Accumulating studies have defined a role for the intestinal microbiota in modulation of host behavior. Research using gnotobiotic mice emphasizes that early microbial colonization with a complex ...microbiota (conventionalization) can rescue some of the behavioral abnormalities observed in mice that grow to adulthood completely devoid of bacteria (germ-free mice). However, the human infant and adult microbiomes vary greatly, and effects of the neonatal microbiome on neurodevelopment are currently not well understood. Microbe-mediated modulation of neural circuit patterning in the brain during neurodevelopment may have significant long-term implications that we are only beginning to appreciate. Modulation of the host central nervous system by the early-life microbiota is predicted to have pervasive and lasting effects on brain function and behavior. We sought to replicate this early microbe-host interaction by colonizing gnotobiotic mice at the neonatal stage with a simplified model of the human infant gut microbiota. This model consortium consisted of four "infant-type" Bifidobacterium species known to be commensal members of the human infant microbiota present in high abundance during postnatal development. Germ-free mice and mice neonatally-colonized with a complex, conventional murine microbiota were used for comparison. Motor and non-motor behaviors of the mice were tested at 6-7 weeks of age, and colonization patterns were characterized by 16S ribosomal RNA gene sequencing. Adult germ-free mice were observed to have abnormal memory, sociability, anxiety-like behaviors, and motor performance. Conventionalization at the neonatal stage rescued these behavioral abnormalities, and mice colonized with Bifidobacterium spp. also exhibited important behavioral differences relative to the germ-free controls. The ability of Bifidobacterium spp. to improve the recognition memory of both male and female germ-free mice was a prominent finding. Together, these data demonstrate that the early-life gut microbiome, and human "infant-type" Bifidobacterium species, affect adult behavior in a strongly sex-dependent manner, and can selectively recapitulate the results observed when mice are colonized with a complex microbiota.
Mood disorders and constipation are often comorbid, yet their shared etiologies have rarely been explored. The neurotransmitter serotonin (5-HT) regulates central nervous system and enteric nervous ...system (ENS) development and long-term functions, including gastrointestinal (GI) motility and mood. Therefore, defects in neuron production of 5-HT might result in brain and intestinal dysfunction. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme in 5-HT biosynthesis. A variant of TPH2 that encodes the R441H substitution (TPH2-R441H) was identified in individuals with severe depression. We studied mice with an analogous mutation (TPH2-R439H), which results in a 60%–80% decrease in levels of 5-HT in the central nervous system and behaviors associated with depression in humans. Feeding chow that contains 5-HTP slow release (5-HTP SR) to TPH2-R439H mice restores levels of 5-HT in the central nervous system and reduces depressive-like behaviors.
We compared the effects of feeding chow, with or without 5-HTP SR, to mice with the TPH2-R439H mutation and without this mutation (control mice). Myenteric and submucosal plexuses were isolated from all 4 groups of mice, and immunocytochemistry was used to quantify total enteric neurons, serotonergic neurons, and 5-HT–dependent subsets of neurons. We performed calcium imaging experiments to evaluate responses of enteric neurons to tryptamine-evoked release of endogenous 5-HT. In live mice, we measured total GI transit, gastric emptying, small intestinal transit, and propulsive colorectal motility. To measure colonic migrating motor complexes (CMMCs), we isolated colons and constructed spatiotemporal maps along the proximodistal length to quantify the frequency, velocity, and length of CMMCs. We measured villus height, crypt perimeter, and relative densities of enterochromaffin and enteroendocrine cells in small intestinal tissue.
Levels of 5-HT were significantly lower in enteric neurons from TPH2-R439H mice than from control mice. TPH2-R439H mice had abnormalities in ENS development and ENS-mediated GI functions, including reduced motility and intestinal epithelial growth. Total GI transit and propulsive colorectal motility were slower in TPH2-R439H mice than controls, and CMMCs were slower and less frequent. Villus height and crypt perimeter were significantly decreased in colon tissues from TPH2-R439H mice compared with controls. Administration of 5-HTP SR to adult TPH2-R439H mice restored 5-HT to enteric neurons and reversed these abnormalities. Adult TPH2-R439H mice given oral 5-HTP SR had normalized numbers of enteric neurons, total GI transit, and colonic motility. Intestinal tissue from these mice had normal measures of CMMCs and enteric epithelial growth
In studies of TPH2-R439H mice, we found evidence for reduced release of 5-HT from enteric neurons that results in defects in ENS development and GI motility. Our findings indicate that neuron production of 5-HT links constipation with mood dysfunction. Administration of 5-HTP SR to mice restored 5-HT to the ENS and normalized GI motility and growth of the enteric epithelium. 5-HTP SR might be used to treat patients with intestinal dysfunction associated with low levels of 5-HT.
Display omitted
The Human Microbiome Project has ushered in a new era for human metagenomics and high-throughput next-generation sequencing strategies.
