Evaluating the impact of alternative antimicrobials on biofilms formed by Clostridium perfringens
Creators: name: Omoigberale, May
Publisher: name: University of Lincoln
Year: 2023
Subjects: C500 - Microbiology
C530 - Bacteriology

description: 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 Azadirachtaindica). 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 cellsusing 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 Acquorsolsolution 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.