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研究生：	韓煥文 Han, Huan-Wen
論文名稱：	葡萄球菌屬細菌之分子鑑定及mecA基因檢測 Molecular Identification of Staphylococcus spp. and Detection of the mecA Gene
指導教授：	張憲彰 Chang, Hsien-Chang 張長泉 Chang, Tsung-Chain
學位類別：	博士 Doctor
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2015
畢業學年度：	104
語文別：	英文
論文頁數：	141
中文關鍵詞：	葡萄球菌、葡萄球菌屬、飛行質譜儀、寡核苷酸晶片、mecA基因
外文關鍵詞：	Staphylococci, Staphylococcus spp., MALDI-TOF, Oligonucleotide array, mecA gene
相關次數：	點閱：113 下載：0
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飛行質譜儀
葡萄球菌屬(Staphylococcus)尤其是金黃色葡萄球菌 (S. aureus) 在臨床上可引起很多感染，包括膿皰、蜂窩組織炎、泌尿道感染、肺炎、心內膜炎、心肌炎、腹膜炎、骨髓炎、菌血症、及食物中毒等。S. aureus很容易鑑定，但以生化特性鑑定其它葡萄球菌，有時並不是很可靠。基質輔助雷射脫附游離飛行時間質譜儀 (matrix-assisted laser desorption ionization-timeofflight mass spectrometry, MALDI-TOF MS) 已廣泛被應用於臨床菌種鑑定，其優點是便宜、快速、正確、及高通量等。根據 Bruker MALDI-TOF MS 軟体，當閾值 (score cutoff value) 大於2.0時，菌株可鑑定到種名，當閾值介於2.0 到 1.7 之間，只有鑑定到屬名，而軟体輸出的種名僅供參考，當閾值在 1.7 以下時，鑑定結果為不可靠。本研究評估 MALDI-TOF MS 對葡萄球菌的鑑定效果。以144株參考菌株 (36種) 及 440 株臨床分離株 (11種) 進行試驗，評估閾值 (2.0 或1.7) 及重複數(1或2重複) 對鑑定率的影響，這些菌株均先以 16S rRNA 基因，或 gap 或 tuf 基因確認種名，並以基因定序結果做為標準。在 440 臨床分離株中，閾值為 1.7 時，菌種、菌屬、及不可靠鑑定率分別為99.3%、0.2%、及0.5%。如果閾值為2.0 ，相對應鑑定率分別為 93.4%、5.7%、及 0.9%。二種閾值比較，閾值1.7對菌種鑑定率顯著性的較高(P< 0.01)，而菌屬鑑定率則顯著性的較低 (P< 0.01)。在參考菌株中，也有同樣的趨勢。二重複和一重複比較且閾值為 2.0 時，前者對菌種鑑定率顯著性的較高 (93.4% vs 83.9%, P< 0.05)；在閾值為1.7時，也有類似結果 (99.3% vs 92.7%, P< 0.05)。總之，使用Bruker MALDI-TOF MS進行葡萄球菌菌種鑑定時, 建議使用閾值1.7及二重複。

寡核苷酸晶片
葡萄球菌屬 (Staphylococcus) 是一群在自然環境中廣泛存在的革蘭氏陽性球菌。早期認為分離到的葡萄球菌中，只有金黃色葡萄球菌 (S. aureus) 具有臨床意義，但近年許多研究指出，凝固酶陰性葡萄球菌 (coagulase-negative staphylococci; CoNS) 會造成臨床個案的感染，而現在大多認為CoNS是院內感染的一個重要來源，在嬰兒、進行移殖手術、及一些免疫不健全的患者中CoNS也會引起感染。葡萄球菌屬中除了S. aureus較容易鑑定外，CoNS菌株的鑑定需操作許多生化反應，有時菌種鑑定結果可靠性不高，另外CoNS菌株常褲帶有多種抗藥基因。本研究發展一種可以同時鑑定30種葡萄球菌及抗藥基因mecA的寡核苷酸晶片，並以129株目標參考菌株和434臨床分離株測試。每株菌皆以16S rRNA，或gap或tuf基因定序以確認種名，並以定序結果之種名做為鑑定標準，而抗藥性則以傳統藥物感受性試驗(紙錠擴散法)結果做為標準。寡核苷酸晶片正確鑑定100% (129/129)參考菌株。而在臨床分離株中，S. aureus的鑑定率為98.9%，CoNS的鑑定率為98%。在臨床分離的S. aureus中，檢測mecA基因對methicillin感受性的靈敏度及專一性分別為99% 及98.9%，而臨床分離的CoNS 菌株中，mecA檢測的靈敏度及專一性分別為97.2% 和93.7%。整體而論，晶片鑑定所需要的時間大約5個小時，可以同時準確地鑑定葡萄球菌種及mecA的存在，在臨床直接檢体的應用值得進一步探討。

MALDI-TOF
Phenotypic identification of species of Staphylococcuscan be difficult. The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a cost-effective and rapid method for microbial identification. However, different score cutoff values have been used for identification of staphylococci. This study aimed toevaluate two score cutoffs (2.0 and 1.7) and replicate number onthe identification of staphylococci. A collection of 440 clinical isolates (11 species) identified by 16S rRNA or gap or tuf gene sequencing was evaluated in duplicate.At a cutoff of 1.7, the rates of species, genus, and unreliableidentifications were99.3%, 0.2%, and0.5% respectively, while the respective values were 93.4%, 5.7%, and 0.9% when a cutoff of 2.0 was used. Comparing the two cutoffs (1.7 vs 2.0), the species identification rate was significantly higher (99.3% vs 93.4%, P<0.05) and the genus identification rate was significantly lower(0.2% vs 5.7%, P<0.05) at the lower cutoff. Furthermore, 144 reference strains (37 species) of staphylococci wereanalyzed. The species identification rates were 88.9% and 77.1% at cutoffs of 1.7 and 2.0 (P< 0.05), respectively, and the respective rates for genus identification were 3.5% and 13.9% (P<0.05).Furthermore, a duplicate test resulted in higher species identification rates than a single test at a cutoff of 1.7 (99.3% vs 92.7%, P<0.05)or 2.0 (93.4% vs 83.9%, P<0.05). In conclusion, a cutoff of 1.7 and a duplicate test are recommended in routine practice for identification of staphylococci using the Bruker MALDI-TOF MS.

