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Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus , by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB , encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405 ). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.

Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes.