Clostridium difficile toxins facilitate bacterial colonization by modulating the fence and gate function of colonic epithelium

The contribution of Clostridium difficile toxin A and B (TcdA and TcdB) to cellular intoxication has been extensively studied, but their impact on bacterial colonization remains unclear. By setting-up two- and three-dimensional in vitro models of polarized gut epithelium, we investigated how C. difficile infection is affected by host cell polarity and whether TcdA and TcdB contribute to such events. Indeed, we observed that C. difficile adhesion and penetration of the epithelial barrier is substantially enhanced in poorly polarized or EGTA-treated cells, indicating that bacteria bind preferentially to the basolateral cell surface. In this context, we demonstrated that sub-lethal concentrations of C. difficile TcdA are able to alter cell polarity by causing redistribution of plasma membrane components between distinct surface domains. Taken together, the data suggest that toxin-mediated modulation of host cell organization may account for the capacity of this opportunistic pathogen to gain access to basolateral receptors leading to a successful colonization of the colonic mucosa.

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.

L'infiammazione intestinale nell'animale sperimentale come modello per lo sviluppo di nuovi farmaci: ruolo della via tachichininergica nelle malattie infiammatorie intestinali

Background: Several lines of evidence showed that inflammation is associated with changes in the expression of tachykinins both in human and animal models. Tachykinins, including substance P (SP), are small peptides expressed in the extrinsic primary afferent nerve fibres and enteric neurons of the gut: they exert their action through three distinct receptors, termed NK1, NK2 and NK3. SP modulates intestinal motility and enteric secretion, acting preferentially through the NK1 receptor. SP neural network and NK1 receptor expression are increased in patients with inflammatory bowel disease, and similar changes were observed in experimental models of inflammation. The 2,4 Dinitrobenzene Sulphonic Acid (DNBS) model of colitis is useful to study innate immunity, non-specific inflammation and wound healing; it has been suggested that the transmural inflammation seen in this model resembles that found in Crohn’s disease and can therefore be used to study what cells and mediators are involved in this type of inflammation. Aim: To test the possible protective effect of the NK1 receptor antagonist SSR140333 on: 1) acute model of intestinal inflammation; 2) reactivation of DNBS-induced colitis in rats. Methods: Acute colitis was induced in male SD rats by intrarectal administration of DNBS (15 mg/rat in 50% ethanol). Reactivation of colitis was induced by intrarectal injections of DNBS on day 28 (7.5 mg/rat in 35% ethanol). Animals were sacrificed on day 6 (acute colitis) and 29 (reactivation of colitis). SSR140333 (10 mg/kg) was administered orally starting from the day before the induction of colitis for 7 days (acute colitis) or seven days before the reactivation of colitis. Colonic damage was assessed by means of macroscopic and microscopic scores, myeloperoxidase activity (MPO) and TNF-α tissue levels. Enzyme immunoassay was used to measure colonic substance P levels. Statistical analysis was performed using analysis of variance (one-way or two-way, as appropriate) with the Bonferroni’s correction for multiple comparisons. Results: DNBS administration impaired body weight gain and markedly increased all inflammatory parameters (p<0.01). Treatment with SSR140333 10 mg/kg significantly counteracted the impairment in body weight gain, decreased macroscopic and histological scores and reduced colonic myeloperoxidase activity (p<0.01). Drug treatment counteracted TNF-α tissue levels and colonic SP concentrations (acute model). Similar results were obtained administering the NK1 receptor antagonist SSR140333 (3 and 10 mg/kg) for 5 days, starting the day after the induction of colitis. Intrarectal administration of DNBS four weeks after the first DNBS administration resulted in reactivation of colitis, with increases in macroscopic and histological damage scores and increase in MPO activity. Preventive treatment with SSR140333 10 mg/kg decreased macroscopic damage score, significantly reduced microscopic damage score but did not affect MPO activity. Conclusions: Treatment with SSR140333 significantly reduced intestinal damage in acute model of intestinal inflammation in rats. The NK1 receptor antagonist SSR140333 was also able to prevent relapse in experimental colitis. These results support the hypothesis of SP involvement in intestinal inflammation and indicate that NK receptor antagonists may have a therapeutic potential in inflammatory bowel disease.

