Screen For The Quorum Sensing Inhibitory Activity From Marine Bacterial Isolates And The Study Of Active Compounds

The problem of bacterial drug resistance is becoming serious day by day. Some pathogenic bacteria are found easily resistant to the traditional antimicrobial drugs. To fundamentally remove the bacterial pathogenicity, it is important to block the expression of the disease-causing genes by blocking or change the integrant pathway during the pathogenic process. This will not only eliminate the bacterial pathogenicity, but also avoid the drug resistance problem. Quorum sensing regulates many physiological functions of bacteria, whereas the most bacterial pathogenicity is also affected by the bacterial quorum sensing system regulation. To establish a scientific screen model and find some natural products with bacterial quorum sensing inhibitive activity through the study of bacterial quorum sensing system, is an effective way in the development of novel antibacterial substances.In this paper, we used Chromobacterium violaceum12472as the report-strain to screen for the quorum sensing inhibitory activity from272bacteria strains isolated from the sponge tissues collected around the San Juan Island. The results showed that, among the active strains, bacterium No.66and No.108had the strongest quorum sensing inhibitory activity. These two bacteria were then identified by using16SrDNA analysis. Through other screen methods, the bacterium No.108was chosen for the further study. The fermentation conditions of No.108were optimized. After that, extract of108active strains were separated by reverse phase decompression column and HPLC, and the bioactive chemicals were identified by NMR and GC-MS. The main conclusions of the study are as follows:(1) Disc diffusion assay, double-layer soft agar assay and pigment produce inhibition assay were combind used to screen for the quorum sensing inhibitory activity from272bacteria strains isolated from the sponge tissues collected around the San Juan Island. The results showed that the extracts of17strains had higher activity in disc diffusion assay, Among them, strains No.66,74,108,552showed highest activity with the radius of inhibtion zone larger than2.0mm. While in double-layer soft agar assay,40positive strains were found. Most of them also showed some activity in disc diffusion assay. The activity of51active isolates were further screened by using pigment produce inhibition assay. Strain No.108had the strongest and stable activity, which inhibition rate reached20%compared to the control. Combind results of three screen methods, strains No.66and108were chosen for identification and strain No.108was chosen for further compound isolation study.(2) The16SrDNA analysis was performed to identify two active strain, No.66and108. The results indicated that they were similar to Brevibacterium epidermidis SW34and Staphy lococcus equorum subsps, respectively.(3) According to the single factor experiment, we optimized the fermentation conditions and medium components of strain No.108. The results showed that the optimum conditions were: ferment time36h, ferment temperature25℃, rotate speed120rpm, inoculating quantity5%, pH7, medium contained peptone15g/L, yeast extract5g/L, NaCl15g/L,(4) Strain No.108was fermented in large scale (45L) using optimized conditions, and2.9g extracts were obtained from the fermentation broth by ethyl acetate-acetone extraction. Base on their polarity, the extracts were primarily separated into three parts, n-hexane phase, dichloromethane (DCM) phase and65%methanol aqueous phase. Disc diffustion assays were carried out to test the quorum sensing inhibitory activity of the three fractions. The results showed that the dichloromethane fraction was the most effective one. Reverse phase decompression column, with the elution gradient of10%、20%、40%、60%、95%and100%methonal in distilled water was performed to separate the active DCM fraction. The60%methonal eluant had the strongest activity, with the inhibition radius of7mm at the test concentration of250μg per disc. Area normalization analysis of HPLC spectrum showed that the purity of the bioactive chemical was15.962%. The structure of the substance was primarly identified as cyclic proline-leucine by blend1H NMR and GC-MS analysis.