"克雷伯氏肺炎桿菌酯多醣 O2ac, O3, O12 基因體區域完整定序及酯多醣基因分型方法的發展"

Abstract

摘要 背景 克雷伯氏肺炎桿菌 (Klebsiella pneumoniae) 是造成院內感染 (肺炎、嗜中性球低下發燒) 以及社區感染 (化膿性肝膿瘍) 的一種重要致病菌。1986年，台灣學者首先注意到造成化膿性肝膿瘍的克雷伯氏肺炎桿菌能夠經由血流擴散而併發遠處感染，尤其是眼內炎或/和中樞神經感染症。這種併發症常導致嚴重殘障甚至死亡。由於細菌侵入眼部/腦部的機轉係經由血流擴散，細菌在血流中對人體血清殺菌力的抵抗性是克雷伯氏肺炎桿菌最重要的致病力特徵之一。基因剔除實驗發現酯多醣 (LPS) 及莢膜多醣 (CPS) 是細菌產生血清抗性的必要因子。目前已知莢膜多醣的K基因型是會影響細菌血清抗性的因子－平均來說，K1基因型的菌株的血清抗性是最強的，其次是K2以及K5/K20/K54基因型的菌株。然而，由於O血清分型的技術操作困難，酯多醣O型別對細菌血清抗性的影響仍不清楚。 研究目的 1. 完成克雷伯氏肺炎桿菌酯多醣O2ac、O3、O12基因體區域完整定序 2. 發展可靠且有效的O基因分型方法 材料及方法 我們自世界衛生組織指定的大腸桿菌及克雷伯氏菌血清分型參考實驗室－丹麥國家血清中心(Staten Serum Institut, Copenhagen, Denmark) 購入9株克雷伯氏菌O血清型的標準菌株，以及77株克雷伯氏菌K血清型 (已用競爭性酵素免疫分析法 (iELISA) 區分出O型別) 標準菌株 (其中51株為克雷伯氏肺炎桿菌)。首先，我們進行O2ac、O3、O12標準菌株的酯多醣基因體區域完整定序。在比較並分析wb基因體區域後，我們發展出O型別特異的聚合酶鏈鎖反應核酸引子 (PCR primers)。我們先於9株O標準菌株中測試O型別特異聚合酶鏈鎖反應引子的特異度，再於已知O血清型的77株K標準菌株中測試O型別特異引子的敏感度及特異度。 結果 我們已完成克雷伯氏肺炎桿菌O2ac (O2ac標準菌株，菌株編號：5053)、O3 (O3標準菌株，菌株編號：636/52) 及O12 (O12標準菌株，菌株編號：702) 酯多醣基因體區域完整定序，並將序列上傳至基因庫 (Genbank) (編號分別為：AB795943, AB795941以及AB795942)。透過基因體區域的比較分析，我們發現O1和O2 (ac) 其實為同一種基因型。我們先於9株O標準菌株確認O1/O2/O2ac、O3、及O12型別特異引子的特異度。接著以iELISA分型方法為黃金標準，測試77株已知O型別 (以iELISA分型) 的WHO K標準菌株，結果顯示：O1/O2/O2ac型別特異引子的敏感度為71.43%，特異度為82.9%，O3型別特異引子的敏感度為84.6%，特異度為100%，O12型別特異引子的敏感度及特異度皆為100%。最後，我們使用核酸序列的定序結果做為新的黃金標準，並於51株克雷伯氏肺炎桿菌標準菌株做測試。發現我們所發展出的O1/O2/O2ac、O3、O12型別特異引子的敏感度及特異度皆為100%，皆高於傳統的競爭性酵素免疫分析血清分型方法。 討論 我們已成功的發展出一種原創性的克雷伯氏肺炎桿菌O基因分型方法，並以世界衛生組織菌株測試證實其高敏感度與高特異度。未來更可利用這個有力的O基因分型方法分析酯多醣在克雷伯氏肺炎桿菌致病力的重要性。

