The commensal infant gut meta-mobilome as a potential reservoir for persistent multidrug resistance integrons

Despite the accumulating knowledge on the development and establishment of the gut microbiota, its role as a reservoir for multidrug resistance is not well understood. This study investigated the prevalence and persistence patterns of an integrase gene (int1), used as a proxy for integrons (which often carry multiple antimicrobial resistance genes), in the fecal microbiota of 147 mothers and their children sampled longitudinally from birth to 2 years. The study showed the int1 gene was detected in 15% of the study population, and apparently more persistent than the microbial community structure itself. We found int1 to be persistent throughout the first two years of life, as well as between mothers and their 2-year-old children. Metagenome sequencing revealed integrons in the gut meta-mobilome that were associated with plasmids and multidrug resistance. In conclusion, the persistent nature of integrons in the infant gut microbiota makes it a potential reservoir of mobile multidrug resistance.

Maternal weaning modulates emotional behavior and regulates the gut-brain axis

Evidence shows that nutritional and environmental stress stimuli during postnatal period influence brain development and interactions between gut and brain. In this study we show that in rats, prevention of weaning from maternal milk results in depressive-like behavior, which is accompanied by changes in the gut bacteria and host metabolism. Depressive-like behavior was studied using the forced-swim test on postnatal day (PND) 25 in rats either weaned on PND 21, or left with their mother until PND 25 (non-weaned). Non-weaned rats showed an increased immobility time consistent with a depressive phenotype. Fluorescence in situ hybridization showed non-weaned rats to harbor significantly lowered Clostridium histolyticum bacterial groups but exhibit marked stress-induced increases. Metabonomic analysis of urine from these animals revealed significant differences in the metabolic profiles, with biochemical phenotypes indicative of depression in the non-weaned animals. In addition, non-weaned rats showed resistance to stress-induced modulation of oxytocin receptors in amygdala nuclei, which is indicative of passive stress-coping mechanism. We conclude that delaying weaning results in alterations to the gut microbiota and global metabolic profiles which may contribute to a depressive phenotype and raise the issue that mood disorders at early developmental ages may reflect interplay between mammalian host and resident bacteria.

Lactobacillus acidophilus CRL 1014 improved gut health in the SHIME® reactor

Background: How to maintain gut health is a goal for scientists throughout the world. Therefore, microbiota management models for testing probiotics, prebiotics, and synbiotics have been developed.Methods: The SHIME® model was used to study the effect of Lactobacillus acidophilus 1014 on the fermentation pattern of the colon microbiota. Initially, an inoculum prepared from human feces was introduced into the reactor vessels and stabilized over 2-wk using a culture medium. This stabilization period was followed by a 2-wk control period during which the microbiota was monitored. The microbiota was then subjected to a 4-wk treatment period by adding 5 mL of sterile peptone water with L. acidophilus CRL1014 at the concentration of 108 CFU/mL to vessel one (the stomach compartment). Plate counts, Denaturing Gradient Gel Electrophoresis (DGGE), short-chain fatty acid (SCFA) and ammonium analyses were carried out for monitoring of the microbial community from the colon compartments.Results: A significant increase (p < 0.01) in the Lactobacillus spp. and Bifidobacterium spp. populations was observed during the treatment period. The DGGE obtained showed changes in the lactobacilli community from the colon compartments of the SHIME® reactor. The (SCFA) concentration increased (p < 0.01) during the treatment period, due mainly to significant increased levels of acetic, butyric, and propionic acids. However, ammonium concentrations decreased during the same period (p < 0.01).Conclusions: This study showed the beneficial influence of L. acidophilus CRL 1014 on microbial metabolism and lactobacilli community composition for improving human health. © 2013 Sivieri et al.; licensee BioMed Central Ltd.

Estrutura, ultraestrutura e histoquímica do aparelho digestório do Prochilodus lineatus. Análise da diversidade da microbiota intestinal de Prochilodus lineatus e Pterygoplichthys anisitsi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da restrição alimentar e do grupo genético sobre a microbiota do trato gastrintestinal, a expressão gênica hepática e a deposição de lipídeos na carcaça de frangos de corte

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

P-215-Quantitative variation of some gut bacteria in Crohn's disease can be related to the patient's gender

