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Background and aims Clostridium difficile infection (CDI) remains a healthcare burden and recurrent CDI (rCDI) still affects 20-30% of patients. Probiotics are live microorganisms that confer a host health benefit, but evidence of their efficacy in CDI prevention/treatment is controversial. Non-toxigenic C. difficile (NTCD) have been used successfully used in animals/humans to reduce CDI. The present study aimed to assess efficacy of two probiotics, Lactobacillus casei Shirota (LcS, Yakult) and NTCD, in preventing simulated CDI in an in vitro human gut model. Methods C. difficile-negative pooled faeces from healthy volunteers (>65 yrs) was used to inoculate the gut model. Two probiotics, LcS (6.2 x 109 cfu) and NTCD (1 x 108 spores), were dosed into separate gut models prior to C. difficile (CD) ribotype 027 spores (RT027, 1 x 108). LcS was chosen given its commercial availability, in addition to studies that report the prevention of antibiotic-associated diarrhoea by LcS, and its role in preventing CDI. Similarly, NTCD was chosen given reports of its antagonism against toxigenic C. difficile. Probiotic dosing was for 28d; LcS once-daily and NTCD spores once-weekly. The rationale for dosing is consistent with literature and the ease of obtaining the probiotic. For example LcS is commercially available whereas NTCD had to be prepared using a rigorous process over a duration of 7 - 10 days. Various antimicrobial groups such as lincosamides, aminopenicillins, fluoroquinolones, and third generation cephalosporins was used to disrupt the gut microflora in separate experiments. Gut model contents were assayed for microflora composition using viable counting techniques and CD cytotoxin production using a Vero cell cytotoxicity assay. Probiotic dosing ceased 14 days before the end of the experiment. Results LcS dosing resulted in marked increases in lactobacilli and bifidobacterial viable counts. However, during clindamycin (DA) dosing these viable counts declined by 4-log10cfu/mL. RT027 spore germination and cell proliferation was observed during/after antimicrobial instillation. Interestingly, another cycle of growth/cytotoxin was observed after LcS dosing ceased. NTCD did not colonise the gut model prior to DA instillation; spores were quiescent and washed out. NTCD spores germinated and vegetative cells multiplied, whereas, RT027 spores did not germinate and no cytotoxin was produced. NTCD remained vegetative until the end of the experiment and isolated cells retained their non-toxigenic phenotype. Conclusions Instillation of NTCD prevented primary CDI in a human gut model, whereas dosing with LcS did not. LcS, if beneficial in the antagonism of CD, is unlikely to be due to nutrient/adhesion competition or production of antimicrobial substances. NTCD may be beneficial not only in treating rCDI but also in the prevention of primary infection. Further work is needed to better understand the protection that NTCD might confer.

Probiotics are live microorganisms which when administered in adequate amounts confer one or more health benefits on the host. Different processing conditions, the acidic condition of the stomach and exposure to hydrolytic enzymes affect the viability and efficacy of probiotic organisms. This study investigated the protective effects of two biopolymers poly-gamma-glutamic acid (γ-PGA) and bacterial cellulose (BC) on probiotics during freeze drying and during exposure to simulated intestinal juices and bile salts. The antibacterial property of Bifidobacterium strains was also investigated against four pathogenic bacteria. γ-PGA, a naturally occurring biopolymer was produced by two bacteria (Bacillus subtilis ATCC 15245 and B. licheniformis ATCC 9945a) in GS and E media, γ-PGA yields of about 14.11g/l were achieved in shake flasks and molecular weight of up to 1620 k Da was recorded, γ-PGA production was scaled up in a fermenter with B. subtilis using GS medium. BC, an edible biopolymer was produced by Gluconacetobacter xylinus ATCC 23770 in HS medium and a modified HS (MHS) medium. A yield of about 1.37g/l was recorded and BC production with MHS medium was used for probiotic application. B. longum NCIMB 8809 B. breve NCIMB 8807 and B. animalis NCIMB 702716 showed the best antimicrobial properties against the investigated pathogens. Survival of Bifidobacterium strains was improved when protected with powdered BC (PBC) although γ-PGA offered better protection than PBC. Viability of B. longum NCIMB 8809, B. breve NCIMB 8807 and B. animalis NCIMB 702716 in simulated gastric juice (SGJ) and simulated intestinal juice with bile salts was improved when protected with 5% γ-PGA and 5% γ-PGA+PBC with a reduction of < 1 Log CFU/ml while a reduction of ≤2 Log CFU/ml was recorded in PBC protected cells. Protecting Bifidobacterium strains with γ-PGA, PBC or a novel γ-PGA + PBC combination is a promising method to deliver probiotic bacteria to the target site in order to confer their health benefits on the host.

THE UNIVERSITY OF MANCHESTER ABSTRACT OF THESIS submitted by Raja Moftah Moman for the Degree of Doctor of Philosophy entitled Interactions of Oral Bacteria with Host Tissues and Allochthonous Microorganism. 15th June 2017. The oral microbiome is a taxonomically diverse microbial community situated principally upon the hard and soft tissue surfaces of the mouth. It represents a readily accessible biofilm community for the investigation of bacteria-bacteria and bacteria-host interactions, which are responsible for some of the main features of oral biofilms in health and disease (colonisation resistance, antimicrobial tolerance, metabolic cross feeding, and other cooperative phenomena). In the oral cavity these relate specifically to cariogenesis and interactions with soft tissue that are responsible for periodontal disease. This doctoral thesis presents a series of investigations that consider processes for which growth in the biofilm phenotype or bacterial-bacteria or bacteria-host cell interaction are responsible. Four distinct methods were used to assess the effect of the biofilm phenotype on susceptibility of eight distinct oral hygiene actives with various modes of action. Bisphenol microbicide triclosan and the bis-biguanide chlorhexidine were most effective. All were markedly more effective against bacteria grown planktonically than the same organisms grown as biofilms illustrating antimicrobial tolerance, an important biofilm characteristic. In studies of interactions between oral isolates, bacteria previously isolated from the saliva and different oral sites of the oral cavity were tested using a modified cross streak method, in all possible pair-wise combinations. The frequency and strength of physical interactions (coaggregation) between these isolates was also assessed. The incidence of positive interactions was higher than the incidence of negative interactions (15.21% vs. 1.04%) and the incidence of coaggregation in bacteria isolated from saliva was significantly lower than for bacteria isolated from oral biofilms. Together, these data suggest that bacterial cooperation plays a greater role in oral biofilm development and maintenance than competition. With respect to putatively beneficial interactions between bacteria and host, the potential of the candidate dental probiotics L. rhamnosus GG, L. reuteri and S. salivarius to protect host tissues from damage by three Gram negative periodontal pathogens were investigated using human oral cells culture and the (invertebrate) G. mellonella model system. All probiotics inhibited the growth of the test pathogens when applied simultaneously, and significantly decreased toxicity (p P. gingivalis > A. actinomycetemcomitans) in two distinct cell lines. Whilst all probiotics conferred protection against the periodontal pathogens, L. rhamnosus GG, had the greatest protective effect, regardless of probiotic or pathogen used, followed by L. reuteri. S. salivarius was the least effective. Prophylactic treatment with probiotics conferred greater protection than treatment concomitant with pathogen challenge. The data presented in this doctoral thesis demonstrate the functional significance of interactions between taxonomically distinct bacteria and between bacteria and host tissues. Such interactions may determine the outcome of exposure to antimicrobials and are, particularly significant in health and through further research, may be harnessed for prevention and treatment of oral disease.

The intestine and associated bacterial microbiota have a central role the physiology and homoeostasis of the host. The understanding of how farming conditions affect the intestine and associated microbiota of fish is the high importance to counteract the potential threats to health and welfare. Thus, this thesis aims to understand the role of stressful husbandry conditions on the intestine and associated microbiota of rainbow trout and Atlantic salmon. Within this context, the role of Pediococcus acidilactici as health promoter was also investigated Chapter 3 investigated the replacement of fishmeal by different plant protein ingredients in rainbow trout. The results of this chapter revealed that the effect of P. acidilactici on the microbiota of distal intestine in rainbow trout was dependent on the ingredients of the diet. The results also showed that the FM substitution induced major changes in the intestinal microbiota. Moreover, the modulation induced by plant-based diets on the microbiota varied according to the ingredients used. Chapter 4 studied the effect of dietary oxytetracycline in the distal intestinal microbiota of rainbow trout and the role of P. acidilactici to ameliorate the impact of antibiotic therapy. Experimental groups fed the diets with oxytetracycline had substantial changes in the distal intestinal microbiota including a decrease in the bacterial diversity. P. acidilactici did not ameliorate the effect of antibiotic therapy in the intestinal microbiota. Chapter 5 used Atlantic salmon during smoltification to study the changes in the microbiota of distal intestine and the role of P. acidilactici to promote intestinal health. The results showed that bacterial communities in the mucosa differed from the digesta. Seawater transfer and P. acidilactici had significant changes in the intestinal microbiota of both mucosa and digesta. However, the modulatory effect of both factors evaluated was larger in the mucosa-associated microbiota than in the digesta-associated microbiota. Furthermore, P. acidilactici induced a significant increase in antiviral-related genes. Chapter 6 investigated the replacement of fish oil by rapeseed oil alone or combined with P. acidilactici on the intestinal health and microbiota of two intestinal regions in Atlantic salmon. Replacement of fish oil by rapeseed oil alone or in combination with P. acidilactici supplementation did not induce major changes in the intestinal health and microbiota. The bacterial communities found were significantly different between the pyloric caeca and mid-intestine. In conclusion, this thesis contributes to new knowledge regarding the effect of dietary supplementation of P. acidilactici and the impact of different potential challenging factors in the health and intestinal microbiota of farmed salmonid species.

Three investigations were conducted to assess the effects of selected bio-active feed additives on tilapia (Oreochromis niloticus) growth performance, feed utilisation, haemato-immunological status, intestinal morphology and microbiology. The first experiment was conducted to evaluate the effects of feeding tilapia semi-practical diets supplemented with exogenous enzymes (phytase, protease or carbohydrase). After six weeks of the dietary supplementation, tilapia fed diets supplemented with exogenous enzymes (phytase and carbohydrase) showed enhanced growth performance and higher microvilli density when compared to the control group. Additionally, the intestinal bacterial community profile of tilapia fed the carbohydrase supplemented diet was significantly altered in contrast to those fed the control diet. In the second experiment, tilapia were fed with practical diets supplemented with exogenous enzymes (phytase, protease or xylanase) for eight weeks. Tilapia fed the xylanase supplemented diet demonstrated significantly higher final body weight (FBW), improved feed conversion ratio (FCR) and better protein efficiency ratio (PER) when compared to the control group. Apparent digestibility coefficients of protein, ash, energy, phosphorus, calcium and sodium were highest in tilapia fed a diet supplemented with phytase. The third experiment was carried out to assess the combined effects of dietary exogenous enzymes (phytase, protease and xylanase) and probiotics (Bacillus subtilis, B. licheniformis and B. pumilus) on tilapia growth performance and health. After seven weeks of feeding, tilapia fed the diet supplemented with both exogenous enzymes and probiotics showed significantly higher FBW, specific growth rate, improved FCR and better PER. The serum lysozyme activity was observed to be significantly higher in tilapia fed the probiotic supplemented diet when compared to the control group. The dietary supplementation with combined exogenous enzymes and probiotics increased intestinal perimeter ratio, microvilli count (density), diameter and subsequently resulted in higher enterocyte absorptive area in tilapia. This study demonstrates that feeding tilapia with dietary exogenous enzymes can enhance growth performance and modulate microbial community profile. In addition, supplementation with both exogenous enzymes and probiotics is capable of improving tilapia growth performance, immune parameters and intestinal morphology.

There is an increasing consumer demand for dairy products which are safe and free from additives. Microbial starter strains, in combination with other factors, were studied for their contribution to the control of unwanted microbes, and maintaining the quality of soft cheese. The technological and functional characteristics of the starter culture strains Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris, and probiotic bacterial strains Bifidobacterium animalis subsp. lactis BB12, Lactobacillus acidophilus LA-5 and Lactobacillus casei Shirota were investigated. The tests included the milk fermentation, resistance to salt and heat, bile and acid resistance, and growth at a range of temperatures. The probiotic strains differed in their resistance to salt, bile salts and acid. Inhibitory interactions between probiotic bacterial strains with each other and with starter culture strains were not detected. The probiotic bacteria and starter culture strains used have an ability to grow together on homofermentative and heterofermentative differential agar and fermentation of fructose in different levels. Non-starter cheese (NSC), cheese with starter strains (SCS), and cheese with starter and probiotic strains (PSC) were manufactured. The levels of mesophilic aerobic and lactic acid bacteria, moulds and yeasts, and Enterobacteriaceae were evaluated in all cheeses. Their contents of fat, total solids, salt and pH value were tested during 21 days of storage at 2-5°C. Starter culture strains contributed to maintaining the quality of all cheeses, through decreasing the viable count of some undesirable microbes. Cheeses differed in the intensity of the crumbliness attribute, and in preference and intensity of colour attribute. The colour of starter soft cheese, which was tested using a colorimeter, was closer to the colour of probiotic soft cheese than those cheeses which were manufactured without starter culture. The microbial status, storage conditions, rancidity, and the sensory characteristics of unripened soft cheese, which was manufactured with starter culture strains only, were determined during the storage for 50 days at 2-5°C, as well as during their shelf life for the product. Modified Atmosphere Packaging (MAP) contributed to slowing the growth of unwanted microbes, and decreased the values of TBA, TVB-N and TMA in soft cheese. Consequently, delaying the undesirable changes and maintaining the quality of the product and extending its shelf life, when compared with vacuum, brine, and air packaging methods, under the same storage conditions. Potential effects of inulin on the cheese quality and sensory characteristics of probiotic soft cheese were investigated. The cheeses differed in their loads of lactic acid bacteria, in addition to the total solids and water activity. The levels of probiotic bacterial strains were higher in probiotic soft cheese that manufactured with inulin than in cheese without inulin, with a potential in the formation synbiotic between the probiotic strains LA-5 and BB12 and inulin. Both cheeses were recorded to have high acceptance in the cheese attributes, in terms of appearance, aroma, colour texture and the overall acceptance. The presence of inulin increased the hardness of cheese under vacuum packaging, after storage for 14 days at 2-5°C.

