- 學位論文
來自膿胸病人檢體中對甲氧苯青黴素具抗藥性的金黃色葡萄球菌之基因型與表現型分析
Molecular Typing and Phenotypes Characterization of Methicillin-Resistant Staphylococcus aureus From Patients With Empyema Thoracis
摘要
研究背景：金黃色葡萄球菌(Staphylococcus aureus，S.aureus)於臨床醫療中可引起許多嚴重的感染，例如菌血症、皮膚軟組織感染、肺炎、骨關節炎，和敗血症等，具有較高的死亡率和併發症發生率。對甲氧苯青黴素具抗藥性的金黃色葡萄球菌(Methicillin-Resistant S. aureus，MRSA)於1960年代被發現，在台灣地區已經造成60%至80%的金黃色葡萄球菌菌株屬於MRSA。文獻曾報導於社區有關的MRSA (CA-MRSA) 和與醫院有關的MRSA菌株 (HA-MRSA) 在基因分型與表現型有明顯的差別，而且在特定地區流行少數特定的MRSA基因群 (clones)。本次研究的目的在於分析引起膿胸的MRSA菌株之基因型和表現型，並比較造成膿胸的MRSA菌株的基因型和表現型之關係。研究方法：依據臨床標準收集23株自胸水檢體分離培養出對甲氧苯青黴素具抗藥性的金黃色葡萄球菌(MRSA)菌株，執行下列表現型與基因分型的鑑定。表現型為執行菌株鑑定、藥物敏感性試驗、瓊脂稀釋法測定對萬古黴素的最低抑菌濃度(MIC)、巨酯類抗生素誘發抗藥性試驗(MLSBi，即D-test)、毒殺白血球毒素基因 (pvl) 測定、葡萄球菌超級抗原毒素因子基因測定；基因分型則執行葡萄球菌攜抗藥性基因染色體卡莢(SCCmec) 型別鑑定、多位址序列分析 (MLST)、葡萄球菌表面蛋白基因多形性鑑定(spa)、附屬調節基因型別鑑定(agr)，和直接重複核苷酸序列(dru)。研究結果：自23位膿胸病人分離出MRSA，經實驗室菌株鑑定確認為19株非重複之MRSA。操作藥物敏感性試驗結果得約74％的MRSA菌株為多重抗藥性菌株；利用瓊脂稀釋法測定對萬古黴素的最低抑菌濃度(MIC)範圍為2-3mg/L；19株MRSA的巨酯類抗生素誘發抗藥性試驗皆為陰性；pvl陽性率只有16％；19株MRSA菌株可分類為9種超級抗原毒素因子組合。屬於醫院有關感染的MRSA(HA-MRSA)佔68％(基因分型為SCCmecII和SCCmecIII)；屬於社區有關感染的MRSA(CA-MRSA)佔32％(基因分型為SCCmecIV和SCCmecVT)。19株自膿胸病人胸水檢體分離培養的非重複MRSA菌株均確認具有mecA基因，共有五種主要的基因群： SCCmecII-ST5-spat002-dru4、SCCmecIII-ST239-spat037-dru14or12or15、 SCCmecIV-ST59-spat437-dru9、SCCmecIV-ST573-spat3523-dru9or8or10、SCCmecVT-ST59-spat437-dru11。pvl基因與SCCmecIV、ST59和spat437相關；多重抗藥性(大於或等於4種以上非β-lactam類抗生素)與SCCmecII、ST5、spa t002、dru4相關；菌株對於萬古黴素具有較高的MIC則與SCCmecIII、 ST239、spa t037、agr group I有關；無任何基因分型與MLSBi表現型有關；共確認9種超級抗原毒素因子基因組合，其中主要的基因分型與毒素因子基因為sea-selk-selq SCCmecIII (5株，26％)。結論：自膿胸病人分離CA-MRSA與HA-MRSA的基因分型與表現型有差異。造成膿胸的MRSA菌株具有有限的基因群，且其表現型各有不同的集中性。需要收集連續時段的菌株執行相關研究以釐清胸水中MRSA菌株的流行病學與可能的演化途徑。
並列摘要
Background: Staphylococcus aureus (S.aureus) can cause many serious infections, such as bacteremia, skin and soft tissue infections, pneumonia, osteoarthritis, and sepsis ,with high mortality rate and complication rate. Methicillin-Resistant S. aureus( MRSA) was discovered in the 1960s, has 60-80% of S. aureus belonging to MRSA in Taiwan. Literature has reported on Community- associated (CA-MRSA) and Healthcare- associated MRSA (HA-MRSA) is significantly different between genotyping and phenotype, and MRSA has a few specific clones in particular endemic. The purpose of this study is to investigate the cause of empyema by MRSA strains of genotype and phenotype of specificity and differences.Methods: Based on clinical criteria collected 23 specimens from pleural effusion isolated with methicillin-resistant Staphylococcus aureus (MRSA) strains of the following organisms phenotype and molecular typing of identification. Phenotype for the strain identification, drug susceptibility testing, minimum inhibitory concentration(MIC) of vancomycin by agar dilution method, induced macrolide-lincosamide-streptogramin B resistance testing (MLSBi, D-test), Panton-Valentine Leukocidin toxin gene (pvl),determination of staphylococcal enterotoxin (SEs) virulence factor gene; molecular biology typing is performed Staphylococcal cassette chromosome mec (SCCmec), multilocus sequence typing (MLST), Staphylococcal protein A (spa), accessory gene regulator (agr), and direct repeat units (dru). Results: From 23 patients with empyema isolated MRSA, confirmed by laboratory analysis was identified as 19 non-duplicate of MRSA. Drug susceptibility test results of approximately 74% of the MRSA strains are multidrug-resistant strains;the range of minimum inhibitory concentration (MIC) to vancomycin using the agar dilution method were 2-3mg / L;19 MRSA strains of the macrolide-lincosamide-streptogramin B resistance testing (MLSBi) were negative; pvl positive rate of only 16%; 19 MRSA strains can be classified into nine kinds of enterotoxins (SEs) virulence factor complex. HA-MRSA accounted for 68% (molecular biology typing to SCCmecII and SCCmecIII); CA-MRSA accounted for 32% (molecular biology typing to SCCmecIV and SCCmecVT). 19 specimens from pleural empyema patient isolation of non-duplicate MRSA strains were having mecA gene, and total of five main genetic groups： SCCmecII-ST5-spat002-dru4, SCCmecIII-ST239-spat037-dru14or12or15, SCCmecIV-ST59-spat437-dru9, SCCmecIV-ST573-spat3523-dru9or8or10, SCCmecVT-ST59-spat437-dru11；pvl gene related with SCCmecIV, ST59 andspat437; multidrug resistance (more than or equal to 4 kinds antibiotics) related with SCCmecII, ST5, spat002, dru4; MRSA strain with high vancomycin MIC is related with SCCmecIII, ST239, spat037 and agr group I；there is no any related between MLSBi and phenotyping or genotyping；the 19 isolates identified into nine kinds of staphylococcal enterotoxins virulence factor gene complex, the major molecular typing of enterotoxins virulence factor genome is sea-selk-selq-SCCmecIII-agrI (5 strains of 26%). Conclusion: Diverse molecular types with relatively high rate of multidrug resistance and elevated MIC to vancomycin were found in 19 MRSA isolates from patient with empyema. More information including demographic and laboratory data of patients with MRSA empyema are needed to correlate these data with molecular and phenotyping results and to identify molecular and phenotypic indicators for outcomes of empyema patients.
參考文獻
延伸閱讀
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