The role of bifidobacteria in newborn health and the intestinal microbial balance

Gut microbial acquisition during the early stage of life is an extremely important event since it affects the health status of the host. In this contest the healthy properties of the genus Bifidobacterium have a central function in newborns. The aim of this thesis was to explore the dynamics of the gut microbial colonization in newborns and to suggest possible strategies to maintain or restore a correct balance of gut bacterial population in infants. The first step of this work was to review the most recent studies on the use of probiotics and prebiotics in infants. Secondly, in order to prevent or treat intestinal disorders that may affect newborns, the capability of selected Bifidobacterium strains to reduce the amount of Enterobacteriaceae and against the infant pathogen Streptococcus agalactiae was evaluated in vitro. Furthermore, the ability of several commercial fibers to stimulate selectively the growth of bifidobacterial strains was checked. Finally, the gut microbial composition in the early stage of life in response to the intrapartum antibiotic prophylaxis (IAP) against group B Streptococcus was studied using q-PCR, DGGE and next generation sequencing. The results globally showed that Bifidobacterium breve B632 strain is the best candidate for the use in a synbiotic product coupled to a mixture of two selected prebiotic fibers (galactooligosaccharides and fructooligosaccharides) for gastrointestinal disorders in infants. Moreover, the early gut microbial composition was affected by IAP treatment with infants showing lower counts of Bifidobacterium spp. and Bacteroides spp. coupled to a decrement of biodiversity of bacteria, compared to control infants. These studies have shown that IAP could affect the early intestinal balance in infants and they have paved the way to the definition of new strategies alternative to antibiotic treatment to control GBS infection in pregnant women.

Like will to like: abundances of closely related species can predict susceptibility to intestinal colonization by pathogenic and commensal bacteria

The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM(con21)). 16S rRNA sequence analysis comparing LCM, LCM(con21) and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri(RR) strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.

Garlic extract induces intestinal P-glycoprotein, but exhibits no effect on intestinal and hepatic CYP3A4 in humans

Garlic extracts have been shown to decrease drug exposure for saquinavir, a P-glycoprotein and cytochrome P450 3A4 substrate. In order to explore the underlying mechanisms and to study the effects of garlic on pre-systemic drug elimination, healthy volunteers were administered garlic extract for 21 days. Prior to and at the end of this period, expression of duodenal P-glycoprotein and cytochrome P450 3A4 protein were assayed and normalized to villin, while hepatic cytochrome P450 3A4 function and simvastatin, pravastatin and saquinavir pharmacokinetics were also evaluated. Ingestion of garlic extract increased expression of duodenal P-glycoprotein to 131% (95% CI, 105-163%), without increasing the expression of cytochrome P450 3A4 which amounted to 87% (95% CI, 67-112%), relative to baseline in both cases. For the erythromycin breath test performed, the average result was 96% (95% CI, 83-112%). Ingestion of garlic extract had no effect on drug and metabolite AUCs following a single dose of simvastatin or pravastatin, although the average area under the plasma concentration curve (AUC) of saquinavir decreased to 85% (95% CI, 66-109%), and changes in intestinal P-glycoprotein expression negatively correlated with this change. In conclusion, garlic extract induces intestinal expression of P-glycoprotein independent of cytochrome P450 3A4 in human intestine and liver.

Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1 week after. The specimens were analyzed by LC-ESI-MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivity.

The continuum of intestinal CD4+ T cell adaptations in host-microbial mutualism

How a mutualistic relationship between the intestinal microbiota and intestinal T cell compartments is established is important, as a breakdown of intestinal T cell homeostasis may cause inflammatory bowel diseases. A number of studies have shown that different bacterial species modulate the intestinal CD4+ T cell compartment in different ways. We performed mechanistic in vivo studies that demonstrated the crucial requirement for regulatory T cells (Treg) and interleukin-10 (IL-10) in the induction of intestinal T cell homeostasis even following colonization with a completely benign microbiota. In the absence of a functional Treg response or IL-10 receptor signaling, the same bacteria that induced a Treg response in wild-type animals now induced T helper type 17 responses, without intestinal inflammation. Therefore, Treg, IL-10 and Th17 are crucial regulatory mechanisms in the intestine not only for controlling inflammation, but also to establish a continuum of CD4+ T cell homeostasis upon commensal colonization.

Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice.