Extreme physical exertion is commonly associated with acute physiological changes in immune variables known to disturb host defences. Likely induced by the production of stress hormones (e.g., cortisol), partaking in ultra-endurance events with accompanying physiological stressors (e.g., environmental extremes, sleep deprivation and compromised hydration and (or) nutritional status) may amplify stress hormone responses and compromise immune status to a greater extent. To date, research investigating the impact of extreme physical exertion (e.g., ultra-marathon events) on physiological variables is extremely limited. More recently, the potential use of probiotics with known immunomodulatory effects may be considered an appropriate nutritional strategy to improve host defences and minimise and (or) prevent sub-clinical or clinically significant outcomes in active populations.

The development of childhood atopy is postulated to be influenced by perinatal microbial exposure. Inadequate numbers of probiotic organisms forming the intestinal microbiota, is an early pre-symptomatic feature linked to the expression of allergy. Perinatal probiotic supplementation is therefore hypothesized to contribute to the microbiota mediated immunomodulatory response determining immune hypo- responsiveness to allergens. Aims: This thesis examined the anti-allergy immunomodulatory effects of probiotic bacteria invitro in the context of the pathophysiology of atopy, as a preamble to perinatal probiotic supplementation. Methods: Cytokine responses were measured after umbilical cord blood mononuclear cells (CBMCs) were co-cultured in the presence of a consortium of probiotic organisms consisting of two strains of lactobacilli and two strains of bifidobacteria. Additionally pregnant women with a familial history of allergic disease from 36 weeks gestation and their infants to age 6 months, were supplemented with probiotics or a placebo daily. The immunomodulatory effects of probiotics on immune function in vivo was then analysed within the peripheral blood of the supplemented neonates. Results: CBMCs co-cultured in the presence of the probiotic consortium generated a dose dependent, monocyte mediated release of the pro-inflammatory cytokines TNF-a, IL- 12p70, IFN-y and the immunosuppressive cytokines IL-10 and TGF-pi. The consortium down regulated PH A and SEB induced IL-13 (a key allergy orchestrating cytokine) while potentiating IFN-y (a key Thl driving cytokine) responses from CBMCs. Interestingly in the probiotic supplemented group the cord blood eosinophil count was significantly reduced. Additionally the IL-12p70 concentrations in microbial stimulated venous blood at age 6 months was significantly elevated in comparison to the placebo supplemented group. Conclusion: The immunomodulatory effect of probiotic bacteria is marked by a capacity to promote a Thl orientation of the immune system. Probiotics administered during pregnancy and early infancy may therefore be effective in the prevention of Th2 mediated atopic disorder.

As the use of probiotics and postbiotics is increasingly gaining ground in the past decade, the possibility of using bacterial strains with postbiotic activity to restore homeostasis in pathologic conditions such as IBD is extensively debated. However, clinical data as far as induction of remission is concerned has not been encouraging so far, while researchers observe that only a small number of treatments which seem promising on in vitro or mouse models translate to significant clinical benefit. This could mean that the models used so far to test potential treatments are not an accurate representation of the human intestine's rather complex micro-environment, and thus there is a need for the development of more relevant models. In this thesis, a novel organ culture model for polarized stimulation of intestinal mucosa is described. In the intestine, apical and basolateral challenge of the mucosal layer can elicit completely different results, and this is one of the issues addressed in this work. First of all, we show that it is possible to keep human intestinal mucosa in polarized culture for at least 24 hours, provided the explants are cultured in an atmosphere that is rich in oxygen. Polarized challenge is achieved by mechanical means, namely by attaching a cave plastic cylinder on the apical side of the mucosal layer, in order to confine the stimuli. We examine the impact of the cylinder and the surgical glue used to attach it on tissue morphology and survival, and show that there is no negative impact. Once optimized, this experimental set-up is used to challenge explants with pathogenic and probiotic bacteria alike and evaluate the immune response. On this model we are able to mimic a classical pathogen infection using a highly invasive Salmonella enterica serovar typhimurium strain, FB62. The response of the tissue to the pathogen was monitored by assessing the phenotype of explants after culture, while cytoine secretion profiles of the tissue were also studied. Salmonella challenge led to a damaged explant phenotype, upregulation of TNF-α and downregulation of IL-10. This response was abolished in the presence of an antiinflammatory postbiotic component, namely culture supernatant of the probiotic Lactobacillus paracasei. Moreover, as far as challenge with pathogens is concerned, we use this novel system to examine the mechanism of Shigella induced apoptosis of epithelial cells. Importantly, no animal models are available for the study of Shigella infection, as these 18 bacteria are poorly virulent in rodents. Thus, in an effort to study the involvement of Gadd45a in the apoptotic route triggered by Shigella on intestinal epitelial cells, HeLa cells, which are however a poor model for an intestinal pathogen were used. Gadd45a participates in the responses to a variety of DNA damaging agents and interacts with proteins such as Cdc2, PCNA and p21. In this work, the authors showed that after infection of HeLa cells with Shigella, Gadd45a is involved in the induction of the apoptotic process. The data was confirmed in a more relevant setting, by applying Shigella on human intestinal mucosa. Finally, we use the novel organ culture platform to test three different strains thought to exert probiotic actions. Surprisingly, we show that this is not the case, and the three strains can exert different activities even on healthy tissue. More specifically, Lactobacillus paracasei and Lactobacillus rhamnosus GG did not significantly alter the phenotype or cytokine secretion profiles of healthy explants, but challenge with Lactobacillus plantarum resulted in a detrimental effect. Of note, all three strains were detrimental for IBD explants when administered as live bacteria, even though one of the strains (L. paracasei) had previously been found to exert a prophylactic effect in a mouse model of colitis. On the contrary, we show that a potent postbiotic (L. paracasei culture supernatant) is able to ameliorate overt inflammation on both ulcerative colitis and Crohn's disease tissues as attested by cytokine secretion profiles of challenged explants. In conclusion, this work introduces a valid alternative system on which to study the interaction of various components (bacterial, pharmacological, and others) with the human intestinal mucosa.

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by eight bacteria (B. subtilis 23856, B. subtilis 23857, B. subtilis 23858 B. subtilis 23859, B. subtilis natto, B. licheniformis 1525, B. licheniformis 6816 and B. licheniformis 9945a) in GS and E media. B. subtilis natto and B. licheniformis 9945a have been investigated extensively for γ-PGA production, however, the remaining six have not previously been used. Using the eight bacteria, yields of up to 22.3 g/l were achieved in shake flasks. On characterization, it was observed that γ-PGA with different properties (crystallinity, acid/salt form and molecular weights ranging from 3,000 Da to 871,000 Da) was produced. Production of γ-PGA by B. subtilis natto in GS medium was scaled up using a fermenter and was tested for novel probiotic applications. The survival of probiotics during freeze drying, storage and ingestion was improved by combining them with a γ-PGA matrix. For L. paracasei, 10% γ-PGA protected the cells significantly better (P < 0.05) than 10% sucrose during freeze drying, whereas for B. longum and B. breve, it showed comparable cryoprotectant activity (P > 0.05) to 10% sucrose. This study also demonstrated the potential use of a non-dairy foodstuff (orange juice) for delivery of probiotics. Two Bifidobacteria strains protected with γ-PGA survived significantly better (P < 0.05) in orange juice for 39 days, with a log reduction in viability of less than 2.99 CFU/ml, when compared to unprotected cells, which showed complete loss in viability by day 20. In addition, γ-PGA protection improved survival of Bifidobacteria in a solution mimicking the environment of the stomach. γ-PGA-protected Bifidobacteria showed little (< 0.47 log CFU/ml) or no loss in viability when stored in simulated gastric juice (pH 2.0) for four hours, whereas unprotected cells died within two hours.

Probiotics have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The beneficial effects of probiotics in the gut are well described and roles including immunomodulation and colonisation resistance have been documented. Recent reports suggest that topical use of probiotic bacteria may be an effective strategy to promote skin health or inhibit disease. Therefore, in this thesis the potential of probiotics to protect skin from pathogenic bacteria was assessed using primary keratinocytes as a model system, and the skin pathogen, Staphylococcus aureus. The ability of three probiotics, L. reuteri ATCC 55730, L. rhamnosus AC413 and L. salivarius UCC118 to inhibit the growth of S. aureus was tested using well-diffusion assays and spot on the lawn assays. All three probiotics inhibited the growth of S. aureus in well-diffusion assays, though this property was dependent on growth medium. Inhibition of S. aureus growth was principally via the production of organic acids rather than bacteriocin production. Next, to determine whether probiotics could protect keratinocytes, confluent normal human epidermal keratinocytes (NHEK) were infected with S. aureus (106 CFU/ml) in the presence or absence of the probiotic (108 CFU/ml). NHEK viability was measured using trypan blue exclusion assays. L. reuteri had a significant protective effect on NHEK when applied 1h prior to (P=0.0003), or simultaneously with S. aureus (P=0.002). L. reuteri did not however protect NHEK when applied 1h after S. aureus addition. There was no change in the number of viable S. aureus in cell culture assays. To determine whether the protective effect was due to the inhibition of adhesion, NHEK were either pre-exposed to the probiotic for 1h, simultaneously exposed to the probiotic and S. aureus for 1h, or exposed to the probiotic 30 minutes after S. aureus addition for 1h. Pre-exposure of NHEK to L. reuteri (exclusion) and simultaneous exposure to L. reuteri and S. aureus (competition) resulted in significantly less staphylococci adhering to NHEK (P=0.03 and P=0.008 respectively). However when L. reuteri was added after S. aureus (displacement), the number of adherent staphylococci was not reduced. The necessity of S. aureus adherence for the inactivation of NHEK was demonstrated using a α5β1 integrin blocking antibody. Finally, to compare the innate response of NHEK to probiotics with S. capitis and S. aureus, TLR-2, antimicrobial peptide (AMP) expression and IL-8 production were measured. TLR-2 protein (but not mRNA) expression was reduced in the presence of S. aureus (P=0.018). NHEK pre-exposed to S. capitis prior to S. aureus infection however, exhibited elevated TLR-2 protein and mRNA expression (P<0.0001 and P=0.009 respectively). NHEK pre-exposed to L. reuteri prior to S. aureus had no significant change in TLR-2 expression compared to untreated controls. ELISAs demonstrated that IL-8 production was significantly increased in NHEK pre-exposed to L. reuteri prior to S. aureus infection (P=0.0001). In conclusion, L. reuteri protected NHEK from the toxic effects of S. aureus at least partly through competitive exclusion of binding sites on NHEK. Finally, NHEK innate responses to probiotic bacteria were akin to those to the skin commensal, S. capitis. L. reuteri induced expression of a neutrophil chemoattractant, suggesting it could be of importance in priming the innate immune response against S. aureus infections. Taken together, these results suggest that probiotic bacteria could be used prophylactically within skin creams and soaps to prevent S. aureus colonisation and infection in skin.

The anaerobic spirochaete Brachyspira pilosicoli causes enteric disease in avian, porcine and human hosts, amongst others. Avian intestinal spirochaetosis (AIS), the resulting disease from colonisation of the caeca and colon of poultry by Brachyspira leads to production losses, with an estimated annual cost of circa £18 million to the commercial layer industry in the United Kingdom. A lack of knowledge of the metabolic capabilities and little genomic information for Brachyspira has resulted in a limited understanding of the pathobiology of this genus. In addition, an emergence of antibiotic resistance in Brachyspira, together with bans on the prophylactic use of antimicrobials in animal feed, drive an urgent requirement for alternative treatment strategies for diseases such as AIS. In the first intra-species genome comparison within the genus Brachyspira, these studies report the whole genome sequence of an avian strain of B. pilosicoli, B2904, and the incomplete genome sequence of a human strain of B. pilosicoli, WesB. Comparisons are made between the de novo sequenced strains and those of B. pilosicoli 95/1000, a pig strain and other available Brachyspira genome sequences from public databases. Furthermore, this study reports the first application of the high-throughput Biolog phenotype screening tool to Brachyspira for detailed phenotypic analysis and confirmation of metabolic deductions made from the genotypic data. Probiotics have been reported as protecting against infection with common enteric pathogens in livestock and in this study investigations into which aspects of the biology of Brachyspira they antagonise were undertaken. Lactobacilli reduced the growth and motility of B. pilosicoli and its ability to adhere and invade epithelial cells in vitro. Following these encouraging results, an in vivo intervention study was performed using a B. pilosicoli challenge model in poultry to elucidate the potential for probiotic intervention against AIS. This study demonstrated that when administered in drinking water, L. reuteri LM1, isolated from a healthy chicken, reduced all aspects of the clinical presentation of AIS.

Genes, pathways & transcription factors involved in probiotic mediated resolution of gut inflammation in IL10-KO mice an animal model of Inflammatory Bowel Disease. An integrated Gene, Protein and Bioinformatics Approach The IL10-KO mouse is a model of human inflammatory bowel disease (IBD), used to study host microbial interactions and potential therapeutics. Affymetrix microarray and proteomics analysis on colon of WT and IL10-KO mice and cecum of IL10-KO and WT mice orally administered with and without probiotic VSL#3 was performed and identified signalling pathways and transcription factors relevant to gut inflammation and anti-inflammatory probiotics. Results were validated by Real-time PCR, immunocytochemistry, proteomics, histopathology and via pathway signature analysis of publicly available microarray data. Changes in metabolically active bacteria in response to VSL#3 were assessed with DGGE. Inflammation in IL10-KO mice was characterised by up-regulation of immune/inflammatory and down-regulation of lipid/xenobiotic metabolism and PPAR signalling. VSL#3 resolved inflammation in the cecum inducing down-regulation of genes in immune/inflammatory pathways, decrease in the number of CCL5 positive T cells and up-regulation of galectin2, known to trigger apoptosis of T cells. VSL#3 induced up-regulation of PPARα/PPAR signalling and lipid/xenobiotic metabolism, antagonistic to NFB signalling and reduced metabolically active bifidobacteria. Analysis of publicly available data showed results were relevant to human IBD, indicated that antigen processing/presentation is up-regulated early on during development of colitis in IL10-KO mice, identified the potential of PPARα/PPAR signalling to induce down-regulation of CCL5, CD3 & antigen processing/presentation, and the potential of the xenobiotic metabolism to induce down-regulation of cytokine-cytokine interaction & mitosis. As VSL#3 treatment of IL10-KO mice induced up-regulation of PPARα/PPAR signalling and xenobiotic metabolism these results provide a possible mechanistic explanation for the VSL#3 induced down-regulation of CCL5, CD3, antigen processing/presentation, cytokine-cytokine interaction and mitosis in the cecum of VSL#3 treated IL10-KO mice.

Probiotics are nonpathogenic microorganisms that have been extensively studied for their ability to prevent various infectious, gastrointestinal, and autoimmune diseases. The mechanisms underlying these probiotic effects have not been elucidated. However, we and other researchers have evidence suggesting that probiotic bacteria secrete metabolites that are antimicrobial and anti-inflammatory. As such, we developed a methodology to collect the secreted metabolites from a probiotic bacterium, Lactobacillus acidophilus, and tested this cell free filtrate (CFF) both in vitro and in vivo. Using this CFF, we have demonstrated that L. acidophilus secretes a molecule(s) that has specific bactericidal activity against the opportunistic pathogen, Pseudomonas aeruginosa. Additionally, we have shown that the CFF inhibits P. aeruginosa biofilm formation, an important virulence factor that contributes to P. aeruginosa’s antibiotic resistance. Importantly, our data show that the CFF eradicates 20 h and 48 h established biofilm. We hypothesized that the CFF could modulate pro-inflammatory mediator secretion. To investigate the role of the CFF on tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) production, we measured TNF-α and IL-8 production from THP-1 monocytes and macrophages. Interestingly, our data show that the CFF could stimulate or reduce inflammatory mediator release, depending on the concentration of CFF and whether the cells had been prestimulated with LPS. To elucidate the mechanism underlying the immunomodulatory activity of the CFF, we conducted luciferase assays using a NF-κB reporter THP-1 cell line. Here, we provide evidence that the CFF modulates inflammatory mediators at least in part by regulating NF-κB activation. In addition, we tested the effects of the CFF in a mouse model of P. aeruginosa wound infection. The CFF cleared P. aeruginosa bacterial load and reduced plasma inflammatory mediator production by 5 days. To investigate the bioactive molecule within the CFF, we performed sequential fractionation steps and LCMS to identify and characterize the bioactive molecule, which has a similar mass to charge ratio (m/z) as known polyunsaturated fatty acids (PUFAs) and modified fatty acids. Taken together, our findings provide evidence that L. acidophilus secretes a bioactive molecule that has bactericidal, anti-biofilm, and immunomodulatory activities.

Aims/hypothesis Modulation of gut microbiota has emerged as a promising strategy to treat or prevent the development of diferent metabolic diseases, including type 2 diabetes and obesity. Previous data from our group suggest that the strain Pediococcus acidilactici CECT9879 (pA1c) could be an efective probiotic for regulating glucose metabolism. Hence, the objectives of this study were to verify the efectiveness of pA1c on glycaemic regulation in diet-induced obese mice and to evaluate whether the combination of pA1c with other normoglycaemic ingredients, such as chromium picolinate (PC) and oat β-glucans (BGC), could increase the efcacy of this probiotic on the regulation of glucose and lipid metabolism. Methods Caenorhabditis elegans was used as a screening model to describe the potential synbiotic activities, together with the underlying mechanisms of action. In addition, 4-week-old male C57BL/6J mice were fed with a high-fat/high-sucrose diet (HFS) for 6 weeks to induce hyperglycaemia and obesity. Mice were then divided into eight groups (n=12 mice/group) according to dietary supplementation: control-diet group; HFS group; pA1c group (1010 colony-forming units/day); PC; BGC; pA1c+PC+BGC; pA1c+PC; and pA1c+BGC. Supplementations were maintained for 10 weeks. Fasting blood glucose was determined and an IPGTT was performed prior to euthanasia. Fat depots, liver and other organs were weighed, and serum biochemical variables were analysed. Gene expression analyses were conducted by real-time quantitative PCR. Sequencing of the V3–V4 region of the 16S rRNA gene from faecal samples of each group was performed, and diferential abundance for family, genera and species was analysed by ALDEx2R package. Results Supplementation with the synbiotic (pA1c+PC+BGC) counteracted the efect of the high glucose by modulating the insulin–IGF-1 signalling pathway in C. elegans, through the reversal of the glucose nuclear localisation of daf-16. In diet-induced obese mice, all groups supplemented with the probiotic signifcantly ameliorated glucose tolerance after an IPGTT, demonstrating the glycaemia-regulating efect of pA1c. Further, mice supplemented with pA1c+PC+BGC exhibited lower fasting blood glucose, a reduced proportion of visceral adiposity and a higher proportion of muscle tissue, together with an improvement in the brown adipose tissue in comparison with the HFS group. Besides, the efect of the HFS diet on steatosis and liver damage was normalised by the synbiotic. Gene expression analyses demonstrated that the synbiotic activity was mediated not only by modulation of the insulin–IGF-1 signalling pathway, through the overexpression of GLUT-1 and GLUT-4 mediators, but also by a decreased expression of proinfammatory cytokines such as monocyte chemotactic protein-1. 16S metagenomics demonstrated that the synbiotic combinations allowed an increase in the concentration of P. acidilactici, together with improvements in the intestinal microbiota such as a reduction in Prevotella and an increase in Akkermansia muciniphila. Conclusions/interpretation Our data suggest that the combination of pA1c with PC and BGC could be a potential synbiotic for blood glucose regulation and may help to fght insulin resistance, diabetes and obesity.

Acute intermittent porphyria (AIP) is a metabolic disorder caused by mutations in the porphobilinogen deaminase (PBGD) gene, encoding the third enzyme of the heme synthesis pathway. Although AIP is characterized by low clinical penetrance (~1% of PBGD mutation carriers), patients with clinically stable disease report chronic symptoms and frequently show insulin resistance. This study aimed to evaluate the beneficial impact of nutritional interventions on correct carbohydrate dysfunctions in a mouse model of AIP that reproduces insulin resistance and altered glucose metabolism. The addition of spores of Bacillus coagulans in drinking water for 12 weeks modified the gut microbiome composition in AIP mice, ameliorated glucose tolerance and hyperinsulinemia, and stimulated fat disposal in adipose tissue. Lipid breakdown may be mediated by muscles burning energy and heat dissipation by brown adipose tissue, resulting in a loss of fatty tissue and improved lean/fat tissue ratio. Probiotic supplementation also improved muscle glucose uptake, as measured using Positron Emission Tomography (PET) analysis. In conclusion, these data provide a proof of concept that probiotics, as a dietary intervention in AIP, induce relevant changes in intestinal bacteria composition and improve glucose uptake and muscular energy utilization. Probiotics may offer a safe, efficient, and cost-effective option to manage people with insulin resistance associated with AIP.

Marine macroalgae (seaweeds) are ecologically and economically important organisms. However, with increasing environmental stress, disease in seaweeds is likely to become more frequent and/or severe, leading to a reduction of both wild and farmed populations. Eukaryotic hosts (e.g., from humans, terrestrial plants to seaweeds) are associated with microbial communities (microbiota) that play critical roles in host health and fitness. However, to what extent microbiota can be harnessed for seaweed disease management and the influence of host immunity on the native microbiota remains underexplored. Here I identified over 30 bacterial isolates that can antagonise opportunistic pathogens responsible for a bleaching disease observed in the red seaweed Delisea pulchra. Subsequent in vivo testing found inoculation with either a Pseudoalteromonas sp. strain PB2-1 or Phaeobacter sp. strain BS52 (later BS52) significantly reduced bleaching occurrence in D. pulchra, even in the presence of a known pathogen. Analysis of the D. pulchra associated bacterial community post-infection suggested that disease protection afforded by BS52 was likely due to its mitigation of dysbiosis rather than direct inhibition of the pathogen. Using comparative genomic analysis of BS52 with closely related commensal strains I identified putative functions such as prophage induction and antibiotic production that may contribute to the protective ability of BS52. Interestingly I found that inoculation with the disease protective bacteria from D. pulchra reduced the risk of tip bleaching disease in the geographically and phylogenetically distinct seaweed Agarophyton vermiculophyllum, suggesting these bacteria could act as general probiotics for seaweeds. Moreover, through the application of different bacterial community assessment tools (amplicon-sequencing, epifluorescence microscopy, and qPCR) I found that elicitation of seaweed immunity reduced epibacterial loads while causing minor changes in the community composition and structure, suggesting the seaweed immunity may also function to control the microbiota. Overall, my thesis demonstrated the feasibility of manipulating microbiota to reduce disease in seaweeds and advanced our understanding of the roles played by both symbiotic bacteria and the host immunity in maintaining holobiont homeostasis. It is hoped that these findings will benefit disease management in seaweeds with future applications for conservation and aquaculture.

Background: Dry eye is considered one of the most common ocular surface diseases. Millions of people throughout the world are affected by this symptomatic condition. Dry eye disease has a direct effect on the quantity and/or quality of the tear film layer and can lead to ocular discomfort, visual disturbance, tear film instability and finally ocular surface inflammation. In addition, it can contribute to a decrease in quality of life and because of its high prevalence, it is a critical public health issue. Emerging research suggests that dry eye disease has many distinguishing features of a chronic inflammatory disorder. The gut microbiome, through the production of metabolites, mucosal mediators, and systemic immune regulation, plays an important role in the regulation of low-grade chronic inflammation in different parts of the body, including the eye. Probiotics (live microorganisms provide health benefits) and prebiotics (non-digestible food ingredients that boost the function of beneficial microorganisms such as probiotics) are increasingly being used to regulate gut dysbiosis associated with inflammatory chronic disease. This thesis aimed to explore the potential of oral probiotics and prebiotics in reducing the severity of signs and symptoms of dry eye disease through systemic and localised (ocular) immune function modulation. Methods: This was a double-masked, randomised controlled clinical trial in which a total of 41 participants with dry eye received either the probiotic and prebiotic supplements, or their respective placebos, for a duration of four months. The participants were followed up at one month and four months after commencing treatment, and again one month after treatment cessation. Dry eye symptoms and signs were evaluated using the Ocular Surface Disease Index (OSDI), Dry Eye Questionnaire 5 (DEQ-5), osmolarity, non-invasive keratograph break-up time (NIKBUT), ocular surface staining, tear meniscus height (TMH), lipid layer thickness, and conjunctival redness. Flush tears were collected and assayed for the bio-inflammatory proteins MMP-9 and TIMP-1, and blood samples for serum C-reactive protein (CRP). Corneal nerve features and dendritic cell (DCs) counts were also derived from images obtained by in-vivo confocal microscopy. Ocular and nasal swabs and faecal samples were collected to assess the microbiome before and after the application of study interventions by culture-dependent and independent methods. Results: After four months, the average OSDI score of the treatment group was significantly better compared to that of the controls. The NIKBUT and TMH did not change significantly with treatment but reduced significantly for controls. There were no significant changes in CRP, MMP-9, TIMP-1, or MMP-9: TIMP-1 ratio between baseline and 4-months in either group. Nerve fibre width was reduced in the treatment group and there was an increased proportion of immature DCs in the control group. In ocular swabs evaluated by culture-dependent method, after administration of oral prebiotics and probiotics the number of CFU/swab and the abundance of staphylococcus genera reduced in the treatment group but not in the controls. The CFU/swab in nasal swabs did not change significantly from the baseline to the fourth month visit in either group. No significant changes were found at the phyla level of the ocular and faecal samples evaluated by the culture-independent method. Conclusion: The results of this study suggest that probiotics and prebiotics may be useful in the management of dry eye disease by reducing symptoms and stabilising clinical signs. This study involved comprehensive monitoring of symptoms, signs, systemic and ocular inflammation, gut, and ocular microbiome for the duration of the study. Future investigations are necessary to explore whether the effect of probiotic and prebiotic can be maximised by changing the dosage, or formulation, and whether they are beneficial in other populations with dry eye disease.