Espressione e ruolo funzionale di interleuchina-33 e del suo recettore, ST2, nelle malattie infiammatorie croniche intestinali

IL-33 is a novel member of the IL-1 family and ligand for the IL-1 receptor-related protein, ST2. Recent evidence suggests that the IL-33/ST2 axis plays a critical role in several autoimmune and inflammatory disorders; however, its role in inflammatory bowel disease (IBD) has not been clearly defined. We characterized IL-33 and ST2 expression and modulation following conventional anti-TNF therapy in Crohn’s disease and ulcerative colitis (UC) patients, and investigated the role of IL-33 in SAMP1/YitFc (SAMP) mice, a mixed Th1/Th2 model of IBD. Our results showed a specific increase of mucosal IL-33 in active UC, localized primarily to intestinal epithelial cells (IEC) and colonic inflammatory infiltrates. Importantly, increased expression of full-length IL-33, representing the most bioactive form, was detected in UC epithelium, while elevated levels of cleaved IL-33 were present in IBD serum. ST2 isoforms were differentially modulated in UC epithelium and sST2, a soluble decoy receptor with anti-inflammatory properties, was also elevated in IBD serum. Infliximab (anti-TNF) treatment of UC decreased circulating IL-33 and increased sST2, while stimulation of HT-29 IEC confirmed IL-33 and sST2 regulation by TNF. Similarly, IL-33 significantly increased and correlated with disease severity, and potently induced IL-5, IL-6 and IL-17 from mucosal immune cells in SAMP mice. Taken together, the IL-33/ST2 system plays an important role in IBD and experimental colitis, is modulated by anti-TNF therapy, and may represent a specific biomarker for active UC.

Ruolo di interleuchina-33 sull'espressione di geni profibrotici e sull'ipertrofia dei miofibroblasti nella fibrosi intestinale

Background: Intestinal fibrosis is a serious complication of IBD, with more than a third of Crohn’s disease (CD) patients developing a fibrostenosing phenotype with formation of strictures that will require surgical intervention. Remarkably, SAMP1/YitFc (SAMP) mice, a spontaneous model of CD, develop gut fibrosis; similar to IBD patients, the pathophysiology of SAMP fibrosis is unknown. IL-33 is a member of the IL-1 cytokine family and increased expression is associated with IBD. Emerging evidence suggests its potential role in liver and cutaneous fibrosis, as well as myofibroblast-associated colonic ulcerations . Aim: The aim of this study was to evaluate the role of IL-33 as a potential mediator of profibrotic events leading to intestinal fibrosis and possible stricture formation. Methods: A detailed histologic time course study, with collagen-specific Masson trichrome staining and IHC for ST2 (IL-33 receptor), was performed on SAMP and control AKR (parental strain) mice. qRT-PCR was done on full-thickness ilea for the profibrogenic genes, collagen (coll)-1, coll-3, connective tissue growth factor (CTGF) and insulin-like growth factor 1 (IGF-1). Exogenous IL-33 (33 μg/kg, i.p.) or vehicle was administered daily for 7d to SAMP and AKR mice (N=6/exp group), and ileal tissues evaluated as above. Finally, microarray analysis was performed on full-thickness ilea from SAMP and AKR mice, and IL-33 stimulated subepithelial myofibroblasts (SEMFs). Results: SAMP mice displayed ileal skip lesions with randomly distributed strictures, preceded by typical pre-stricture dilations of the ileum. Ileal wall was visibly thickened with hypertrophy of the serosa, muscularis mucosa, muscularis propria, within which intense collagen deposition was observed, and inflammatory infiltrates in segments showing strictures. Interestingly, intense ST2 staining was present within the inflamed lamina propria of SAMP, notably localized to SEMFs. Fibrosis was first observed at 20 wks, and reached its peak by 50 wks of age. mRNA expression of coll-1 (4.74±0.69-fold; P=0.001), coll-3 (4.92±1.05-fold; P=0.01), IGF1 (12.9±3.45; P=0.006), and CTGF (3.29±0.69; P=0.004) was dramatically elevated in SAMP vs. AKR ilea. IL-33 treatment of AKR mice induced a marked increase in muscle fiber/myofibroblast cellularity and hypertrophy of the muscularis propria (4.13±0.74-fold; P<0.0001), and mRNA expression of coll-1 (5.16±0.89-fold; P=0.0009), coll-3 (1.97±0.14-fold; P=0.01), IGF-1 (9.32±2.27-fold; P=0.004), and CTGF (1.43±0.31-fold; P=0.006) vs. vehicle controls. Microarray data from SAMP ilea and IL-33-treated SEMFs confirmed these trends, displaying a global increase in profibrogenic gene expression. Conclusion: These data suggest an important role for IL-33 in intestinal fibrosis, and may represent a potential target for the treatment of IBD-associated fibrosis and stricture formation.