Generation and analysis of a mouse intestinal metatranscriptome through Illumina based RNA-sequencing

With the advent of high through-put sequencing (HTS), the emerging science of metagenomics is transforming our understanding of the relationships of microbial communities with their environments. While metagenomics aims to catalogue the genes present in a sample through assessing which genes are actively expressed, metatranscriptomics can provide a mechanistic understanding of community inter-relationships. To achieve these goals, several challenges need to be addressed from sample preparation to sequence processing, statistical analysis and functional annotation. Here we use an inbred non-obese diabetic (NOD) mouse model in which germ-free animals were colonized with a defined mixture of eight commensal bacteria, to explore methods of RNA extraction and to develop a pipeline for the generation and analysis of metatranscriptomic data. Applying the Illumina HTS platform, we sequenced 12 NOD cecal samples prepared using multiple RNA-extraction protocols. The absence of a complete set of reference genomes necessitated a peptide-based search strategy. Up to 16% of sequence reads could be matched to a known bacterial gene. Phylogenetic analysis of the mapped ORFs revealed a distribution consistent with ribosomal RNA, the majority from Bacteroides or Clostridium species. To place these HTS data within a systems context, we mapped the relative abundance of corresponding Escherichia coli homologs onto metabolic and protein-protein interaction networks. These maps identified bacterial processes with components that were well-represented in the datasets. In summary this study highlights the potential of exploiting the economy of HTS platforms for metatranscriptomics.

Bacterial colitis increases susceptibility to oral prion disease

Dietary exposure to prion-contaminated materials has caused kuru and variant Creutzfeldt-Jakob disease in humans and transmissible spongiform encephalopathies (TSEs) in cattle, mink, and felines. The epidemiology of dietary prion infections suggests that host genetic modifiers and possibly exogenous cofactors may play a decisive role in determining disease susceptibility. However, few cofactors influencing susceptibility to prion infection have been identified. In the present study, we investigated whether colitis might represent one such cofactor. We report that moderate colitis caused by an attenuated Salmonella strain more than doubles the susceptibility of mice to oral prion infection and modestly accelerates the development of disease after prion challenge. The prion protein was up-regulated in intestines and mesenteric lymph nodes of mice with colitis, providing a possible mechanism for the effect of colitis on the pathogenesis of prion disease. Therefore, moderate intestinal inflammation at the time of prion exposure may constitute one of the elusive risk factors underlying the development of TSE.

Host responses to intestinal microbial antigens in gluten-sensitive mice

BACKGROUND AND AIMS: Excessive uptake of commensal bacterial antigens through a permeable intestinal barrier may influence host responses to specific antigen in a genetically predisposed host. The aim of this study was to investigate whether intestinal barrier dysfunction induced by indomethacin treatment affects the host response to intestinal microbiota in gluten-sensitized HLA-DQ8/HCD4 mice. METHODOLOGY/PRINCIPAL FINDINGS: HLA-DQ8/HCD4 mice were sensitized with gluten, and gavaged with indomethacin plus gluten. Intestinal permeability was assessed by Ussing chamber; epithelial cell (EC) ultra-structure by electron microscopy; RNA expression of genes coding for junctional proteins by Q-real-time PCR; immune response by in-vitro antigen-specific T-cell proliferation and cytokine analysis by cytometric bead array; intestinal microbiota by fluorescence in situ hybridization and analysis of systemic antibodies against intestinal microbiota by surface staining of live bacteria with serum followed by FACS analysis. Indomethacin led to a more pronounced increase in intestinal permeability in gluten-sensitized mice. These changes were accompanied by severe EC damage, decreased E-cadherin RNA level, elevated IFN-gamma in splenocyte culture supernatant, and production of significant IgM antibody against intestinal microbiota. CONCLUSION: Indomethacin potentiates barrier dysfunction and EC injury induced by gluten, affects systemic IFN-gamma production and the host response to intestinal microbiota antigens in HLA-DQ8/HCD4 mice. The results suggest that environmental factors that alter the intestinal barrier may predispose individuals to an increased susceptibility to gluten through a bystander immune activation to intestinal microbiota.

Regulation of hepatic cytochrome P450 expression in mice with intestinal or systemic infections of citrobacter rodentium.

We reported previously that infection of C3H/HeOuJ (HeOu) mice with the murine intestinal pathogen Citrobacter rodentium caused a selective modulation of hepatic cytochrome P450 (P450) gene expression in the liver that was independent of the Toll-like receptor 4. However, HeOu mice are much more sensitive to the pathogenic effects of C. rodentium infection, and the P450 down-regulation was associated with significant morbidity in the animals. Here, we report that oral infection of C57BL/6 mice with C. rodentium, which produced only mild clinical signs and symptoms, produced very similar effects on hepatic P450 expression in this strain. As in HeOu mice, CYP4A mRNAs and proteins were among the most sensitive to down-regulation, whereas CYP4F18 was induced. CYP2D9 mRNA was also induced 8- to 9-fold in the C57BL/6 mice. The time course of P450 regulation followed that of colonic inflammation and bacterial colonization, peaking at 7 to 10 days after infection and returning to normal at 15 to 24 days as the infection resolved. These changes also correlated with the time course of significant elevations in the serum of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, as well as of interferon-gamma and IL-2, with serum levels of IL-6 being markedly higher than those of the other cytokines. Intraperitoneal administration of C. rodentium produced a rapid down-regulation of P450 enzymes that was quantitatively and qualitatively different from that of oral infection, although CYP2D9 was induced in both models, suggesting that the effects of oral infection on the liver are not due to bacterial translocation.

