The development of Clostridium difficile genetic systems

Clostridum difficile is a major cause of healthcare-associated disease in the western world, and is particularly prominent in the elderly. Its incidence is rising concomitant with increasing longevity. More effective countermeasures are required. However, the pathogenesis of C. difficile infection is poorly understood. The lack of effective genetic tools is a principal reason for this ignorance. For many years, the only tools available for the transfer of genes into C. difficile have been conjugative transposons, such as Tn916, delivered via filter mating from Bacillus subtilis donors. They insert into a preferred site within the genome. Therefore, they may not be employed for classical mutagenesis studies, but can be employed to modulate gene function through the delivery of antisense RNA. Attempts to develop transformation procedures have so far met with little success. However, in recent years the situation has been dramatically improved through the demonstration of efficient conjugative transfer of both replication-proficient and replication-deficient plasmids from Escherichia coli donors. This efficient transfer can only be achieved in certain strains through negation of the indigenous restriction barrier, and is generally most effective when the plasmid employed is based on the replicon of the C. difficile plasmid, pCD6.