Background: Imbalance in bacterial species composition of the gut microbiota is one of the factors associated with the cause or complication of the symptoms of Crohn's disease (CD). This disequilibrium consists in the reduction of biodiversity, decrease of genus such as Bifidobacterium and elevation of species such as Escherichia coli. Human microbiota varies among subjects of a same population irrespective of their health condition and among individuals living in distinct geographic locations. In animal models, sex related differences could also be observed in gut bacterial species composition under some pathological conditions. Experiments conducted with mice have demonstrated that the manifestation of type 1 diabetes (T1D) could be under the influence of the animal sex and its serum level of testosterone, which in turn could be modulated by a particular gut microbiota. Considering the existence of similar features between T1D and CD, such as strong genetic component and malfunctioning of the immune system, we investigated whether differences could be observed in the gut microbiota dysbiosis of male and female CD patients. Methods: Fifty and 5 gut mucosal biopsies from 25 adult CD patients (11 males and 14 females) and 43 specimens of an equivalent clinical material from 22 control subjects (11 males and 11 females) were screened for bacterial biodiversity by analyzing sequences of 16SrDNA V6 region. A number of 2-3 samples each from distinct gut segments (from ileum to rectum) were taken from each subject. The 16SrDNA sequences were obtained by sequencing PCR amplicons of the corresponding gene in the Ion torrent PGM sequencer. Identification and classification of the bacterial groups followed the Ribosomal Database Project (RDP) website pipeline. The relationships of the bacterial taxa with each of the study parameters was performed by compiling the data in a MS Excel and the level of statistical significance determined by the Chi-square test. Results: A total of 3203 16SrDNA sequences were detected in the 98 biopsies samples, the majority of which matching Proteobacteria, Firmicutes, Bacterioidetes, and Actinobacteria. The percentage of DNA sequences for each of these phyla found in Male control subjects/Male CD patients was 40.5/33, 32.7/32.4, 20.8/24.5, and 4.4/4,4 for Proteobacteria, Firmicutes, Bacterioidetes, and Actinobacteria, respectively. In Female comparisons, these values were 35.6/42, 39.2/26.3, 19.8/23.3, 5.2/7. Both Male and Female CD patients presented higher numbers of sequences of Actinobacteria and Bacterioidetes than those of control subjects of the same gender. Case-control differences for Firmicutes could be observed only in female comparisons and, for Proteobacteria, although case-control differences were observed in both genders, the nature of difference was distinct, since while in CD female patients a higher number of sequences matching this phylum was detected, in males a reduced number was observed, in comparison with controls. The species responsible for the Proteobacteria variation in both gender was Escherichia coli. Conclusions: The data presented above suggest that any analysis of dysbiosis in CD must take in account the patient's gender, an observation particularly relevant for Escherichia coli, whose association with CD has been most intensively investigated and for which the present study shows a reverse quantitative variation regarding the patients' gender.

Influência do suco de laranja na microbiota intestinal humana

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Microbiota benefits after inulin and partially hydrolized guar gum supplementation - a randomized clinical trial in constipated women

Introduction: Prebiotics positively affect gut microbiota composition, thus improving gut function. These properties may be useful for the treatment of constipation. Objectives: This study assessed the tolerance and effectiveness of a prebiotic inulin/partially hydrolyzed guar gum mixture (I-PHGG) for the treatment of constipation in females, as well as its influence on the composition of intestinal microbiota and production of short chain fatty acids. Methods: Our study enrolled 60 constipated female health worker volunteers. Participants reported less than 3 bowel movements per week. Volunteers were randomized to treatment with prebiotic or placebo. Treatment consisted of 3 weeks supplementation with 15 g/d I-PHGG (fiber group) or maltodextrin (placebo group). Abdominal discomfort, flatulence, stool consistency, and bowel movements were evaluated by a recorded daily questionnaire and a weekly interview. Changes in fecal bacterial population and short chain fatty acids were assessed by real-time PCR and gas chromatography, respectively. Results: There was an increased frequency of weekly bowel movements and patient satisfaction in both the fiber and placebo groups with no significant differences. Total Clostridium sp significantly decreased in the fiber group (p = 0.046) and increased in the placebo group (p = 0.047). There were no changes in fecal short chain fatty acid profile. Conclusions: Consumption of I-PHGG produced clinical results comparable to placebo in constipated females, but had additional protective effects on gut rnicrobiota by decreasing the amount of pathological bacteria of the Clostridium genera.

