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.

Atmospheric plasma processes for microbial inactivation: food applications and stress response in Listeria monocytogenes

This PhD thesis is focused on cold atmospheric plasma treatments (GP) for microbial inactivation in food applications. In fact GP represents a promising emerging technology alternative to the traditional methods for the decontamination of foods. The objectives of this work were to evaluate: - the effects of GP treatments on microbial inactivation in model systems and in real foods; - the stress response in L. monocytogenes following exposure to different GP treatments. As far as the first aspect, inactivation curves were obtained for some target pathogens, i.e. Listeria monocytogenes and Escherichia coli, by exposing microbial cells to GP generated with two different DBD equipments and processing conditions (exposure time, material of the electrodes). Concerning food applications, the effects of different GP treatments on the inactivation of natural microflora and Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli on the surface of Fuji apples, soya sprouts and black pepper were evaluated. In particular the efficacy of the exposure to gas plasma was assessed immediately after treatments and during storage. Moreover, also possible changes in quality parameters such as colour, pH, Aw, moisture content, oxidation, polyphenol-oxidase activity, antioxidant activity were investigated. Since the lack of knowledge of cell targets of GP may limit its application, the possible mechanism of action of GP was studied against 2 strains of Listeria monocytogenes by evaluating modifications in the fatty acids of the cytoplasmic membrane (through GC/MS analysis) and metabolites detected by SPME-GC/MS and 1H-NMR analyses. Moreover, changes induced by different treatments on the expression of selected genes related to general stress response, virulence or to the metabolism were detected with Reverse Transcription-qPCR. In collaboration with the Scripps Research Institute (La Jolla, CA, USA) also proteomic profiles following gas plasma exposure were analysed through Multidimensional Protein Identification Technology (MudPIT) to evaluate possible changes in metabolic processes.

Influenza della tipologia di cereale e di fermentazione sulle possibili proprietà salutistiche del pane

Il rinnovato interesse da parte dei consumatori per le caratteristiche nutrizionali degli alimenti incentiva la ricerca in campo agroalimentare a sviluppare nuovi prodotti per il mercato attuale sempre più attento a salute e benessere. È in questo contesto che nasce a livello europeo il progetto Bake4Fun mirato allo sviluppo di soluzioni biotecnologiche innovative per la messa a punto di nuovi prodotti da forno funzionali. Nell’ambito di questo progetto europeo, lo scopo dello studio oggetto di questo elaborato di laurea è stato quello di valutare le proprietà antiossidanti e antinfiammatorie di diverse tipologie di pani creati ad hoc con farina di frumento o di farro e sottoposti a fermentazione convenzionale o con pasta madre (sourdough). Il farro appartiene alla famiglia dei cosiddetti “cereali antichi” e grazie al suo elevato profilo nutrizionale si presenta come un promettente candidato per la produzione di alimenti dalle proprietà benefiche. Similmente anche diverse tipologie di fermentazione sembrano in grado di possedere caratteristiche salutistiche. Pertanto, nell’ambito di B4F sono stati formulati e prodotti diversi tipi di pane e se ne sono voluti studiare i possibili effetti antiossidanti ed antiinfiammatori in vivo sul modello sperimentale del suino. Dopo 30 giorni di dieta arricchita con i diversi prodotti sperimentali miscelate in un rapporto di 1:1 con una dieta standard è stato valutato lo stato redox plasmatico mediante l’analisi di tre indicatori del danno ossidativo, quali TAC, GSH e TBARS. Il grado d’infiammazione è stato invece valutato attraverso l’analisi di otto citochine (IFNα, IFNγ, IL-1β, IL-4, IL-6, IL-8, IL-10 e TNFα) utilizzando un saggio multiparametrico di tipo ELISA. Tutti gli animali impiegati godevano di buona salute ed i loro plasmi sono stati analizzati sia all’inizio (T0) sia alla fine (T30) dell’esperimento. Sebbene i risultati conseguiti in relazione allo status ossidativo ed infiammatorio nei suini impiegati non abbiano evidenziato particolari differenze tra i diversi tipi di diete sperimentali, non si esclude che eventuali variazioni delle condizioni sperimentali possano portare a conclusioni diverse o che gli effetti siano maggiormente visibili a livello di altri parametri quali, ad esempio, la variazione della microflora intestinale o l’indice glicemico dei prodotti. Questa possibilità unitamente all’importanza degli effetti salutistici dei cereali antichi e delle diverse tipologie di fermentazione, giustificate da altre ricerche in letteratura, sottolineano la necessità di ulteriori studi in questo ambito.

Uso di acqua elettrolizzata per la decontaminazione di mele

Negli ultimi decenni le campagne di incoraggiamento del consumo di almeno 5 porzioni di frutta e verdura al giorno ha determinato un aumento da parte della popolazione del consumo di vegetali freschi. È ben noto infatti che i prodotti freschi sono un’importante risorsa di nutrienti, vitamine e fibre. Tra le varie tecniche di trattamento per diminuire la carica microbica in questi prodotti l’uso dell’ipoclorito di sodio rimane una delle soluzioni più utilizzate per la sua efficacia ed il basso costo. La domanda sempre più elevata da parte dei consumatori di prodotti di alta qualità sicuri ed economici è stato uno dei principali motivi che ha spinto l’industria verso lo sviluppo di tecniche di sanitizzazione alternative ai trattamenti tradizionali. Tra le varie tecniche messe a punto e studiate vi è l’acqua elettrolizzata che non sembra avere problemi di tossicità come altri sanitizzanti quali l’ipoclorito di sodio, la formaldeide e la glutaraldeide e tra i numerosi vantaggi presenta un ridotto tempo di pulizia, un facile utilizzo ed il basso costo. In questo lavoro si è voluto valutare se soluzioni di acqua elettrolizzata neutra (AE) possano essere impiegate come sanitizzanti per la decontaminazione di mele Golden Delicious. A tale scopo si sono effettuati lavaggi delle mele per immersione in AE a differente contenuto di cloro attivo (50, 100 e 200 ppm) verificando la riduzione della contaminazione superficiale rispetto sia alla microflora naturalmente presente, sia ad un patogeno deliberatamente inoculato sul prodotto, Listeria monocytogenes ceppo 56 Ly. A tale proposito si è voluto anche valutare l’effetto del livello di contaminazione iniziale di L. monocytogenes sull’efficacia dei lavaggi confrontandola con quella ottenuta con soluzioni di ipoclorito di sodio. Infine si è verificato se il potere antiossidante ed il contenuto in fenoli totali della buccia subiscono modificazioni a seguito dei lavaggi con AE neutra od ipoclorito di sodio alle condizioni adottate. I risultati di questa sperimentazione, sebbene preliminari, hanno evidenziato che l’acqua elettrolizzata può essere considerata una tecnica di decontaminazione promettente: infatti oltre ad avere un’azione sanitizzante simile all’ipoclorito nei confronti della microflora naturale, e di ridurre il rischio associato alla presenza di L. monocytogenes, non ha indotto modificazioni significative di un parametro qualitativo che caratterizza tale frutto quale l’attività antiossidante determinata, in larga misura, dall’elevato contenuto in fenoli.

Stabilizzazione di succo di melagrana mediante trattamenti termici

L’interesse da parte dell’industria alimentare verso il melograno sta aumentando in virtù delle sue caratteristiche nutrizionali, come ad esempio l’alto contenuto di composti fenolici, che rendono tale frutto interessante per la produzione di succhi funzionali. Studi riportano che il microbiota di tali frutti è rappresentato principalmente da lieviti, muffe, batteri mesofili e lattici che possono proliferare durante la conservazione dei succhi. L’obiettivo di questa tesi è stato quello di valutare l’effetto di trattamenti di pastorizzazione di sul livello di contaminazione microbica di succo di melagrana ottenuto da arilli di due cultivar. A tale scopo si è valutata l’efficacia delle condizioni di trattamento adottate nell’inattivare la microflora naturalmente presente nei succhi di frutta e la sua capacità di recuperare durante la fase di conservazione a temperature sia di refrigerazione, che ambiente. Inoltre si è realizzato un challenge test in cui i succhi sono stati deliberatamente contaminati con S. cerevisiae, L. plantarum e diversi patogeni. I risultati hanno mostrato come il succo di melagrana sia un prodotto a breve shelf-life (5 giorni) quando conservato a temperatura ambiente poichè soggetto ad un rapido sviluppo della microflora. Infatti, sebbene i livelli di contaminazione iniziale rilevati fossero complessivamente bassi, i lieviti hanno raggiunto rapidamente la soglia critica di spoilage (6 Log UFC/ml) nei succhi freschi. Le condizioni di trattamento termico adottate hanno portato ad una significativa riduzione della microflora a livelli inferiori al limite di rilevabilità, e le cellule sopravvissute al trattamento non sono state in grado di proliferare nel succo conservato a 4°C per quasi 2 mesi. Quando conservati a temperatura ambiente, i succhi esposti al processo più blando hanno presentato una shelf-life di circa 25 giorni, mentre questa è aumentata fino oltre 32 giorni nei prodotti trattati più a lungo.

Short-term effects of systemic antibiotics during periodontal healing

OBJECTIVES: To investigate the short-term effects of nonsurgical therapy (scaling and root planing, SRP) on the subgingival microbiota in chronic (CP) and aggressive (AP) periodontal disease. METHOD AND MATERIALS: Ninety-seven CP and AP subjects underwent full-mouth SRP on 2 consecutive days. AP patients were randomly assigned to either receive systemic metronidazole plus amoxicillin (AP+AB) or were treated mechanically alone (AP). Pathogens were identified with 16S rRNA oligodeoxynucleotide probes and dot-blot hybridization before and at days 2, 3, 4, 7, 10, and 21 of healing. CP subjects were treated by scaling and root planing along with placebo tablets. RESULTS: Initially, AP cell counts were 69.9- (Porphyromonas gingivalis), 10.2- (Aggregatibacter actinomycetemcomitans), 5.7- (Tannerella forsythia), and 3.3-fold (Prevotella intermedia) enhanced compared to CP cell counts. Following SRP, immediate elimination occurred in single individuals of all three treatment groups at day 2. After SRP plus antibiotic therapy (AP+AB), the prevalence scores dropped beyond the levels of AP and CP, beginning at day 7, and remained low until day 21 (P =or< .05). Clinical healing statistically benefited from SRP with no differences among the three treatment groups. CONCLUSION: Nonsurgical therapy resulted in both a suppression and early elimination of single taxa immediately after completion of active treatment. Systemic antibiotics significantly accelerate the suppression of the periodontal microflora, but have limited effect on the elimination of target isolates during healing.

Comparison of real-time polymerase chain reaction and DNA-strip technology in microbiological evaluation of periodontitis treatment

The impact of a semiquantitative commercially available test based on DNA-strip technology (microIDent®, Hain Lifescience, Nehren, Germany) on diagnosis and treatment of severe chronic periodontitis of 25 periodontitis patients was evaluated in comparison with a quantitative in-house real-time PCR. Subgingival plaque samples were collected at baseline as well as at 3, 6, and 12 months later. After extracting DNA, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and several other periodontopathogens were determined by both methods. The results obtained by DNA-strip technology were analyzed semiquantitatively and additionally quantitatively by densitometry. The results for the 4 major periodontopathogenic bacterial species correlated significantly between the 2 methods. Samples detecting a high bacterial load by one method and negative by the other were always found in less than 2% of the total samples. Both technologies showed the impact of treatment on microflora. Especially the semiquantitative DNA-strip technology clearly analyzed the different loads of periodontopathogens after therapy and is useful in microbial diagnostics for patients in dental practices.

Xenobiotic metabolomics: major impact on the metabolome

Xenobiotics are encountered by humans on a daily basis and include drugs, environmental pollutants, cosmetics, and even components of the diet. These chemicals undergo metabolism and detoxication to produce numerous metabolites, some of which have the potential to cause unintended effects such as toxicity. They can also block the action of enzymes or receptors used for endogenous metabolism or affect the efficacy and/or bioavailability of a coadministered drug. Therefore, it is essential to determine the full metabolic effects that these chemicals have on the body. Metabolomics, the comprehensive analysis of small molecules in a biofluid, can reveal biologically relevant perturbations that result from xenobiotic exposure. This review discusses the impact that genetic, environmental, and gut microflora variation has on the metabolome, and how these variables may interact, positively and negatively, with xenobiotic metabolism.

Innate and adaptive immunity in host-microbiota mutualism

Healthy individuals live in peaceful co-existence with an immense load of intestinal bacteria. This symbiosis is advantageous for both the host and the bacteria. For the host it provides access to otherwise undigestible nutrients and colonization resistance against pathogens. In return the bacteria receive an excellent nutrient habitat. The mucosal immune adaptations to the presence of this commensal intestinal microflora are manifold. Although bacterial colonization has clear systemic consequences, such as maturation of the immune system, it is striking that the mutualistic adaptive (T and B cells) and innate immune responses are precisely compartmentalized to the mucosal immune system. Here we summarize the mechanisms of mucosal immune compartmentalization and its importance for a healthy host-microbiota mutualism.

Comparisons of bacterial patterns present at implant and tooth sites in subjects on supportive periodontal therapy. I. Impact of clinical variables, gender and smoking

OBJECTIVE: (I) To compare the oral microflora at implant and tooth sites in subjects participating in a periodontal recall program, (II) to test whether the microflora at implant and tooth sites differ as an effect of gingival bleeding (bleeding on probing (BOP)), or pocket probing depth (PPD), and (III) to test whether smoking and gender had an impact on the microflora. MATERIAL AND METHODS: Data were collected from 127 implants and all teeth in 56 subjects. Microbiological data were identified by the DNA-DNA checkerboard hybridization. RESULTS: PPD> or =4 mm were found in 16.9% of tooth, and at 26.6% of implant sites (P<0.01). Tooth sites with PPD> or =4 mm had a 3.1-fold higher bacterial load than implant sites (mean difference: 66%, 95% confidence interval (CI): 40.7-91.3, P<0.001). No differences were found for the red, orange, green, and yellow complexes. A higher total bacterial load was found at implant sites with PPD> or =4 mm (mean difference 35.7 x 10(5), 95% CI: 5.2 (10(5)) to 66.1 (10(5)), P<0.02 with equal variance not assumed). At implant sites, BOP had no impact on bacterial load but influenced the load at tooth sites (P<0.01). CONCLUSION: BOP, and smoking had no impact on bacteria at implant sites but influenced the bacterial load at tooth sites. Tooth sites harbored more bacteria than implant sites with comparable PPD. The 4 mm PPD cutoff level influenced the distribution and amounts of bacterial loads. The subject factor is explanatory to bacterial load at both tooth and implant sites.

Comparison of bacterial plaque samples from titanium implant and tooth surfaces by different methods

Studies have shown similarities in the microflora between titanium implants or tooth sites when samples are taken by gingival crevicular fluid (GCF) sampling methods. The purpose of the present study was to study the microflora from curette and GCF samples using the checkerboard DNA-DNA hybridization method to assess the microflora of patients who had at least one oral osseo-integrated implant and who were otherwise dentate. Plaque samples were taken from tooth/implant surfaces and from sulcular gingival surfaces with curettes, and from gingival fluid using filter papers. A total of 28 subjects (11 females) were enrolled in the study. The mean age of the subjects was 64.1 years (SD+/-4.7). On average, the implants studied had been in function for 3.7 years (SD+/-2.9). The proportion of Streptococcus oralis (P<0.02) and Fusobacterium periodonticum (P<0.02) was significantly higher at tooth sites (curette samples). The GCF samples yielded higher proportions for 28/40 species studies (P-values varying between 0.05 and 0.001). The proportions of Tannerella forsythia (T. forsythensis), and Treponema denticola were both higher in GCF samples (P<0.02 and P<0.05, respectively) than in curette samples (implant sites). The microbial composition in gingival fluid from samples taken at implant sites differed partly from that of curette samples taken from implant surfaces or from sulcular soft tissues, providing higher counts for most bacteria studied at implant surfaces, but with the exception of Porphyromonas gingivalis. A combination of GCF and curette sampling methods might be the most representative sample method.

Immediate bacterial colonization of titanium implants

Background: The information on bacterial colonization immediately after dental implant insertion is limited. Aims: (1) to assess the early colonization on titanium implants immediately post placement through the first12 post-surgical weeks , (2) to compare the microflora at interproximal subgingival implant and adjacent tooth sites. Material and Methods: Subgingival plaque samples from implant and neighbouring teeth were studied by checkerboard DNA-DNA hybridization before, 30 min. after implant placement , 1 week, 2 weeks, 4 weeks, 8 weeks, and 12 weerks after surgery. Results: Comparing bacterial loads at implant sites between 30 min. after placement with one week data showed that only the levels of V.parvula (p<0.05) differed with higher loads at week 1. Week 12 data demonstrated significantly higher bacterial loads for 15/40 species at tooth sites compared to pre-surgery (p < values varying between 0.05 and 0.01). Between immediately post-surgery and week 12 at implant sites 29/40 species were more commonly found at week 12. Included among these bacteria at implant sites were P.gingivalis (p< 0.05), T.forsythia, (p < 0.01), and T denticola (p<0.001). Immediately post-surgery 5.9% of implants, and 26.2% of teeth and at week 12, 15.0 % of implants, and 39.1% of teeth harbored S.aureus. Comparing tooth and implant sites, significantly higher bacterial loads were found at tooth sites for 27/40 species at the 30 minutes after placement interval. This difference increased to 35/40 species at week 12. Conclusions: The colonization of bacteria occurs within 30 minutes. Colonization patterns differed between implants and tooth surfaces.

Upregulation of toll-like receptors in chronic enteropathies in dogs

Background: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown. Hypothesis: That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. Animals: Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. Methods: Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs. Results: There were significant differences (P small intestine >/= colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores. Conclusions and Clinical Importance: Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.

The mucosal immune system at the gastrointestinal barrier

The immune system faces a considerable challenge in its efforts to maintain tissue homeostasis in the intestinal mucosa. It is constantly confronted with a large array of antigens, and has to prevent the dissemination and proliferation of potentially harmful agents while sparing the vital structures of the intestine from immune-mediated destruction. Complex interactions between the highly adapted effector cells and mechanisms of the innate and adaptive immune system generally prevent the luminal microflora from penetrating the intestinal mucosa and from spreading systemically. Non-haematopoietic cells critically contribute to the maintenance of local tissue homeostasis in an antigen-rich environment by producing protective factors (e.g. production of mucus by goblet cells, or secretion of microbicidal defensins by Paneth cells) and also through interactions with the adaptive and innate immune system (such as the production of chemotactic factors that lead to the selective recruitment of immune cell subsets). The complexity of the regulatory mechanisms that control the local immune response to luminal antigens is also reflected in the observation that mutations in immunologically relevant genes often lead to the development of uncontrolled inflammatory reactions in the microbially colonized intestine of experimental animals.

Potent and broad-spectrum antimicrobial activity of CXCL14 suggests an immediate role in skin infections

The skin is constantly exposed to commensal microflora and pathogenic microbes. The stratum corneum of the outermost skin layer employs distinct tools such as harsh growth conditions and numerous antimicrobial peptides (AMPs) to discriminate between beneficial cutaneous microflora and harmful bacteria. How the skin deals with microbes that have gained access to the live part of the skin as a result of microinjuries is ill defined. In this study, we report that the chemokine CXCL14 is a broad-spectrum AMP with killing activity for cutaneous gram-positive bacteria and Candida albicans as well as the gram-negative enterobacterium Escherichia coli. Based on two separate bacteria-killing assays, CXCL14 compares favorably with other tested AMPs, including human beta-defensin and the chemokine CCL20. Increased salt concentrations and skin-typical pH conditions did not abrogate its AMP function. This novel AMP is highly abundant in the epidermis and dermis of healthy human skin but is down-modulated under conditions of inflammation and disease. We propose that CXCL14 fights bacteria at the earliest stage of infection, well before the establishment of inflammation, and thus fulfills a unique role in antimicrobial immunity.