Limosilactobacillus reuteri ATCC 6475 metabolites upregulate the serotonin transporter in the intestinal epithelium.

The serotonin transporter (SERT) readily takes up serotonin (5-HT), thereby regulating the availability of 5-HT within the intestine. In the absence of SERT, 5-HT remains in the interstitial space and has the potential to aberrantly activate the many 5-HT receptors distributed on the epithelium, immune cells and enteric neurons. Perturbation of SERT is common in many gastrointestinal disorders as well as mouse models of colitis. Select commensal microbes regulate intestinal SERT levels, but the mechanism of this regulation is poorly understood. Additionally, ethanol upregulates SERT in the brain and dendritic cells, but its effects in the intestine have never been examined. We report that the intestinal commensal microbe Limosilactobacillus (previously classified as Lactobacillus) reuteri ATCC PTA 6475 secretes 83.4 mM ethanol. Consistent with the activity of L. reuteri alcohol dehydrogenases, we found that L. reuteri tolerated various levels of ethanol. Application of L. reuteri conditioned media or exogenous ethanol to human colonic T84 cells was found to upregulate SERT at the level of mRNA. A 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+) uptake assay confirmed the functional activity of SERT. These findings were mirrored in mouse colonic organoids, where L. reuteri metabolites and ethanol were found to upregulate SERT at the apical membrane. Finally, in a trinitrobenzene sulphonic acid model of acute colitis, we observed that mice treated with L. reuteri maintained SERT at the colon membrane compared with mice receiving phosphate buffered saline vehicle control. These data suggest that L. reuteri metabolites, including ethanol, can upregulate SERT and may be beneficial for maintaining intestinal homeostasis with respect to serotonin signalling.

Diacylglycerol kinase synthesized by commensal Lactobacillus reuteri diminishes protein kinase C phosphorylation and histamine-mediated signaling in the mammalian intestinal epithelium.

Lactobacillus reuteri 6475 (Lr) of the human microbiome synthesizes histamine and can suppress inflammation via type 2 histamine receptor (H2R) activation in the mammalian intestine. Gut microbes such as Lr promote H2R signaling and may suppress H1R proinflammatory signaling pathways in parallel by unknown mechanisms. In this study, we identified a soluble bacterial enzyme known as diacylglycerol kinase (Dgk) from Lr that is secreted into the extracellular milieu and presumably into the intestinal lumen. DgK diminishes diacylglycerol (DAG) quantities in mammalian cells by promoting its metabolic conversion and causing reduced protein kinase C phosphorylation (pPKC) as a net effect in mammalian cells. We demonstrated that histamine synthesized by gut microbes (Lr) activates both mammalian H1R and H2R, but Lr-derived Dgk suppresses the H1R signaling pathway. Phospho-PKC and IκBα were diminished within the intestinal epithelium of mice and humans treated by wild-type (WT) Lr, but pPKC and IκBα were not decreased in treatment with ΔdgkA Lr. Mucosal IL-6 and systemic interleukin (IL)-1α, eotaxin, and granulocyte colony-stimulating factor (G-CSF) were suppressed in WT Lr, but not in ΔdgkA Lr colonized mice. Collectively, the commensal microbe Lr may act as a "microbial antihistamine" by suppressing intestinal H1R-mediated proinflammatory responses via diminished pPKC-mediated mammalian cell signaling.

GABA-producing Bifidobacterium dentium modulates visceral sensitivity in the intestine.

BACKGROUND: Recurrent abdominal pain is a common and costly health-care problem attributed, in part, to visceral hypersensitivity. Increasing evidence suggests that gut bacteria contribute to abdominal pain perception by modulating the microbiome-gut-brain axis. However, specific microbial signals remain poorly defined. γ-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter and a key regulator of abdominal and central pain perception from peripheral afferent neurons. Although gut bacteria are reported to produce GABA, it is not known whether the microbial-derived neurotransmitter modulates abdominal pain. METHODS: To investigate the potential analgesic effects of microbial GABA, we performed daily oral administration of a specific Bifidobacterium strain (B. dentiumATCC 27678) in a rat fecal retention model of visceral hypersensitivity, and subsequently evaluated pain responses. KEY RESULTS: We demonstrate that commensal Bifidobacterium dentium produces GABA via enzymatic decarboxylation of glutamate by GadB. Daily oral administration of this specific Bifidobacterium (but not a gadB deficient) strain modulated sensory neuron activity in a rat fecal retention model of visceral hypersensitivity. CONCLUSIONS & INFERENCES: The functional significance of microbial-derived GABA was demonstrated by gadB-dependent desensitization of colonic afferents in a murine model of visceral hypersensitivity. Visceral pain modulation represents another potential health benefit attributed to bifidobacteria and other GABA-producing species of the intestinal microbiome. Targeting GABAergic signals along this microbiome-gut-brain axis represents a new approach for the treatment of abdominal pain.

