Thesis (PhD (Microbiology))--Stellenbosch University, 2008. / One of the key selection criteria for potential probiotics is the ability to adhere and colonise the host gastrointestinal tract (GIT). Probiotics compete for receptor sites at the host intestinal surface, preventing the colonisation of pathogens, thereby protecting the host from infection. In addition, several important intestinal functions are mediated by the binding of probiotics to host tissue. However, the molecular mechanisms and genotypic characterization of adhesive elements have not received as much attention as other aspects of probiotic research. The present study aims to contribute to this area of research. The first part of the study focused on monitoring the expression of mucus adhesion genes mub, mapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, as well as mub, surface layer protein (slp) and EF-Tu of Lactobacillus acidophilus ATCC 4356 when grown in the presence of mucin, bile, pancreatin and at low pH. Real time PCR was used. mub, mapA and EF-Tu of strain 423 were up-regulated in the presence of mucus and expression increased under increasing concentrations of mucus. Expression of mapA was up-regulated under normal gut conditions (0.3%, w/v, bile; 0.3%, w/v, pancreatin; pH 6.5) and at higher levels of bile (1.0%, w/v) and pancreatin (1.0%, w/v). Expression of mub was downregulated in the presence of bile and pancreatin at pH 6.5, whilst the expression of EFTu and plaA remained unchanged. At pH 4.0, the expression of mub and mapA remained unchanged, whilst EF-Tu and plaA were up-regulated. Expression of mapA was down-regulated in the presence of 0.1% (w/v) cysteine, suggesting that the gene is regulated by a mechanism of transcription attenuation that involves cysteine. In the case of L. acidophilus ATCC 4356, none of the genes were up-regulated under increasing concentrations of mucin, whilst only slp and EF-Tu were up-regulated under normal and stressful gut conditions in vitro. In the second part of the study, male Wistar rats were used to evaluate which section of the gastrointestinal tract are colonised by L. plantarum 423 and Enterococcus mundtii ST4SA and determine the effect of adhesion. Fluorescent in situ hybridization (FISH) incorporating strain specific oilgonucleotide probes indicated strong fluorescent signals for L. plantarum 423 along the intestinal lining of the ileum and the cecum. L. plantarum 423 did not colonise the colon as indicated by real timePCR. Fluorescent signals were recorded for E. mundtii ST4SA across the epithelial barrier of cecum and colonic tissue, suggesting that translocation took place. Real time PCR revealed highest cell numbers of strain ST4SA in the cecum and the colon. Haemotoxylin eosin staining of rat tissue revealed no change in morphology or any toxic effects induced upon adhesion of the strains. 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE) revealed a decrease in enterobacterial species whilst the lactic acid bacterial content remained unchanged. Strains 423 and ST4SA agglutinated yeast cells in vitro, indicating the possible presence of mannose receptors. It is well known that these receptors play a crucial role in the elimination of type 1 fimbriated strains of E. coli. It is thus safe to speculate that mannose receptors may have played a role in diminishing the enterobacterial content in the gut. The third part of the study encompassed characterization of cell surface proteins of L. plantarum 423 and their role in adhesion to Caco-2 cell lines. The strain lacks the typical surface layer protein whilst a multifunctional “intracellular” protein, elongation factor Tu (EF-Tu) and glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) were detected. Removal of surface proteins reduced adherence of strain 423 to Caco-2 cell lines by 40%, suggesting that these proteins play a role in adhesion. The ability of strain 423 to competitively adhere, exclude and displace Clostridium sporogenes LMG 13570 and Enterococcus faecalis LMG 13566 from Caco-2 cell lines, was studied. Adhesion of C. sporogenes LMG 13570 and E. faecalis LMG 13566 was inhibited by 70% and 90%, respectively. Strain 423 excluded C. sporogenes LMG 13570 from Caco-2 cells by 73% and displaced the pathogen by 80%. E. faecalis LMG 13566 was excluded by 60% and displaced from Caco-2 cells by 90%. Despite removal of the surface proteins, L. plantarum 423 was still capable of competitively adhering to Caco-2 cells and reduced adherence of C. sporogenes LMG 13570 by 50% and E. faecalis LMG 13566 by 70%.