滤纸片法筛选抑制嗜水气单胞菌群体感应的乳酸菌及抑制作用分析

Screening of Lactic Acid Bacteria with Inhibitory Activity Against Quorum Sensing of Aeromonas hydrophila Using Filter Paper Method and Their Inhibitory Effects

采用滤纸片法从传统发酵蔬菜中筛选抑制嗜水气单胞菌群体感应的乳酸菌菌株,应用96孔板法测定其对生物膜的抑制率,光学显微镜和扫描电镜观察其对嗜水气单胞菌生物膜形成的影响,同时以蛋白酶、嗜铁素、群集及泳动为指标研究其对嗜水气单胞菌毒力作用的影响。结果表明:来源于辽宁锦州酸菜的乳酸菌菌株SCT-2对嗜水气单胞菌群体感应有明显抑制作用,当其代谢产物粗提物质量浓度为8 mg/mL时,对嗜水气单胞菌生物膜抑制率为45.16%,光学显微镜下显示菌株SCT-2粗提物可有效抑制嗜水气单胞菌生物膜的形成,扫描电镜结果进一步表明菌株SCT-2粗提物不仅降低了嗜水气单胞菌生物膜的生成量,而且使其生物膜断裂。8 mg/mL的SCT-2粗提物可使嗜水气单胞菌蛋白酶和嗜铁素的分泌量分别减少27.18%和22.11%,对信号分子的降解率为32.27%,且对嗜水气单胞菌的群集和泳动现象抑制明显。经生理生化和16S rRNA鉴定菌株SCT-2为植物乳杆菌(Lactobacillus plantarum)。从传统东北酸菜中获得对嗜水气单胞菌群体感应有抑制作用的植物乳杆菌SCT-2,为开发一种抑制嗜水气单胞菌群体感应的乳酸菌生物制剂提供了理论支持。

Lactic acid bacteria（LAB） with inhibitory activity against quorum sensing of Aeromonas hydrophila were screened from traditional fermented vegetables using the filter paper method. The biofilm inhibitory activity of the LAB isolates was measured by a 96-well microtiter plate reader, and their effect on the biofilm formation of A. hydrophila was observed by light microscopy and scanning electron microscopy（SEM）. The proteolytic activity, siderophore production, swarming and swimming were used as indicators to evaluate the effects of these quorum sensing inhibitors（QSI） on the virulence of A. hydrophila. In this study, a strain designated SCT-2, with inhibitory activity toward quorum sensing of A. hydrophila, was isolated from pickled cabbage from Jinzhou, Liaoning province. Crude metabolite extract from strain SCT-2 at 8 mg/mL inhibited the biofilm formation of A. hydrophila by 45.16%. Light microscopy revealed that the crude extract pronouncedly inhibited the biofilm formation of A. hydrophila. The SEM images also revealed that the crude extract not only decreased the production of biofilm of A. hydrophila, but also destroyed the biofilm. The secretion amount of proteolytic and siderophore in A. hydrophila treated with 8 mg/mL of the crude extract was decreased by 27.18% and 22.11%, respectively. In addition, the crude extraction degraded 32.27% of the N-acylhomoserine lactones（AHLs） of A. hydrophila. It also inhibited obviously the swarming and swimming of A. hydrophila. Strain SCT-2 was identified as Lactobacillus plantarum by physiological and biochemical tests and 16 S rRNA sequence analysis. This study may pave the foundation for the development LAB-based biological agent for inhibiting quorum sensing of A. hydrophila.