Identification and characterization of bacteriocins active against clinically important strains of Clostridium difficile

The aim of this project is to identify novel bacteriocins produced by clostridia and other bacteria from environmental samples, establish their host-range by testing against a range of clinically relevant C. difficile isolates including the hypervirulent PCR-ribotype 027 strains and determine the mechanisms of bacteriocin import and activity against sensitive cells.

The strategy has involved isolating spore-forming and non-spore forming bacteria form environmental samples such as sewage, soil material and animal matter. All bacterial isolates were then tested for the ability to produce active bacteriocins against strains of Clostridium difficile by a biological activity assay. Zones of inhibitory activity around the producing organisms were indicative of a positive result and the producing organisms were identified and characterised further using Gram stain, 16S RNA sequencing and whole genome sequencing. The presence of bacteriocins in the genome were investigated further by in silico data mining of the genome, and novel bacteriocins genes were cloned into expression plasmids and the recombinant bacteriocins were expressed. Gel overlay assays have shown activity of the recombinant proteins. Additional work will involve engineering mutations in the bacteriocin genes to determine loss of biological activity and confirm association of activity with the known bacteriocin