v.s. 33.2%, p=0.01). Multivariate analysis revealed that septic shock (Odds ratio, 15.95; 95% confidence interval, 1.41-180.89) and bacteremia originated from lower respiratory tract infection (OR, 5.78; 95% CI, 1.10-30.28) were two independent risk factors for 30-day mortality. Multidrug resistant ST45 strains with high-level ciprofloxacin resistance (100% resistant, MICs range: 8-128 μg/ml) have replaced ST59 as the predominant clone since 2012. No major clone of ST45 isolates was detected by pulsed-field gel electrophoresis. Our results showed that the emerging multidrug resistant ST45 have predominated ST59 in BORSA isolates. We also analyzed MRSA isolates obtained from nursing home surveillance in central and southern Taiwan. The results showed that ST45 was the second common clone colonizing nursing home residents following ST59. Our study revealed that a multidrug resistant CA-MRSA clone emerged in recent year and replaced the original predominant ST59 clone in BORSA isolates. Further study is mandated for better understanding the virulence, competition ability over ST59 and drug resistant character of ST45 clone in Taiwan.

誌謝 I
中文摘要 II
英文摘要 IV
第一章、緒論 1
第二章、實驗架構設計 5
2.1 FIRST PART 5
2.2 SECOND PART 5
2.3 THIRD PART 5
第三章、材料與方法 6
3.1 FIRST PART 6
Study design and population 6
Antimicrobial susceptibility testing 6
Multilocus sequence typing and spa typing 7
Pulsed-field gel electrophoresis 7
Determination of drug resistance genes 7
3.2 SECOND PART 8
Study design and population 8
Data collection and definition 8
Isolation, identification and antimicrobial susceptibility testing of S. aureus 9
Molecular typing 10
Statistical analysis 10
3.3 THIRD PART 11
Study design and population 11
第四章、實驗結果 12
4.1 FIRST PART 12
Colonization rate during rifampin containing anti-tuberculosis treatment 12
Susceptibility testing 13
Molecular typing of colonized isolates 13
Genetic mutations of rifampin and fusidic acid 14
 
4.2 SECOND PART 15
Annual incidence of BORSA bacteremia 15
Patients and clinical features 15
Molecular typing and genetic relatedness of BORSA isolates 16
Antimicrobial susceptibility testing 16
Genetic mutations in mecA promoter region andgryA/grlAgenes 17
Comparison of clinical presentations between ST59 and ST45 BORSA bacteremia 17
Risk factors associated with 30-day mortality of BORSA bacteremia 17
4.3 PART THREE 18
第五章、討論 19
第六章、表 25
表一：Demographic data of enrolled TB patients receiving rifampin and their status of colonization at enrollment. 25
表二：Susceptibilities and typing of MRSA nasal colonization isolates from 4 TB patients receiving rifampin. 26
表三：Comparison of demographic data, clinical presentations and outcome for patients with methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) and borderline-oxacillin resistant mecA-positive S. aureus (BORSA) bacteremia 27
表四：Antimicrobial susceptibilities of BORSA bacteremia isolates and comparison of the two major ST types; ST45 and ST59 30
表五：Univariate analysis of clinical characteristics comparing ST59 and ST45 BORSA bacteremia 31
表六：Univariate and multivariate analysis of risk factors associated with 30-day mortality in patients with BORSA bacteremia 32
 
第七章、圖 33
圖一：Pulsed-field gel electrophoresis of the 30 S. aureus isolatesfrom surveillance for nasal swab culture in TB patients receiving rifampin. 33
圖二：Annual prevalence of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) and borderline-oxacillin resistant mecA-positive S. aureus (BORSA) bacteremia per 1000 emergency department admission during 2001-2015. 34
圖三：Cases distribution of sequence-types of 64 BORSA isolates during 2001-2015.ST45 replaced ST59 and became the predominant clone in BORSA bacteremia isolates. 35
圖四：Pulsed-field gel electrophoresis of 25 ST45 BORSA isolates showing no major clone detected 36
圖五：Kaplan-Meier survival curve at 30 days for BORSA bacteremia patients infected by ST45 and ST 59. 37
圖六：Case distribution of MLST typing for MRSA isolates from central (a) and southern (b) Taiwan nursing home residents. 38
圖七：Growth curve of three randomly selected ST45(a) and three ST59 (b) compared with S. aureus ATCC29213. 39
參考文獻 40
附錄 45

