RESUMO
Oral administration of probiotics has been proposed as a promising biotherapy to prevent and treat different diseases related to gastrointestinal disorders, such as irritable bowel syndrome (IBS). Due to the increasing research area on the characterisation of new probiotic bacterial strains, it is necessary to perform suitable in vitro experiments, using pertinent cellular models, in order to establish appropriate readout profiles based on IBS symptoms and subtypes. In this work, a collection of 30 candidate strains, belonging mainly to the Lactobacillus and Bifidobacterium genera, were screened using three different sets of in vitro experiments with different readouts to identify promising probiotic strains with: (1) the ability to inhibit the synthesis of IL-8 production by TNF-α stimulated HT-29 cells, (2) immunomodulatory properties quantified as increased IL-10 levels in peripheral blood mononuclear cell (PBMCs), and (3) the ability to maintain epithelial barrier integrity by increasing the trans-epithelial/endothelial electrical resistance (TEER) values in Caco-2 cells. Based on these criteria, three strains were selected: Lactobacillus gasseri PI41, Lacticaseibacillus rhamnosus PI48 and Bifidobacterium animalis subsp. lactis PI50, and tested in a murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS), which mimics some of the symptoms of IBS. Among the three strains, L. gasseri PI41 improved overall host well-being by preventing body weight loss in DNBS-treated mice and restored gut homeostasis by normalising the intestinal permeability and reducing pro-inflammatory markers. Therefore, the potential of this strain was confirmed in a second murine model known to reproduce IBS symptoms: the neonatal maternal separation (NMS) model. The PI41 strain was effective in preventing intestinal permeability and reducing colonic hypersensitivity. In conclusion, the set of in vitro experiments combined with in vivo assessments allowed us to identify a promising probiotic candidate strain, L. gasseri PI41, in the context of IBS.
Assuntos
Síndrome do Intestino Irritável , Probióticos , Probióticos/administração & dosagem , Probióticos/farmacologia , Síndrome do Intestino Irritável/terapia , Síndrome do Intestino Irritável/microbiologia , Humanos , Animais , Camundongos , Células CACO-2 , Células HT29 , Modelos Animais de Doenças , Leucócitos Mononucleares/imunologia , Lactobacillus/fisiologia , Interleucina-8/metabolismo , Bifidobacterium/fisiologia , Interleucina-10 , Lactobacillus gasseri , Lacticaseibacillus rhamnosus/fisiologia , Masculino , Bifidobacterium animalis/fisiologiaRESUMO
The developmental origin of health and disease highlights the importance of the period of the first 1000 days (from the conception to the 2 years of life). The process of the gut microbiota establishment is included in this time window. Various perinatal determinants, such as cesarean section delivery, type of feeding, antibiotics treatment, gestational age or environment, can affect the pattern of bacterial colonization and result in dysbiosis. The alteration of the early bacterial gut pattern can persist over several months and may have long-lasting functional effects with an impact on disease risk later in life. As for example, early gut dysbiosis has been involved in allergic diseases and obesity occurrence. Besides, while it was thought that the fetus developed under sterile conditions, recent data suggested the presence of a microbiota in utero, particularly in the placenta. Even if the origin of this microbiota and its eventual transfer to the infant are nowadays unknown, this placental microbiota could trigger immune responses in the fetus and would program the infant's immune development during fetal life, earlier than previously considered. Moreover, several studies demonstrated a link between the composition of placental microbiota and some pathological conditions of the pregnancy. All these data show the evidence of relationships between the neonatal gut establishment and future health outcomes. Hence, the use of pre- and/or probiotics to prevent or repair any early dysbiosis is increasingly attractive to avoid long-term health consequences.
Assuntos
Disbiose/microbiologia , Microbioma Gastrointestinal/fisiologia , Imunidade/fisiologia , Intestinos/crescimento & desenvolvimento , Complicações na Gravidez/microbiologia , Antibacterianos/efeitos adversos , Cesárea/efeitos adversos , Disbiose/tratamento farmacológico , Disbiose/etiologia , Disbiose/prevenção & controle , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Lactente , Fenômenos Fisiológicos da Nutrição do Lactente/fisiologia , Recém-Nascido , Intestinos/microbiologia , Exposição Materna , Placenta/microbiologia , Prebióticos/administração & dosagem , Gravidez , Complicações na Gravidez/tratamento farmacológico , Probióticos/administração & dosagemRESUMO
Some clinical studies have suggested a relationship between allergic diseases and gut microbiota. We aimed to study bifidobacterial colonization at species and strain levels in ten allergic French infants included at their first clinical consultation and 20 controls matching for age at sampling, mode of delivery, per partum antibiotics, type of feeding and antibiotics in the first weeks of life. The faecal microbiota was analyzed by culture methods and TTGE. Bifidobacterial species and strains were identified using multiplex PCR and Box-PCR fingerprinting. No differences were observed between groups in the number of colonized infants or in the levels of colonization by the main aerobic and anaerobic genera. All infants were colonized with high levels of Bifidobacterium except for one in each group. One to 5 Bifidobacterium species and 1 to 7 strains were observed per subject independently of allergic status and age at sampling. Our study showed the infants to be colonized by several species and strains, including several strains from the same species. This diversity in Bifidobacterium colonization was not related with the allergic status and showed that the link between Bifidobacterium colonization and allergic diseases is complex and cannot be restricted to the role attributed to Bifidobacterium species.
