犬蛔蟲抗原蛋白對格蘭氏陽性菌與陰性菌具抑菌與殺菌效果之研究

本計畫為「犬蛔蟲抗原蛋白對格蘭氏陽性菌與陰性菌具抑菌與殺菌效果之研究」。前人的研究顯示，豬蛔蟲及豬鞭蟲等蠕蟲的蟲體所分泌之抗原性蛋白能夠對格蘭氏陽性菌及陰性菌造成抑菌或殺菌的效果。犬蛔蟲成蟲(Toxocara canis)寄生於狗的小腸內，與豬蛔蟲同屬於蛔蟲屬，生存的環境亦相近，然而對T. canis蟲體本身或所分泌的抗原成分是否有類似抑菌或殺菌的能力至今仍不明確。本計畫使用犬蛔蟲成蟲各種不同器官之抗原蛋白[腸道(TAI)、體腔液(TAF)、生殖器官(雄TMG、雌TFG)、表皮(TAC)]和幼蟲不同的抗原蛋白[體抗原(TLS)、排泌性抗原(TES)]來對A.)格蘭氏陽性菌：金黃色葡萄球菌( Staphylococcus aureus )及糞腸球菌( Enterococcus faecalis )與B.)格蘭氏陰性菌：大腸桿菌( Escherichia coli )及腸道型沙門氏桿菌( Salmonella enterica sub sp.)。利用濾紙圓片擴散法（disk diffusion technique）進行感受性試驗(susceptibility experiment)；最低濃度抑制試驗(MIC)做抑菌試驗(antibacterial experiment)；最低濃度殺菌試驗(MBC)進行殺菌試驗(bactericidal experiment)。研究發現：犬蛔蟲成蟲的不同抗原蛋白均可以對S. aureus 具備殺菌效果；對E. faecalis 僅有部分抗原(TAI及TAC)具抑菌但無殺菌效果；幼蟲的TLS蛋白及TES蛋白都對S. aureus 及E. faecalis 具有殺菌效果。然而不論是成蟲或幼蟲抗原蛋白，皆無法對格蘭氏陰性菌具備抑菌或殺菌的效果。利用高效能液相層析儀(high performance liquid chromatography, HPLC)進一步確認抑菌及殺菌的機轉，發現在犬蛔蟲3kDa分子量以下的不同抗原蛋白中，均具有和四環黴素標準品 (Tetracycline hydrochloride, 480 mw)差不多的分子量，又因為抗原蛋白出現的peak時間較標準品晚，因此推論其分子量大小應較標準品的大。綜合上述結果推論犬蛔蟲抗原蛋白的抗菌成份應具有較複雜的結構，分子量約在500mw上下，且與單一抗陽性菌的抗生素較類似。進一步推論可能和較複雜的penicillin類藥物有相似的殺菌機制，例如：雙氯西林dicloxacillin、美洛西林mezlocillin等藥物之殺菌機轉為阻礙細菌細胞壁的合成，導致細菌在低滲透壓環境下膨脹破裂後死亡。

關鍵字

犬蛔蟲；抗原蛋白；殺菌MBC ；抑菌MIC ；格蘭氏陽性菌與陰性菌； HPLC

並列摘要

This study intends to investigate on the antibacterial and bactericidal effects of Toxocara canis antigenic proteins on Gram-positive and -negative bacteria. Previous researchers have indicated that antigenic proteins released from adult Ascaris suum or Trichuris suis showed potent inhibitory or bactericidal effects on Gram-positive and -negative bacteria. T. canis is like A. suum that resides in the dog's small intestine which belong to Ascaris genus and their habitat environments are also very similar to that of A. suum. Nevertheless, it is still unclear on whether antigenic proteins released from T. canis have the same inhibitory or bactericidal ability as seen in A. suum. Present study utilizes various antigenic proteins prepared from different organs or fluids of worm's body cavity e.g., intestinal (TAI), fluid (TAF), genital organs (male, TMG; female, TFG), cuticle (TAC) as well as larval somatic (TLS) and excretory-secretory antigens (TES) to evaluate their susceptible, inhibition, and bactercidal ability to Gram-positive e.g., Staphylococcus aureus, Enterococcus faecalis and Gram-negative bacteria e.g., Escherichia coli, Salmonella enterica sub sp as assessed by tests of susceptibility, minimal inhibitory concentration, and minimal bactericidal concentration, respectively. Our findings indicated that all of 7 antigenic proteins are capable of killing S. aureus but only TAI and TAC have the inhibitory effects on E. faecalis. Whereas, TLS as well as TES poses bactericidal ability to both bacterial strains of S. aureus and E. faecalis. However, all of 7 antigenic proteins are unable to kill Gram-negative bacterial strains of E. coli and S. enterica sub sp. The mechanisms of inhibition and bactericidal ability for those effective antigenic proteins with low molecular weight (mw) of less than 3 kDa were found to be very similar to that of Tetracycline hydrochloride (TH) with 480 mw as assessed by high performance liquid chromatography (HPLC), nevertheless its molecular weight may be higher than 480 mw because longer time for peak presence in Toxocara proteins than that of TH was found by HPLC.

並列關鍵字

Toxocara canis ； antigenic proteins ； MIC ； MBC ； Gram-positive and –negative

參考文獻

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犬蛔蟲抗原蛋白對格蘭氏陽性菌與陰性菌具抑菌與殺菌效果之研究

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