Room temperature liquid metals (LMs) are an emerging class of materials that shows metallic conductivity and maintains liquid-like properties, resulting in applications ranging from soft electronics to biomedicine. However, LMs usage for biomedical applications has been hindered due to their high surface tension that leads to low colloidal stability – a critical parameter for their integration into biological systems. As such, interfacial engineering of LMs is an active area of research. Recently, surface engineering using natural phenolics has become an intriguing strategy to modify and incorporate new properties into functional systems. Furthermore, phenolic compounds have been at the forefront of various surface functionalization methods because of their natural abundance and diverse physicochemical properties. This thesis will focus on the combination of natural phenolic compounds and LMs, harnessing their synergistic properties to create nanostructured films for biomedical applications. First, LMs were used to trigger the oxidative self-polymerization of phenolics. The study shows that LMs trigger the generation of highly active radical species causing the oxidation of phenolics and their deposition on different substrates. Such nanocoatings demonstrate excellent antioxidant and antibacterial properties. Second, the self-assembly properties of phenolic compounds are used to prepare and stabilize LMs nano-dispersions. This stabilization possibly occurs by coordination complexation and oxidative polymerization of phenolic compounds. Moreover, the resulting composite can be incorporated into a porous insulating matrix such as cellulose and can be transformed into conductive paper with potential applications in electronics. In the third project, a substrate-independent coating strategy using a natural-phenolic complex and LMs nanoparticles is developed. The coating network is formed via the coordination crosslinking of the phenolic by group (IV) transition metal ions, and the LMs nanoparticles are encapsulated in situ into the coatings as a Ga3+ reservoir. The obtained nanocoatings displayed high biocompatibility, antioxidant, and antibacterial properties. Finally, this thesis presents a cell-mediated catalytic process to engineer nanoshells on individual living cells such as probiotics. This process leverages the cell environment to induce oxidative polymerization of phenolic compounds, and simultaneously assemble to form phenolic nanocoatings. Moreover, the nanoencapsulated probiotics display improved gastric tolerance, enhanced adhesion, and high antioxidant activity.

Feeding probiotics has been shown to improve the health and production characteristics of dairy calves. The objective of this trial was to evaluate the effect of a probiotic fed daily on health outcomes, average daily gain, frame growth, feed efficiency, and intake when fed to Holstein heifers from birth to 180 d. A total of 324 Holstein heifer calves from a California dairy were enrolled within 48 h of birth into two treatment groups: 1) control group (CON: 176 calves) were given 0.5 g of lactose added to milk once daily from birth to weaning (60 d) and then 0.75 g in grain from weaning to 180 d and 2) probiotic group (PRO: 153 calves) were given 0.5 g (1.1 × 1010 CFU2/g) of probiotic in milk once daily from birth to weaning and then 0.75 g (1.65 × 1010 CFU2/g) of probiotic in grain from weaning to 180 d of a B. subtilis, B. lichenformis, L. animalis, and P. freudenreichii probiotic (Bovamine Dairy Plus, Chr. Hansen, Milwaukee, WI). The lactose powder was given to CON to balance the lactose content in the probiotic treatment and to ensure no difference in the handling of milk bottles. The PRO ADG was lower during the hutch period (1 - 91 d) and higher fecal shedding of Clostridium at 21 d and 42 d collections. There were no differences in all other health and performance outcomes.

The transition period is a crucial stage in the lactation cycle and can lead to significant metabolic changes in cows. The use of nutritional interventions, such as direct-fed microbials, may assist cows during the transition from pregnancy to early lactation, reducing the occurrence of metabolic disorders and improving overall health. The primary goal of this study was to assess the effects of a commercial rumen-derived directfed microbial (DFM) product (Galaxis™ Frontier (GF) Native Microbials Inc., CA, USA) on various factors such as performance, blood biomarkers, rumen fermentation, and bacterial population in dairy cows during the transition period up to 100 d in milk (DIM). Overall, the results showed that rumen derived DFM supplementation could promote positive responses on performance, such as milk yield and feed efficiency. In the rumen, the DFM product contributed to increased butyrate and valerate, accompanied by an improved ruminal abundance of lactic acid-utilizing bacteria. Moreover, DFM supplementation may have influenced lipid metabolism, leading to greater oxidative stress and inflammation within non-pathological levels. In conclusion, our study suggests that supplementing DFM during the transition period to mid-lactation can positively impact lactation performance and the rumen environment, indicating an overall beneficial effect.

The current work describes the capability of casein-chitosan microparticles to encapsulate Lactobacillus plantarum (CECT 220 and WCFS1 strains) and evaluates their ability to target the distal areas of the gut and to stimulate the immune system. Microparticles were prepared by complex coacervation, between sodium caseinate and chitosan in an aqueous suspension of the bacteria, and dried by spray-drying. In order to increase the survival rate of the loaded bacteria, microparticles were cross-linked with one of the following cross-linkers: tripolyphosphate, calcium salts or vanillin. Overall, microparticles displayed a mean size of about 7.5 μm with a bacteria loading of about 11 Log CFU/g, when cross-linked with vanillin (MP-LP-V). For conventional microparticles, the payload was 10.12 Log CFU/g. The storage stability study at 25 ◦C/60% RH, MP-LP-V offered the highest degree of protection without signif- icant modification of the payload in 260 days. Compared with control (aqueous suspension of bacteria), MP-LP-V also displayed a significantly higher degree of protection against probiotic inactivation in simulated gastric and intestinal fluids. In vivo results evidenced that microparticles, orally administered to rats, were able to reach the distal ileum and colon in about 4 h post-administration. Additionally, the effect of the daily administration of 107 CFU/mouse of MP-LP-V, for 3 weeks, induced an immunomodulatory effect characterized by an important enhancement of Th1 and Th17 responses. In conclusion, these microparticles seem to be a promising strategy for increasing survival and efficacy of probiotics, allowing the formulation of cost-effective and more stable and effective probiotic-based nutraceuticals.

The composition of the gastrointestinal microbiota has profound influences on overall health and on host-microbe interactions within humans. Dietary interventions, and consumption of health-promoting components, such as probiotics and prebiotics, are of interest for their influence on microbiome composition. Functional dietary components are of special importance in the diet of infants because they can be fundamental to establishing a healthy microbiome from the onset of life. An infant diet consisting of mother’s-own breastmilk is considered to the ‘gold-standard’ for infants, however, for many complex and personal reasons, not all infants are able to breastfeed. Breastfed infants and formula fed infants differ not only in their gastrointestinal microbiota but can also differ in developmental benchmarks and long-term health outcomes. Therefore, there is a scientific need for both researchers and industry professionals to identify components within breastmilk to be synthesized and incorporated into infant formulas for their protective, immunological, or bioactive roles, and to ultimately generate products that are more similar to human breastmilk. In this thesis we assess two functional components of diet, live microbe-containing fermented foods and the milk fat globule membrane for their impact on gastrointestinal composition and possible effects on human health. Advisors: Jennifer M. Auchtung and Robert W. Hutkins

Abstract: Introduction Patient engagement as a major component of patient-oriented research (POR) emphasizes that patients should be engaged not only as participants, but also as research partners. Having patient perspectives to inform the content and relevance of patient-reported outcome measures (PROMs) is now a requirement in many regulatory body guidelines. Increasing use of PROMs in pediatric populations has been documented frequently. As such, child and adolescent involvement to establish content validity of pediatric instruments has been recommended. Minimal important difference (MID) is defined as the smallest difference/change which patients perceive as important. Historically, healthcare providers have had the sole role in MID estimation. However, over time, calls to incorporate patient perspectives have emerged. Recent calls to determine MID from the patient’s perspective are especially relevant for therapies that are accessed by consumers without a prescription (e.g., probiotics). According to the latest Cochrane systematic review, probiotics have moderate protective effects in preventing antibiotic-associated diarrhea (AAD) in children. However, no studies reported seeking the perspective of children or parents about the MID of probiotics in preventing AAD and there is no specific validated instrument available to measure the incidence and severity of pediatric AAD. The objective of this thesis was to investigate whether and how children and/or parents have been engaged in developing PROMs and determining MID, and to use patient-centered approaches to 1) inform the MID of probiotics in prevention of pediatric AAD and 2) to validate an instrument for the measurement of incidence and severity of AAD in children. Methods Two review studies were conducted. In chapter 2, the method, stage, and level of engagement of the studies developing a new PROM in the pediatric population which engaged children and/or their parents in the process of development were described. In chapter 3, characteristics and findings of the studies reporting patient involvement in determining MID of any intervention were described. In the survey study described in chapter 4, parents of children presenting to the emergency department of a children’s hospital and pediatricians were approached to develop parent and pediatrician-derived MID. In chapter 5, with the help of a patient advisory group, an instrument was developed to measure the incidence and severity of pediatric AAD. The internal consistency and convergent validity of the instrument were examined in a prospective observational study in children (birth to 17 years old) newly prescribed antibiotics or on antibiotics for ≤ 7 days. Results Both review studies showed scarcity of evidence in engagement of children/parents either in developing pediatric PROMs or determining MID. The survey study showed good agreement between parents and clinicians regarding MID of probiotics in preventing pediatric AAD. Half of the participants in both groups reported they would use probiotics if it reduced the risk of AAD by 39%. The most important outcomes to parents and clinicians in measurement of pediatric AAD were identified. In the PAAD instrument development study, we found a broad range of incidence risk of AAD (27%-83%) by applying four different definitions of diarrhea. Cronbach’s α for the severity scale was less than 0.7. A high correlation was found between the PAAD severity score and numerical rating score of diarrhea severity reported by parents (r>0.5). Conclusion This doctoral dissertation showed that existing evidence regarding child/family engagement in PROM development and MID determination is sparse and at a preliminary stage. The estimated MID of probiotics in preventing pediatric AAD will help in sample size calculation and interpretation of results of future randomized clinical trials. Lastly, the PAAD instrument developed, and validity tested in this thesis will enable accurate measurement of pediatric AAD in future studies.

Since 1945, the market for broiler production has evolved from a collection of smallholder poultry farms into an industry worth billions of dollars. Poultry production has not gone without challenges. Lameness in young broilers is presently a serious issue in the poultry industry for reasons of animal health and welfare, as well as significant lost production and revenue. Bacterial chondronecrosis with osteomyelitis (BCO) is a major form of lameness in broilers. This dissertation contains information about (i) the influence of Staphylococcus agnetis, Staphylococcus saprophyticus, Staphylococcus epidermidis and Enterococcus faecalis on inducing BCO lameness, (ii) evaluation of of probiotics for reducing lameness in a BCO challenge model through promoting gut integrity, and (iii) evaluation of commercial symbiotic for decreasing the number of broiler chickens that develop of BCO lameness. The discovery of dietary supplements that have the potential to promote gut integrity could be crucial to the prevention of BCO. We demonstrated that certain probiotics could reduce the risk of lameness. We investigated one human isolate of Staphylococcus aureus as well as four types of bacteria that were recovered from lame birds: S. agnetis, S. saprophyticus, S. epidermidis, and Enterococcus faecalis. All these bacteria were tested for their ability to induce lameness on wire flooring. Birds that are reared on litter floors are susceptible to developing lameness when even a small amount of bacterial pathogen is introduced into their drinking water. It's possible that the litter-flooring model can more accurately represent broiler operations. Commercial symbiotic have been put through a series of tests to determine whether or not they can decrease the number of broiler chickens that develop BCO lameness. The chickens in this trial were divided into five treatment groups: a control group; three groups receiving symbiotic; and one group raised on wire flooring to stimulate BCO lameness. Probiotics and other feed supplements were demonstrated to reduce lameness in broiler chickens by 20–25 percent in these trials.

Bacterial vaginosis (BV) is a condition in which healthy lactobacilli are replaced by an overabundance of pathogenic bacteria in the female reproductive tract. Current antibiotic treatments often fail to “cure” infection, resulting in recurrence in more than 50% of women, 6 months post-treatment. Recently, probiotics have demonstrated promise to restore vaginal health; however, as with other active agents, delivery requires once-to-twice daily administration. Recently, three-dimensional (3D)-bioprinting has enabled the fabrication of well-defined cell-laden architectures with tunable agent release, thereby presenting a novel approach with which to deliver probiotics. One promising bioink, gelatin alginate, was selected for study, due to its ability in other work to provide structural stability, host compatibility, viable probiotic incorporation, and nutrient diffusion. The focus of this study was to formulate and characterize 3D-bioprinted Lactobacillus crispatus (L.cr.)-containing gelatin alginate scaffolds for reproductive health applications. Different weight to volume (w/v) ratios of gelatin alginate were bioprinted to determine the formulation with the highest printing resolution, and different crosslinking reagents were evaluated for effect on scaffold integrity, via mass loss and swelling measurements. Additionally, post-print viability, sustained-release, and vaginal keratinocyte cytotoxicity assays were conducted. A 10:2 (w/v) gelatin alginate formulation was selected based on line continuity and resolution, while degradation and swelling experiments demonstrated the greatest structural stability with dual-crosslinking, showing minimal mass loss and swelling over 28 days. Last, 3D-bioprinted L.cr.-containing scaffolds demonstrated sustained-release of therapeutically-relevant levels of probiotics over 28 days, while maintaining the viability of vaginal epithelial cells. For the first time, this study shows that 3D-bioprinted scaffolds may provide a new alternative to sustain probiotic delivery with future goals to help maintain or restore female reproductive health after BV infection.

