Browsing by Author "Van Zyl, Winschau Fayghan"

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    Fluorescence and bioluminescence imaging of the intestinal colonization of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in mice infected with Listeria monocytogenes EGde
    (Stellenbosch : Stellenbosch University, 2015-04) Van Zyl, Winschau Fayghan; Deane, Shelly May; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Lactic acid bacteria (LAB) are common inhabitants of the human gastro-intestinal tract (GIT). Some LAB, especially lactobacilli, are well known for their application in fermented foods and probiotic properties. These microorganisms exert many beneficial effects on human health, such as digestion and assimilation of food and preventing pathogens colonising the GIT. Furthermore, some selected probiotic strains are believed to perform a critical role in the treatment of gastro-intestinal disorders, lactose intolerance and in the stimulation of the immune system. Despite the ever increasing consumer interest in probiotic LAB, the mechanisms by which they exert their beneficial effects and the activities of probiotics in the GIT often remain poorly understood. Understanding survival mechanisms of LAB in the GIT, especially the interaction between LAB and pathogens, would be facilitated by the direct in vivo monitoring of these processes. Using the mCherry fluorescence gene, we successfully constructed Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA reporter strains. With this study we showed that fluorescence imaging can be used to detect Lb. plantarum 423 and Ent. mundtii ST4SA in the GIT of mice. The two species colonized the cecum and colon for at least 24 h after one oral administration. To our knowledge, this is the first report on fluorescence imaging of LAB expressing mCherry in a mouse model. Using a bioluminescence marker system, we evaluated the impact of Lb. plantarum 423 and Ent. mundtii ST4SA on orally acquired Listeria monocytogenes infection and the ability of the probiotics to compete with the pathogen in the GIT of mice. Challenging Lb. plantarum 423 and Ent. mundtii ST4SA that were already established in the GIT of mice with L. monocytogenes EGDe had no effect on the survival and persistence of the probiotic strains. We demonstrated that the colonization of mice with Lb. plantarum 423 and Ent. mundtii ST4SA, or a combination of the strains, protected the animals against colonization of the GIT by L. monocytogenes EGDe. Enterococcus mundtii proved more effective than Lb. plantarum 423 in reducing the number of L. monocytogenes EGDe in the mouse model.
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    Gastrointestinal persistence of the probiotic bacteria Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA, and their anti-listerial activity
    (Stellenbosch : Stellenbosch University, 2018-12) Van Zyl, Winschau Fayghan; Deane, S. M.; Dicks, Leon Milner Theodore; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Gastrointestinal diseases, and in particular those caused by bacterial infections, are a major cause of morbidity and mortality worldwide. Treatment is becoming increasingly difficult due to the increase in number of species developing resistance to antibiotics. Different treatment strategies need to be developed. Probiotic lactic acid bacteria (LAB) have considerable potential as alternatives to antibiotics, both in prophylactic and therapeutic applications. Lactic acid bacteria have a long history of safe use in food and therapeutic products and is increasingly recognised for their beneficial effects. However, the underlying mechanisms by which probiotic LAB enhance the health of the consumer have not been fully elucidated. Demonstrating key antimicrobial and protective probiotic mechanisms in vivo will allow for industry and consumers to choose scientifically validated probiotics for the prevention or treatment of targeted gastrointestinal diseases. The present study aimed to contribute to this area of probiotic research. The first part of this study focus on monitoring the survival, metabolic activities and persistence of Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA in the gastrointestinal tract (GIT) of mice using a bioluminescence imaging (BLI) system. The route and destination of both probiotic strains in the GIT were determined after single and multiple doses. Both strains prominently colonized the cecum and colon. Enterococcus mundtii ST4SA persisted in the GIT and faeces the longest and in higher numbers while also actively colonizing the small intestine. This is the first report of in vivo and ex vivo BLI of E. mundtii ST4SA in a murine model. The second part of the study encompassed the development of a novel system that facilitates the rapid and efficient isolation of double-crossover integration or deletion mutants of L. plantarum 423 and E. mundtii ST4SA. The system was useful in the construction of L. plantarum 423 and E. mundtii ST4SA bacteriocin and adhesion gene mutants. The newly described method expands the LAB molecular research genetic toolkit and has significant potential to allow genetic modification of most, if not all LAB species. This provides the unique opportunity to study the role of specific probiotic LAB genes in complex environments using reverse genetics analysis. In the final part of the study, the ability of L. plantarum 423 and E. mundtii ST4SA to competitively exclude L. monocytogenes EGDe, an intestinal pathogen, from the GIT of mice was proven. Valuable insight was gained on the molecular modes of action of the two probiotic strains. Plantaricin 423 and mundticin ST are bacteriocins produced by L. plantarum 423 and E. mundtii ST4SA, respectively. Bacteriocin-negative mutants of L. plantarum 423 and E. mundtii ST4SA failed to exclude L. monocytogenes EGDe from the GIT, confirming in situ bacteriocin production as an anti-infective mediator. Additional confirmation of in situ bacteriocin production as a mechanism of action was provided by using variant strains of L. monocytogenes EGDe expressing the immunity genes of plantaricin 423 and mundticin ST, respectively, which provided resistance to the respective bacteriocins. Furthermore, the exclusion of L. monocytogenes EGDe from the GIT was reduced when mice were administered with L. plantarum 423 and E. mundtii ST4SA adhesion gene knockout strains. These results substantiate our understanding of the functional attributes of probiotics currently available to consumers and the improvement of future probiotic products.