Metagenomic trajectory of gut microbiome in the human lifespan

Co-evolving with the human host, gut microbiota establishes configurations, which vary under the pressure of inflammation, disease, ageing, diet and lifestyle. In order to describe the multi-stability of the microbiome-host relationship, we studied specific tracts of the bacterial trajectory during the human lifespan and we characterized peculiar deviations from the hypothetical development, caused by disease, using molecular techniques, such as phylogenetic microarray and next-generation sequencing. Firstly, we characterized the enterocyte-associated microbiota in breast-fed infants and adults, describing remarkable differences between the two groups of subjects. Successively, we investigated the impact of atopy on the development of the microbiome in Italian childrens, highlithing conspicuous deviations from the child-type microbiota of the Italian controls. To explore variation in the gut microbiota depending on geographical origins, which reflect different lifestyles, we compared the phylogenetic diversity of the intestinal microbiota of the Hadza hunter-gatherers of Tanzania and Italian adults. Additionally, we characterized the aged-type microbiome, describing the changes occurred in the metabolic potential of the gut microbiota of centenarians with respect to younger individuals, as a part of the pathophysiolology of the ageing process. Finally, we evaluated the impact of a probiotics intervention on the intestinal microbiota of elderly people, showing the repair of some age-related dysbioses. These studies contribute to elucidate several aspects of the intestinal microbiome development during the human lifespan, depicting the microbiota as an extremely plastic entity, capable of being reconfigured in response to different environmental factors and/or stressors of endogenous origin.

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.

Acquisition of a multifunctional IgA+ plasma cell phenotype in the gut

The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and differentiate to become plasma cells. However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells.

Sex Differences in the Gut Microbiome Drive Hormone-Dependent Regulation of Autoimmunity

Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipient's microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.

Dietary inclusion of feathers affects intestinal microbiota and microbial metabolites in growing Leghorn-type chickens.

Feather pecking in laying hens is a serious behavioral problem that is often associated with feather eating. The intake of feathers may influence the gut microbiota and its metabolism. The aim of this study was to determine the effect of 2 different diets, with or without 5% ground feathers, on the gut microbiota and the resulting microbial fermentation products and to identify keratin-degrading bacteria in chicken digesta. One-day-old Lohmann-Selected Leghorn chicks were divided into 3 feeding groups: group A (control), B (5% ground feathers in the diet), and C, in which the control diet was fed until wk 12 and then switched to the 5% feather diet to study the effect of time of first feather ingestion. The gut microbiota was analyzed by cultivation and denaturing gradient gel electrophoresis of ileum and cecum digesta. Short-chain fatty acids, ammonia, and lactate concentrations were measured as microbial metabolites. The concentration of keratinolytic bacteria increased after feather ingestion in the ileum (P < 0.001) and cecum (P = 0.033). Bacterial species that hydrolyzed keratin were identified as Enterococcus faecium, Lactobacillus crispatus, Lactobacillus reuteri-like species (97% sequence homology), and Lactobacillus salivarius-like species (97% sequence homology). Molecular analysis of cecal DNA extracts showed that the feather diet lowered the bacterial diversity indicated by a reduced richness (P < 0.001) and shannon (P = 0.012) index. The pattern of microbial metabolites indicated some changes, especially in the cecum. This study showed that feather intake induced an adaptation of the intestinal microbiota in chickens. It remains unclear to what extent the changed metabolism of the microbiota reflects the feather intake and could have an effect on the behavior of the hens.

Microbiota-mediated fine-tuning of the threshold of intestinal inflammasome activation in host-microbial mutualism

The inflammasome is a complex of proteins that controls the activity of caspase-1, pro-IL-1b and pro-IL-18. It acts in inflammatory processes and in pyropoptosis. The lower intestine is densely populated by a community of commensal bacteria that, under healthy conditions, are beneficial to the host. Some evidence suggests that the gut microbiota influences regulation of the inflammasome. Components of inflammasomes have been shown to have a protective function against development of experimental colitis, dependent on IL-18 production. However the precise mechanisms and the role of the inflammasome in maintaining a healthy host-microbial mutualism remains unknown. To address this question, we have performed axenic (GF) and gnotobiotic in vivo experiments to investigate how the inflammasome components mainly at the level of intestinal epithelial cells (IECs) are regulated under different hygiene conditions. We have established that gene expression of the inflammasome components NLRC4, NLRP3, NLRP6, NLRP12, caspase-1, ASC and IL-18 do not differ between germ-free and colonised conditions under steady-state. In contrast, induction in IL-18 was observed following infection with the pathobiont Segmented Filamentous Bacteria or the pathogen C. rodentium. Additional preliminar findings suggest that a more diverse intestinal flora, like specific pathogen-free (SPF) flora, is more efficient in inducing basal activation of the inflammasome and especially production of IL-18 by IECs, shortly after colonisation. We are also in the process of testing if basal activation of the inflammasome upon intestinal colonization with commensal bacteria helps to protect the host from potential pathobiont bacteria, like C. rodentium, SFB, Prevotella and TM7.