Microbial life is all around us and inside us in an unseen vast and varied ecosystem. For the past several decades, the microbiome field has attempted to characterize this microscopic web of life using next generation sequencing technology. It turns out that the number of host organism cells is generally outnumbered by the number of microbial cells that live on and within them. Moreover, these microbes collectively have two orders of magnitude more genes than their host organism. While the host organism’s genome stays constant during its life, the microbial community and its associated genes change much more frequently. Since microbial lifespans are sometimes measured in minutes, selective pressures in the host micro-environments can rapidly affect the evolution and gene content of the microorganisms present. Over the course of a lifetime, the host winds up with a unique set of microbes selected to fit their unique individual environment, from immune system quirks to specialized diets to biological rhythms. However, despite biological rhythms having always played a role in host behavior and survival, the impact of time on health has been a widely neglected realm until recently. The basic argument of this thesis is that time is an underappreciated but critical variable that impacts both experimental study design as well as host health.

Chicken is the preferred animal protein (meat) source worldwide, surpassing pork in production in 2021 and projected to continue in 2022. However, greenhouse gas (GHG) emissions have increased 11% from 2018 to 2019, with 55% coming from agriculture production. Another increasing environmental contaminant is ammonia, produced by birds due to the overconsumption of protein containing nitrogen. Changing the microbiota of birds could help to reduce the production of ammonia. The microbiota is influenced by many factors including age, disease, antibiotic usage, and diet; with the largest impact via the diet. Alterations to the microbiota can be achieved by a variety of feed additives, including prebiotics, organic acids, phytogenics, enzymes, bacteriophages, and probiotics while simultaneously improving performance and digestibility. Despite being commonplace as an animal feed additive, there is limited data showing how long layers and broilers need to be given probiotics to become established in the bird’s gut microbiota. Therefore, the goal of this study was to determine if a yeast strain (Saccharomyces cerevisiae) improved the effects of a probiotic mixture (Lactobacillus plantarum and L. rhamnosus) through an increase in production, a reduction of ammonia/nitrogen levels in the blood and manure, and an increase in digestibility. Additionally, results may highlight the week in which improvements were observed. Findings could help determine the minimum length of time for which a probiotic should be provided. One-day-old Cobb 500 broiler chicks (600) were fed one of the following diets: no alterations to the basal diet as a control (CON); probiotic S. cerevisiae, inclusion rate at 4.26 × 106 CFU/kg of feed (SCY); probiotics L. plantarum and L. rhamnosus, inclusion rate at 4.35 × 108 CFU/kg of feed for each (LPR); and a combination of L. plantarum and L. rhamnosus at 4.35 × 108 CFU/kg of feed for each, and S. cerevisiae at an inclusion rate of 4.26 × 106 CFU/kg of feed (SWL). The experimental design was completely randomized with pens as the experimental unit. There were four treatments, with five replicates, each with 30 broilers per pen (replicate). Performance was measured weekly as feed consumption, weight gain, body weight (BW), and feed conversion ratio (FCR) over six weeks. Accompanying biochemical analyses included lipase activity of the pancreas, liver weight, and uric acid (UA) concentration in liver on days 15, 29, and 43. In addition, albumin, total protein, UA, ammonia, and blood urea nitrogen (BUN) were measured in serum. The ammonium ion from manure and apparent ileal digestibility from digesta were also measured. Significance was determined at P < 0.05. Probiotics supplements produced no significant differences in biochemical analyses; however, there were significant temporal changes in performance. Feed consumption increased over time for all treatments (P = 2.00 × 10- 16). CON had lower weight gain in week 2 (P = 0.013). CON had the lowest BW in week 5 (P = 0.0008) and week 6 (P = 0.0124) compared to SWL. Future research into specific probiotic strains, with well-defined inclusion rates, and surrounding environmental analyses of present microbes are needed to ascertain effects of probiotics. Ultimately, work on confirmation of probiotics present in the digesta/ceca, how the probiotics alter the microbiota within the GI tract, and how probiotics alter the serum heterophil:lymphocyte ratio to further examine potential immune response to probiotics is needed.

Probiotics and botanical extracts are investigated as potential feed additives for weaned pigs to strengthen the intestinal function or overall immunity by improving nutrient digestibility and immunity in weaned pigs. Under proper manipulation, gut microbiota can additionally offer great physiological and immunological benefits to growing pigs, however, gut microbiota yet tends to be overlooked when assessing the viability of potential feed additives to weaned pigs under diarrheal stress. Therefore, three experiments were conducted to determine the impacts of various feed additives (Bacillus (B.) subtilis, B. amyloliquefaciens, and botanical blends) on gut microbiota of newly weaned pigs using a disease challenge model. The first study investigated modifications in fecal and intestinal microbiota of weaned pigs challenged with enterotoxigenic Escherichia coli (ETEC) and compared the gut microbiota when supplemented with B. subtilis DSM 25841 or carbadox, an antibiotic growth promoter. ETEC infection decreased (P < 0.05) microbial richness in feces on d 7 post-inoculation (PI) compared with d 0 before ETEC infection. Relative abundance of Firmicutes was decreased (P < 0.05) while Proteobacteria and Enterobacteriaceae were increased (P < 0.05) in fecal samples of pigs challenged with ETEC on d 7 PI than on d 0. Supplementation of carbadox reduced (P < 0.05) the relative abundance of Lactobacillaceae in feces on d 0 and 21 PI. Lactobacillus was more (P < 0.05) abundant on d 7 PI and Lactobacillaceae was greater (P < 0.05) in relative abundance on d 21 PI in feces from pigs supplemented with B. subtilis than with carbadox. Among intestinal segments, Enterobacteriaceae was more (P < 0.05) abundant in ileum than in jejunum and colon of weaned pigs on d 21 PI. Pigs supplemented with B. subtilis increased (P < 0.05) the relative abundance of gram-positive bacteria than pigs supplemented with carbadox, including Lactobacillaceae and Bifidobacteriaceae in ileal digesta on d 21 PI. Overall, the present study indicated that B. subtilis supplementation modulated the fecal and intestinal microbiota differently from carbadox. The modulation of gut microbiota might contribute to the reduced diarrhea and enhanced intestinal health of ETEC infected pigs. The second study investigated the supplementation of B. amyloliquefaciens on growth performance, diarrhea, systemic immunity, and intestinal microbiota of weaned pigs infected with ETEC and compared the efficacy of B. amyloliquefaciens vs. carbadox on weaned pigs. ETEC infection reduced (P < 0.05) average daily gain (ADG) from d 0 and 21 PI, gain:feed from d 14 and 21 PI, increased (P < 0.05) frequency of diarrhea during the entire experiment and white blood cell and lymphocyte counts on d 7 and 21 PI when positive control was compared with negative control. B. amyloliquefaciens or carbadox supplementation did not affect diarrheal frequency throughout the experiment. However, carbadox supplementation enhanced (P < 0.05) ADG from d 0 to 21 PI than positive control. Pigs fed with carbadox had greater (P < 0.05) monocyte percentage in serum samples on d 7 and 21 PI but lower (P < 0.05) neutrophil percentage on d 21 PI than pigs fed with B. amyloliquefaciens under ETEC challenge. Pigs supplemented with B. amyloliquefaciens had greater (P < 0.05) Shannon index in ileal digesta than pigs fed with carbadox on d 21 PI. Positive control pigs had a greater (P < 0.05) abundance of Bacteroidota and Proteobacteria and lower (P < 0.05) Firmicutes in ileal digesta than negative control on d 21 PI. Carbadox addition increased (P < 0.05) the relative abundances of Firmicutes and Clostridiaceae but reduced (P < 0.05) Bifidobacterium in ileal digesta compared with B. amyloliquefaciens on d 21 PI. The results of the present study indicated that supplementation of B. amyloliquefaciens tended to increase ADG and had limited effects on diarrhea of ETEC infected pigs. However, pigs fed with B. amyloliquefaciens exhibit milder systemic inflammation than control. B. amyloliquefaciens differently modified intestinal microbiota of weaned pigs, compared with carbadox. The third study characterized the impacts of two types of botanical blends on gut microbiota composition and serum and ileal mucosa metabolites in weaned pigs challenged with ETEC. Botanical blends (BB1 and BB2) were comprised of 0.3% capsicum oleoresin and 12% garlic oil extracted from different sources. Throughout the experiment, the relative abundance of Lachnoclostridium was decreased (P < 0.05) in feces as the age of pigs was increased from d -7 to d 21 PI. ETEC infection reduced (P < 0.05) the relative abundance of fecal Faecalibacterium and Prevotella on d 5 and 21 PI when positive control was compared with negative control. On d 21 PI, the relative abundance of Firmicutes and Lactobacillaceae were lower (P < 0.05) in feces of pigs fed with 100 ppm BB1 than pigs in positive control. Pigs supplemented with 100 ppm BB2 had a greater (P < 0.05) relative abundance of Lachnospiraceae in feces than pigs fed with 100 ppm BB1 on d 21 PI. Pigs in positive control had the highest (P < 0.05) Shannon and Chao1 indices in ileal digesta on d 5 PI. Pigs supplemented with 100 ppm BB1 had greater (P < 0.05) abundances of Enterobacteriaceae and Escherichia-Shigella in ileal digesta than pigs in positive control on d 5 PI. The relative abundance of Prevotellaceae was greater (P < 0.05) in cecum of pigs supplemented with 100 ppm BB1 than positive control pigs on d 5 PI. The metabolomics analysis revealed that ETEC infection downregulated pinitol, malic acid, and methionine on d 4 PI and upregulated methionine and guanosine on d 21 PI when positive control was compared with negative control. Supplementation of 100 ppm BB2 upregulated serum pinitol on d 5 PI and serum cholesterol and aminomalonic acids on d 21 PI compared with positive control. In ileal mucosa, asparagine was downregulated by 50 ppm BB2 in comparison to positive control. The present study indicated that supplementation of botanical blends modulated ileal microbiota and serum metabolomics profiles in weaned pigs under ETEC challenge. Overall, feed additives, including probiotics and botanical blends modified gut microbiota of weaned pigs challenged with ETEC to different extent, which may potentially contribute to reduced diarrhea and enhanced intestinal integrity and health of newly weaned pigs when these feed additives were supplemented into the diet.

Oral dysbiosis is an imbalance in the oral microbiome and is associated with a variety of oral and systemic diseases, including periodontal disease, caries, and head and neck/oral cancer. Although antibiotics can be used to control this dysbiosis, they can lead to adverse side effects and superinfections. Thus, novel strategies have been proposed to address these shortcomings. One strategy is the use of probiotics as antimicrobial agents, since they are considered safe for humans and the environment. Specifically, the gram-positive Lactococcus lactis, an endemic species to the oral and gut microflora, is able to produce nisin, which has been used worldwide for food preservation. Therefore, the objective of this study was to test whether the probiotic can promote a healthier oral microbiome in pathogen-spiked oral biofilms. We found that L. lactis can prevent oral biofilm formation, abrogate 48h pre-formed biofilms. Finally, we demonstrate that both treatments return the relative abundance and diversity levels back to healthy control levels. Thus, nisin and its probiotic (nisin-producing L. lactis) can be used to treat peri-implantitis and promote healthier oral biofilms, which may be useful for improving patient oral health.

Obesity and diabetes incidence rates are increasing dramatically, reaching pandemic proportions. Therefore, there is an urgent need to unravel the mechanisms underlying their pathophysiology. Of particular interest is the close interconnection between gut microbiota dysbiosis and obesity and diabetes progression. Hence, microbiota manipulation through diet has been postulated as a promising therapeutic target. In this regard, secretion of gut microbiota–derived extracellular vesicles is gaining special attention, standing out as key factors that could mediate gut microbiota-host communication. Extracellular vesicles (EVs) derived from gut microbiota and probiotic bacteria allow to encapsulate a wide range of bioactive molecules (such as/or including proteins and nucleic acids) that could travel short and long distances to modulate important biological functions with the overall impact on the host health. EV-derived from specific bacteria induce differential physiological responses. For example, a high-fat diet–induced increase of the proteobacterium Pseudomonas panacis–derived EV is closely associated with the progression of metabolic dysfunction in mice. In contrast, Akkermansia muciniphila EV are linked with the alleviation of high-fat diet–induced obesity and diabetes in mice. Here, we review the newest pieces of evidence concerning the potential role of gut microbiota and probiotic-derived EV on obesity and diabetes onset, progression, and management, through the modulation of inflammation, metabolism, and gut permeability. In addition, we discuss the role of certain dietary patterns on gut microbiota–derived EV profile and the clinical implication that dietary habits could have on metabolic diseases progression through the shaping of gut microbiota–derived EV.

