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The production of antagonistic substance by bacterium present in the infected root canal system (RCS) probably is an important ecological factor for its successful colonization of the local. The objective of this study was to partially characterize an antagonistic substance produced by a Clostridium butyricum isolated from infected RCS. Production of inhibitory compound was evaluated by the agar double layer diffusion technique using Fusobacterium nucleatum and Bifidobacterium adolescentis asdoi:10.1590/s1517-83822007000200014 fatcat:dkp2gjbt6nbtliheptedsrrxja