Effects of different nutritional strategies on intestinal inflammation in pigs

Intestinal health is essential for the health of the body since the gastro-intestinal mucosa is the main site of interaction with the external environment, as well as the major area colonized by the microbiota. Intestinal health relies on proper barrier function, epithelial integrity and related mechanisms of protection (mucous layer, tight junctions, immune and inflammatory system). In pigs, during the weaning transition, intestinal inflammation and barrier integrity play a crucial role in regulating intestinal health and, consequently, pig’s health, growth and productivity. The aim of the project was to assess the impact of different nutritional strategies on the intestinal health of weaning piglets with reference to the inflammatory status and epithelial integrity. Therefore, in vivo trials were conducted to test the in-feed supplementation with zinc, tributyrin, or organic acids and nature-identical compounds (NIC) to weaning piglets. All the dietary interventions positively impacted the intestinal inflammatory status and, as a consequence, improved epithelial integrity by modulating tight junctions proteins (zinc or tributyrin) or by enhancing barrier properties measured with Ussing chambers (organic acids and NIC). These findings highlight that intestinal inflammation and barrier function are strictly linked, and that the control of inflammation is essential for adequate barrier function. In addition, in zinc trial and organic acids and NIC trial, better intestinal health could successfully result in better growth performance, as aimed for pig production improvement. To conclude, this work shows that dietary supplementation with bio-active substances such as zinc, tributyrin or organic acids and NIC may improve intestinal health of weaning piglets modulating intestinal inflammatory stress and barrier integrity and allowing better piglet’s health, growth and productivity.

Probiotics for the prevention/treatment of human diseases and ecological study of the intestinal microbiota

The interest in human intestinal microbiota has increased in the last 20 years and significant advances have been achieved with regard to its composition and functions. The gut microbiota contributes to the maintenance of the host health status and, since alterations in the gut microbiota have been involved in the pathogenesis/progression of some diseases, several studies have focused on the manipulation of its composition. Probiotics are a strategy to maintain/restore the correct balance of gut microbial population and to prevent/treat diseases. The aim of this thesis was to explore the possibility of probiotic supplementation for the prevention/treatment of human diseases and the related study of the intestinal microbial environment. After reviewing studies concerning the use of Bifidobacterium breve as probiotic in paediatric diseases, the effectiveness of a probiotic formulation consisting of two strains of B. breve was assessed in paediatric subjects for the prevention or alleviation of gastrointestinal disorders, including coeliac disease and paediatric obesity. As the emerging role of gut microbiota in neurological diseases, the intestinal microbial environment in amyotrophic lateral sclerosis patients compared to healthy controls and the effects of a probiotic administration were examined. Considering the role of viruses in shaping gut microbiota, gut bacteriophages and bacterial community of preterm infants were investigated. The results evidenced differences in gut microbial composition of healthy controls and diseased subjects in coeliac and amyotrophic lateral sclerosis patients. The probiotic approach was effective in restoring the microbial composition in the former, whereas, in the latter, the influence was focused only on some microbial groups. The probiotic intervention was effective in improving the glyco-insulinemic profile in obese children and in preventing gastrointestinal disorders in healthy newborns. The study of the bacterial and phage composition in preterm infants suggested a transkingdom interplay between bacteria and viruses with a reciprocal influence on their composition.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Molecular characterization of the human gut microbiota: the effect of aging

Age-related physiological changes in the gastrointestinal tract, as well as modification in lifestyle, nutritional behaviour, and functionality of the host immune system, inevitably affect the gut microbiota. The study presented here is focused on the application and comparison of two different microarray approaches for the characterization of the human gut microbiota, the HITChip and the HTF-Microb.Array, with particular attention to the effects of the aging process on the composition of this ecosystem. By using the Human Intestinal Tract Chip (HITChip), recently developed at the Wageningen University, The Netherland, we explored the age-related changes of gut microbiota during the whole adult lifespan, from young adults, through elderly to centenarians. We observed that the microbial composition and diversity of the gut ecosystem of young adults and seventy-years old people is highly similar but differs significantly from that of the centenarians. After 100 years of symbiotic association with the human host, the microbiota is characterized by a rearrangement in the Firmicutes population and an enrichment of facultative anaerobes. The presence of such a compromised microbiota in the centenarians is associated with an increased inflammation status, also known as inflamm-aging, as determined by a range of peripheral blood inflammatory markers. In parallel, we overtook the development of our own phylogenetic microarray with a lower number of targets, aiming the description of the human gut microbiota structure at high taxonomic level. The resulting chip was called High Taxonomic level Fingerprinting Microbiota Array (HTF-Microb.Array), and was based on the Ligase Detection Reaction (LDR) technology, which allowed us to develop a fast and sensitive tool for the fingerprint of the human gut microbiota in terms of presence/absence of the principal groups. The validation on artificial DNA mixes, as well as the pilot study involving eight healthy young adults, demonstrated that the HTF-Microb.Array can be used to successfully characterize the human gut microbiota, allowing us to obtain results which are in approximate accordance with the most recent characterizations. Conversely, the evaluation of the relative abundance of the target groups on the bases of the relative fluorescence intensity probes response still has some hindrances, as demonstrated by comparing the HTF.Microb.Array and HITChip high taxonomic level fingerprints of the same centenarians.