This review describes evolving strategies in metagenomics, with ...a special emphasis on the core technology of DNA pyrosequencing. The challenges of microbial identification in the context of microbial populations are discussed. The development of next-generation pyrosequencing strategies and the technical hurdles confronting these methodologies are addressed. Bioinformatics-related topics include taxonomic systems, sequence databases, sequence-alignment tools, and classifiers. DNA sequencing based on 16S rRNA genes or entire genomes is summarized with respect to potential pyrosequencing applications.
Both the approach of 16S rDNA amplicon sequencing and the whole-genome sequencing approach may be useful for human metagenomics, and numerous bioinformatics tools are being deployed to tackle such vast amounts of microbiological sequence diversity. Metagenomics, or genetic studies of microbial communities, may ultimately contribute to a more comprehensive understanding of human health, disease susceptibilities, and the pathophysiology of infectious and immune-mediated diseases.
Neonates are at high risk for central line associated bloodstream infections (CLABSI). Biofilm formation is universal on indwelling catheters but why some biofilms seed the bloodstream to cause ...CLABSI is not clearly understood. With the objective to test the hypothesis that catheter biofilm microbiome in neonates with CLABSI differs than those without infection, we prospectively enrolled neonates (n = 30) with infected and uninfected indwelling central catheters. Catheters were collected at the time of removal, along with blood samples and skin swabs at the catheter insertion sites. Microbiomes of catheter biofilms, skin swabs and blood were evaluated by profiling the V4 region of the bacterial 16S rRNA gene using Illumina MiSeq sequencing platform. The microbial DNA load was higher from catheter biofilms of CLABSI patients without differences in alpha diversity when compared to that of the non-CLABSI neonates. Proteus and unclassified Staphylococcaceae were more abundant in infected catheter biofilms while Bradyrhizobium, Cloacibacterium, and Sphingomonas were more abundant in the uninfected catheters. A blood microbiome was detected in uninfected samples. The blood microbiome in CLABSI neonates clustered separately from the uninfected blood samples in beta diversity plots. We found that the microbiome signature in catheter biofilm and blood of neonates with CLABSI is different than the microbiomes of non-CLABSI neonates.
Preclinical and clinical evidence supports the concept of bidirectional brain-gut microbiome interactions. We aimed to determine if subgroups of irritable bowel syndrome (IBS) subjects can be ...identified based on differences in gut microbial composition, and if there are correlations between gut microbial measures and structural brain signatures in IBS.
Behavioral measures, stool samples, and structural brain images were collected from 29 adult IBS and 23 healthy control subjects (HCs). 16S ribosomal RNA (rRNA) gene sequencing was used to profile stool microbial communities, and various multivariate analysis approaches were used to quantitate microbial composition, abundance, and diversity. The metagenomic content of samples was inferred from 16S rRNA gene sequence data using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). T1-weighted brain images were acquired on a Siemens Allegra 3T scanner, and morphological measures were computed for 165 brain regions.
Using unweighted Unifrac distances with hierarchical clustering on microbial data, samples were clustered into two IBS subgroups within the IBS population (IBS1 (n = 13) and HC-like IBS (n = 16)) and HCs (n = 23) (AUROC = 0.96, sensitivity 0.95, specificity 0.67). A Random Forest classifier provided further support for the differentiation of IBS1 and HC groups. Microbes belonging to the genera Faecalibacterium, Blautia, and Bacteroides contributed to this subclassification. Clinical features distinguishing the groups included a history of early life trauma and duration of symptoms (greater in IBS1), but not self-reported bowel habits, anxiety, depression, or medication use. Gut microbial composition correlated with structural measures of brain regions including sensory- and salience-related regions, and with a history of early life trauma.
The results confirm previous reports of gut microbiome-based IBS subgroups and identify for the first time brain structural alterations associated with these subgroups. They provide preliminary evidence for the involvement of specific microbes and their predicted metabolites in these correlations.
Gastrointestinal problems affect the health and quality of life of individuals with Rett syndrome (RTT) and pose a medical hardship for their caregivers. We hypothesized that the variability in the ...RTT phenotype contributes to the dysbiosis of the gut microbiome and metabolome in RTT, predisposing these individuals to gastrointestinal dysfunction.
We characterized the gut bacterial microbiome and metabolome in girls and young women with RTT (n = 44) and unaffected controls (n = 21), and examined the relation between the composition of the microbiome and variations in the RTT phenotype.
Demographics and clinical information, including growth and anthropometric measurements, pubertal status, symptoms, clinical severity score, bowel movement, medication use, and dietary intakes were collected from the participants. Fecal samples were collected for analysis of the gut microbiome using Illumina MiSeq-based next-generation sequencing of the 16S rRNA gene followed by bioinformatics analysis of microbial composition, diversity, and community structure. Selected end-products of microbial protein metabolism were characterized by liquid chromatography-mass spectrometry.