1.Enright, M.C., et al., Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol, 2000. 38(3): p. 1008-15.
2.Planet, P.J., et al., Architecture of a Species: Phylogenomics of Staphylococcus aureus. Trends Microbiol, 2017. 25(2): p. 153-166.
3.Hung, W.C., et al., Molecular Evolutionary Pathways toward Two Successful Community-Associated but Multidrug-Resistant ST59 Methicillin-Resistant Staphylococcus aureus Lineages in Taiwan: Dynamic Modes of Mobile Genetic Element Salvages. PLoS One, 2016. 11(9): p. e0162526.
4.International Working Group on the Classification of Staphylococcal Cassette Chromosome, E., Classification of staphylococcal cassette chromosome mec (SCCmec): guidelines for reporting novel SCCmec elements. Antimicrob Agents Chemother, 2009. 53(12): p. 4961-7.
5.Chuang, Y.Y. and Y.C. Huang, Molecular epidemiology of community-associated meticillin-resistant Staphylococcus aureus in Asia. Lancet Infect Dis, 2013. 13(8): p. 698-708.
6.Sekiguchi, J., et al., Emergence of rifampicin resistance in methicillin-resistant Staphylococcus aureus in tuberculosis wards. J Infect Chemother, 2006. 12(1): p. 47-50.
7.Feil, E.J., et al., eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol, 2004. 186(5): p. 1518-30.
 
8.Huang, Y.H., et al., Clonal spread of SCCmec type IV methicillin-resistant Staphylococcus aureus between community and hospital. Clin Microbiol Infect, 2007. 13(7): p. 717-24.
9.Tsao, F.Y., H.W. Kou, and Y.C. Huang, Dissemination of methicillin-resistant Staphylococcus aureus sequence type 45 among nursing home residents and staff in Taiwan. Clin Microbiol Infect, 2015. 21(5): p. 451-8.
10.Enright, M.C., et al., The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci U S A, 2002. 99(11): p. 7687-92.
11.Tan, C.K., et al., Increased rifampicin resistance in blood isolates of meticillin-resistant Staphylococcus aureus (MRSA) amongst patients exposed to rifampicin-containing antituberculous treatment. Int J Antimicrob Agents, 2011. 37(6): p. 550-3.
12.Tan, C.K., et al., Vancomycin plus rifampicin for methicillin-resistant Staphylococcus aureus pneumonia benefits only those who have no development of rifampicin resistance during treatment. Crit Care Med, 2010. 38(8): p. 1754; author reply 1754-5.
13.Forrest, G.N. and K. Tamura, Rifampin combination therapy for nonmycobacterial infections. Clin Microbiol Rev, 2010. 23(1): p. 14-34.
14.Russell, C.D., et al., Adjunctive rifampicin may improve outcomes in Staphylococcus aureus bacteraemia: a systematic review. J Med Microbiol, 2014. 63(Pt 6): p. 841-8.
15.Septimus, E.J. and M.L. Schweizer, Decolonization in Prevention of Health Care-Associated Infections. Clin Microbiol Rev, 2016. 29(2): p. 201-22.
 
