Establishment and development of infant gut microbiota

The nascent gut microbiota at birth is established in concert with numerous developmental parameters. Here, in the INFAMTET study, we chronicled the impact of some factors which are key determinants of the infant gut microbiota, namely; mode of birth, gestational age, and type of feeding. We determined that the aggregated microbiota profile of naturally delivered, initially breastfed infants are relatively stable from one week to six months of age and are not significantly altered by increased duration of breastfeeding. Contrastingly, there is significant development of the microbiota profile of C-section delivered infants, and this development is significantly influenced by breastfeeding duration. Preterm infants, born by either mode of birth, initially have a high proportion of Proteobacteria, and demonstrate significant development of the gut microbiota from week 1 to later time-points. The microbiota is still slightly, but significantly, affected by birth mode at one year of age although no specific genera were found to be significantly altered in relative abundance. By two years of age, there is no effect of either birth mode or gestational age. However this does not preclude the possibility that symptoms developed later in life, which are associated with preterm or C-section birth, are as a result of the early perturbation of the neonatal gut microbiota. It is likely that the combination of relatively low exposure (breast fed), high exposure (formula fed) or delayed exposure (C-section and preterm) to specific antigens and the resulting inflammatory responses, in this crucial window of host-microbiota interaction, influence systemic health of the individual throughout life.

Effect of room temperature transport vials on DNA quality and phylogenetic composition of faecal microbiota of elderly adults and infants

Background: Alterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults. Results: Use of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week's storage compared to immediately extracted samples. After 2 weeks' storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2. The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p

Impact of salt reduction on biogenic amines, fatty acids, microbiota, texture and sensory profile in traditional blood dry-cured sausages

Meat industry needs to reduce salt in their products due to health issues. The present study evaluated the effect of salt reduction from 6% to 3% in two Portuguese traditional blood dry-cured sausages. Physicochemical and microbiological parameters, biogenic amines, fatty acids and texture profiles and sensory panel evaluations were considered. Differences due to salt reduction were perceptible in a faint decline of water activity, which slightly favoured microbial growth. Total biogenic amines content ranged from 88.86 to 796.68 mg kg 1 fresh matter, with higher amounts, particularly of cadaverine, histamine and tyramine, in low-salt products. Still, histamine and other vasoactive amines remained at low levels, thus not affecting consumers’ health. Regarding fatty acids, no significant differences were observed due to salt. However, texture profile analysis revealed lower resilience and cohesiveness in low-salt products, although no textural changes were observed by the sensory panel. Nevertheless, low-salt sausages were clearly preferred by panellists.

Caracterização bioquímica de proteínas aderidas à fibra do bagaço de cana-de-açúcar obtidas de conteúdo ruminal de ovinos.

A conversão da celulose e da hemicelulose do bagaço de cana-de-açúcar em bioetanol tem utilizado enzimas comerciais importadas, tornando o processo caro, justificando a busca e a produção de enzimas nacionais. O presente trabalho objetivou caracterizar as proteínas aderidas à fibra do bagaço de cana-de-açúcar (BCA), obtidas do conteúdo ruminal de ovinos Morada Nova, quanto à resistência térmica e à variação de pH.

Avaliação do impacto de eventos comerciais de milho Bt sobre a microbiota do solo.

2016

Impacto do manejo agrícola sobre a microbiota do solo.

Para avaliar o efeito do manejo agrícola de solo sobre os parâmetros biológico, bioquímico e químico, três propriedades agrícolas de Suzano/SP foram selecionadas contendo os seguintes tratamentos: áreas de manejo biodinâmico com o cultivo de hortaliças, áreas de manejo convencional com o cultivo de milho, área de pastagem (8 anos) e área de mata nativa. Amostras de solo foram submetidas as analises química, bioquímica e biológica. Os resultados mostraram que alguns parâmetros biológicos (bactérias esporulantes, actinomicetos, microrganismos celulolíticos), químico (pH, CTC, MO) e bioquímico (desidrogenase, polissacarídeos) foram superiores nas áreas de manejo biodinâmico em relação ao manejo convencional.

Impacto de mudanças climáticas globais sobre a microbiota terrestre.

2008

Impacto de xenobióticos e metais pesados na microbiota do solo.

2008

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.

