Fluorescence and bioluminescence imaging of the intestinal colonization of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 in mice infected with Listeria monocytogenes EGde

Van Zyl, Winschau Fayghan (2015-04)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

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.

AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) kom algemeen in die mens se spysverteringkanaal (SVK) voor. Verskeie MSB, veral lactobacilli, is bekend vir hul gebruik in gefermenteerde voedsel en as probiotika. Die bakterieë het baie eienskappe wat die mens se gesondheid kan bevoordeel, insluitend vertering en assimilasie van voedsel en voorkoming van kolonisering van die SVK deur patogeniese bakterieë. Sekere probiotiese rasse speel ook ʼn belangrike rol in die behandeling van SVK versteurings, laktose intoleransie en die stimulering van die immuun stelsel. Alhoewel die belangstelling in probiotiese bakterieë toeneem, is daar min inligting bekend oor die meganismes wat MSB gebruik om hulle voordelige eienskappe in die SVK uit te voer. Die oorlewing van MSB in die SVK, veral die interaksies tussen MSB en patogene, kan met behulp van ʼn in vivo moniteringsisteem bestudeer word. Deur die mCherry fluorisensie geen in Lactobacillus plantarum 423 en Enterococcus mundtii ST4SA te kloneer, het ons daarin geslaag om ʼn effektiewe verklikker sisteem te ontwikkel en kon die voorkoms en migrasie van die twee spesies in die SVK van muise bestudeer word. Lactobacillus plantarum 423 en Ent. mundtii ST4SA het veral die blindederm en kolon gekoloniseer. Beide rasse het na ʼn enkele mondelingse toediening vir ten minste 24 h in die SVK oorleef. Sover ons kennis strek, is hierdie die eerste verslag van fluoriserende MSB wat met behulp van die mCherry geenproduk in die SVK bestudeer is. Deur gebruik te maak van ʼn bioliggewende verklikker sisteem, het ons die vermoë van Lb. plantarum 423 en Ent. mundtii ST4SA om met Listeria monocytogenes in die SVK te kompeteer, bestudeer. Listeria monocytogenes het geen invloed gehad op die kolonisering van Lb. plantarum 423 en Ent. mundtii ST4SA nie. Deur die muise vooraf met Lb. plantarum 423 en Ent. mundtii ST4SA te koloniseer (in kombinasie of met net een van die twee rasse), kon ons daarin slaag om kolonisering van die SVK met L. monocytogenes te voorkom. In die muis model wat in hierdie studie gebruik is, was Ent. mundtii ST4SA meer effektief as Lb. plantarum 423 in die verlaging van Listeria selgetalle.

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