"克雷伯氏肺炎桿菌酯多醣 O1, O5, O8 基因體區域完整定序及酯多醣基因分型方法的發展"

Abstract

背景： 克雷伯氏肺炎桿菌是革蘭氏陰性腸內桿菌，為台灣化膿性肝膿瘍的主要致病菌。有一部份化膿性肝膿瘍患者的克雷伯氏肺炎桿菌會進一步經由血流擴散侵入眼睛或中樞神經系統，引發嚴重的菌性眼內炎或中樞神經感染，而合併災難性的後遺症。過去研究指出莢膜多醣K1基因型為化膿性肝膿瘍引發眼內炎或中樞神經感染的獨立危險因子。由於酯多醣O血清分型在方法學上存在一定程度的困難，酯多醣型別在化膿性肝膿瘍引發的眼內炎或中樞神經感染所扮演的角色目前仍未曾被探討過。 目的： 1. O1、O5與O8標準菌株酯多醣完整基因體區域完整定序。 2. 開發準確且方便的O基因分型方法。 材料與方法： 九株克雷伯氏菌O標準菌株及77株克雷伯氏菌K標準菌株 (其中51株為克雷伯氏肺炎桿菌) ，由丹麥國家血清中心 (Statens Serum Institut, Copenhagen, Denmark) 參考實驗室購入。首先對 O1、O5、O8 標準菌株進行酯多醣基因體區域完整定序。經由基因體序列比較分析發展O1、O5、O8型別特異聚合酶鏈反應引子 (polymerase chain reaction primers)，然後對77株業經競爭性酵素免疫分析 (competitive enzyme-linked immunosorbent assay, iELISA) 血清分型法測定酯多醣O血清型的K標準菌株進行型別特異引子的敏感度與特異度測試。

結果: 本研究完成了酯多醣O1 (O1標準菌株，菌株編號: Friedlander 204)、O5 (O5標準菌株，菌株編號:5710/52)、O8 (O8標準菌株，菌株編號:889) 基因體區域的完整定序，並已將序列資料上傳至全球基因資料庫Genbank (accession number: AB819964, AB819962, AB819963)。經過基因體序列比較分析，我們發現O1和O2是相同的基因型。型別特異引子在9株O標準菌株的測試中確認具有特異度。以核苷酸序列定序結果作為黃金標準，在51株克雷伯氏肺炎桿菌標準菌株中，O1、O5、O8型別特異的聚合酶鏈鎖反應引子的敏感度分別為100%, 100% 和100% , 特異度分別為100%, 100% 和100%。而競爭性酵素免疫分析血清分型方法的敏感度分別僅有83.9%, 100%, 50%，特異度則分別僅有95.0%, 100%, 100%。 結論: 我們成功地開發出準確而方便的聚合酶鏈反應克雷伯氏肺炎桿菌O基因分型方法，其敏感度與特異度優於傳統的競爭性酵素免疫分析血清分型方法。此基因分型方法可應用於探討酯多醣在克雷伯氏肺炎桿菌化膿性肝膿瘍併發菌性眼/中樞神經系統致病機轉中的角色。

Background Klebsiella pneumoniae, a gram-negative enteric bacillus, is a major cause of pyogenic liver abscess (PLA) in Taiwan. From PLA, K. pneumoniae may further invade the distant eye or central nervous system (CNS) to cause septic ocular/CNS complications, such as endophthalmitis and meningitis, with catastrophic outcomes. Previous studies indicate that capsular polysaccharide genotype K1 is an independent risk factor for septic ocular/CNS complication from PLA. The role of the lipopolysaccharide (LPS) type, nevertheless, has not yet been studied because of the methodological difficulties in LPS O serotyping. Aims 1. Complete sequencing of lipopolysaccharide O1, O5, and O8 genomic region of K. pneumoniae reference strains 2. Development of an accurate and convenient O genotyping method that can be performed by polymerase chain reaction (PCR) Materials and Methods We obtained nine Klebsiella serotype O reference strains and 77 Klebsiella serotype K reference strains (51 of them are K. pneumoniae) from the Statens Serum Institut (Copenhagen, Denmark). We sequenced the lipopolysaccharide genomic region of WHO O reference strains O1, O5, and O8. We developed O1, O5, and O8 type-specific PCR primers through comparative genomic analysis, and confirmed their specificity among 9 Klebsiella O reference strains. Finally, we tested the sensitivity and specificity of the primers among K. pneumoniae K reference strains with O serotypes that had been previously determined by competitive enzyme-linked immunosorbent assay (iELISA). Results We completed the sequencing of LPS O1 (O1 reference strains, strain no: Friedlander 204), O5 (O5 reference strains, strain no: 5710/52), and O8 (O8 reference strains, strain no: 889) genomic regions, and submitted the sequences to GenBank (accession no: AB819964, AB819962, and AB819963, respectively). Through comparative genomic analyses, we determined that O1 and O2 are identical genotypes. Testing against 9 O serotype references, we confirmed the specificity of the primers. When using nucleotide sequencing as the gold standard for LPS O type determination among 51 WHO K. pneumoniae K reference strains, the sensitivity of O1/O2-, O5-, and O8-specific PCR primers is 100%, 100%, and 100%, respectively; and the specificity is 100%, 100%, and 100%, respectively. In comparison, the sensitivity of iELISA is only 83.9%, 100%, and 50% for O1/O2, O5 and O8, respectively; and the specificity is 95.0%, 100%, and 100%, respectively. Conclusions We have developed an accurate and convenient PCR K. pneumoniae O genotyping method, with sensitivity and specificity that is superior to that of the traditional iELISA serotyping method. This method can be applied in the future to investigate the role of LPS in the risk of septic ocular/CNS complications from K. pneumoniae PLA.