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.

Mycobacterium avium Subsp. avium infection in four veal calves: differentiation from intestinal tuberculosis

Mycobacterium avium subsp. avium (Maa) is an intracellular pathogen belonging to the Mycobacterium avium-intracellulare complex (MAC). Reservoirs of MAC are the natural environment, wildlife and domestic animals. In adult bovine, MAC infections are typically caused by Mycobacterium avium subsp. paratuberculosis (Map). Maa infections in bovine are rarely reported but may cause clinical disease and pathological lesions similar to those observed in paratuberculosis or those induced by members of the Mycobacterium tuberculosis complex (MTBC). Therefore, differentiation of MAC from MTBC infection should be attempted, especially if unusual mycobacterial lesions are encountered. Four veal calves from a fattening farm dying with clinical signs of otitis media, fever, and weight loss were submitted for necropsy. Samples from affected organs were taken for histologic investigation, bacteriologic culture, and bacterial specification using PCR. Macroscopic thickening of the intestinal mucosa was induced by granulomatous enteritis and colitis. Intracytoplasmic acid-fast bacteria were detected by Ziehl-Neelsen stains and PCR revealed positive results for Mycobacterium avium subsp. avium. Clinical and pathological changes of Maa infection in veal calves had features of Mycobacterium avium subsp. paratuberculosis and the MTBC. Therefore, Mycobacterium tuberculosis complex infection should be considered in cases of granulomatous enteritis in calves.

Bacterial infections in cirrhosis: a position statement based on the EASL Special Conference 2013.

Bacterial infections are very common and represent one of the most important reasons of progression of liver failure, development of liver-related complications, and mortality in patients with cirrhosis. In fact, bacterial infections may be a triggering factor for the occurrence of gastrointestinal bleeding, hypervolemic hyponatremia, hepatic encephalopathy, kidney failure, and development of acute-on-chronic liver failure. Moreover, infections are a very common cause of repeated hospitalizations, impaired health-related quality of life, and increased healthcare costs in cirrhosis. Bacterial infections develop as a consequence of immune dysfunction that occurs progressively during the course of cirrhosis. In a significant proportion of patients, infections are caused by gram-negative bacteria from intestinal origin, yet gram-positive bacteria are a frequent cause of infection, particularly in hospitalized patients. In recent years, infections caused by multidrug-resistant bacteria are becoming an important clinical problem in many countries. The reduction of the negative clinical impact of infections in patients with cirrhosis may be achieved by a combination of prophylactic measures, such as administration of antibiotics, to reduce the occurrence of infections in high-risk groups together with early identification and management of infection once it has developed. Investigation on the mechanisms of altered gut microflora, translocation of bacteria, and immune dysfunction may help develop more effective and safe methods of prevention compared to those that are currently available. Moreover, research on biomarkers of early infection may be useful in early diagnosis and treatment of infections. The current manuscript reports an in-depth review and a position statement on bacterial infections in cirrhosis.

Keeping bugs in check: The mucus layer as a critical component in maintaining intestinal homeostasis

In the mammalian gastrointestinal tract the close vicinity of abundant immune effector cells and trillions of commensal microbes requires sophisticated barrier and regulatory mechanisms to maintain vital host-microbial interactions and tissue homeostasis. During co-evolution of the host and its intestinal microbiota a protective multilayered barrier system was established to segregate the luminal microbes from the intestinal mucosa with its potent immune effector cells, limit bacterial translocation into host tissues to prevent tissue damage, while ensuring the vital functions of the intestinal mucosa and the luminal gut microbiota. In the present review we will focus on the different layers of protection in the intestinal tract that allow the successful mutualism between the microbiota and the potent effector cells of the intestinal innate and adaptive immune system. In particular, we will review some of the recent findings on the vital functions of the mucus layer and its site-specific adaptations to the changing quantities and complexities of the microbiota along the (gastro-) intestinal tract. Understanding the regulatory pathways that control the establishment of the mucus layer, but also its degradation during intestinal inflammation may be critical for designing novel strategies aimed at maintaining local tissue homeostasis and supporting remission from relapsing intestinal inflammation in patients with inflammatory bowel diseases.

The outer mucus layer hosts a distinct intestinal microbial niche

The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.