LC-MS/MS检测水产品源致病菌和腐败菌群体感应AHLs信号分子

为分析水产品源致病菌和腐败菌中群体感应信号分子AHLs的含量和种类,实验建立液相色谱-串联质谱(LC-MS/MS)同时检测细菌群体感应11种AHLs类信号分子的技术,并与生物报告菌法比较。结果显示,报告菌紫色杆菌CV026和根癌农杆菌A136检测发现嗜水气单胞菌和2株铜绿假单胞菌是AHLs产生菌。建立的LC-MS/MS能完全分离和检测11种AHLs,并发现假单胞菌和嗜水气单胞菌产生的AHLs信号分子含量较高,其中ATCC 9027和ATCC 15692产生3-oxo-C12-HSL,嗜水气单胞菌A2产生C4-HSL,而腐败希瓦氏菌XH4分泌的C4-HSL信号分子含量较低,沙门氏菌ATCC 14028-3、大肠杆菌O157:H7和3种弧菌的AHLs很低。随着细菌浓度的增加,铜绿假单胞菌ATCC 9027产生的AHLs含量逐步增加,在12 h达到最高。研究表明,LC-MS/MS可用于多种AHLs信号分子的定性和定量检测,具有灵敏性与准确性更高的优点。在11株水产品源致病菌和腐败菌中,假单胞菌和气单胞菌分泌的AHLs含量最高。

Detection of N-acylated-L-homoserine lactones of quorum sensing secreted by pathogen and spoilage bacteria from aquatic product using liquid chromatography-mass spectrometry/mass spectrometry

To investigate the level and kinds of quorum sensing signal molecules N-acylated-L-homoserine lactones (AHLs) secreted by pathogen and spoilage bacterial from aquatic product, AHLs were determined by biosensors C.violacceum CV026 and A.tumefaciens A136. Furthermore, the method of LC-MS/MS detecting simultaneously the AHLs was developed. Biofilm was determined by crystal violet assay. The changes of the level of AHLs and biofilm were evaluated in P.aeruginosa ATCC 9027. The results showed that A.hydrophila A2 and two kinds of P.aeruginosa could secret AHLs by biosensors. Qualitative and quantitative analysis of 11 kinds of AHLs were completed by optimized LC-MS/MS parameters. High level of AHLs with 3-oxo-C12-HSL and C4-HSL were produced by P.aeruginosa and A.hydrophila, respectively. Low content of AHLs secreted by S.putrefaciens XH4 were C4-HSL. However, no AHLs were detected in Salmonella, E.coli O157:H7 and four kinds of Vibro. The production of AHLs and biofilm formation of P.aeruginosa ATCC 9027 increased with the growth of bacteria, and the highest level of AHLs was observed after 12h culture. Thus, LC-MS/MS was used to qualitative and quantitative analysis of profiling of AHLs, which was highly accurate and sensitive. The kinds and level of AHLs secreted by pathogen and spoilage bacterial from aquatic product differed, in which P.aeruginosa and A.hydrophila produced the high AHLs signal molecule. The present results provided the good foundation to explore the mechanism and control of pathogen and spoilage bacterial.