Abstract: Background Short bowel syndrome (SBS) is the leading cause of intestinal failure (IF), wherein either the function of the intestines is suboptimal, or length has been reduced due to congenital or acquired conditions, such that parenteral nutrition (PN) is required for >6 weeks. The most common etiology in neonates is necrotizing enterocolitis (NEC), a condition resulting from disruption or poor establishment of the microbiota that allows inflammatory species to proliferate and cause tissue damage. Repeated rounds of antibiotics to treat central line infections and small intestinal bacterial overgrowth are necessary, but further exacerbate dysbiosis. Sepsis with gut-derived organisms and IF-associated liver disease, which is partly caused by bacterial translocation, are now the leading causes of mortality in children with SBS. Treatment for dysbiosis must replace beneficial organisms, rather than simply remove harmful ones, in order to support normal growth and development of children with SBS. Probiotics are one option that have had success in preventing NEC, but some Lactobacilli have been associated with inhibition of adaptation in some instances of single species supplementation, and in others with an increase in sepsis. In this experiment, we looked at using fecal microbial transplant (FMT) as a way of restoring a healthy diverse microbiota in a single treatment in neonatal piglets with SBS. Methods Neonatal male Duroc piglets aged 3-7 days were assigned to saline (SAL, n=12), fecal microbial transplant (FMT, n=12), or sow-fed control (SOW, n=6) treatment groups. SAL and FMT underwent 75% distal SBS surgery on day 0, and treatment on day 2 (100 mL saline or FMT material, respectively). Donor FMT material was collected from a healthy female gilt, filtered, stored at -80 °C, and thawed at 4 °C overnight before use. SOW piglets remained with their litter, and were terminated on day 7 along with their treatment piglet littermates. Tissue samples were collected at baseline and termination surgeries, as well as daily stool samples pre-treatment, post-treatment, and on the mornings of day 3-6. Microbial DNA was extracted, analyzed for 16S rRNA, and compared to established databases for taxonomy assignment. Percent relative composition of bacterial taxa was analyzed and compared between groups, as well as alpha and beta diversities. Results No piglets developed sepsis or experienced disease-specific mortality, and FMT was well-tolerated. FMT piglets showed increased Actinobacteria and Firmicutes families, and increased alpha diversity, compared with SAL post-treatment and on day 3, but these increases in general did not persist afterwards. FMT and SAL piglets had significantly different beta diversity post and day 3, but the Donor material was significantly different at all time points. SOW piglet term tissue sample alpha diversity in jejunum and colon, and beta diversity in the colon, was significantly higher than treatment piglets. Conclusion Piglets in this trial demonstrated no disease-specific mortality and no increase in sepsis rates compared with our previous SBS trials. Differences in bacterial taxa were measurable in stool samples only through the day after FMT treatment. It is possible this was due to inadequate dietary fiber sources for establishment of the donor communities, given the neonatal piglets were fed enteral nutrition (EN) that contained only simple carbohydrates. Differences in beta diversity between SAL and FMT were present in the colonic mucosal-associated bacteria at termination, indicating small numbers of some species were able to establish and survive. Supplementation with complex fiber sources accessible by butyrate-producing species should be added to EN in the future to aid in persistence of beneficial species.

Immediately post-hatch, broiler chicks are exposed to microbes that begin colonizing the gut, including environmental pathogens. One of the costliest enteric diseases in broiler production is necrotic enteritis (NE), caused by the ubiquitous opportunistic bacteria Clostridium perfringens (CP). With the worldwide reduction in antibiotic growth-promoters (AGPs), there is increased interest in natural alternatives to reduce disease and improve broiler health. The overall objective of the studies described herein was to apply probiotics or synbiotics to birds by in ovo application or orally before they leave the hatchery, then evaluate bird performance and various intestinal responses. Data were analyzed in JMP with LS Means to separate means with significance assigned at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. The first 21-day (D) study used 480 male Cobb 500 broilers randomly divided into one of four treatments using a 2x2 factorial design: a no-additive control (CTRL), a one-time oral application of synbiotic at the hatchery fed a basal diet (HS), an oral application of water at the hatchery with dietary synbiotics (DS), and a hatchery synbiotic plus dietary synbiotic (HSDS). Performance was measured on day-of-hatch (DOH), D3, D7, D14, and D21. mRNA abundance of various intestinal markers was measured at D7 and D21, including tight junction proteins ZO-1, ZO-2, and CLD-1; nutrient transporters SGLT1 and PepT1; and immune response markers TLR2, TLR4, and IL-10. HS lowered feed intake (FI) and feed conversion ratio (FCR) without lowering body weight (BW) from D14-21. There was greater abundance of PepT1 mRNA (P ≤ 0.1) and IL-10 mRNA (P ≤ 0.05) on D21 in HSDS. Second, a 21-day pilot study with 480 male and female Cobb 500 broilers was conducted to determine the optimum in ovo dosage level of a probiotic or synbiotic (PROB or SYNB) applied at embryonic day 18 (E18) with subsequent NE challenge using seven treatments: in ovo application of sterile water (CTRL), low (PROB-L or SYNB-L: 1x105 CFU), medium (PROB-M or SYNB-M: 1x106 CFU), or high (PROB-H or SYNB-H: 1x107 CFU) probiotic or synbiotic doses dissolved in sterile water. Performance measurements were taken on DOH, D4, D8, D14 and D21. On D8, NE lesion scores were not impacted by treatment. D8 ileal samples were taken for mRNA abundance of TLR4, IL-10, IL-1β, AvBD8, AvBD10, and AvBD13. SYNB-H had higher abundance of AvBD10 mRNA compared to CTRL (P ≤ 0.1), and higher IL-1β mRNA compared to SYNB-L (P ≤ 0.05). PROB-H and SYNB-H had better performance than the low and medium doses, but were not better than the CTRL. The high doses were chosen for subsequent studies. Third, a longer 42-day study using 1,630 Ross 708 male and female broilers was conducted consisting of the following six treatments. A negative control (NC): sterile water in ovo fed basal corn/soybean meal mash diet without NE challenge; antibiotic growth-promoter (AGP+): sterile water in ovo fed basal diet with virginiamycin (0.5 kg/MT) as an AGP with NE challenge; NC+: same as NC plus NE challenge; SI+: synbiotic in ovo fed the basal diet and NE challenged; SD+: sterile water in ovo fed basal diet supplemented with synbiotic (0.5 kg/MT feed) and NE challenged; and SID+: synbiotic in ovo fed basal diet with synbiotic (0.5 kg/MT feed) with NE challenge. Cumulatively, SID+ had lower FI and FCR than NC+, but no change in BW or BWG. The combination treatment (SID+) often had an additive effect compared to SD+ or SI+ alone on mRNA abundance and D7 cecal fatty acid profiles. SD+ and SID+ also had higher D42 lean:fat ratios compared to NC+. Last, a 42-day study was conducted using 1,630 male and female Ross 708 broilers and the in ovo application of probiotics and subsequent NE challenge with five treatments. NC: sterile water in ovo, fed basal corn/soybean meal mash diet without NE challenge; AGP+: sterile water in ovo, fed basal diet with virginiamycin (0.5 kg/MT of feed) as AGP with NE challenge; NC+: NC treatment, with NE challenge; PI+: probiotic in ovo, fed basal diet, with NE challenge; PD+: sterile water in ovo, fed basal diet supplemented with probiotic (1.3 kg/MT of feed), with NE challenge. The use of probiotics in this study had little effect on performance, lean:fat ratios, and cecal fatty acid profiles, but PD+ increased mRNA abundance of D14 TLR2, D14 TNF-α, and D42 LEAP2 in cecal tonsils compared to controls. PI+ increased mRNA abundance of D7 and D42 MUC2, D7 LEAP2, and D42 TNF-α in the ileum. PI+ increased mRNA abundance in the cecal tonsils of D7 TLR2 and D42 TNF-α. These studies yielded interesting results about probiotics and synbiotics during a NE challenge by evaluating performance, intestinal immune responses, and fatty acid profiles in the ceca of broilers. In conclusion, the probiotic in this study did not improve broiler health during a NE challenge, but synbiotic use in ovo and continuation in the feed showed improvement over in ovo or dietary application alone. Synbiotic improved FCR over a challenged control, and altered mRNA abundance in the small intestine.

Perturbations to the gut microbiome may boost the use of addictive substances such as cocaine. In animals, antibiotic-induced gut microbiome depletion heightens cocaine-seeking, although adolescent rats appear less sensitive than adults to these perturbations. The purpose of this study was to use adolescent vs. adult male Wistar rats to test two hypotheses: 1) probiotics will reduce cocaine-seeking that has been heightened by antibiotic treatment; and 2) probiotics administered throughout behavioral testing will reduce cocaine-taking and/or -seeking. Rats were catheterized and self-administered cocaine (0.36mg/kg i.v.), then tested for extinction and reinstatement of cocaine-seeking after abstinence. Some rats received antibiotics during self-administration followed by probiotics during abstinence, while others received probiotics throughout testing. Probiotics during abstinence attenuated antibiotic-heightened cocaine-seeking in adults but did not decrease adolescent responses. Prophylactic probiotics did not attenuate cocaine behaviors significantly. Probiotics may be useful in addiction treatment, but these results do not support their use in prevention.

Results from the present study found for the first time that the use of bacterial strains of Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium thermophilum, and Enterococcus faecium from the commercial product PrimaLac® had the potential to act as a possible probiotic for juvenile red drum. The addition of PrimaLac® probiotics [whether as a water-soluble probiotic (WSP) or in a probiotic enhanced starter feed (PESF)] reduced mortality (%M), enhanced growth rates (MW, LT, SGR, and DGR), improved feed conversion efficiencies (FCEs), and physical condition factors in the juvenile red drum. Improvement was quantified using external morphological condition indices (MCIs) and blood physiological condition indices (PCIs). Results showed a strong positive relationship between MCIs and PCIs, which suggested that probiotics treated fish were in better health (lower MCIs) with lower fasting blood glucose and lactate levels than control fish. Addition of probiotics also resulted in improved water quality (lower nitrate, nitrite, and ammonia) in the treatment tanks compared to controls. Application of PrimaLac® probiotics on the morphology and histology of three different regions of the intestine (proximal, mid-and distal) improved intestinal length (Li), mass (Mi), and digesta mass (Md). In 5μm histological sections examined for differences among treatments (probiotics vs. controls), five variables within each intestinal region were also quantified: (i) perimeter ratio (PR) between the internal perimeter (IP) of the intestinal lumen and the external perimeter (EP) of the intestine where, PR = IP/EP; (ii) width of lamina propria (wLP); (iii) thickness of the muscle layer (tM); (iv) the number of goblet cells per segments (GC); and (v) height of enterocytes (hE). Overall, Li and Mi significantly increased by 13.4 ± 1.2% and 11.8 ± 0.9%, respectively, and Md decreased by 33.2 ± 1.21% compared to controls. Significant increases in microvilli heights, PR (by 21.3 ± 1.2%), tM (by 18.4 ± 0.9%), and increased numbers of GC (by 35.1 ± 2.8%) provided evidence that bacteria species from PrimaLac® (whether in WSP or PESF) affected the intestinal histomorphology of juvenile red drum. Collectively, these changes were partially responsible for increased feed assimilation (supported by reduced digesta) and improved overall juvenile red drum growth and condition. These results will add significantly to the limited database on how probiotics, as possible standard practice at the fish farm, may offer significant benefits for fish production and fish health. Addition of probiotics will also be of benefit to most aquaculture species and not only for red drum, but also for other high-value carnivorous species, such as Atlantic salmon, without threat to natural populations or damaging fisheries-based ecosystems that are already on the edge of collapse from over fishing and pollution.

Salmonella and Campylobacter are leading bacterial agents both nationally and globally, making them high public health concerns. Both are significant important zoonotic pathogens commonly found in livestock. Poultry and poultry products are the most common sources of human infections. Infected poultry show little to no clinical signs, and risk the chance of entering the food system, potentially contaminating consumers. Direct links between the high load of bacteria in the chicken intestinal tract and the high contamination of poultry carcasses support the need for pre-harvest control. Previously, antibiotics were used to control bacterial infections and growth; but increasing occurrence of antibiotic resistant bacteria has caused laws and practices to shift. Targeting the control of foodborne pathogens in the pre-harvest stage can improve animal welfare and public health. Alternative treatment methods are needed to combat Salmonella and Campylobacter in production animals, improve antibiotic stewardship, and subsequently strengthen the economy.We discovered the antimicrobial efficacy of Lactobacillus acidophilus (LA), Lactobacillus rhamnosus GG (LGG), and Bifidobacterium animalis subsp. lactis (Bb12) in vitro. More importantly, we showed LGG significantly reduced Salmonella in the chicken cecum by 1.9 logs (PSalmonella serovars in an agar well diffusion assay. Characterization of antibacterial activity of LGG revealed that although organic acids are present, the most crucial part of inhibition are the heat and protease stable peptides that were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS).After which, we tested the efficacy of Lactobacillus rhamnosus GG (LGG) derived small peptides (P1-NPSRQERR, P2- PDENK, and P4-MLNERVK) against Salmonella Typhimurium (ST) in chickens and examined their antibacterial effects against Salmonella Enteritidis (SE) and other Salmonella serovars in vitro. The antagonistic effects of peptides were heat and protease resistant and antibacterial activity is likely due to their effect on Salmonella membranes. Although, P1, P2, and P4 inhibited ST, SE, and other Salmonella serovars in vitro at 18mM, 15mM, and 18mM respectively, they did not affect the growth of Gram-positive commensal bacteria. P1and P2 at 50mg/kg successfully reduced the colonization of ST by 2.2 logs and 1.8 logs, respectively (P< 0.05), in chickens 7 days post infection. P1, P2, or P4 had no adverse effects on chicken cecum microbial communities, but birds not treated and challenged with Salmonella showed differences in microbial richness and evenness compared to the unchallenged birds. Confocal and transmission electron microscopy revealed that peptides attacked the membrane of Salmonella causing its flaccidity, sloughing, or disruption.Lastly, we analyzed the ability of Salmonella Typhimurium derived recombinant attenuated Salmonella vaccines (RASV) to reduce the colonization of C. jejuni in chickens. One-day-old Salmonellaand Campylobacter free chickens were orally immunized with RASV constructs (2x108 CFU/ bird), and a booster was given 10 days later. Birds were challenged with a cocktail of 5 C. jejuni strains on day 18. Colonization load was determined by enumerating C. jejuni in the cecum of the birds. Four trials were conducted analyzing the RASVs effect on Campylobacter load in the cecum. Our data suggested that vaccine efficacy was dependent on amount of challenge dose and antigens present in vaccine. RASV90+201 and RASV131 showed consistent reduction across trials 2,3, and 4. The best reduction in Campylobacter load occurred in trial 3 with 103 Campylobacter challenge dose. At 17 days post infection, pG8R-86 (cjaA), pG8R-90+201 (FlaA), and pG8R-131 (FlaA) low challenge dose groups yielded significant reductions in Campylobacter load (approximately 3.7, 2.4, and 1.7 logs, respectively, compared to vector control). Additionally, in multiple trials RASV-90 showed a significant induction of IgY antibody titers compared to the untreated and unchallenged vector control group. Overall, these studies revealed that antibiotic independent novel therapeutics and vaccines are promising alternatives to control Salmonella and Campylobacter in chickens.