Characterization of novel probiotics and prebiotics

The role of the human gut microbiota in impacting host’s health has been widely studied in the last decade. Notably, it has been recently demonstrated that diet and nutritional status are among the most important modifiable determinants of human health, through a plethora of presumptive mechanisms among which microbiota-mediated processes are thought to have a relevant role. At present, probiotics and prebiotics represent a useful dietary approach for influencing the composition and activity of the human gut microbial community. The present study is composed of two main sections, aimed at elucidating the probiotic potential of the yeast strain K. marxianus B0399, as well as the promising putative prebiotic activity ascribable to four different flours, naturally enriched in dietary fibres content. Here, by in vitro studies we demonstrated that K. marxianus B0399 possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotics. Successively, we investigated the impact of a novel probiotic yoghurt containing B. animalis subsp. lactis Bb12 and K. marxianus B0399 on the gut microbiota of a cohort of subjects suffering from IBS and enrolled in a in vivo clinical study. We demonstrated that beneficial effects described for the probiotic yoghurt were not associated to significant modifications of the human intestinal microbiota. Additionally, using a colonic model system we investigated the impact of different flours (wholegrain rye and wheat, chickpeas and lentils 50:50, and barley milled grains) on the intestinal microbiota composition and metabolomic output, combining molecular and cellular analysis with a NMR metabolomics approach. We demonstrated that each tested flour showed peculiar and positive modulations of the intestinal microbiota composition and its small molecule metabolome, thus supporting the utilisation of these ingredients in the development of a variety of potentially prebiotic food products aimed at improving human health.

Decorso delle coliti severe: un confronto tra colite ulcerosa e di Crohn

Pochi sono i dati disponibili sul decorso clinico della malattia di Crohn del colon severa(CD). L'obiettivo è quello di descrivere il decorso clinico della colite di Crohn severa (CC) in una coorte di pazienti isolata con CD del colon o ileocolica, e di confrontarlo con il decorso clinico di pazienti affetti da colite ulcerosa severa (UC). 34 pazienti con CC severa sono stati identificati retrospettivamente nella nostra coorte di 593 pazienti ricoverati (2003-2012) attraverso la valutazione di CDAI score e HBI. 169 pazienti con UC severa sono stati identificati retrospettivamente in una coorte di 449 pazienti ricoverati (2003-2012) attraverso la valutazione del score di Lichtiger e di Truelove-Witts. Abbiamo valutato questi risultati: risposta agli steroidi, risposta ai farmaci biologici, tasso di colectomia acuta, tasso di colectomia durante il follow-up, megacolon e tasso di infezione da citomegalovirus. Non abbiamo trovato differenze significative nella risposta agli steroidi e biologici, della percentuale di infezione da citomegalovirus e di megacolon, mentre il tasso di colectomia in acuto è risultato essere maggiore nei pazienti con CC rispetto ai pazienti con UC; anche la differenza tra i tassi di colectomia alla fine del follow-up è risultata non significativa. Con l'analisi univariata la giovane età alla diagnosi è associata ad un aumentato rischio di colectomia in assoluto (p = 0,024) e in elezione (p = 0.022), ma non in acuto. Il tasso globale di colectomia nei pazienti con CC severa è superiore a quella dei pazienti con UC severa , ma questo dato non è supportato da una diversa risposta clinica alla terapia steroidea o terapia di salvataggio con biologici. Il vero decorso clinico della colite di Crohn severa necessita di essere chiarito da studi prospettici che includano un numero maggiore di pazienti con questo sottogruppo di malattia.