16.Ammerlaan, H.S., et al., Eradication of methicillin-resistant Staphylococcus aureus carriage: a systematic review. Clin Infect Dis, 2009. 48(7): p. 922-30.
17.Falagas, M.E., I.A. Bliziotis, and K.N. Fragoulis, Oral rifampin for eradication of Staphylococcus aureus carriage from healthy and sick populations: a systematic review of the evidence from comparative trials. Am J Infect Control, 2007. 35(2): p. 106-14.
18.Wannet, W.J., et al., Widespread dissemination in The Netherlands of the epidemic berlin methicillin-resistant Staphylococcus aureus clone with low-level resistance to oxacillin. J Clin Microbiol, 2004. 42(7): p. 3077-82.
19.Chen, C.J. and Y.C. Huang, New epidemiology of Staphylococcus aureus infection in Asia. Clin Microbiol Infect, 2014. 20(7): p. 605-23.
20.Qi, W., et al., Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Zurich, Switzerland (2003): prevalence of type IV SCCmec and a new SCCmec element associated with isolates from intravenous drug users. J Clin Microbiol, 2005. 43(10): p. 5164-70.
21.Chen, F.J., et al., mecA-positive Staphylococcus aureus with low-level oxacillin MIC in Taiwan. J Clin Microbiol, 2012. 50(5): p. 1679-83.
22.Ho, C.M., et al., Methicillin-resistant Staphylococcus aureus isolates with SCCmec type V and spa types t437 or t1081 associated to discordant susceptibility results between oxacillin and cefoxitin, Central Taiwan. Diagn Microbiol Infect Dis, 2016. 86(4): p. 405-411.
23.Chen, C.J., et al., Molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus bloodstream isolates in Taiwan, 2010. PLoS One, 2014. 9(6): p. e101184.
 
24.Shopsin, B., et al., Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol, 1999. 37(11): p. 3556-63.
25.Wichelhaus, T.A., et al., Molecular characterization of rpoB mutations conferring cross-resistance to rifamycins on methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother, 1999. 43(11): p. 2813-6.
26.Chen, H.J., et al., Fusidic acid resistance determinants in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother, 2010. 54(12): p. 4985-91.
27.Chen, S.Y., et al., Methicillin-resistant Staphylococcus aureus (MRSA) staphylococcal cassette chromosome mec genotype effects outcomes of patients with healthcare-associated MRSA bacteremia independently of vancomycin minimum inhibitory concentration. Clin Infect Dis, 2012. 55(10): p. 1329-37.
28.Rangel-Frausto, M.S., et al., The natural history of the systemic inflammatory response syndrome (SIRS). A prospective study. JAMA, 1995. 273(2): p. 117-23.
29.Milheirico, C., D.C. Oliveira, and H. de Lencastre, Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob Agents Chemother, 2007. 51(9): p. 3374-7.
30.Gillet, Y., et al., Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients. Lancet, 2002. 359(9308): p. 753-9.
31.Chen, F.J., et al., Role of the mecA gene in oxacillin resistance in a Staphylococcus aureus clinical strain with a pvl-positive ST59 genetic background. Antimicrob Agents Chemother, 2014. 58(2): p. 1047-54.
 
32.Takahata, M., et al., Mutations in thegyrA and grlA genes of quinolone-resistant clinical isolates of methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother, 1996. 38(3): p. 543-6.
33.Watanabe, Y., et al., Impact of rpoB mutations on reduced vancomycin susceptibility in Staphylococcus aureus. J Clin Microbiol, 2011. 49(7): p. 2680-4.
34.Baek, K.T., et al., Stepwise decrease in daptomycin susceptibility in clinical Staphylococcus aureus isolates associated with an initial mutation in rpoB and a compensatory inactivation of the clpX gene. Antimicrob Agents Chemother, 2015. 59(11): p. 6983-91.
35.Chen, C.J., et al., Reduced susceptibility to vancomycin in isogenic Staphylococcus aureus strains of sequence type 59: tracking evolution and identifying mutations by whole-genome sequencing. J Antimicrob Chemother, 2014. 69(2): p. 349-54.
36.Wang, J.L., et al., Comparison of both clinical features and mortality risk associated with bacteremia due to community-acquired methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus. Clin Infect Dis, 2008. 46(6): p. 799-806.