Ruolo del Microbiota Intestinale Nella Risposta al Trattamento con Anticorpi anti Checkpoints Immunitari in Pazienti Affetti da Linfoma

L’interazione tra il sistema immunitario dell’ospite e la cellula tumorale rappresenta uno degli elementi cardine dello sviluppo del clone neoplastico: la capacità della cellula cancerosa di evadere il controllo immunitario sfruttando meccanismi fisiologici come i checkpoint immunitari è alla base di diverse neoplasie, incluse le sindromi linfoproliferative. Lo sviluppo di anticorpi monoclonali che bloccano selettivamente l’interazione tra il recettore trans-membrana PD-1 (programmed death -1) ed i propri ligandi (PD-L1 e PD-L2), rappresenta una delle scoperte terapeutiche più promettenti in ambito onco-ematologico. Nonostante l’importante efficacia antitumorale degli anticorpi anti checkpoint immunitari dimostrata dai differenti studi clinici condotti sia in ambito oncologico che ematologico, una parte dei pazienti, a parità di patologia e di farmaco ricevuto, non risponde alla terapia o sviluppa eventi avversi immuno-relati. La comprensione della variabilità di risposta dimostrata dai pazienti con stessa patologia, sottoposti a stesso trattamento rappresenta pertanto un punto chiave allo scopo di identificare strategie che possano potenziare l’efficacia terapeutica di tali anticorpi, riducendone gli effetti collaterali. Studi recenti hanno evidenziato il ruolo del microbiota intestinale (MI) nel modellare la risposta immunitaria sistemica e, nel contesto neoplastico, nel modificare e mediare l’attivazione del sistema immunitario ad agenti chemio-immunoterapici. È noto che il MI sia un ecosistema plastico che può riorganizzare funzionalità e composizione in maniera adattativa in risposta a diversi fattori ambientali. La struttura individuale del MI e la sua dinamicità temporale possono, pertanto, influenzare l’outcome delle chemio-immunoterapie onco-ematologiche, modulandone l’efficacia e la tossicità. In questo scenario, ipotizziamo che la caratterizzazione longitudinale (pre, durante e post-terapia) del MI di pazienti affetti da linfoma trattati con anticorpi anti-checkpoint inibitori e la sua correlazione con la risposta al trattamento e con lo sviluppo di eventi avversi possa avere un ruolo nel delineare l’outcome di tali pazienti e nell’identificare nuovi criteri di stratificazione del rischio.

Can gut microbiota analysis be beneficial for ex-situ and in-situ conservation of threatened animal species?

Ex-situ conservation and the in-situ conservation of natural habitats are the tools to conserve biodiversity. Habitats and ecosystems have been becoming altered by human activities and a growing number of species requires form of management to ensure their survival. Conservation queries become more complex and urgent. Developing scientifically based and innovative approaches to ex-situ conservation is necessary. Recent studies underline importance of gut microbiome in animal health with implications for animal conservation and management. Animal and human studies have demonstrated that environmental factors can impact gut microbiome composition. Within this scenario, the present work focused on species belonging to different taxa, reptiles and mammals: Aldabrachelys gigantea, the giant tortoise of the Seychelles islands and Indri indri, the greatest leaving lemur of Madagascar. The Seychelles giant tortoise is vulnerable species with declining population, whereas the indri is a critically endangered species that could reach the extinction within 25 years. Both need research to help them to survive. Tortoises live for very long time and to observe how they can afford the environmental changes is very difficult. Indris, instead, are able to survive only in a small area of the Madagascar forest, with a very strong link between the species’ survival and the environment. The obtained results underline importance of environmental factors, both in-situ and ex-situ, for species conservation. Microbiome could help the organisms to respond on a short timescale and cope with, environmental changes. However, species with long generation time might not be able to adapt to fast changes but bacteria with a short generation time can adapt on a shorter timescale allowing the host to cope with fluctuating environment. Gut microbiome plays an important role in an animal’s health and has the potential to improve the management of individuals under human care for conservation purposes.

Effetto sul microbiota intestinale della sostituzione dei nitriti con antiossidanti naturali in salami sperimentali

Recentemente il consumo di carne ha attirato l'attenzione per motivi salutistici, ambientali ed etici; pertanto è sempre più urgente considerare formulazioni di prodotti a base di carne più salutari e sostenibili inclusi in una dieta sana e nutriente. In questa tesi sono stati valutati gli effetti in vitro sul microbiota intestinale umano di salami riformulati sostituendo i nitriti con acido ascorbico e antiossidanti vegetali. A questo scopo è stato utilizzato il modello intestinale in vitro MICODE, nella versione che include la digestione gastro-duodenale seguita dalla fermentazione colonica. Gli effetti sul microbiota intestinale sono stati valutati attraverso tecniche di analisi quantitativa Real Time qPCR in base agli shift di alcune popolazioni microbiche, sia quelle benefiche (come Lactobacillales, Bifidobacteriaceae, Clostridium gruppo IV), sia quelle opportuniste (come Enterobacteriaceae, Clostridium gruppo I) e infine il rapporto Firmicutes/Bacteroidetes (importante indice di eubiosi del microbiota intestinale dell’ospite). Inoltre, sono state analizzate, tramite SPME-GC-MS, le molecole volatili prodotte in seguito alla fermentazione colonica, sia positive, come gli SCFA, sia negative, come fenolo e cresolo. I risultati ottenuti hanno mostrato che le formulazioni innovative promuovono una generale eubiosi del microbiota intestinale e una riduzione di popolazioni microbiche negative, in confronto ai controlli. Inoltre, l’analisi del volatiloma evidenzia una maggiore produzione di molecole benefiche e una maggiore riduzione delle molecole negative per l’ospite. Sebbene le formulazioni innovative non abbiano dato benefici nettamente superiori a quelli dei controlli, i risultati ottenuti sono promettenti, in quanto gli antiossidanti utilizzati in sostituzione hanno dato risultati comparabili a quelli ottenuti con il formulato tradizionale.