Probiotics are certain living microorganisms that have to be in the human body for health benefits. The administration of probiotics can treat different metabolic disorders and other health issues. Bacteria are sensitive to their environment. The viability of probiotics is affected by adverse conditions such as pH and temperature. Probiotic delivery can be in different modes. Nowadays, food products containing probiotic mixtures are of great interest by conumers. These products are for the administration of probiotics to improve the gut microflora. Oral administration of probiotics can improve not only the gut health, but it is a holistic approach of health improvement also. The immunomodulation ability of probiotics is beneficial for treatments of a number disorders and skin improvement. Objective of this research was to study the effects of a novel food gel(NG) on the viability of Lactobacillus acidophilus and antimicrobial activity against acne causing bacteria Propionibacterium acnes. An acne treatment ointment had taken as a control to compare the microbial analysis. The results showed, a negative effect of gel on the viability of Lactobacillus acidophilus, but a positive result was obtained for the antimicrobial activity of gel against Propionibacterium acnes. The flow characteristics were depended on the temperature and was suitable for a topical gel formulation. Further studies must be done for formulate new food gel as a topical acne treatment ointment.

Recent research has highlighted the connection between dysbiosis of the gut microbiome and its role in the development of Irritable Bowel Syndrome (IBS). Over the last few years, probiotics have grown in popularity as a potential treatment option in IBS. However, most current probiotic studies are limited due to small study populations, older median age, and short study time duration. The proposed study will be a multicenter, randomized, double-blinded placebo controlled study comparing the multi-strain probiotic Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Escherichia coli DSM17252 to placebo in patients diagnosed with IBS based on Rome IV criteria over a 3-months duration. The study participants will have a baseline evaluation and a final evaluation at the end of the 3-months duration. The primary outcome will be the IBS Symptom Severity Score and the secondary outcomes will the individual components of the IBS Symptom Severity Score. The results of this study will begin to fill gaps in the current knowledge of the use of probiotics in the treatment of IBS.

Bifidobacterium taxa are associated with the breastfed infant gut, namely, due to the bifidogenic nature of human milk’s oligosaccharide fraction. This association provides a model of resource-driven microbial assemblies as human milk oligosaccharides (HMO) are accessible to microbes found in the infant gut but the infant cannot use HMO as a nutrient resource. The catabolism strategy (intracellular versus extracellular) and breadth of HMO structures utilized by a Bifidobacterium microbe impacts fitness and competitive nature in a microbial community. Previous work focused on profiling Bifidobacterium genetic diversity and in vitro metabolism of human milk oligosaccharides (HMO). Chapter 1, a mini-review, attempts to synthesize research, focusing on how functional capabilities provide a fitness advantage to certain Bifidobacterium species when applied to an infant gut. The addendum contains primary research on how Bifidobacterium pseudocatenulatum strains forage for fucosylated HMO.The principal goal of this dissertation was to understand if a Bifidobacterium population could be enriched in a complex microbial community and if persistence could be achieved through introduction of an HMO nutrient resource, 2’-fucosyllactose (2’FL). Chapter 2 introduces how supplementation of 2’FL provides a fitness advantage to a Bifidobacterium pseudocatenulatum strain possessing a fucosylated HMO gene cluster in a mouse model. Even when high colonization resistance was present, 2’FL, a privileged nutrient resource, facilitated persistence and ameliorated a DSS-induced colitis model. Chapter 3 further characterizes the Bifidobacterium pseudocatenulatum persistence mouse model. Bifidobacterial metabolite products from 2’FL catabolism were found locally and systemically, although enrichment of those metabolites was dependent on high levels of B. pseudocatenulatum persisting in the mouse gut. This study also introduces the concept of mice categorized as responders and non-responders in this work. Additional Bifidobacterium species were tested for persistence in a mouse model. These strains evaluated hypotheses surrounding catabolism strategies and the necessity of HMO utilization genes. In Chapter 4 subjects were categorized as responder and non-responder depending on Bifidobacterium persistence success, which was varied. This variance could not be determined by the presence of any one bacterial taxa at baseline. However, future studies could assess the functional capacity of the baseline microbiota to test if certain niches were open in mice categorized as responders. This work furthers our understanding of Bifidobacterium strain-specific fitness differences in complex microbial communities. When choosing bacterial species with the objective of persistence or engraftment, researchers should first understand the molecular mechanisms for resource capture and catabolism, and the baseline microbial community’s functional capabilities.

In this study, a novel value-added dairy-based health formulation was developed using whey protein hydrolysate and probiotic organisms. In the first part of the study, different forms of whey proteins, concentrate WPC80, isolate WPI90, and hydrolysates WPH10, WPH15 and WPH20 were screened for bioactivities (antimicrobial, antioxidant, and antihypertensive activity). Hydrolysate WPH10, exhibiting the highest bioactivities was conjugated with maltodextrin. A batch of 2L conjugated solution was spray dried in a Niro drier with an inlet and outlet temperature of 200⁰C and 90⁰C, and alternatively, freeze dried at −80 °C under 50 mTorr vacuum. The bioactivities of the conjugated samples were then assessed. Overall, the hydrolysates showed significantly higher bioactivities as compared to concentrate and isolate. The conjugated WPH10 solution demonstrated higher antimicrobial, and antioxidant activity, whereas no significant difference in the antihypertensive activity was observed, as compared to WPH10 alone. Subsequent spray and freeze drying of the conjugate solution exhibited even higher antimicrobial, and antioxidant activities, while retaining the antihypertensive activity. Overall, the results indicate the ability of the WPH-maltodextrin to retain the bioactivities after conjugating with a reduced carbohydrate. In the second part of the first study, conjugated whey protein hydrolysate (WPH10), was used as an encapsulant to entrap probiotic cultures; Bifidobacterium animalis subsp. lactis ATCC27536 and Lactobacillus acidophilus ATCC4356, through a spray drying process. Each culture was added in the conjugated solution at ratio of 1:1, with spiking level of 10 log CFU/mL. The mixture was spray dried in 2L batches using a Niro drier with an inlet and outlet temperature of 200⁰C and 90⁰C, respectively. Following drying, scanning electron microscopy was used to observe the particle structure of the WPH10 conjugate. Following, the dried formulation was stored in airtight bottles for 16 weeks at 4°, 25° and -18°C, to monitor cell viability, moisture, and functionality. From the micro images it was observed that subsequent drying, nonconjugated WPH10 presented link bridges between the powder particles, whereas in case of conjugated WPH10, the particles presented a round surface with pores on it. The mean counts in conjugated WPH10 matrix before and after drying were 10.59 log CFU/g and 8.98 log CFU/g, respectively. At the end of 10 weeks, the counts were 7.18 log CFU/g at 4°C and 7.87 log CFU/g at -18°C, whereas at 25°C the viability significantly decreased to 3.97 log CFU/g. At this point, the wetting time increased from 47min to 61min, and solubility decreased from 90% to 82%. In the second study, whole-genome sequencing was done to determine the genotypic variants and associate with phenotypic expression in Listeria strains. Genomic DNA was extracted from the strains (Listeria innocua 634-2; Listeria innocua 634-34-S- 5; Listeria innocua 634-34-S-6; Listeria welshimeri 634-3; Listeria welshimeri 634-253- S-5, and Listeria monocytogenes 315-S-1), isolated from the dairy processing environment and run through Whole-genome sequencing on the Ilumina MiSeq and Nanopore sequencing platform. The genome was fed into the RAST database to develop the annotations. The results revealed 13 types of phenotypic responses related to the genotypic variants. Overall, the genotypic variants varied with the species and within the strains. Some of the common variants and features identified in the isolates were virulence (DNase, phage terminase, tRNA-Arg-ACG), cell signaling (NAG-IIA, NAGIIB), phage immunity (CRISPR proteins), osmotic stress (CadA), oxidative stress (YRKL), and antibiotic resistance (BaiE, Lde). L. monocytogenes was positive for all the phenotypic characteristics, allowing it to be more persistent, whereas L. innocua and L. welshimeri isolates lacked variants for motility (ActA), biofilm formation (AgD), and acid tolerance (AdiA), making them more sporadic strains.

Abstract: Fermented dairy products such as kefir have long been associated with a host of health benefits; however, the role that microbial composition has in these benefits has not been properly examined. This research aimed to examine how kefir impacts host metabolic health, as well as how alterations to the microbial populations of the kefir impacts these health benefits and the fermentation dynamics. In order to examine how kefir impacts metabolic health, four traditional and one commercial kefir were administered to mice using a high fat diet-induced obesity model. Two traditional kefirs decreased weight gain and plasma cholesterol levels in mice and one also lowered liver triglycerides. Conversely, commercial kefir had no beneficial effect. Improvements to liver triglycerides corresponded with decreases in the expression of fatty acid synthase, a gene involved in liver lipogenesis. This study shows that traditional kefir has the potential for improving metabolic dysfunction associated with obesity and indicates that differences in kefir microbial populations may influence the ability of traditional kefir to positively impact host metabolic health. As the metabolic health benefits associated with kefir were only associated with traditional examples, we set out to create a kefir product that utilized microbes from traditional kefir while maintaining a production method that is viable on an industrial scale. This was accomplished through the isolation of a large collection of organisms from a variety of traditional kefirs representing the most abundant species. Characterization of the isolates was carried out, and individual isolates were selected for use in the pitched kefir based on the characteristics of both the isolate and the kefir from which it was isolated. This proof of concept study showed that it is possible for traditional kefir organisms to ferment milk outside of a kefir grain, and that the resulting product resembles kefir in microbial density and pH. Following the development of the novel pitched kefir, we investigated the ability of this pitched culture kefir to recapitulate health benefits observed with a specific traditional kefir. This was accomplished using a mouse model of diet induced obesity. Additionally, we examined how differences in the microbial composition of kefir impacted these benefits. Both the traditional kefir and its pitched culture equivalent decreased plasma cholesterol and liver triglyceride levels by similar amounts when compared to commercial kefir. Furthermore, a pitched kefir produced without yeast and pitched kefir produced without lactobacilli did not show cholesterol-lowering effects. The traditional and pitched kefir with the full complement of microbes were able to impart corresponding decreases in the expression of multiple cholesterol and lipid metabolism genes in the liver. These results show that traditional kefir organisms can be utilized to create a more health promoting commercial kefir, while also highlighting the importance of microbial interactions during fermentation in the ability of fermented functional foods to beneficially impact host health. To better understand how alterations to the microbial composition of kefir fermentations impact the dynamics of the fermentation, we used two-dimensional gas chromatography and time-of-flight mass spectrometry (GCxGC-TOFMS) to examine the metabolite profiles of traditional and pitched culture kefir, including pitched culture kefirs that had the yeast or lactobacilli removed. Interestingly, despite similarities in their ability to improve host metabolic health, the Pitch and traditional kefir differed significantly in the profile of metabolites present, especially in yeast associated metabolites such as ethanol and esters. In addition, the removal of lactobacilli from the Pitch fermentation resulted in lower levels of organic acid formation during fermentation, while the removal of yeast appeared to result in little alteration in the metabolite profile. This thesis provides insight into how microbial composition impacts kefir fermentation, and presents a new strategy for the development of fermented functional foods.

The “mycobiome" refers to the composition of both bacterial and fungal communities in the human gut microbiome and has been the focus of disease-state correlations investigated by researchers and pursued with commercial interests by biotech startups. A microbiome startup currently sells direct-to-consumer at-home microbiome sequencing kits and probiotics that aim to balance the gut biofilm that contributes to the dysbiosis-associated conditions. This company has expressed an interest in developing new business strategies to leverage their intellectual and technical strengths.This thesis is composed of two parts:The first section is a scientific and technical investigation of the micro- and myco-biome, sequencing techniques and strategies (16S, ITS, WGS, and Shotgun Metagenomic Sequencing) that play a role in the characterization and identification of fungal and bacterial colonies in the gut. These strategies aim to overcome challenges in characterizing and quantifying microbiota composition. Next, this sequencing data can form a robust database of patient data that plays a role in disease identification, and this thesis identifies some of the bioinformatic analyses to achieve this goal. The section concludes with how insights derived from patient data can be used in the optimization of cohort design in clinical trials for various diseases.The second section investigates three different business models that a microbiome startup has expressed interest in exploring for future development: (1) medical foods; (2) a therapeutic pipeline; and (3) a data-licensing and discovery platform for drug development. A detailed analysis of the market dynamics, competitive landscape, regulatory issues, and other nascent concerns was performed for each potential vertical as a foundation to develop future business strategy of a microbiome-related startup. The thesis is concluded on a holistic analysis of the scientific and technical assets and business opportunities and strategies necessary for microbiome startups to successfully implement new business development. The final section serves as a final assessment of the holistic analysis and provides considerations regarding sustainable business growth for a microbiome-based biotech startup.