Development of a porcine lactation model for the evaluation of mammary transfer of exogenous molecules - A contribution from the conception project

Around 5 million women give birth each year in Europe and, while breastfeeding, the majority of them may need to take medications, either occasionally or continuously. Unfortunately, there is often scarce evidence of trustworthy information about how a specific molecule might affect the physiology of lactation. This is the reason that brought a European public-private partnership to fund the development of a reliable platform to provide women and health-care professionals a helpful instrument to reduce uncertainty about the effects of medication used during breastfeeding. On April 1st 2019, the ConcePTION project (Grant Agreement n°821520) started to develop such envisaged platform. The 3rd Work Package was in charge of the validation of in vitro, in vivo and in silico lactation models. Between the numerous species currently used in preclinical studies, pigs’ similarities with humans’ anatomy, physiology and genomics make them extremely useful as translational models, when proper veterinary expertise is applied. The ASA team from the University of Bologna, went first to characterize the translational lactation model using the swine species, chosen upon literature review. The aim of this work was to lay the foundations of a porcine lactation model that could be suitable for application within pharmaceutical tests, to study drug transfer through milk prior approval and commercialization. The obtained results highlighted both strengths and critical points of the study design, allowing a significant improvement in the knowledge of pharmacokinetic physiology in lactating mammals. Lastly, this project allowed the assessment of microbial changes in gut resident bacteria of newborns through an innovative in vitro colonic model. Indeed, even if there were no evident adverse effects determined by drug residues in milk, possible alterations in the delicate microbial ecology of newborns’ gastrointestinal tract was considered pivotal, giving its possible impact on the individual health and growth.

Gut microbiota perturbation induced by emerging dietary habits: an in vitro colon model to predict their effects by a microbiomic and metabolomic approach

Over the past 30 years, unhealthy diets and lifestyles have increased the incidence of noncommunicable diseases and are culprits of diffusion on world’s population of syndromes as obesity or other metabolic disorders, reaching pandemic proportions. In order to comply with such scenario, the food industry has tackled these challenges with different approaches, as the reformulation of foods, fortification of foods, substitution of ingredients and supplements with healthier ingredients, reduced animal protein, reduced fats and improved fibres applications. Although the technological quality of these emerging food products is known, the impact they have on the gut microbiota of consumers remains unclear. In the present PhD thesis, the recipient work was conducted to study different foods with the substitution of the industrial and market components to that of novel green oriented and sustainable ingredients. So far, this thesis included eight representative case studies of the most common substitutions/additions/fortifications in dairy, meat, and vegetable products. The products studied were: (i) a set of breads fortified with polyphenol-rich olive fiber, to replace synthetic antioxidant and preservatives, (ii) a set of Gluten-free breads fortified with algae powder, to fortify the protein content of standard GF products, (iii) different formulations of salami where nitrates were replaced by ascorbic acid and vegetal extract antioxidants and nitrate-reducers starter cultures, (iv) chocolate fiber plus D-Limonene food supplement, as a novel prebiotic formula, (v) hemp seed bran and its alkalase hydrolysate, to introduce as a supplement, (vi) milk with and without lactose, to evaluate the different impact on human colonic microbiota of healthy or lactose-intolerants, (vii) lactose-free whey fermented and/or with probiotics added, to be introduced as an alternative beverage, exploring its impact on human colonic microbiota from healthy or lactose-intolerants, and (viii) antibiotics, to assess whether maternal amoxicillin affects the colon microbiota of piglets.