Assuntos
Bifidobacterium/genética , Trato Gastrointestinal/microbiologia , Lactente , Bifidobacterium/classificação , Bifidobacterium/crescimento & desenvolvimento , Bifidobacterium/isolamento & purificação , Estudos de Casos e Controles , Pré-Escolar , Fezes/microbiologia , França , Humanos , Hipersensibilidade/diagnóstico , Complexo Antígeno L1 Leucocitário/análise , Modelos Logísticos , Reação em Cadeia da Polimerase/métodos , RNA Bacteriano/genética , RNA Ribossômico 16S/genéticaRESUMO
Despite years of investigation, pathogenesis of necrotizing enterocolitis (NEC) remains elusive. Bacterial metabolites were implicated by several authors but their roles remain controversial. The aim of our study was to investigate the role of SCFAs and polyamines through a kinetic study of histological and macroscopical digestive lesions in monobiotic quails. Germ-free quails, inoculated with a Clostridium butyricum strain involved in a NEC case, were fed or not with a diet including lactose (7%). Quails were sacrificed at various times between D7 and D24 after bacterial inoculation. NEC-like lesions, i.e. thickening, pneumatosis, and hemorrhages, occurred only in lactose-fed quails and increased with time. The main histological characteristics were infiltrates of mononuclear cells, then heterophilic cells, then gas cyst and necrosis. The first event observed, before histological and macroscopical lesions, is a high production of butyric acid, which precedes an increase of iNOS gene expression. No difference in polyamines contents depending on the diet was observed. These results show the major role of butyric acid produced by commensal bacteria in the onset of the digestive lesions.
Assuntos
Ceco , Enterocolite Necrosante/fisiopatologia , Ácidos Graxos/metabolismo , Vida Livre de Germes , Lactose/administração & dosagem , Poliaminas/metabolismo , Codorniz , Animais , Ceco/metabolismo , Ceco/microbiologia , Ceco/patologia , Clostridium butyricum/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/microbiologia , Humanos , Cinética , Lactose/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
The gut of preterm neonates is colonised with a paucity of bacterial species originating more from the environment than from the mother. Furthermore, a delayed colonisation by bifidobacteria promotes colonisation by potentially pathogenic bacteria. This may contribute towards the development of neonatal necrotising enterocolitis (NEC). The physiopathology of NEC is still unclear but immaturity of the gut, enteral feeding and bacterial colonisation are all thought to be involved. None of the current preventive treatments are considered satisfactory. Modulating the autochthonous microflora by probiotics or prebiotics could be a more reliable approach to prevention. Using gnotobiotic quails as an experimental model of NEC we have shown that onset of intestinal lesions requires a combination of low endogenous lactase activity, lactose in diet, and colonisation by lactose-fermenting bacteria such as the clostridia. The protective role of bifidobacteria was demonstrated in this model through a decrease in clostridial populations and in butyric acid. Oligofructose dietary supplementation was shown to enhance this effect with an increase in the bifidobacterial level and consequently a greater decrease in clostridia. However, oligofructose was unable to promote a bifidobacterial acquisition when the microflora was initially deprived of this group. Nevertheless, oligofructose can act as an anti-infective agent and decrease the occurrence or severity of the lesions depending on the bacteria involved. According to these results and to the fact that oligosaccharides are a major component of breast milk, the addition of oligofructose in formula milks may be a nutritional approach to favouring colonisation by a beneficial flora.
Assuntos
Enterocolite Necrosante/prevenção & controle , Recém-Nascido Prematuro , Intestinos/microbiologia , Oligossacarídeos/uso terapêutico , Animais , Antibiose , Bifidobacterium/metabolismo , Ceco/microbiologia , Clostridium/metabolismo , Modelos Animais de Doenças , Enterocolite Necrosante/microbiologia , Vida Livre de Germes , Humanos , Lactente , Recém-Nascido , CodornizRESUMO
The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79--685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79--685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins.
Assuntos
Proteínas de Bactérias/genética , Clostridioides difficile/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Clostridioides difficile/química , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta , Homologia de SequênciaRESUMO
Previous results have demonstrated that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp groEL gene is highly conserved between various C. difficile isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Escherichia coli in fusion with glutathione S:-transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione-Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited C. difficile cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.