Generation and validation of a universal perinatal database and biospecimen repository: PeriBank.

OBJECTIVE: There is a dearth of biospecimen repositories available to perinatal researchers. In order to address this need, here we describe the methodology used to establish such a resource. STUDY DESIGN: With the collaboration of MedSci.net, we generated an online perinatal database with 847 fields of clinical information. Simultaneously, we established a biospecimen repository of the same clinical participants. RESULTS: The demographic and clinical outcomes data are described for the first 10 000 participants enrolled. The demographic characteristics are consistent with the demographics of the delivery hospitals. Quality analysis of the biospecimens reveals variation in very few analytes. Furthermore, since the creation of PeriBank, we have demonstrated validity of the database and tissue integrity of the biospecimen repository. CONCLUSION: Here we establish that the creation of a universal perinatal database and biospecimen collection is not only possible, but allows for the performance of state-of-the-science translational perinatal research and is a potentially valuable resource to academic perinatal researchers.

Randomised clinical trial: gut microbiome biomarkers are associated with clinical response to a low FODMAP diet in children with the irritable bowel syndrome.

BACKGROUND: A low fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAP) diet can ameliorate symptoms in adult irritable bowel syndrome (IBS) within 48 h. AIM: To determine the efficacy of a low FODMAP diet in childhood IBS and whether gut microbial composition and/or metabolic capacity are associated with its efficacy. METHODS: In a double-blind, crossover trial, children with Rome III IBS completed a 1-week baseline period. They then were randomised to a low FODMAP diet or typical American childhood diet (TACD), followed by a 5-day washout period before crossing over to the other diet. GI symptoms were assessed with abdominal pain frequency being the primary outcome. Baseline gut microbial composition (16S rRNA sequencing) and metabolic capacity (PICRUSt) were determined. Metagenomic biomarker discovery (LEfSe) compared Responders (≥50% decrease in abdominal pain frequency on low FODMAP diet only) vs. Nonresponders (no improvement during either intervention). RESULTS: Thirty-three children completed the study. Less abdominal pain occurred during the low FODMAP diet vs. TACD [1.1 ± 0.2 (SEM) episodes/day vs. 1.7 ± 0.4, P < 0.05]. Compared to baseline (1.4 ± 0.2), children had fewer daily abdominal pain episodes during the low FODMAP diet (P < 0.01) but more episodes during the TACD (P < 0.01). Responders were enriched at baseline in taxa with known greater saccharolytic metabolic capacity (e.g. Bacteroides, Ruminococcaceae, Faecalibacterium prausnitzii) and three Kyoto Encyclopedia of Genes and Genomes orthologues, of which two relate to carbohydrate metabolism. CONCLUSIONS: In childhood IBS, a low FODMAP diet decreases abdominal pain frequency. Gut microbiome biomarkers may be associated with low FODMAP diet efficacy. ClinicalTrials.gov identifier: NCT01339117.

Identification of a proton-chloride antiporter (EriC) by Himar1 transposon mutagenesis in Lactobacillus reuteri and its role in histamine production.

The gut microbiome may modulate intestinal immunity by luminal conversion of dietary amino acids to biologically active signals. The model probiotic organism Lactobacillus reuteri ATCC PTA 6475 is indigenous to the human microbiome, and converts the amino acid L-histidine to the biogenic amine, histamine. Histamine suppresses tumor necrosis factor (TNF) production by human myeloid cells and is a product of L-histidine decarboxylation, which is a proton-facilitated reaction. A transposon mutagenesis strategy was developed based on a single-plasmid nisin-inducible Himar1 transposase/transposon delivery system for L. reuteri. A highly conserved proton-chloride antiporter gene (eriC), a gene widely present in the gut microbiome was discovered by Himar1 transposon (Tn)-mutagenesis presented in this study. Genetic inactivation of eriC by transposon insertion and genetic recombineering resulted in reduced ability of L. reuteri to inhibit TNF production by activated human myeloid cells, diminished histamine production by the bacteria and downregulated expression of histidine decarboxylase cluster genes compared to those of WT 6475. EriC belongs to a large family of ion transporters that includes chloride channels and proton-chloride antiporters and may facilitate the availability of protons for the decarboxylation reaction, resulting in histamine production by L. reuteri. This report leverages the tools of bacterial genetics for probiotic gene discovery. The findings highlight the widely conserved nature of ion transporters in bacteria and how ion transporters are coupled with amino acid decarboxylation and contribute to microbiome-mediated immunomodulation.