Oligonucleotid Array
AlthoughS. arureus is an important pathogen in clinical settings, coagulase-negative staphylococci (CoNS) can causea wide spectrum of infections inimmunosuppressed patients, neonates, and people with prosthetic implants. Phenotypic identification of CoNScan be not reliable and treatment of infections caused by CoNS is challenging as many species in this group carry genes for multiple antibiotic resistances. The aim of the study was to develop an oligonucleotide array to identify 30 species of staphylococci and to detect mecA gene in them. A total of 129 target reference strains and 434 clinical isolates of staphylococci were evaluated by the array. The species names of all reference strains and clinical isolates were reconfirmed by sequencing of genes of 16S rRNA, gaportuf.Species names determined by gene sequencing and methicillin susceptibility determined by the disc diffusion methodwere considered the gold standard. The array correctly identified100% (129/129) of allstaphylococcal reference strains, and 98.9% (186/188) of clinical isolates of S. aureus and 98% (241/246) of CoNS. Comparing with the disc diffusion method, mecAdetection by the array had a sensitivity of 99% and a specificity of 98.9% in clinical isolatesof S. aureus and the respective values inclinical isolates of CoNS were 97.2% and 93.7%. In conclusion, the current array with a turnaround time of 5 hhas a good performance for the identification of staphylococci and for the detection of mecA. Application of the array in direct clinical specimensneeds further investigation.

中文摘要..........I
ABSTRACT..........III
Chapter 1. INTRODUCTION..........1
Chapter 2. Paper review
2.1 Characterization of the staphylococci..........3
2.2 Taxonomy of staphylococci..........4
2.3 Infections caused by Staphylococcus spp...........5
2.4 Phenotype methods for identification of staphylococci	..........8
2.5 Molecular methods for identification of staphylococci	..........11
2.6 Commercial kits..........15
2.7 MALDI-TOF..........18
2.8 Drug resistance gene in staphyococci..........20
Chapter 3. MATERIALS AND METHODS
Reference strains..........23
Clinical isolates..........24
MALDI-TOF MS analysis..........24
Data analysis of MALDI-TOF MS..........25
Statistical analysis of MALDI-TOF MS..........26
DNA extraction..........26
PCR Amplification of ITS, 16S rRNA gene, gap and tuf..........27
ITS cloning..........28
Probe design..........28
Array fabrication and probe immobilization..........29
Hybridization test..........30
Methicillin drug susceptibility testing..........30
Limit of detection..........31
RESULTS
Identification of reference strains by the array..........32
Specificity of the array	34
Identification of clinical isolates by the array..........34
Comparison of the array and MALDI-TOF MS for identification of staphylococcal referencestrains and clinical isolates..........35
Detection of mecA by the Array..........36
Limit of detection of the array..........38
DISCUSSION..........39
REFERENCES..........49

FIGURES
Figure 1. History of staphylococci classification...........68
Figure 2. Virulence factors of staphylococcal infections..........69
Figure 3. Gram Stain..........70
Figure 4. Selective media..........71
Figure 5. Biochemical characterization of staphylococci..........72
Figure 6. Staphylococcal identification to species..........73
Figure 7. Layout of oligonucleotide probes on the array..........74
Figure 8. Hybridization results for 30 species of staphylococci and detection of mecA in several selected strains...........75
Figure 9. Hybridization patterns of non-target reference strains by the array...........76

TABLES
Table 1. The 16S rRNA cluster group of staphylococci and their clinical source..........77
Table 2. Coagulase test in common species of staphylococci..........80
Table 3. Target reference strains and clinical isolates analyzed by the array..........81
Table 4. Non-target strains used for specificity test of the array..........87
Table 5. Primers used for PCR..........89
Table 6. Oligonucleotide probes used to identify staphylococci..........90
Table 7. Analysis of 4 reference strains with discrepant identifications by gene sequencing..........93
Table 8. Identification of reference strains of staphylococci by the array..........94
Table 9. Identification of clinical isolates of staphylococci by the array..........96
Table 10. Analysis of 7 clinical isolates with discrepant identification between the array and culture..........97
Table 11. Results of identification of reference strains and clinical isolates 
by the array..........99
Table 12. Performance comparison between the array and MALDI-TOF (score cutoff value 2.0) for specieslevel identification of staphylococci...........100
Table 13. Performance between the array and MALDI-TOF (score cutoff value 1.7) for species identification of staphylococci..........101
Table 14. Comparison of the array and drug susceptibility testing for detection of methicillin resistance in staphylococci..........102
Table 15. Analysis of 13 strains with discrepant methicillin resistance between the array and drug susceptibility testing...........104
Table 16. Species distribution of methicillin-resistant clinical isolates of staphylococci determined by the array and drug susceptibility testing..........105
Table 17. Limits of detection of the array for identification of several staphylococcal species and for detection of mecA..........106
Appendix 1. Identification, methicillin susceptibility, and mecA and mecC PCR of each staphylococcal isolate	..........107
                                    

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