Selection of candidate probiotic strains from human microbial niches, for the development of tailored foods designed for specific consumers

My PhD project was intended, throughout the selection of probiotics from human milk and healthy vaginal environment, for the development of tailored fermented foods. According to this aim, several activities were carried out. The first one, concerning the isolation of Lactobacillus and Bifidobacterium strains from human milk to find new probiotic candidates to be included in food products showed promising results. Probiotics have been also proposed to improve female genital health and microbial strains isolated and connected with healthy vaginal ecosystem could be used to prevent or treat vaginal dysbiosis. In this context vaginal lactobacilli previously characterized for their technological features and antagonistic activity against several female uro-genital pathogens were investigated for their metabolic aptitude and additional probiotic features, showing interesting results hypothesizing their inclusion in foods. In addition, in order to preserve vaginal strains viability during food processing/digestion it was also evaluated the potential of microencapsulation by spray-drying. In this framework the results obtained were highly promising from the perspective of using encapsulated powders in food formulations. Another activity connected with the main idea to develop a food strategy for the administration of these vaginal strains was carried out. Lactobacillus crispatus BC4, was supplemented in a Squacquerone cheese, and its digestive fate was evaluated adopting SHIME® system. The results showed that during colonic fermentation, L. crispatus BC4 was metabolically active. Additionally, although probiotic delivery to humans has traditionally been associated with fermented dairy foods, recently the demand for non-dairy-alternatives as potential probiotics carrier is increasing. In this framework, my latest activity was connected with the development of fermented soy milks with encapsulated and non-encapsulated L. crispatus BC4 and L. gasseri BC9. The same fermented soy milks were also investigated for their nutritional qualities and after in vitro digestion for their specific functionality on post-menopausal fecal microbiota and protein bioaccessibility.

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.

Colic surgery in the equine patient: novel biomarkers, postoperative management, microbiotal influence and epidemiological analysis on the italian territory

The aim of this work was to investigate novel diagnostic and prognostic tools, postoperative treatments and epidemiologic factors impacting the outcome of surgical cases of colic. To make a more accurate diagnosis and establish a prognosis, several biomarkers have been investigated in colic patients. In this study we evaluated peritoneal PCT and blood ADMA and SDMA in SIRS positive and negative colic patients to be used as prognostic biomarkers. Our results highlighted the limits of these biomarkers in detection and the lack of specificity. In fact PCT was not detectable and even if ADMA and SDMA significantly increased in colic horses, they are not diagnostic nor prognostic markers for SIRS. Fluid therapy has been described to be crucial for the outcome of colic patients, nevertheless no guidelines have been established. Overhydration was the common practice in post surgical management. We compared cases with an extended fluid therapy protocol and cases with a restricted protocol. Results showed that survival rate and postoperative complications were similar between the groups, despite costs being significantly lower in the restricted group. The possible correlation between intestinal microbiota and colics has gained interest. In this study, cecal and colonic content from horses undergoing laparotomy were collected, and the microbiota analized. Results showed some differences in microbiota between discharged and non discharged patients, and between strangulating and non strangulating types of colic, that might suggest some influence of hind gut microbiota on the disease. A multicentric study involving three veterinary teaching hospitals on the italian territory was conducted investigating factors affecting postoperative survival and complications in colics. Results showed that the influence of age, PCV, TPP, blood lactate, reflux, type of disease, type of lesion, presence of anastomosis, duration of surgery and surgeons, were in line with literature. Amount of crystalloids used could affected the outcome.