Strategie nutrizionali finalizzate alla modulazione del microbiota intestinale del cane e del gatto

Il microbiota intestinale riveste un ruolo importantissimo nell’influenzare la salute dell’ospite. È stato dimostrato come la composizione della dieta possa condizionare lo stato di benessere dell’animale, inducendo importanti cambiamenti tra le popolazioni batteriche che coabitano l’intestino; l’uso di prebiotici rappresenta una delle strategie maggiormente impiegate per modulare positivamente la composizione ed il metabolismo dell’ecosistema gastroenterico. Il presente progetto di dottorato si è proposto di indagare gli effetti sul microbiota intestinale del cane e del gatto di diete a diverso tenore proteico e contenenti proteine di diversa digeribilità in presenza o meno di sostanze prebiotiche. Inoltre, sono stati valutati gli effetti della presenza di un estratto di Yucca schidigera e di tannini sulla microflora intestinale del gatto. In ultima istanza, sono state valutate le conseguenze di dosi crescenti di lattosio sul benessere intestinale del cane. I risultati del presente studio hanno rilevato come le sostanze prebiotiche influiscono sulla composizione e sul metabolismo della microflora del cane e del gatto, e come l’impiego di diete ricche di proteine possa avere conseguenze negative sull’ambiente intestinale. Tuttavia, la presenza di oligosaccaridi non sembra contrastare gli effetti negativi che diete ad alto tenore proteico potrebbero avere sull’ecosistema intestinale dell’animale. Nella successiva prova è stato evidenziato come l’inclusione nella dieta di estratti di Yucca e tannini possa contribuire a mitigare l’emanazione di sostanze maleodoranti dalle deiezioni degli animali da compagnia. Nel corso dell’ultima prova, nonostante non siano state osservate differenze tra le popolazioni microbiche intestinali, la somministrazione di dosi crescenti di lattosio ha indotto una certa riduzione delle fermentazioni proteolitiche microbiche. Ulteriori studi sono necessari per stabilire in che misura la dieta e gli alimenti “funzionali” possano influire sul microbiota intestinale del cane e del gatto e come queste informazioni possono essere utilizzate per migliorare miratamente l’alimentazione e lo stato di salute degli animali da compagnia.

Pathogenetic mechanisms of gastrointestinal symptoms in a mouse model of Fabry disease: insights on the microbiota-gut-brain axis

Fabry disease (FD), X-linked metabolic disorder caused by a deficiency in α-galactosidase A activity, leads to the accumulation of glycosphingolipids, mainly Gb3 and lyso-Gb3, in several organs. Gastrointestinal (GI) symptoms are among the earliest and most common, strongly impacting patients’ quality of life. However, the origin of these symptoms and the exact mechanisms of pathogenesis are still poorly understood, thus the pressing need to improve their knowledge. Here we aimed to evaluate whether a FD murine model (α-galactosidase A Knock-Out) captures the functional GI issues experienced by patients. In particular, the potential mechanisms involved in the development and maintenance of GI symptoms were explored by looking at the microbiota-gut-brain axis involvement. Moreover, we sought to examine the effects of lyso-Gb3 on colonic contractility and the intestinal epithelium and the enteric nervous system, which together play important roles in regulating intestinal ion transport and fluid and electrolyte homeostasis. Fabry mice revealed visceral hypersensitivity and a diarrhea-like phenotype accompanied by anxious-like behavior and reduced locomotor activity. They reported also an imbalance of SCFAs and an early compositional and functional dysbiosis of the gut microbiota, which partly persisted with advancing age. Moreover, overexpression of TRPV1 was found in affected mice, and partial alteration of TRPV4 and TRPA1 as well, identifying them as possible therapeutic targets. The Ussing chamber results after treatment with lyso-Gb3 showed an increase in Isc (likely mediated by HCO3- ions movement) which affects neuron-mediated secretion, especially capsaicin- and partly veratridine-mediated. This first characterization of gut-brain axis dysfunction in FD mouse provides functional validation of the model, suggesting new targets and possible therapeutic approaches. Furthermore, lyso-Gb3 is confirmed to be not only a marker for the diagnosis and follow-up of FD but also a possible player in the alteration of the FD colonic ion transport process.

The predictive role of the human gut microbiota in covid-19: implications for disease severity and post-covid syndrome

The COVID-19 pandemic, sparked by the SARS-CoV-2 virus, stirred global comparisons to historical pandemics. Initially presenting a high mortality rate, it later stabilized globally at around 0.5-3%. Patients manifest a spectrum of symptoms, necessitating efficient triaging for appropriate treatment strategies, ranging from symptomatic relief to antivirals or monoclonal antibodies. Beyond traditional approaches, emerging research suggests a potential link between COVID-19 severity and alterations in gut microbiota composition, impacting inflammatory responses. However, most studies focus on severe hospitalized cases without standardized criteria for severity. Addressing this gap, the first study in this thesis spans diverse COVID-19 severity levels, utilizing 16S rRNA amplicon sequencing on fecal samples from 315 subjects. The findings highlight significant microbiota differences correlated with severity. Machine learning classifiers, including a multi-layer convoluted neural network, demonstrated the potential of microbiota compositional data to predict patient severity, achieving an 84.2% mean balanced accuracy starting one week post-symptom onset. These preliminary results underscore the gut microbiota's potential as a biomarker in clinical decision-making for COVID-19. The second study delves into mild COVID-19 cases, exploring their implications for ‘long COVID’ or Post-Acute COVID-19 Syndrome (PACS). Employing longitudinal analysis, the study unveils dynamic shifts in microbial composition during the acute phase, akin to severe cases. Innovative techniques, including network approaches and spline-based longitudinal analysis, were deployed to assess microbiota dynamics and potential associations with PACS. The research suggests that even in mild cases, similar mechanisms to hospitalized patients are established regarding changes in intestinal microbiota during the acute phase of the infection. These findings lay the foundation for potential microbiota-targeted therapies to mitigate inflammation, potentially preventing long COVID symptoms in the broader population. In essence, these studies offer valuable insights into the intricate relationships between COVID-19 severity, gut microbiota, and the potential for innovative clinical applications.