Year

2005

Degree Name

Master of Science (Research)

Department

School of Biological Sciences - Faculty of Science

Abstract

Increasing awareness that the human intestinal flora is a major factor in health and disease has led to different strategies to manipulate the flora to promote health. These approaches include changes to the diet by inclusion of prebiotics and probiotics. Prebiotics are non-digestible polysaccharide food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the gastrointestinal tract (GIT). Probiotics on the other hand are viable culture of bacteria, which applied to animals or humans, beneficially affect the host by improving the properties of the indigenous microflora. One mechanism of action of probiotics is the production of antimicrobial substances called bacteriocins, such as colicins, that inhibit the growth of their competitors. The experiments described in this thesis examined the potential use of prebiotics and probiotics to manipulate GIT bacteria. A crude polysaccharide extract (HW) from the medicinal mushroom Ganoderma lucidium was prepared by extracting the fruiting body with boiling water. The extract was then purified by ethanol precipitation resulting in the hot water-ethanol (HWE) extract. Groups of mice were fed these extracts over a period of three weeks at a concentration of 150 ��g/ml in sterile drinking water and the mice then euthanised after three weeks. Changes in population dynamics of lumen bacteria were determined in the duodenum, ileum, colon and faeces by rigorous washing of excised segments while adherent bacteria were released with the non-ionic detergent Triton X100, which does not affect the viability of the bacteria. The prevalence of haemolytic colonies was assessed by plating washouts onto blood agar. Total colony forming units were enumerated on bacteriological media selective for Enterobacteriaceae, Streptococci, Enterococci and Lactic acid Bacteria (LAB). Results showed that there was little change in population dynamics elicited by extract feeding. The exception was a significant reduction in haemolytic lumen bacteria and increase in LAB lumen bacteria recovered from the colon of HWE treated mice. A multiplex PCR was optimized and applied to survey the prevalence of eight common colicin genes (Colicins A, D, E1, E2, E6, E7, Ia and V) in Escherichia coli (E. coli) isolates. The study focused on 39 clinical isolates from humans and 68 isolates from pigs with post-weaning diarrhoea. In addition, 152 porcine commensal E. coli isolates obtained from different compartments of the GIT (duodenum, ileum, colon and faeces) were included in the PCR analysis. Six individual colicins (E1, E2, E6, E7, Ia and V) and four dual colicin combinations (E1/E2, E1/E7, E2/E7, & E2/Ia) were detected. Approximately 28.2 % of the human pathogenic isolates had at least one colicin gene with colicins D, E1, E7 and V occurring at frequencies of 5.1 % each. Colicins E6, Ia and the dual colicin, E2/Ia, were less frequent and were found in about 2.6 % of clones. Only 4 % of the porcine pathogenic isolates possessed a colicin gene and these were exclusively E1 and V. In contrast, there was a significantly higher carriage (36.2%) of colicin genes in commensal porcine E. coli. Of these, E1, E7 and Ia accounted for 87 % of all colicin genes detected. Six of the commensal strains possessed multiple types of colicins with the most common being the E2/E7 combination. Furthermore, there appeared to be differences in the type of colicins found in commensal E. coli isolates recovered from different intestinal compartments. Seven porcine commensal E. coli strains producing standard colicins were evaluated for inhibitory activity against five pathogenic E. coli of human and porcine origin. The experiment utilized a kinetic inhibitory microtitre assay (KIMA) to assess inhibition using non-induced supernatants and supernatants induced with 0.2 ��g/ml of mitomycin C to stimulate colicin production. The level of inhibition was found to be variable with most of the commensal porcine E. coli strains showing little or no inhibitory action against the five pathogenic strains. However, two commensal strains, ILC33 and CC89 were found to highly inhibitory to three porcine pathogenic E. coli strains of serotypes O141:K85, 0141:K88 and 0149:K88. The findings of this thesis suggest that purified polysaccharide extracts (HWE) from Ganoderma lucidium have the potential to be used in further studies as prebiotics in view of their positive effects on beneficial LAB. In addition, the use of colicin-bearing strains as probiotic bacteria is justifiable because of the low incidence of colicin genes in pathogenic E. coli compared to commensals. Finally, these findings indicate that potential probiotic bacterial strains have to be scrutinised for their inhibitory activity against individual pathogenic strains prior to being subjected to further assessments.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.