Heat shock increases conjugation efficiency in Clostridium difficile()

Clostridium difficile infection has increased in incidence and severity over the past decade, and poses a unique threat to human health. However, genetic manipulation of C. difficile remains in its infancy and the bacterium remains relatively poorly characterised. Low-efficiency conjugation is currently the only available method for transfer of plasmid DNA into C. difficile. This is practically limiting and has slowed progress in understanding this important pathogen. Conjugation efficiency varies widely between strains, with important clinically relevant strains such as R20291 being particularly refractory to plasmid transfer. Here we present an optimised conjugation method in which the recipient C. difficile is heat treated prior to conjugation. This significantly improves conjugation efficiency in all C. difficile strains tested including R20291. Conjugation efficiency was also affected by the choice of media on which conjugations were performed, with standard BHI media giving most transconjugant recovery. Using our optimised method greatly increased the ease with which the chromosome of R20291 could be precisely manipulated by homologous recombination. Our method improves on current conjugation protocols and will help speed genetic manipulation of strains otherwise difficult to work with.