黄精与土霉素协同抑制副溶血弧菌研究

Synergistic Antibacterial Effects of Polygonatum sibiricum Red.with Oxytetracycline on Vibrio parahaemolyticus

为了抑制对虾养殖急性肝胰脏坏死病(AHPND)的主要病原菌副溶血弧菌(Vibrio parahaemolyticus),本论文用药食同源的天然草本黄精和8种水产用抗生素联合抑制副溶血弧菌,通过棋盘法测定黄精和抗生素的最低抑菌浓度(minimum inhibitory concentration,MIC)和联合抑菌效果,研究了黄精等对细菌细胞膜电导率、膜电位和存活率的作用,采用RT-PCR研究了黄精等对细菌外排泵相关基因表达的影响。结果表明,黄精和土霉素具有较好的协同抑菌效果,最佳配比为1/8 MIC黄精+1/4 MIC土霉素,此时分级抑菌浓度(fractional inhibitory concentration,FIC)为0.38,但在羰基氰化物间氯苯腙(CCCP)的存在下,这种协同作用转为相加甚至无关作用。电导率、膜电位和碘化丙啶染色证明黄精能破坏副溶血弧菌细胞外膜,提高其渗透性,导致细胞内膜去极化,膜两侧质子梯度消失。黄精显著抑制副溶血弧菌5个外膜蛋白、5个反向协同运输子和11个RND外排转运子的表达,其中vpa0096(OmpW)下调497.51倍,vp0425(TolC)、vp2072(Na^(+)/H^(+)反向协同运输泵)、vp2665(Na^(+)/Ca^(2+)交换蛋白)、vp1092、vp0941、vp1178、vpa0344和vpa0809分别下调27.03倍、73.53、55.56、100.00、35.71、37.74、49.02和56.18倍。黄精降低TCA循环关键酶苹果酸脱氢酶(MDH)和琥珀酸脱氢酶(SDH)酶活,从而抑制副溶血弧菌体内TCA循环。土霉素是vp0425(TolC)的底物之一,黄精属于天然外排泵抑制剂,能将土霉素阻留在细胞内,提高土霉素的抑菌效果。黄精和土霉素协同作用于水产养殖的弧菌病,大幅度缩减土霉素使用浓度、残留风险和耐药性弧菌的出现,从而促进水产养殖的健康发展。

In this paper,Polygonatum sibiricum Red.and 8 kinds of aquatic antibiotics were used to inhibit Vibrio parahaemolyticus,the main pathogen of acute hepatopancreas necrosis disease(AHPND)in shrimp culture.The minimum inhibitory concentration(MIC)of P.sibiricum Red.and antibiotics were determined by chessboard method.The effects of Polygonatum on the membrane conductivity,membrane potential and survival rate of bacteria were studied.The effects of Polygonatum sibiricum Red.on the expression of efflux pump related genes were studied by RT-PCR.The results showed that the combination of P.sibiricum Red.and oxytetracycline had the best synergistic antibacterial effect with the optimum combination of 1/8 MIC P.sibiricum Red.+1/4 MIC oxytetracycline and the FIC of 0.38.The synergy turned into additive or even irrelevant effects in the presence of CCCP.Conductivity,membrane potential and propidium iodide staining proved that P.sibiricum Red.could destroy the cell membrane of V.parahaemolyticus,improving its permeability,leading to the depolarization of the cell membrane and the disappearance of proton gradient on both sides of the membrane.P.sibiricum Red.significantly inhibited the expression of five outer membrane proteins,five reverse synergistic transporters and 11 RND efflux transporters of V.parahaemolyticus,of which vpa0096(OmpW)was down-regulated by 497.51 times,while vp0425(TolC),vp2072(Na^(+)/H^(+)),vp2665(Na^(+)/Ca^(2+)exchanger),vp1092,vp0911,vp1178,vpa0344,and vpa0809 were down-regulated by 27.03,73.53,55.56,100.00,35.71,37.74,49.02,and 56.18 times,respectively.P.sibiricum Red.reduced the activities of malate dehydrogenase(MDH)and succinate dehydrogenase(SDH)of TCA cycle,thereby inhibiting the TCA cycle of V.parahaemolyticus.Oxytetracycline is one of the substrates of vp0425(TolC).Polygonatum sibiricum Red.is a natural efflux pump inhibitor,which can keep oxytetracycline in cells and improve the antibacterial effect of oxytetracycline.The synergistic effect of Polygonatum sibiricum Red.and oxytetracycline on vibriosis in aquaculture can greatly reduce the concentration of oxytetracycline,residual risk and the emergence of drug-resistant Vibrio,thus promoting the healthy development of aquaculture.