AIM: To establish Caenorhabditis elegans based in vivo method for screening bioactives from marine sponge associated bacteria (SAB) against Vibrio species. METHODS AND RESULTS: About 256 SAB isolates were screened for their ability to rescue C. elegans infected with Vibrio species. The chloroform extract of the positive isolate was subjected to column fractionation and purity of the active fraction was analysed using HPLC. Further, the components were elucidated using GC/MS. The active fraction was tested for its in vivo rescue activity, antibacterial and anti-QS activity. In vivo colonization reduction and biofilm inhibition efficiency were assessed using GFP-tagged V. alginolyticus using confocal laser scanning microscopy (CLSM). The ability of the active fraction in modulating expression of V. alginolyticus quorum sensing (QS) regulators luxT and lafK was measured using real-time PCR. The results indicated that the chloroform extract of SAB4.2 displayed significant rescue activity against V. alginolyticus by inhibiting the QS pathway. HPLC analysis of the active fraction revealed a single major peak and GC/MS analysis suggested Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) as the major constituent. The potent bacterial isolate was identified as Alcaligenes faecalis. CONCLUSIONS: In vivo screening using C. elegans identified a marine isolate that inhibits the virulence of V. alginolyticus by interrupting the QS pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a C. elegans based in vivo screening method for identifying bioactives from natural resources by overcoming the disadvantages of traditional in vitro plate assays.
AIM: To establish Caenorhabditis elegans based in vivo method for screening bioactives from marine sponge associated bacteria (SAB) against Vibrio species. METHODS AND RESULTS: About 256 SAB isolates were screened for their ability to rescue C. elegans infected with Vibrio species. The chloroform extract of the positive isolate was subjected to column fractionation and purity of the active fraction was analysed using HPLC. Further, the components were elucidated using GC/MS. The active fraction was tested for its in vivo rescue activity, antibacterial and anti-QS activity. In vivo colonization reduction and biofilm inhibition efficiency were assessed using GFP-tagged V. alginolyticus using confocal laser scanning microscopy (CLSM). The ability of the active fraction in modulating expression of V. alginolyticus quorum sensing (QS) regulators luxT and lafK was measured using real-time PCR. The results indicated that the chloroform extract of SAB4.2 displayed significant rescue activity against V. alginolyticus by inhibiting the QS pathway. HPLC analysis of the active fraction revealed a single major peak and GC/MS analysis suggested Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) as the major constituent. The potent bacterial isolate was identified as Alcaligenes faecalis. CONCLUSIONS: In vivo screening using C. elegans identified a marine isolate that inhibits the virulence of V. alginolyticus by interrupting the QS pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a C. elegans based in vivo screening method for identifying bioactives from natural resources by overcoming the disadvantages of traditional in vitro plate assays.
Authors: Siti N Fatin; Tan Boon-Khai; Alexander Chong Shu-Chien; Melati Khairuddean; Amirul Al-Ashraf Abdullah Journal: Front Microbiol Date: 2017-11-16 Impact factor: 5.640