Análise da microbiota intestinal em adultos com hábitos alimentares distintos e de associações com a inflamação e resistência à insulina

Introdução: A microbiota intestinal possui grande diversidade de bactérias, predominantemente dos filos Bacteroidetes e Firmicutes, com múltiplas funções. A alimentação pode alterar sua composição e função. Alto teor de gordura saturada altera a permeabilidade intestinal, eleva os lipopolissacarídeos e predispõe à inflamação subclínica crônica. Dieta rica em fibras, como a vegetariana, induz elevação de ácidos graxos de cadeia curta e benefícios metabólicos. Objetivos: Para analisar a composição da microbiota intestinal de adventistas com diferentes hábitos alimentares e associá-los à inflamação subclínica e resistência à insulina, esta tese incluiu: 1) revisão dos mecanismos que associam a alimentação à microbiota intestinal e ao risco cardiometabólico; 2) verificação da composição da microbiota intestinal segundo diferentes hábitos alimentares e de associações com biomarcadores de doenças cardiometabólicas; 3) avaliação da associação entre a abundância de Akkermansia muciniphila e o metabolismo da glicose; 4) análise da presença de enterótipos e de associações com características clínicas. Métodos: Este estudo transversal incluiu 295 adventistas estratificados segundo hábitos alimentares (vegetariano estrito, ovo-lacto-vegetariano e onívoro). Foram avaliadas associações com dados clínicos, bioquímicos e inflamatórios. O perfil da microbiota foi obtido por sequenciamento do gene 16S rRNA (Illumina® Miseq). Resultados: 1) Há evidências de que as relações entre dieta, inflamação, resistência à insulina e risco cardiometabólico são em parte mediadas pela composição da microbiota intestinal. 2) Vegetarianos apresentaram melhor perfil clínico quando comparados aos onívoros. Confirmou-se maior abundância de Firmicutes e Bacteroidetes, que não diferiram segundo a adiposidade corporal. Entretanto, vegetarianos estritos apresentaram mais Bacteroidetes, menos Firmicutes e maior abundância do gênero Prevotella quando comparados aos outros dois grupos de hábitos alimentares. Entre os ovo-lactovegetarianos verificou-se maior proporção de Firmicutes especialmente do gênero Faecalibacterium. Nos onívoros, houve super-representação do filo Proteobacteria (Succinivibrio e Halomonas) comparados aos vegetarianos. 3) Indivíduos normoglicêmicos apresentaram maior abundância de Akkermansia muciniphila que aqueles com glicemia alterada. A abundância desta bactéria correlacionou-se inversamente à glicemia e hemoglobina glicosilada. 4) Foram identificados três enterótipos (Bacteroides, Prevotella e Ruminococcaceae), similares àqueles previamente descritos. As concentrações de LDL-C foram menores no enterótipo 2, no qual houve maior frequência de vegetarianos estritos. Discussão: 1) Conhecimentos sobre participação da microbiota na fisiopatologia de doenças poderão reverter em estratégias para manipulá-la para promover saúde. 2) Apoia-se a hipótese de que hábitos alimentares se associam favorável ou desfavoravelmente a características metabólicas e inflamatórias do hospedeiro via alterações na composição da microbiota intestinal. Sugerimos que a exposição a alimentos de origem animal possa impactar negativamente nas proporções de comunidades bacterianas. 3) Sugerimos que a abundância da Akkermansia muciniphila possa participar do metabolismo da glicose. 4) Reforçamos que a existência de três enterótipos não deva ser específica de certas populações/continentes. Apesar de desconhecido o significado biológico destes agrupamentos, as correlações com o perfil lipídico podem sugerir sua utilidade na avaliação do risco cardiometabólico. Conclusões: Nossos achados fortalecem a ideia de que a composição da microbiota intestinal se altera mediante diferentes hábitos alimentares, que, por sua vez, estão associados a alterações nos perfis metabólicos e inflamatórios. Estudos prospectivos deverão investigar o potencial da dieta na prevenção de distúrbios cardiometabólicos mediados pela microbiota.