Clostridium perfringens is a Gram-positive, anaerobic bacterium that is a pathogen in humans
and animals. C. perfringens has been associated with humans causing gas gangrene, food
poisoning, necrotizing enterocolitis (NEC) in very premature neonates and necrotic enteritis
(NE) in poultry. Although it is well established that pathogenic bacteria form biofilms
extensively in nature to survive and resist adverse conditions by attaching to each other and
to a substrate with extracellular polymeric substance (EPS). Biofilm formation in anaerobic
organisms is under-researched.
One of the objectives of this study was to compare the biofilm-forming potential of C.
perfringens isolates from broiler chickens, free-range poultry environments and neonatal
humans using the traditional protocols of crystal violet staining assays in microtiter plates. In
addition, the susceptibility of C. perfringens to a range of veterinary or human antimicrobial
drugs in conventional or biofilm modes were tested using the broth microdilution method. All
fifty-four (54) C. perfringens isolates tested from a variety of sources were shown to form
biofilms. 7/54 (13%) were strong biofilm producers, 31/54 (57%) were moderate biofilm
producers and 16/54 (30%) were weak biofilm producers according to current criteria. There
was no significant difference observed in the density of biofilms formed by isolates from
different sources but out of the 9 isolates tested previously for the presence of adhesin genes
(virulence factors/appendages that facilitates bacteria attachment to host cell/surfaces),
moderate biofilm producers harbored multiple adhesin genes whilst weak biofilm producers
harbored only one adhesin gene each. As anticipated, the formation of biofilms significantly
protected C. perfringens from the action of the antibiotics and resistance ranged between 83%
to 100%.
Another objective of this study was to determine the in vitro activity of silver and gold
nanoparticles against C. perfringens established biofilm. The antibiofilm activity of silver and
gold nanoparticles alone and the enhanced antibiotic effect of antibiotics in the presence of
nanoparticles were determined by enumerating viable cells on agar plates after antimicrobial
treatment. Treatment of C. perfringens with silver nanoparticles resulted in 19% to 58% (1.3log
to 4.8log) biofilm reduction while treatment with gold nanoparticles recorded 11% to 39%
(0.7log to 3.2log) biofilm reduction. Combination of antibiotic and nanoparticles improved
biofilm reduction compared to antibiotic alone, but this was not generally significant at the
concentration tested.
The third objective of this study was to test the antibiofilm potential of the ethanolic leaf extract
of three medicinal plants (Vernonia amygdalina, Ocimum gratissimum and Azadirachta
indica). The study showed that the plant’s extracts inhibited or reduced biofilm differently for
each tested C. perfringens isolate at the concentration tested. Using the viable count method
to determine the effect of treatment showed minimal (2%/0.1 log) to good (28% /2.5 log) biofilm
inhibition and reduction.
The final part of this study was aimed at testing the antibacterial and antibiofilm activity of a
recently developed antimicrobial (QAC) solution (Acquorsol) on C. perfringens. The
antibacterial and antibiofilm activity of Acquorsol was determined by enumerating viable cells
using total viable count assay. 50% to 0.1% of Acquorsol solution completely inhibited the
growth of four C. perfringens planktonic grown strains while 50% to 0.78% of Acquorsol
solution completely prevented the growth of four C. perfringens biofilm grown strains. 50% to
6.25% of Acquorsol inhibited the growth of 100% of tested planktonic strains while 50%
concentration of Acquorsol prevented the formation of C. perfringens biofilm in 91% (30 out of
33) of tested strains.
In summary, the results in this thesis show that C. perfringens grown in biofilm mode
independent of the source of isolation could reduce the effective susceptibility of
antimicrobials. Furthermore, it showed that alternative therapeutic strategies have the
potential to control biofilm related contamination and infections in animals and people. These
alternative bacteriocides could be useful for decontaminating surfaces in hospital and may
have a role in decontamination of skin surfaces and may be used in poultry environments as
well as other public places