Lactobacillus reuteri-specific immunoregulatory gene rsiR modulates histamine production and immunomodulation by Lactobacillus reuteri.

Human microbiome-derived strains of Lactobacillus reuteri potently suppress proinflammatory cytokines like human tumor necrosis factor (TNF) by converting the amino acid l-histidine to the biogenic amine histamine. Histamine suppresses mitogen-activated protein (MAP) kinase activation and cytokine production by signaling via histamine receptor type 2 (H2) on myeloid cells. Investigations of the gene expression profiles of immunomodulatory L. reuteri ATCC PTA 6475 highlighted numerous genes that were highly expressed during the stationary phase of growth, when TNF suppression is most potent. One such gene was found to be a regulator of genes involved in histidine-histamine metabolism by this probiotic species. During the course of these studies, this gene was renamed the Lactobacillus reuteri-specific immunoregulatory (rsiR) gene. The rsiR gene is essential for human TNF suppression by L. reuteri and expression of the histidine decarboxylase (hdc) gene cluster on the L. reuteri chromosome. Inactivation of rsiR resulted in diminished TNF suppression in vitro and reduced anti-inflammatory effects in vivo in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of acute colitis. A L. reuteri strain lacking an intact rsiR gene was unable to suppress colitis and resulted in greater concentrations of serum amyloid A (SAA) in the bloodstream of affected animals. The PhdcAB promoter region targeted by rsiR was defined by reporter gene experiments. These studies support the presence of a regulatory gene, rsiR, which modulates the expression of a gene cluster known to mediate immunoregulation by probiotics at the transcriptional level. These findings may point the way toward new strategies for controlling gene expression in probiotics by dietary interventions or microbiome manipulation.

Identification of Lactobacillus strains with probiotic features from the bottlenose dolphin (Tursiops truncatus).

AIMS: In order to develop complementary health management strategies for marine mammals, we used culture-based and culture-independent approaches to identify gastrointestinal lactobacilli of the common bottlenose dolphin, Tursiops truncatus. METHODS AND RESULTS: We screened 307 bacterial isolates from oral and rectal swabs, milk and gastric fluid, collected from 38 dolphins in the U.S. Navy Marine Mammal Program, for potentially beneficial features. We focused our search on lactobacilli and evaluated their ability to modulate TNF secretion by host cells and inhibit growth of pathogens. We recovered Lactobacillus salivarius strains which secreted factors that stimulated TNF production by human monocytoid cells. These Lact. salivarius isolates inhibited growth of selected marine mammal and human bacterial pathogens. In addition, we identified a novel Lactobacillus species by culture and direct sequencing with 96·3% 16S rDNA sequence similarity to Lactobacillus ceti. CONCLUSIONS: Dolphin-derived Lact. salivarius isolates possess features making them candidate probiotics for clinical studies in marine mammals. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to isolate lactobacilli from dolphins, including a novel Lactobacillus species and a new strain of Lact. salivarius, with potential for veterinary probiotic applications. The isolation and identification of novel Lactobacillus spp. and other indigenous microbes from bottlenose dolphins will enable the study of the biology of symbiotic members of the dolphin microbiota and facilitate the understanding of the microbiomes of these unique animals.

Genotypic and phenotypic studies of murine intestinal lactobacilli: species differences in mice with and without colitis.

