降解嗜水气单胞菌群体感应AHLs信号分子的乳酸菌筛选及淬灭作用

Screening and Quorum Quenching of Lactic Acid Bacteria with Degrading Aeromonas hydrophila Quorum Sensing AHLs Activity

AHLs是一类介导革兰氏阴性菌群体感应的氮酰基高丝氨酸内酯类信号分子,从自然界中寻找对AHLs具有降解活性的乳酸菌,探究应用乳酸菌群体感应抑制剂控制致病菌的新途径。采用96孔板法从传统发酵食品和海水鱼肠道中分离的156株乳酸菌中初筛到56株对嗜水气单胞菌群体感应AHLs具有降解作用的菌株,初筛率为35.90%。采用琼脂扩散法对初筛菌株复筛得到12株降解活性较强的乳酸菌,其中来源于牙鲆肠道的菌株YP 4-1-1降解活性接近100%。GC-MS分析表明:菌株YP 4-1-1对嗜水气单胞菌C4-HSL和C6-HSL信号分子的降解活性分别为98.31%和81.81%。经生理生化和16S rRNA序列分析确定菌株YP 4-1-1为清酒乳杆菌。菌株YP 4-1-1酶解AHLs的活性来源于其粗细胞提取物的可溶性部分,可能是一种AHLs-酰基转移酶或氧化还原酶。YP 4-1-1粗提物对紫色杆菌CV026和嗜水气单胞菌的MIC分别为2.0 mg/mL和4.0 mg/mL,3个不同亚MIC(0.25 MIC,0.50 MIC,0.75 MIC)对紫色杆菌素的降解作用具有剂量依赖性。在体外共培养试验中,菌株YP 4-1-1可减弱嗜水气单胞菌溶血性、蛋白酶、DNA酶和嗜铁素等毒力因子的表达,并对其群体感应的群集和泳动行为具有抑制活性。本文从自然生态环境中获得对革兰氏阴性菌AHLs具有降解活性的乳酸菌,为研发乳酸菌生物制剂控制革兰氏阴性菌导致的水产动物疾病奠定理论和应用基础。

AHLs are a kind of signal molecules that mediate quorum sensing of gram-negative bacteria.Screening microorganisms with degradative activity on AHLs from nature as a quorum sensing inhibitor is a new strategy for controlling pathogenic bacteria.56 strains of degrading Aeromonas hydrophila quorum-sensing AHLs signaling molecules were rapidly screened out from 156 strains of lactic acid bacteria that isolated from traditional fermented foods and marine fish intestines using a 96-well plate method.The initial screening rate was 35.90%.56 strains of lactic acid bacteria screened out 12 strains with strong degradation activity by agar diffusion method.The degradation activity of strain YP 4-1-1 derived from the intestine of marine fish gingiva was close to 100%.Analysis by GC-MS showed that the activity of the strain YP 4-1-1 degrading Aeromonas hydrophila C4-HSL and C6-HSL was 98.31%and 81.06%,respectively.The physiological and biochemical reactions and 16S rRNA sequence analysis confirmed that strain YP 4-1-1 was Lactobacillus sakei.Further studies showed that the quorum quenching enzyme activity of strain YP 4-1-1 is present in the soluble fraction of the crude cell extract,and may be an AHLs-acyltransferase or oxidoreductase.The MICs of YP 4-1-1 crude extracts against Chromobacterium violaceum CV026 and Aeromonas hydrophila were 2.0 mg/mL and 4.0 mg/mL,and three different sub-MICs(1/4 MIC,1/2 MIC and 3/4 MIC)dose-dependent degradation of the purple pigment.In vitro co-culture experiments,strain YP 4-1-1 attenuated the expression of virulence factors such as hemolysin,DNase and lipase of Aeromonas hydrophila,and had inhibitory activity on quorum-sensing clustering and migration.In this paper,strains with degrading activity against gram-negative bacteria AHLs signal molecules are obtained from lactic acid bacteria in the natural environment,which lays the theoretical and application foundation for the development of lactic acid bacteria biological agents that control quorum sensing.