A study of Clostridium difficile in biofilm mode of growth using the Sorbarod filter system

In all ecosystems, bacteria are likely to grow in biofilms, and the organisms making up the natural flora of the colon are considered to be no exception to this. One of the most important hospital-acquired infections is antibiotic associated diarrhoea, with the causative agent being the Gram-positive anaerobe, Clostridium difficile. This organism elaborates two exotoxins, A and B, which cause disease by interaction with the enterocytes of the colon. Treatment of the condition can be difficult, with treatment failures not uncommon. As the organism produces a heat stable spore, it can survive with ease in the environment. There are no previous reports concerned with the study of anaerobes in an in vitro biofilm system. The work described here is a study of C. docile, using the Sorbarod filter system. This was to determine its suitability in establishing continuous culture biofilms of the organism. It was shown that C. difficile readily established itself on the filters, maintained a titre in excess of 109 cfu/filter for at least 72h. The lack of sporulation in the system confirmed that vegetative growth was being maintained. In the filters, the organism exhibited elongated forms, sometimes in excess of 10 microns in length. Electron microscopy showed that this was probably due to lack of cell separation at cell division. Toxin production appeared to be higher when C. difficile was grown in biofilm in comparison with growth in broth and biofilm effluent. The susceptibility of the organism to benzylpenicillin, vancomycin, metronidazole and rifampicin was determined in broth culture, biofilm and biofilm effluent. Broth minimum inhibitory and minimum bacteriocidal (MIC/MBC) experiments showed, that apart from rifampicin, the organism was tolerant to the antibiotics. This was essentially repeated in the filter system. Rifampicin had a significantly better bactericidal activity against the organism, and synergistic killing was achieved when rifampicin was combined with vancomycin. The growth of C. difficile in biofilm with other common members of the bowel flora was investigated in combination experiments with Bacteroidesfragilis, Enterococcus faecalis and Escherichia coli. No antagonistic effect was demonstrated and antimicrobial susceptibility experiments using C. difficile and E. coli in combination showed resistance of both organisms to broad-spectrum antibiotics. Screening for bacteriophages and other biological agents with activity against C. difficile identified an isolate of Lactobacillus paracasei, which had marked activity against the clostridium as shown by the "sloppy agar" method, but there was a very variable effect in broth and the Sorbarod biofilms. Interestingly, the bacteriocin-like agent had activity against not only a number of other clostridial species, but also against Streptococcus pneumoniae. The work here is thus a novel investigation of an important infection control problem, and is the basis for further detailed work examining the growth and pathogenic properties of C. difficile in biofilm.