Lactobacilli represent components of the commensal mammalian gastrointestinal microbiota and are useful as probiotics, functional foods, and dairy products. This study includes systematic polyphasic analyses of murine intestinal Lactobacillus isolates and correlation of taxonomic findings with data from cytokine production assays. Lactobacilli were recovered from mice with microbiota-dependent colitis (interleukin-10 [IL-10]-deficient C57BL/6 mice) and from mice without colitis (Swiss Webster and inducible nitric oxide synthetase-deficient C57BL/6 mice). Polyphasic analyses were performed to elucidate taxonomic relationships among 88 reference and murine gastrointestinal lactobacilli. Genotypic tests included single-locus analyses (16S ribosomal DNA sequencing and 16S-23S rRNA intergenic spacer region PCR) and genomic DNA profiling (repetitive DNA element-based PCR), and phenotypic analyses encompassed more than 50 tests for carbohydrate utilization, enzyme production, and antimicrobial resistance. From 20 mice without colitis, six Lactobacillus species were recovered; the majority of the mice were colonized with L. reuteri or L. murinus (72% of isolates). In contrast, only, L. johnsonii was isolated from 14 IL-10-deficient mice. Using an in vitro assay, we screened murine isolates for their ability to inhibit tumor necrosis factor alpha (TNF-alpha) secretion by lipopolysaccharide-activated macrophages. Interestingly, a subpopulation of lactobacilli recovered from mice without colitis displayed TNF-alpha inhibitory properties, whereas none of the L. johnsonii isolates from IL-10-deficient mice exhibited this effect. We propose that differences among intestinal Lactobacillus populations in mammals, combined with host genetic susceptibilities, may account partly for variations in host mucosal responses.

Molecular evidence of Helicobacter cinaedi organisms in human gastric biopsy specimens.

One hundred twenty-six urease-negative gastric biopsy specimens were evaluated for the presence of Helicobacter genus-specific 16S ribosomal DNA (rDNA) and H. pylori-specific glmM DNA sequences by PCR. The species specificity of the glmM PCR assay was demonstrated, as H. pylori was the only Helicobacter species that yielded the expected glmM amplicon. Most urease-negative specimens (118 of 126 specimens) lacked Helicobacter DNA. However, 8 of 126 urease-negative specimens contained Helicobacter 16S rDNA. In order to identify the Helicobacter species present in urease-negative gastric biopsy specimens, 16S rDNA amplicons were cloned and sequenced. Sequence comparisons were performed by analyses of the sequences in public sequence databases. Two samples contained 16S rDNA that was identified as H. cinaedi with 100% identity and that spanned approximately 400 bp (398 and 398 bp, respectively). In contrast, multiple differences (97% identity; 390 of 398 bp) were observed with H. pylori 16S rDNA in this region. This finding was verified by sequencing an overlapping 537-bp fragment within the 5' portion of 16S rDNA. Although the clinical findings were consistent with H. pylori infection (e.g., duodenal ulcer disease), rapid urease testing and DNA sequence analyses suggested the presence of H. cinaedi organisms and the absence of H. pylori in two human antral biopsy specimens. This study represents the first report of an enteric urease-negative helicobacter in the human stomach. Although these organisms were previously associated with extragastric infections, the roles of these organisms in the pathogenesis of chronic gastritis or peptic ulcer disease remain unclear.

Molecular resistance testing of Helicobacter pylori in gastric biopsies.

OBJECTIVE: To evaluate simultaneous diagnosis of infection and molecular resistance testing of Helicobacter pylori. METHODS: Gastric biopsies were obtained from 26 rapid urease-positive and 51 rapid urease-negative test kits used to diagnose H pylori infection. Following glass bead-assisted DNA isolation, amplification of H pylori 16S ribosomal DNA (rDNA), glmM, and 23S rDNA target genes was performed. RESULTS: Helicobacter pylori DNA was successfully amplified from 100% (26/26) of urease-positive and 3.9% (2/51) of urease-negative gastric biopsies. Subsequent restriction enzyme-mediated digestion of 23S rDNA amplification products revealed that 17% (4/24) of urease-positive and H pylori DNA-positive biopsy specimens contained point mutations (A2142G or A2143G) associated with clarithromycin resistance. Helicobacter pylori DNA from gastric biopsies was successfully amplified 8 weeks following rapid urease testing. CONCLUSION: Helicobacter pylori genotyping may be used to detect macrolide-resistant H pylori in individuals prior to initiation of therapy or in patients refractory to anti-H pylori therapy. Two urease-negative specimens yielded Helicobacter DNA distinct from that of H pylori and indicated the need for further investigations of Helicobacter species present in the human stomach.

