Use of Bacillus amyloliquefaciens for the prophylactic treatment of Clostridium difficile infections

Clostridium difficile is a spore forming, antibiotic resistant pathogen which is one of the main causes of antibiotic associated diarrhea and the major cause of pseudomembranous colitis. Incidences of C. difficile infections (CDI) are rising and standard antibiotic therapy using broad spectrum antibiotics seems to be less and less efficient. C. difficile can only colonize the gut after disruption of the normal microbiota, mainly due to the consumption of antibiotics. Probiotics could be very helpful to prevent or treat CDI if the bacterial strain produces antimicrobial peptides that act against C. difficile but not to strains of the indigenous microbiota. A Bacillus amyloliquefaciens strains isolated in our laboratory showed high antimicrobial activity against a collection of 24 different C. difficile ribotypes but not to a series of strains isolated from the healthy gut microbiota. It was tested in an in vivo mouse model for its prophylactic use against CDI. Results show that it exhibits very interesting probiotic properties for use against CDI. We purified the antimicrobial compound responsible for the antibacterial activity against C. difficile. Antibacterial activity in the cell free supernatant (CFSN) of a B. amyloliquefaciens culture was not affected by treatment with different proteases and showed minimal loss of activity after heating up to 100°C for 30 min. However, adjustment of the pH to a pH lower than 4 resulted in complete loss of activity. Antibacterial activity could be restored by collecting the obtained precipitate and neutralizing the pH. Taking into account these results, the antimicrobial peptide comprises all characteristics associated with lipopeptides. Purification of this antimicrobial peptide was accomplished by acid precipitation of CFSN followed by reversed phase high performance liquid chromatography (RP-HPLC). The collected antimicrobial fractions will be further investigated using electrospray ionization mass spectrometry in the near future