Relazione tra carica virale, reazione immunitaria dell'ospite, microbiota e decorso clinico della bronchiolite acuta nei bambini con età inferiore ai 12 mesi (RMB2018)

Il principale motivo di ospedalizzazione del lattante è la bronchiolite acuta, causata in primis dal Virus Respiratorio Sinciziale (VRS). In questo ambito appaiono controversi i risultati in letteratura riguardo: carica del VRS, risposta immunitaria ed infiammatoria dell’ospite, microbiota del paziente (durante l’infezione e per successivo sviluppo di wheezing e asma). In questo studio di coorte prospettico monocentrico vengono arruolati lattanti peraltro sani ricoverati per primo episodio di bronchiolite acuta, da VRS o da altro agente, per valutare primariamente la relazione tra decorso clinico e carica del VRS, secondariamente l’associazione con specifiche composizioni e modifiche nel tempo del microbiota nasofaringeo ed intestinale durante la fase acuta e nel lungo termine in relazione a sviluppo di wheezing ricorrente. Nello studio sono stati arruolati finora 89 pazienti, di cui 68 con bronchiolite da VRS (76.4%), con analisi della carica virale su 41 lattanti (60.3%) e del microbiota su 20 (29.4%). L’analisi della carica del VRS non ha riscontrato associazione tra outcome di severità clinica quali necessità e durata di ossigenoterapia, nonché durata di ricovero. La presenza di trend di associazione tra carica virale all’ingresso e picco del VRS-RNA con necessità di ossigenoterapia ad alto flusso (HFNC) e l’associazione significativa di clearance di VRS con HFNC (p = 0.03) suggeriscono che la carica virale potrebbe influenzare la severità della bronchiolite da VRS. Le analisi del microbiota evidenziano numerosi genera mai descritti finora in letteratura a nostra conoscenza (es. Alloiococcus e Leptotrichia su aspirato, nonché tutti i dati emersi dallo studio della saliva, prima volta in letteratura) ed un generale aumento della diversity a 6 mesi dalla dimissione. Occorrerà completare l’analisi della carica del VRS con quella del sistema infiammatorio dell’ospite, nonché studiare il microbiota sui campioni del follow-up a lungo termine per la verifica di eventuali associazioni con sviluppo di wheezing e asma.

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.

Different dietary fibres included in the gestation and lactation diet shape sow's microbiota, modify colostrum and milk composition and affect piglet growth and health

This thesis reports three experimental studies that may contribute to understand how the sources or types of dietary fibres (DFs) included in sow diet with similar level of total DFs influence the composition of colostrum and milk and their related effects on offspring performance and gut microbiota. The first study showed that decreasing the level of hemicelluloses (HCs) in sow’s lactation diet increased the proportion of butyrate and the concentration of volatile fatty acids (VFAs), copper and threonine in milk. Simultaneously, the post-weaning growth of low birthweight piglets was improved, and the diarrhoea occurrence was reduced during the second week post-weaning. The second study showed that the level of HCs in the diet of lactating sows affected their faecal microbiota, modified the VFA profile in sow’s faeces during lactation and barely impacted the faecal microbiota of slow and fast growing piglets. The third study showed that replacing a source soluble DFs by one of insoluble DFs in sow’s diet during late gestation and lactation reduced farrowing duration, increased total VFAs and lactoferrin concentrations in colostrum, improved growth performance from birth to 1 day of lactation, during the post-weaning period and throughout the study, and reduced diarrhoea occurrence during the first week post-weaning. Finally, a fourth study proposed a workflow to analyse low biomass samples from the umbilical cord blood aiming at investigating the existence of a pre-birth microbiota with no substantial findings to confirm this hypothesis. Overall, the results of these studies confirmed that, besides the level of DFs, the sources, and the types of DFs included in the sow's diet shape the sow's microbiota, influence the composition of colostrum and milk, and improve offspring performance, but with limited impacts on the microbiota of piglets.