中文關鍵字：克雷伯氏肺炎桿菌；酯多醣；基因分型；基因體區域；完整定序；O抗原

Abstract Background Klebsiella pneumoniae is an important pathogen that causes both nosocomial infections (pneumonia, febrile neutropenia) and community-acquired infections (pyogenic liver abscess). Since1986, researchers in Taiwan have noticed that K. pneumoniae strains that cause pyogenic liver abscesses can further cause distant septic complications, especially endophthalmitis and/or central nervous system infection, through hematogenous spread. The outcome for the patient is usually catastrophic. Because trafficking to the eye/brain is via a hematogenous route, resistance to the cytolytic activity of human serum while in the bloodstream is an important trait in bacterial pathogenesis. Lipopolysaccharide (LPS) and capsular polysaccharide (CPS) have been shown to be essential for bacterial serum resistance in gene knockout studies. The K-antigen genotypes of CPS have been shown to affect the bacterial serum resistance. Typically, genotype K1 strains are the most serum-resistant, followed by the genotype K2 and K5/K20/K54 strains. However, the role of the LPS O-antigen serotype in bacterial serum resistance has not been investigated due to technical difficulties in serotyping the O-antigen. Aims 1. Sequence the complete K. pneumoniae LPS O2ac, O3, and O12 genomic regions (wb gene clusters) of multiple reference strains. 2. Develop a reliable and valid O-antigen genotyping method. Materials and methods Nine Klebsiella O-antigen serotype reference strains and 77 Klebsiella K-antigen serotype reference strains with previously established O-antigen serotype data verified via inhibition enzyme-linked immunosorbent assay (iELISA), 51 of which were K. pneumoniae, were purchased from the Staten Serum Institut (Copenhagen, Denmark), the World Health Organization Escherichia coli and K. pneumoniae serotyping reference laboratory. We first completely sequenced the wb genomic regions in the O2ac, O3, and O12 serotype reference strains. Using comparative genomic analyses of the wb regions, we developed O-antigen type-specific PCR primers. We subsequently tested the specificity of the O-antigen type-specific PCR primers on nine O-antigen reference strains. We then determined the sensitivity and specificity of the O-antigen type-specific PCR primers against the 77 K-antigen reference strains with known O-antigen serotypes. Results We completely sequenced the K. pneumoniae lipopolysaccharide O2ac (O2ac reference strain, strain no. 5053), O3 (O3 reference strain, strain no. 636/52), and O12 (O12 reference strain, strain no. No.702) genomic regions and submitted these sequences to GenBank (accession numbers AB795943, AB795941, and AB795942, respectively). Using comparative genomic analyses, we discovered that the O1 and O2(ac) serotypes have the same genotype. We confirmed the specificity of the O1/O2/O2ac, O3, and O12 type-specific primers against the nine O-antigen reference strains. We then tested the 77 WHO K-antigen reference strains with known iELISA O-antigen serotypes using these same type-specific primers, which showed a sensitivity of 71.4% and a specificity of 82.9% for the O1/O2/O2ac type-specific primers, a sensitivity of 84.6% and a specificity of 100% for the O3 type-specific primers, and a sensitivity of 100% and a specificity of 100% for the O12 type-specific primers, using iELISA as the gold standard. When we used our nucleotide sequencing results as the gold standard, the sensitivity and specificity of the type-specific primers were better overall than the results obtained using the traditional iELISA serotyping method, with a sensitivity and specificity of 100% against the 51 K. pneumoniae reference strains. Conclusions We have successfully developed an original K. pneumoniae O-antigen genotyping method and have validated its sensitivity and specificity using the established WHO K. pneumoniae reference strains. This O-antigen genotyping method can serve as a powerful tool in studying the biological significance of LPS in K. pneumoniae pathogenicity in the future. Key words: Klebsiella pneumoniae, Lipopolysaccharide, Genotyping, Genomic region, Complete sequencing, O-antigen