Isolation of an Antibacterial Substance against Tetracycline Resistant Gene, tetB, from Poncirus trifoliata

Abstract

장염비브리오균은 식중독을 유발하는 병원성 세균으로 최근 조사에 의하면 어류 양식장에서 분리된 장염비브리오균의 97% 이상이 특정 항생제에 내성을 가지고 있을 정도로 내성이 심각한 수준으로 알려져 있다. 따라서 본 연구에서는 우선적으로 양식어장에서 많이 사용되고 있는 tetracycline계 항생제에 내성을 가지고 있는 장염비브리오균을 분리하고 이들 내성균에서 tetracycline에 내성을 부여하는 관련 유전자들의 클로닝을 시도하여 관련 유전자의 클로닝에 성공하였다. Tetracycline 내성균주에 대한 대처치료제를 개발하기 위한 노력으로 몇몇 약재를 가지고 항균활성을 평가하였는데, 그 중 탱자의 MeOH 추출물이 가장 항균활성이 우수하였다. 탱자의 MeOH 추출물과 그 획분의 항균활성 실험 결과 EtOAc 획분> n-BuOH 획분 순으로 항균활성이 나타으며 n-hexane 획분, CH₂Cl₂ 획분, H₂O 획분에서는 항균활성이 나타나지 않았다. EtOAc 획분을 silica gel column chromatography와 thin layer chromatography (TLC)에 의해 6개의 분획물을 얻었다. EtOAc 획분 중 fraction PE1-F02의 항균활성이 가장 우수하였다 그들을 다시 실리카겔 컬럼 크로마토그래피를 수행하여 12개의 fraction을 얻었다. 그들 중 fraction PE2-F012이 tetracycline 내성 균주에 대해 항균활성을 가지고 있었으며, 다시 13개의 fraction으로 분획하였다. 다시 fractions PE3-F013을 12개의 fraction으로 분획하였다. 그들 중 fraction PE4-F02과 PE4-F04을 Sephadex LH-20 column chromatography을 실시하여 화합물 1과 2를 분리하였다. 화합물 1과 2는 ¹H-, ^(13)C-NMR 의 분광학적 분석방법에 의해 각각 poncirin과 naringin 임을 확인하였다.

A tetracycline resistant V. parahaemoliticus, capable of growing on TCBS medium containing tetracycline, was isolated from cultivated fishes. A gene responsible for the tetracycline resistance was cloned from the chromosomal DNA of V. parahaemolyticus 0854 using E. coli KAM3, which lacks major multidrug efflux pumps (△acrB) as host cells. It was determined to be an open reading frame (ORF) for tetracycline resistance protein (TetB) by the nucleotide sequence. In order to characterize the antibiotic resistance of TetB originated from V. parahaemolyticus 0854, the tetB gene was sub-cloned into the plasmid pSTV28. The resulting plasmid was designated as pSTVTetB and transfomated into KAM3. KAM3 cells harboring the recombinant plasmid pSTVTetB is able to grow on plates containing tetracycline and oxytetracycline but doxycycline, indicating that the tetB gene originated from V. parahaemolyticus 0854 confer the tetracycline- and oxytetracycline-resistance to the host cells. Next, it was designed an in vivo screening assay using KAM3/pSTVTetB to discover new drug(s) or specific anti-substance(s) against tetracycline resistant gene or protein. Several medicinal plants were evaluated for its inhibitory activity against KAM3/pSTVTetB in the presence of tetracycline and chloramphenicol. The methanolic extract of Poncirus trifoliata (L.) Raf. exhibited significant inhibitory activity against the cells. To perform more detail investigation on the inhibitory activity, the methanolic extract was further participated with several organic solvents to yield n-hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol fractions. The inhibitory activity of each fraction against KAM3/pSTV TetB was assayed by using a paper disc method in the presence of tetracycline. Among them, EtOAc fraction showed the highest inhibitory activity. The EtOAc fraction was subjected to column chromatography over a silica gel with n-hexane : EtOAc (from 1:1 to 1:5 v/v) gradually, yielding 6 subfractions. PE1-F02 fraction showed the strongest growth inhibitory activity against KAM3/pSTVTetB cells and were further separated into thirteen fraction. Among them, fractions PE3-F013 were re-separated into twelve fraction. Among twelve fraction, PE4-F02 (compound 1) and PE4-F04 (compound 2) fractions were obtained by Sephadex LH-20 column chromatography. In the fraction, two major compounds were observed by thin layer chromatography (TLC). Compound 1 and 2 were identified to be poncirin and naringin by ¹H-NMR and ^(13)C-NMR, respectively.