O'Connell, Nuala H. (2003) A study of Clostridium difficile in biofilm mode of growth using the Sorbarod filter system. MD thesis, University of Warwick.

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Abstract

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.

Item Type:	Thesis (MD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Clostridium difficile, Biofilms
Official Date:	April 2003
Dates:	Date Event April 2003 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	MD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Struthers, J. Keith ; Dowson, Chris
Extent:	xxi, 203 p.
Language:	eng

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