In vitro model development for novel prevention measures against foodborne pathogens

Foodborne disease prevention can benefit from improved antimicrobial applications and targeted approaches to investigating phenomena that drive the presence and absence of pathogens in our foods. The use of suboptimal methodologies for these investigations remains a hurdle to furthering preventative capabilities in many food and health systems. My dissertation research has featured the development of methods for better modeling the interactions between pathogens and their environment, so that antimicrobial measures can be discovered and validated more accurately. Interest in the use of probiotics to prevent microbial infection is growing rapidly, particularly considering the global rise in antibiotic resistance of various pathogens. Probiotics are microorganisms that promote health when consumed, via a number of possible mechanisms including the prevention of pathogen carriage and infection. Adhesion to intestinal cells has been widely used as a criterion for the selection of probiotics. This is in part due to the assumption that adhesion of probiotics to host tissues prevents pathogen attachment and thereby pathogenesis. However, unlike antimicrobial production, adhesion has not been well characterized as a competitive exclusion mechanism. Mechanisms of adhesion to host tissue have been intensively studied in numerous pathogens, such as Escherichia coli and Pseudomonas aeruginosa, and many are mediated by attachment to oligo- and polysaccharide components found on the mucosal surfaces of host tissues. However, the role these types of glycans play in the adhesion of non-pathogens has only recently begun to be investigated. In Chapter 1, I detail the potential contributions of muco-adhesion to the probiotic effect of lactobacilli and highlight the importance of glycan-mediated interactions that are often overlooked in in vitro characterization of organisms. Specific adhesion mechanisms are still poorly understood, largely due to the prohibitive costs of complex oligosaccharides or glycomimetics. Studies of probiotic adhesion are also often confounded by multiple potential mechanisms at play and even when investigating glycan-specific models, whole-cell interactions have shown limited success. Effective control strategies for pathogens within production animal reservoirs could significantly limit human foodborne disease. Poultry comprise one such prominent reservoir, the primary source of Campylobacteri jejuni in developed nations. Fucosylated oligosaccharides have been implicated in the competitive exclusion of C. jejuni from host gastrointestinal tracts, suggesting that glycan-mediated adhesion facilitates colonization and infection. However, such mechanisms are difficult to model in vitro independent of confounding interactions, especially in a way that maintains wild-type functionality and facilitates cell manipulation further. I developed a flexible glycan-specific affinity chromatography method to address this constraint, by simplifying and isolating bacteria-glycan interactions. Chapter 2 details the use and validation of this model by demonstrating the adhesion of E. coli and C. jejuni to specific carbohydrate structures, namely mannose and 2-fucosyllactose respectively. Subpopulations of E. coli, for example, could be separated based on the predominance of mannose-binding fimbriae. The mechanism by which C. jejuni adheres to fucose remains to be determined, but the development of this model serves as a step towards understanding novel mechanisms that can be exploited for establishing and optimizing competitive interactions between target pathogens and probiotics. In pursuit of a more applied approach to food safety, I also sought to address a major concern of Hispanic-style fresh cheeses, which are prone to carriage of one of the deadliest known foodborne pathogens, Listeria monocytogenes. Soft cheeses such as queso fresco are linked to approximately a quarter of US listeriosis cases annually, largely due to a lack of intrinsic factors within these cheeses (e.g. high acidity or salt, low moisture) that would prevent listerial growth. Given the ability of L. monocytogenes to grow under refrigeration, and the negative impact of thermal or high-pressure processing on product quality, additional measures for preventing listerial proliferation are highly desirable. I developed a miniature laboratory-scale queso fresco model to accommodate the assessment of novel antimicrobial compounds that are often available only in limited amounts. This model also allows for greater control over production parameters and biocontainment relative to alternative methods, opening the door to answer questions of the interaction between bacterial contaminants, antimicrobial measures, and their food environment. As described in Chapter 3, we initially validated the model by assessing the antilisterial efficacy of the food-grade bacteriocin nisin, as well as ferulic acid. Both compounds limited the growth of Listeria over a three-week refrigerated shelf life, but were unable to completely halt the growth of contaminants. In search for more effective means of killing L. monocytogenes, bacteriophage endolysins were identified to have considerable potential for overcoming limitations of other antimicrobials in foods such as fresh cheeses. Endolysins are peptidoglycan hydrolases that lyse bacteria as part of the bacteriophage lytic cycle. They do not promote bacterial resistance like bacteriophages, bacteriocins, or antibiotics, still demonstrate specificity for target organisms, should not impact food quality like organic acids and extracts might, and have activity within higher pH ranges as well. There has been much recent investigation of endolysins as antimicrobials, which has resulted in the documentation of numerous ways in which they may be optimized for antimicrobial efficacy via bioengineering. Chapter 4 discusses intrinsic and extrinsic variables that affect the antimicrobial potential of an endolysin, including several elements of endolysin structure, environmental conditions, and target cell physiology. However, many of these variables have not been well characterized, particularly the relationships between cell physiology, sensitivity to lysis, and environmental factors relevant to food applications. To characterize factors that contribute to enzymatic activity and cell sensitivity, I observed the interaction of the recombinant endolysin PlyP100 with Listeria under various experimental conditions in vitro. Chapter 5 shows that PlyP100 exhibits environmental tolerances compatible with application in fresh cheeses, which was confirmed in the miniaturized queso fresco model. Treatment of PlyP100 in cheeses contaminated with L. monocytogenes completely prevented growth over four weeks under refrigeration. Furthermore, I show that endolysin sensitivity is dependent upon cell history with regards to growth conditions, cell cycle, and likely growth rate

Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants.

BackgroundIndividuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the 'secretor' gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2'-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance.ResultsOn average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2'-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ.ConclusionsInfants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants

Lactobacillus Adhesion to Mucus

Mucus provides protective functions in the gastrointestinal tract and plays an important role in the adhesion of microorganisms to host surfaces. Mucin glycoproteins polymerize, forming a framework to which certain microbial populations can adhere, including probiotic Lactobacillus species. Numerous mechanisms for adhesion to mucus have been discovered in lactobacilli, including partially characterized mucus binding proteins. These mechanisms vary in importance with the in vitro models studied, which could significantly affect the perceived probiotic potential of the organisms. Understanding the nature of mucus-microbe interactions could be the key to elucidating the mechanisms of probiotic adhesion within the host

Glycan-specific whole cell affinity chromatography: A versatile microbial adhesion platform

We have sought a universal platform for elucidating and exploiting specificity of glycan-mediated adhesion by potentially uncharacterized microorganisms. Several techniques exist to explore microbial interactions with carbohydrate structures. Many are unsuitable for investigating specific mechanisms or uncharacterized organisms, requiring pure cultures, labeling techniques, expensive equipment, or other limitations such as questionable stability, stereospecificity, or scalability. We have adapted an affinity chromatography resin as a model to overcome these drawbacks, among others. It readily allows for the quantification, selection, and manipulation of target organisms based on interactions with glycan ligands. To maximize its utility as a selective screening method, we have constructed the tool such that it: • Promotes whole-cell interactions using viable, unaltered cells. • Provides robust spatial interactions with target glycans, presented with controlled stereo-specificity, for high affinity/avidity interactions that reflect a complex in vivo matrix. • Has the ability to utilize any reducing glycan, is quick, efficient, safe, and affordable to construct, and is scalable and reusable for multiple applications

Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants.

BackgroundIndividuals with inactive alleles of the fucosyltransferase 2 gene (FUT2; termed the 'secretor' gene) are common in many populations. Some members of the genus Bifidobacterium, common infant gut commensals, are known to consume 2'-fucosylated glycans found in the breast milk of secretor mothers. We investigated the effects of maternal secretor status on the developing infant microbiota with a special emphasis on bifidobacterial species abundance.ResultsOn average, bifidobacteria were established earlier and more often in infants fed by secretor mothers than in infants fed by non-secretor mothers. In secretor-fed infants, the relative abundance of the Bifidobacterium longum group was most strongly correlated with high percentages of the order Bifidobacteriales. Conversely, in non-secretor-fed infants, Bifidobacterium breve was positively correlated with Bifidobacteriales, while the B. longum group was negatively correlated. A higher percentage of bifidobacteria isolated from secretor-fed infants consumed 2'-fucosyllactose. Infant feces with high levels of bifidobacteria had lower milk oligosaccharide levels in the feces and higher amounts of lactate. Furthermore, feces containing different bifidobacterial species possessed differing amounts of oligosaccharides, suggesting differential consumption in situ.ConclusionsInfants fed by non-secretor mothers are delayed in the establishment of a bifidobacteria-laden microbiota. This delay may be due to difficulties in the infant acquiring a species of bifidobacteria able to consume the specific milk oligosaccharides delivered by the mother. This work provides mechanistic insight into how milk glycans enrich specific beneficial bacterial populations in infants and reveals clues for enhancing enrichment of bifidobacterial populations in at risk populations - such as premature infants