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The equine gastro-intestinal tract (GIT) is a relatively unexplored niche concerning the presence of natural microbiota. Studies have shown that disruption of the microbial population naturally present in the GIT leads to the onset of several forms of gastro-intestinal disorders. To maintain a balanced microbiota, probiotic bacteria need to be administered at specific levels. Beneficial microorganisms assist with digestion of the feed, absorption of nutrients from the GIT, strengthens the immune system and improves the animal‟s growth. Various combinations of lactic acid bacteria (LAB) have been administered to horses, but have failed to benefit the host in any of the latter criteria. The screening for alternative strains with probiotic properties is thus necessary. Two strains (Lactobacillus equigenerosi Le1 and Lactobacillus reuteri Lr1) were originally isolated from horse faeces. Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, both bacteriocin-producing strains, were isolated from sorghum beer and soy beans, respectively. All four strains survived growth at acidic conditions (pH 3) and the presence of 0.5%, 1.0% and 1.5% (w/v) bile salts. L. reuteri Lr1 was the most resistant to these conditions. All strains adhered to buccal (cheek) epithelium cells sampled from horses. L. equigenerosi Le1 and E. mundtii ST4SA, however, invaded the cells, but without visible signs of disrupting the cells. None of the strains contained genes encoding adhesion to collagen (Ace), resistance to vancomycin A, B and C, or, production of aggregation substance (AS), cytolysin (Cyl) and, non-cytolysin (β hemolysin III), suggesting that they are non-virulent. Of all strains, L. equigenerosi Le1 competed the best with Clostridium sp. C6 for adherence to epithelial cells. L. equigenerosi Le1 and L. reuteri Lr1, showed the highest level of co-aggregation with Clostridium sp. C6. When the four strains were administered to horses over a period of 10 days, L. reuteri Lr1 was retained the longest (8 days) in the GIT. The numbers of viable cells of Clostridium spp. and Salmonella spp. remained constant during administration of the four strains. Blood analyses showed no negative effects from administering the strains. Total white blood cell counts remained unchanged. However, a small but tentative increase in neutrophil and eosinophil cell numbers has been recorded, suggesting that the LAB may have elicited a mild, transient, intolerance reaction. The glucose, lactate and urea levels decreased during administration with the four LAB strains. / AFRIKAANSE OPSOMMING: Die spysverteringstelsel (SVS) van die perd is 'n relatief onbekende nis wat die voorkoms van natuurlike mikrobiota betref. Studies het getoon dat versteuring van die natuurlike mikrobiese populasie in die SVS aanleiding kan gee tot die ontwikkeling van menige vorms van gastro-intestinale ongesteldhede. Om 'n gebalanseerde mikrobiota te verseker, moet probiotiese bakterieë teen 'n spesifieke vlak toegedien word. Voordelige mikroorganismes bevorder vertering en absorpsie van nutriënte vanaf die SVS, versterk die immuunsisteem en bevorder die groei van die dier. Verskeie kombinasies van melksuurbakterieë is reeds aan perde toegedien, maar sonder ooglopende voordele vir die dier. Die soeke na alternatiewe stamme met probiotiese eienskappe is dus noodsaaklik. Twee melksuurbakterieë (Lactobacillus equigenerosi Le1 en Lactobacillus reuteri Lr1) is oorspronklik uit perdemis geïsoleer. Lactobacillus plantarum 423 en Enterococcus mundtii ST4SA, beide bakteriosienproduserende stamme, is afsonderlik van sorghumbier en sojabone geïsoleer. Al vier spesies groei by lae pH (pH 3) en in die teenwoordigheid van 0.5%, 1.0% en 1.5% (m/v) galsoute. L. reuteri Lr1 is die mees bestand onder hierdie toestande. Al vier stamme het aan wang epiteelselle van perde geheg. L. equigenerosi Le1 en E. mundtii ST4SA het egter die epiteelselle binnegedring, maar sonder opsigtelike vernietiging van die selle. Nie een van die stamme besit gene wat kodeer vir aanhegting aan kollageen (Ace), bestandheid teen vankomisien A, B en C, of produksie van, sel-aggregasie (AS), sitolisien (Cyl) en nie-sitolisien (β-hemolisien III), wat daarop dui dat hulle nie-virulent is. Van al die stamme het L. equigenerosi Le1 die beste met Clostridium sp. C6 vir aanhegting aan epiteelselle gekompeteer. L. equigenerosi Le1 en L. reuteri Lr1, het die beste vlak van ko-aggregasie met Clostridium sp. C6 getoon. Met die toediening van 'n kombinasie van die vier stamme aan die perde oor 'n periode van 10 dae, het L. reuteri Lr1 die langste retensie (8 dae) in die SVS getoon. Die aantal lewende selle van Clostridium spp. en Salmonella spp. het konstant gebly tydens toediening van die vier stamme. Toediening van die vier stamme het geen negatiewe effek getoon met resultate verkry van bloed analises nie. Die totale witbloed seltellings het onveranderd gebly. 'n Klein, maar tentatiewe, toename in neutrofiel- en eosinofiel selgetalle is waargeneem, wat daarop dui dat die melksuurbakterieë 'n geringe allergiese reaksie teweeggebring het. Die glukose, laktaat en ureum vlakke het gedaal tydens die toediening van die vier melksuurbakterie stamme.