- 學位論文
比較不同第一型線毛相的沙門氏菌血清型在生物膜形成、吸附、入侵以及誘發炎症反應的能力
Comparison of biofilm forming, adherence, invasion, and inflammation-inducing ability from Salmonella serovars with different type 1 fimbrial phenotypes
摘要
線毛是位於細菌外膜上毛髮樣的蛋白結構物，對於細菌吸附於宿主腸上皮細胞上扮演重要的角色。在公共衛生所關注的食源性病原菌沙門氏菌，於菌體表面普遍具有第一型線毛結構，此線毛對於甘露醣具有專一性的結合能力。根據過去文獻中曾提及，有高達80％的沙門氏菌分離株具有第一型線毛的構造，但對於沙門氏菌與歸屬於何種血清型，與細菌生物膜形成能力、吸附細胞能力強弱以及促發炎症反應之間連結的相關研究仍然有限。在本研究中透過酵母菌凝集試驗，篩選自不同來源共187個沙門氏菌臨床分離株，這些菌株涵蓋68種不同血清型，檢測細菌在固態培養基或靜態培養的液態培養基中產生第一型線毛的能力，並根據培養條件的不同，進一步區分為四種線毛表現相 (agar / broth：Fim+/Fim+, Fim-/Fim+、Fim+、Fim-或Fim- /Fim-)。我們的研究結果顯示，僅有S. Choleraesuis、S. Pullorum、S. Paratyphi A、S. Paratyphi B、S. Cerro、S. Dressau和S. Arizonae等在上述兩種培養條件下呈現線毛陰性反應 (Fim-)。而其他血清型包括S. Typhimurium、S. Enteritidis、S. Derby及S. Albany等，固態培養基或靜態培養的液兩種條件下，則有產生有第一型線毛的產生 (Fim+)。在結晶紫染色法中發現隸屬於Fim +組的沙門氏菌，在上述任一條件下皆具有的較強的生物膜形成能力。許多研究中表明宿主適應性血清型與廣泛宿主範圍血清型的沙門氏菌，對於腸上皮細胞結合能力的差異，可能與點突變造成fimH等位基因變異有關。我們研究中針對四種第一型線毛表現相代表菌株之fimH序列進行定序並比對，發現特定胺基酸殘基的取代與線毛的表現相並無顯著的關聯性。而在Caco-2腸上皮細胞株中使用S. Newport進行吸附試驗，證實第一型線毛與吸附能力具有正相關性，但在入侵試驗中則有相反的結果。推測第一型線毛的表現促使細菌在宿主中被排出，利於細菌在多樣性的環境中進行傳播。
並列摘要
Fimbriae are hair-like structures present on the outer membrane of bacteria and are implicated in adherence to the host epithelial cells. Type 1 fimbriae with the binding specificity to mannose residue is the most commonly found fimbrial type in Salmonella, a food borne pathogen with public health concern. While previous reports indicated that 80% of Salmonella isolates possessed type 1 fimbriae, information regarding the correlation between the specific serovars and the capacity to produce such fimbriae, biofilm, adhere to epithelial cells and the ability to trigger inflammatory response remains limited. In the present study, 187 Salmonella isolates from different sources comprising 68 serovars were screened by yeast agglutination test to detect the ability to produce type 1 fimbriae on agar or in static broth and classified into four type 1 fimbrial phenotypes (agar / broth: Fim+ / Fim+, Fim- / Fim+, Fim+ / Fim-, or Fim- / Fim-). Our findings revealed that S. Choleraesuis, S. Pullorum, S. Paratyphi A and B, S. Cerro, S. Dressau, and S. Arizonae were the only serovars that were fimbriae-negative (Fim-) in both culture conditions, while other serovars including S. Typhimurium, S. Enteritidis, S. Derby, and S. Albany, just to name a few, produced type 1 fimbriae (Fim+) when incubated either in static broth, solid agar or both culture conditions. The Salmonella in the Fim+ group in either condition demonstrated a stronger biofilm forming capacity determined by crystal violet staining method. Several lines of evidence suggested differential binding capacity of host-adapted and broad-host-range Salmonella serovars to intestinal cell may reside in allelic variants of fimH caused by point mutation. Herein fimH alleles from representative isolates of four different type 1 fimbrial phenotypes were sequenced and compared, nevertheless, connection between specific residual replacement and fimbrial phenotypes was not significant in our study. The adherence assay conferred by the S. Newport with different type 1 fimbrial phenotypes were investigated using Caco-2 epithelial cell line cells. The presence of type 1 fimbriae in S. Newport has been shown to correlate with adhesive capacity, but the invasion assay turned out just the opposite. We speculate that the expression of type 1 fimbriae will facilitate the shedding of bacteria to promote Salmonella transmission in the diverse milieu.