The objective of the outlined research was to determine how a fermented seaweed extract (SWO) and seaweed plus terrestrial plants (SWP) extract influenced ruminant health, productivity, environmental impacts (i.e. enteric methane and urinary N excretions), and the foraging ecology of young livestock. This was conducted over seven experiments. Chapter 3 implemented an in vitro methodology to determine the dose effect of SWO on fermentation parameters. The lowest dose implemented, which would relate to 5-mL/hd/d, reduced ammonia production by 6%, which may indicate lower urinary N excretions in vivo. Chapter 4 applied the determined dose (5-mL/d) to dairy cows during the last third of gestation with either SWO, SWP, or water (CON), and then the dose was increased to 100-mL per d after calving during the early lactation portion of the experiment. It was determined that the cows receiving SWO and SWP had reduced oxidative stress prior to calving and lower oxidative and metabolic stress 3-d after calving compared with the CON cows. Cows provided SWP after calving had 18% lower urinary N excretion, which is described in chapter 5. In chapter 6, pregnant ewes were either provided no supplement (CON-) or a grain based supplement with no plant extracts (CON+), with SWO (10-mL/hd/d), or SWP (10-mL/hd/d). At peak lactation (28 days-in-milk) grain supplementation (CON+) increased oxidative stress compared with CON-, and this effect was negated by SWO and SWP. Grain supplementation has been shown to induce oxidative stress in several animal models at peak lactation. In chapter 7, ewes were managed to lamb as yearlings, with their offspring precluded from consuming the same treatment supplements as in chapter 6. While SWO and SWP showed no benefit to oxidative stress of the ewes, possibly due to the low metabolic stress experienced, the lambs whose dams were provided SWO and SWP had lower oxidative stress one day after weaning. This indicates a greater maternal transmission of antioxidants from the SWO and SWP dams providing defense against oxidative stress induced from the physiological stress associated with weaning. The ram lambs born during chapter 6 continued to receive the supplement treatments of their dams until the start of the experiment described in chapter 8. The ram lambs were allocated to spatially separated strips sown to ryegrass (Lolium perenne), chicory, plantain, lucerne, and dock. Previous exposure to SWP reduced dietary neophobia, whereas the SWO and CON lambs exhibited substantial dietary neophobia to chicory and plantain. For chapter 9, the lambs born during chapter 7 were placed in the same paddocks as the ram lambs used in chapter 8. Similar to chapter 8, it was determined that lambs born to ewes provided SWP showed less dietary neophobia to chicory and lucerne compared with the lambs born to ewes provided SWO and CON. This chapter determined that dietary experience was obtained either from in utero or maternal milk exposure. Collectively, these experiments show the benefit of fermented plant extracts to improve animal health, reduce environmental impacts, and how foraging decisions of ruminants can be manipulated by previous exposure.

Acne vulgaris is a chronic inflammatory skin disorder, which affects the pilosebaceous units of the skin. Although this disease is normally self-limiting, it affects over 50 million Americans and results in lost productivity and treatment costs exceeding 1.2 billion dollars annually.1 While the exact cause of acne is unknown, its pathogenesis is classified into four factors: increased sebum production, abnormal keratinization, hypercolonization by Propionibacterium acnes, and inflammation.2 Despite numerous treatment options, increasing resistance to antibiotics3 and significant side effect profiles of many other therapeutics has created a need for safer, gentler treatment options. There has been renewed interest in the gut-skin-brain axis theory and the therapeutic role of oral probiotics, as new evidence suggests that gut dysbiosis plays an important role in mediating cutaneous skin inflammation and acne lesions.2 Specifically, strains of Lactobacillus acidophilus and Bifidobacterium bifidum, two strains commonly found in probiotics, have been found to decrease gut permeability and reduce systemic inflammation associated with acne.4 Additionally, in small clinical trials, these strains decreased acne severity and had an supplemental effect on clinical efficacy when used with oral antibiotics.5 Despite these promising results, the lack of large randomized controlled trials with standardized outcome measurements has prevented the adoption of oral probiotics as a viable treatment option by domestic and international dermatological societies. Therefore, this study proposes a randomized control trial to determine whether the adjunctive use of oral probiotics with antibiotics significantly reduces acne severity compared to oral antibiotics alone. We hypothesize that there will be a statistically significant decrease in acne severity scores among those receiving oral probiotics and antibiotics compared to those receiving only antibiotics among adolescents between the ages of 12-24 with moderate to severe acne. We hope that these study results will help provide evidence to incorporate oral probiotics into practice as a safe and efficacious treatment option to those suffering from moderate to severe acne, while advancing our understanding of how mediating gut microbiota can improve acne outcomes.

Gastrointestinal disease in cystic fibrosis (CF) begins In Utero and continues throughout life. Intestinal dysbiosis and inflammation have been observed in children with CF, however, the mechanisms linking them, and clinical implications are poorly understood. The intestinal tract as a therapeutic target is relatively underexplored. The overarching hypothesis is the intestinal milieu (bacteria, viruses and host-expressed proteins) is altered in children with CF compared to healthy controls (HC), and these changes have implications for intestinal inflammation and clinical outcomes. This thesis presents a series of observational studies characterising alterations in the intestinal milieu in children with CF, with a focus on identifying mechanisms for inflammation, disease, influences on clinical outcomes and potential therapeutic strategies. This thesis presents a novel research program commenced to address the limitations of current microbiome research and explore the gut-lung and gut-brain axes. This thesis demonstrates that in children with CF: (i) the intestinal milieu is structurally and functionally altered compared to HC, (ii) pro-inflammatory microbiota and bacteriophage lysins are increased in abundance, (iii) Ruminococcaceae may be beneficial for growth and lung function, (iv) upstream regulators for intestinal inflammation include interferon gamma, tumour necrosis factor (TNF) 5 and transglutaminase 2, and (v) xanthine dehydrogenase/oxidase (generates reactive oxygen species) negatively correlate with growth measures. Several potential strategies warranting further investigation for intestinal disease in children with CF include: (i) probiotics, (ii) short-chain fatty acid sources, (iii) antioxidants (e.g. glutathione), (iv) lactotransferrin, (v) TNF inhibitors, and (vi) amino acids (e.g. phenylalanine). A systematic review of probiotics for people with CF revealed low-quality evidence suggestive that probiotics may be beneficial to the health of children and adults with CF, however they are associated with a small number of adverse events. A novel probiotic randomised controlled trial was commenced and its design addresses several key gaps in our understanding of probiotics for children with CF. This thesis contributes to our understanding of the clinical relevance of the structural and functional alterations in the intestinal milieu of children with CF. It provides future directions for both observational and interventional research studies in children with CF.

Soy protein isolate (SPI) and probiotics can serve as a functional ingredient and beneficial supplement, respectively, to food formulations, but few studies focus on the compatibility of these two ingredients. The objective of this study was to prepare soy protein isolate (SPI) from defatted soybean flour and identify an optimal hydrolysis protocol to create soy protein hydrolysates (SPH) with improved solubility and functional properties and achieve an optimum encapsulation technique for probiotics and to determine the in vitro viability of encapsulated probiotic cells with SPH in a gastrointestinal environment. The SPI was prepared using an alkaline extraction procedure for solubility within a beverage-specific pH range. The SPH was prepared from aqueous extracted soy protein isolate using pepsin and freeze-dried. The physicochemical properties of the SPH were investigated for solubility, degree of hydrolysis, surface hydrophobicity, and electrophoresis. Alginate was used as an encapsulation agent for L. acidophilus cultures to create freeze-dried microcapsules. The dried SPH and encapsulated probiotics with alginate in a dry powder mixture were tested for its gastrointestinal resistance and probiotic viability under in vitro simulated diges-tion. The experimental groups were a control of free probiotic cells, encapsulated probiotics only, free probiotics with SPH, and encapsulated probiotics with SPH. Hydrolysates from 2, 2.5, and 3 hr. of hydrolysis time achieved a suitable degree of hydrolysis (DH) between 2.5-5.0%. The 2.5- and 3-hour hydrolysates had higher solubilities of 86 and 88%, respectively, in comparison to SPI with 74% at pH 6.5 (p < .05). Surface hydrophobicity of the hydrolysates ranged from 15 to 20 So units, with surface hydrophobicity generally decreasing over time. Microcapsules measured 1 mm in size using environmental scanning electron microscopy (ESEM). Probiotic cultures with mean log CFU of 7.74 were obtained before gastrointestinal resistance. Approximately 1-log decrease was observed for all experimental groups after simulated digestion ranged with the lowest value of 6.19 log CFU for free probiotics. No significant difference was observed among experimental groups for probiotic viability (p = .445). The findings of this research provide an understanding of improved formulation for more sustainable soy protein hydrolysate and viability of encapsulated probiotics within gastrointestinal environments.

Abstract: The application of probiotics is considered an effective approach to improve overall health conditions and growth performance in swine. Understanding the properties of these probiotics is a prerequisite for the selection of probiotics for pigs. Here, a rational selection criteria of swine probiotics is discussed mainly based on divergent probiotic effects of different strains and practical purposes in swine production (Chapter 2). The systematically investigated ecological history rather than the source of isolates should be regarded as the natural origin of probiotic strains, which helps to correct the inconsistencies arising from incorrect identification of the source. Host-adapted probiotic strains are likely to associated with exclusive colonization while the nomadic or environmental strain exert better immune stimulating functions. Strains with potent enzymatic activity are fitter for grower pigs favoring feed digestion and enhancing growth performance. Enterotoxigenic Escherichia coli (ETEC) is known to be a major pathogen in swine, however, this group of pathogens has a variable profile. Monitoring this profile of ETEC virulence factors could supply novel strategies to develop ETEC vaccines or fimbriae receptor analogues. In this thesis, a multiplex high-resolution melting curves-based qPCR (HRM-qPCR) assay was developed which enabled quantification of predominant ETEC fimbriae types and additionally monitored the presence or absence of other fimbriae types related to post-weaning diarrhea. Inclusion of host-adapted L. reuteri effectively reduced ETEC abundance in swine intestine (Chapter 4 and 5). In contrast, nomadic L. fermentum and L. casei did not show inhibitory effects on ETEC but eliminated Clostridium spp.. Furthermore, probiotic intervention showed limited alteration of commensal gut microbiota. The feed transition after weaning also induces an increasing ability of intestinal microorganisms to harvest energy from dietary carbohydrates. To unravel the adaptation of the swine microbiome to the dietary shift after weaning, a total of 360 high-quality microbial genomes were assembled as the first metagenomic-assembled-genomic reference for swine intestinal microbiota (Chapter 6). The reconstructed gut microbiome allowed identification of key microbial contributors to the degradation of starch, fructans, and lactose. Starch is a substrate for colonic microbiota and its metabolism is dependent on metabolic cooperativity between Firmicutes and Bacteroidetes. The functional prediction of the most abundant extracellular starch degrading enzyme in Firmicutes was further validated by protein purification and activity assays (Chapter 7). Fructans and lactose are fermented by simple enzymatic systems present in Bacteroides and Lactobacillus spp., respectively. The improved understanding of carbohydrate fermentation in the swine intestine enables the development of feeding models with higher feed efficiency and better pathogen control for weanling pigs.

Prolonged exposure to opioids results in analgesic tolerance, drug overdose, and death. The mechanism underlying morphine analgesic tolerance still remains unresolved. Currently, there are studies focusing on the role of the gut microbiota and its interactions with gut and brain (the microbiota-gut-brain axis) in substance abuse, especially opioid abuse. The microbiota-gut-brain axis is a bidirectional communication between central nervous system and gastrointestinal system. Emerging literature provides evidence that the gut microbiota orchestrates neurogenesis, brain development and function, as well as blood-brain-barrier integrity, and host behavior through vagal afferents, gut hormones, cytokines, and microbial metabolites. Gut dysbiosis disrupts the homeostasis between brain and gut in neurodegenerative disorder, central nervous system injury, and exacerbates disease progression. In opioid abuse subjects, increased comorbidity and behavioral changes are found to be associated with impaired gut integrity and bacterial translocation. We show that morphine analgesic tolerance was significantly attenuated in germ-free (GF) and in pan-antibiotic-treated (ABX) mice. Reconstitution of GF mice with naïve fecal microbiota reinstated morphine analgesic tolerance. We further demonstrated that tolerance was associated with microbial dysbiosis with selective depletion in Bifidobacteria and Lactobacillaeae. Probiotics, enriched with these bacteria, attenuated analgesic tolerance in morphine-treated mice. These results suggest that probiotics therapy during morphine administration may be a promising, safe, and inexpensive treatment to prolong morphine’s efficacy and attenuate analgesic tolerance. We hypothesize a vicious cycle of chronic morphine tolerance: morphine-induced gut dysbiosis leads to gut barrier disruption and bacterial translocation, initiating local gut inflammation through TLR2/4 activation, resulting in the activation of pro-inflammatory cytokines, which drives morphine tolerance.