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Title:Colonization dynamics of oro-gastrointestinal microbes critical to infant health
Author(s):Hoeflinger, Jennifer Leigh
Director of Research:Miller, Michael J.
Doctoral Committee Chair(s):Donovan, Sharon M.
Doctoral Committee Member(s):Hoyer, Lois L.; Lee, Youngsoo
Department / Program:Food Science & Human Nutrition
Discipline:Food Science
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):gut microbiota
infant nutrition
human milk oligosaccharides
carbohydrate utilization
necrotizing enterocolitis
Cronobacter sakazakii
host-microbe interaction
Abstract:The gastrointestinal microbiota is intimately tied to a person’s health and well-being. This collection of bacteria, archaea, and fungi is assembled during a person’s infancy and achieves the stability of an “adult-like” microbiota in early childhood. Research has begun to uncover several host and external factors that influence the rate and course of assemblage. Historically, the immune system was considered the primary driver of microbial balance in the gastrointestinal tract. While the immune system is certainly a key component, research has now identified the importance of, among others, an infant’s route of delivery, gestational age, early-life diet, and exposure to antibiotics. This dissertation has evaluated the modulation of the gut microbiota following exposure to fermentable carbohydrates and explored the pathogenesis of two important infantile pathogens. At birth, an infant’s gastrointestinal microbiota is largely devoid of bifidobacteria, and within the first week, this bacterial genus dominates the community. It is well-known that breastmilk contains oligosaccharides that promote the growth of bifidobacteria. However, formula-fed infants lack access to these carbohydrates and have a delayed establishment of bifidobacteria. Therefore, infant formula companies began supplementing bovine and soy-based infant formulas with prebiotics. Prebiotics, non-digestible carbohydrates that promote bacterial growth, stimulate the growth of bifidobacteria and lactobacilli in infants. Microbial analysis of infants fed prebiotics is restricted to measuring alterations in the fecal microbiota. Typically, prebiotics are fermented in the ascending colon, and the fecal microbiota may not be an appropriate representative of the colonic microbiota. In Chapter 2, the impact of galactooligosaccharides and polydextrose on the ileal and ascending colon microbiota of neonatal piglets was evaluated. More specifically, hundreds of lactobacilli were isolated and sequenced to characterize the lactobacilli community composition. Additionally, these lactobacilli isolates were evaluated for their ability to ferment galactooligosaccharides and polydextrose. It was hypothesized that exposure to galactooligosaccharides and polydextrose would promote a lactobacilli community more capable of fermenting these prebiotics in vitro. While a significant increase of lactobacilli in the ascending colon of prebiotic-exposed piglets was observed, the lactobacilli community was not more capable of fermenting galactooligosaccharides and polydextrose than lactobacilli isolated from piglets never exposed to these prebiotics. Therefore, it was concluded that the observed increase was not due to a direct ability to ferment the provided prebiotics, but rather due to factors not directly measured. An additional concern with prebiotic supplementation in infant formula is the promotion of pathogenic microorganisms. In preterm infants, the gastrointestinal tract lacks the maturity of term infants. A severe consequence of an immature gastrointestinal tract is an increased risk of local and disseminated infections. A common secondary complication is consumption of infant formula, lacking human milk oligosaccharides, rather than breastmilk. As observed in Chapter 2, the exposure to prebiotics did not directly stimulate lactobacilli in vivo. A potential mechanism is a cooperative breakdown of prebiotics in the gastrointestinal tract by other microbes. Several commensal microbes are strongly glycolytic and liberate mono- and disaccharides from fermentable fibers and the gastrointestinal mucin layer. These liberated carbohydrates are available for any microorganisms capable of utilization. Therefore, the presence of prebiotics in the gastrointestinal tract may lead to an increase in pathogenic microorganisms. The study presented in Chapter 3 explored the ability of several Enterobacteriaceae, a bacterial family that includes common neonatal pathogens, to ferment a variety of prebiotics and human milk oligosaccharides. The ability to utilize prebiotics for growth was strain-dependent. Galactooligosaccharides supported the growth of many Enterobacteriaceae, whereas fructooligosaccharides were less fermentable. Fortunately, Enterobacteriaceae were incapable of fermenting the intact human milk oligosaccharides tested. Conversely, several Enterobacteriaceae were capable of fermenting the mono- and disaccharides that comprise the human milk oligosaccharides. Taken together, the inclusion of prebiotics and human milk oligosaccharides in infant formula can increase health-promoting microorganisms while conversely, promoting pathogenic microorganisms. While prebiotics and human milk oligosaccharides can support the growth of health-promoting microbes, exposure to pathogenic microorganisms is inevitable. Once in an infant’s gastrointestinal tract, pathogenic microbes upregulate virulence traits and begin an infection. In Chapter 4, the mechanism of autoaggregation in the neonatal pathogen, Cronobacter sakazakii ATCC 29544 (wild-type) is described. Upon isolation of two independent nonautoaggregating clonal variants, two unique single nucleotide polymorphisms in the flagella proteins, FlhA and FliG, were identified. Furthermore, it was hypothesized that structurally intact and functional flagella were required for autoaggregation in C. sakazakii ATCC 29544. Several gene knockouts were constructed to target the flagella structure (ΔflhA, ΔfliG, ΔfliC, ΔflaA, and ΔfliCΔflaA) and function (ΔmotAB). A loss in autoaggregation in ΔflhA, ΔfliG, and ΔfliC gene knockouts was observed, whereas ΔflaA and ΔmotAB retained the ability to autoaggregate. Complementation of FliC restored autoaggregation to the ΔfliC and ΔfliCΔflaA strains. Therefore, it was hypothesized that a direct interaction between FliC filaments of neighboring cells allowed autoaggregation to proceed. Autoaggregation was disrupted following the addition of detached wild-type flagella in a dose-dependent manner. It was concluded that flagellation with FliC mediates direct interactions between neighboring C. sakazakii ATCC 29544 cells which promote autoaggregation. Further experiments utilizing animal models will need to be conducted to determine if autoaggregation in C. sakazakii ATCC 29544 is necessary for its pathogenesis in vivo. In Chapter 5, a model for Candida albicans colonization in neonatal piglets was developed. C. albicans is a commensal fungus for which colonization mechanisms are understudied. A surveillance analysis identified a C. albicans–negative piglet population at the University of Illinois-Urbana Champaign, which provided the basis for the work. Neonatal piglets were orally inoculated with either a laboratory strain, piglet, or human isolate of C. albicans. C. albicans was present on swabs of the mouth, rectum, and environment for at least 14 days post-inoculation demonstrating stable colonization of the animals. Necropsy showed that C. albicans most readily colonized the piglet esophagus. This model provides an opportunity for subsequent studies of C. albicans colonization mechanisms.
Issue Date:2016-11-29
Rights Information:Copyright 2016 Jennifer Hoeflinger
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12

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