The gut bacterial microbiome differed within the RTT cohort based on pubertal status (p<0.02) and clinical severity scores (p<0.02) of the individuals and the type of diet (p<0.01) consumed. Although the composition of the gut microbiome did not differ between RTT and unaffected individuals, concentrations of protein end-products of the gut bacterial metabolome, including γ-aminobutyric acid (GABA) (p<0.001), tyrosine (p<0.02), and glutamate (p<0.06), were lower in the RTT cohort. Differences in the microbiome within RTT groups, based on symptomatic anxiety, hyperventilation, abdominal distention, or changes in stool frequency and consistency, were not detected.
Although variability in the RTT phenotype contributes to the dysbiosis of the gut microbiome, we presently cannot infer causality between gut bacterial dysbiosis and gastrointestinal dysfunction. Nevertheless, alterations in the gut metabolome may provide clues to the pathophysiology of gastrointestinal problems in RTT.
Fecal microbiota transplantation (FMT) is a highly effective treatment for refractoryClostridium difficileinfections. However, concerns persist about unwanted cotransfer of pathogenic microbes such ...as viruses. Here we studed FMT from a single healthy human donor to three pediatric ulcerative colitis patients, each of whom received a course of 22 to 30 FMT treatments. Viral particles were purified from donor and recipient stool samples and sequenced; the reads were then assembled into contigs corresponding to viral genomes or partial genomes. Transfer of selected viruses was confirmed by quantitative PCR. Viral contigs present in the donor could be readily detected in recipients, with up to 32 different donor viral contigs appearing in a recipient sample. Reassuringly, none of these were viruses are known to replicate on human cells. Instead, viral contigs either scored as bacteriophage or could not be attributed taxonomically, suggestive of unstudied phage. The two most frequently transferred gene types were associated with temperate-phage replication. In addition, members ofSiphoviridae, the group of typically temperate phages that includes phage lambda, were found to be transferred with significantly greater efficiency than other groups. On the basis of these findings, we propose that the temperate-phage replication style may promote efficient phage transfer between human individuals. In summary, we documented transfer of multiple viral lineages between human individuals through FMT, but in this case series, none were from viral groups known to infect human cells.
Transfer of whole communities of viruses between humans has rarely been studied but is of likely medical importance. Here we studied fecal microbiota transplantation (FMT), a highly successful treatment for relapsingClostridium difficileinfection and, potentially, other gastrointestinal (GI) diseases. We investigated the transfer of viral communities during FMT and documented transfer of multiple viral lineages between humans. None of these were viruses that replicated on animal cells or that are known to be pathogenic. We found that temperate bacteriophage, which form stable associations with their hosts, were significantly more likely to be transferred during FMT. This supports a model in which the viral temperate replication style may have evolved in part to support efficient viral transmission between environments.
Decreased consumption of dietary fibers, such as cellulose, has been proposed to promote the emergence of inflammatory bowel diseases (IBD: Crohn disease CD and ulcerative colitis UC) where ...intestinal microbes are recognized to play an etiologic role. However, it is not known if transient fiber consumption during critical developmental periods may prevent consecutive intestinal inflammation. The incidence of IBD peaks in young adulthood indicating that pediatric environmental exposures may be important in the etiology of this disease group. We studied the effects of transient dietary cellulose supplementation on dextran sulfate sodium (DSS) colitis susceptibility during the pediatric period in mice. Cellulose supplementation stimulated substantial shifts in the colonic mucosal microbiome. Several bacterial taxa decreased in relative abundance (e.g., Coriobacteriaceae p = 0.001), and other taxa increased in abundance (e.g., Peptostreptococcaceae p = 0.008 and Clostridiaceae p = 0.048). Some of these shifts persisted for 10 days following the cessation of cellulose supplementation. The changes in the gut microbiome were associated with transient trophic and anticolitic effects 10 days following the cessation of a cellulose-enriched diet, but these changes diminished by 40 days following reversal to a low cellulose diet. These findings emphasize the transient protective effect of dietary cellulose in the mammalian large bowel and highlight the potential role of dietary fibers in amelioration of intestinal inflammation.
Gut microbes can synthesize multiple neuro-active metabolites. We profiled neuro-active compounds produced by the gut commensal Bacteroides ovatus in vitro and in vivo by LC-MS/MS. We found that ...B. ovatus generates acetic acid, propionic acid, isobutyric acid, and isovaleric acid. In vitro, B. ovatus consumed tryptophan and glutamate and synthesized the neuro-active compounds glutamine and GABA. Consistent with our LC-MS/MS-based in vitro data, we observed elevated levels of acetic acid, propionic acid, isobutyric acid, and isovaleric acid in the intestines of B. ovatus mono-associated mice compared with germ-free controls. B. ovatus mono-association also increased the concentrations of intestinal GABA and decreased the concentrations of tryptophan and glutamine compared with germ-free controls. Computational network analysis revealed unique links between SCFAs, neuro-active compounds, and colonization status. These results highlight connections between microbial colonization and intestinal neurotransmitter concentrations, suggesting that B. ovatus selectively influences the presence of intestinal neurotransmitters.
Display omitted
•Bacteroides ovatus modulates key neuro-active compounds in vivo in gnotobiotic animals
Neuroscience; Microbiology; Microbiome
PDF