The generation of narrative interpretations in laboratory medicine: a description of service-specific sign-out rounds.

The logistical details for organizing effective interpretive rounds in a laboratory medicine subspecialty must be carefully established so that expert opinions are provided in a timely fashion in a patient-specific report, rather than as a collection of fixed comments associated with a particular laboratory result generated by a computer This report describes the test batteries for interpretations, the billing for interpretations, clinical examples of interpretations, and interpretations for which billing is not typically performed in several clinical or laboratory areas in our institution. These include coagulation disorders, hemoglobin and anemia evaluations, autoimmune disorders, serum protein analysis, toxicology, molecular diagnostics, and transfusion medicine. The information in this report should provide sufficient detail to allow development of interpretive services with successful billing for the areas in laboratory medicine described.

Frameshift mutations in rdxA and metronidazole resistance in North American Helicobacter pylori isolates.

In Helicobacter pylori, the oxygen-insensitive nitroreductase RdxA is likely to activate metronidazole (Mtz) by reduction and formation of cytotoxic intermediates. Mutations in rdxA have been associated with Mtz resistance in H. pylori. In vitro Mtz susceptibilities of 17 randomly selected H. pylori isolates were determined by the agar dilution method. DNA sequence analysis of rdxA alleles of eight susceptible isolates (MIC range: 0.25-1.0 mg/L) and nine resistant isolates (MIC range: 16-256 mg/L) showed that six of nine Mtz-resistant H. pylori isolates contained insertion or deletion mutations ('indel' mutations). One isolate contained a substitution mutation at codon position 148 that resulted in the introduction of a premature stop codon. Creation of stop codons within the rdxA coding sequence by either frameshift or substitution mutations resulted in premature translation termination and expression of putatively truncated RdxA polypeptides.

Helicobacter aurati sp. nov., a urease-positive Helicobacter species cultured from gastrointestinal tissues of Syrian hamsters.

A novel helicobacter with the proposed name Helicobacter aurati (type strain MIT 97-5075c) has been isolated from the inflamed stomachs and ceca of adult Syrian hamsters. The new species is fusiform with multiple bipolar sheathed flagella and periplasmic fibers; it contains urease and gamma-glutamyl transpeptidase. By 16S rRNA sequencing and repetitive element PCR-based DNA fingerprinting, it was found that H. aurati represents a distinct taxon and clusters with Helicobacter muridarum, Helicobacter hepaticus, and Helicobacter sp. MIT 94-022. H. aurati was recovered from hamsters housed in various research and vendor facilities. Further studies are necessary to define its association with disease and other microbiota in hamsters, as well as its impact on research projects involving hamsters. H. aurati (GenBank accession number AF297868) can be used in animal experiments to define the factors that are important for gastric helicobacter pathogenesis.

Point mutations in the 23S rRNA gene of Helicobacter pylori associated with different levels of clarithromycin resistance.

Fifty-four of 59 (91.5%) clarithromycin-resistant isolates of Helicobacter pylori from different patients possessed either the A2143G (formerly A2058G) or the A2144G (formerly A2059G) mutation in the gene encoding 23S rRNA. The A2143G mutation was significantly more likely to occur in isolates with MICs exceeding 64 mg/L (65% versus 30% with the A2144G mutation; P = 0.01). The majority (26 of 31; 83.9%) of isolates with the A2143G mutation had MICs exceeding 64 mg/L. Peptic ulcer disease recurred in a substantial proportion of patients infected with H. pylori strains containing either the A2143G or the A2144G mutation.

A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rRNA gene mutations in clarithromycin-resistant Helicobacter pylori.

We have developed a rapid PCR-oligonucleotide ligation assay that can discriminate single base substitutions that are associated with clarithromycin resistance in Helicobacter pylori. Susceptible isolates were wild type at positions 2143 and 2144 (cognate to 2058 and 2059 in Escherichia coli), while 93% of the resistant isolates contained A-to-G mutations at either position and 7% of the isolates contained A-to-C mutations at position 2143. In addition, the MIC for 86% of the resistant isolates with an A2143 mutation was > or = 64 micrograms per ml, and that for 89% of the resistant isolates with an A2144 mutation was < or = 32 micrograms per ml.

Molecular typing of Helicobacter pylori isolates from a multicenter U.S. clinical trial by ureC restriction fragment length polymorphism.

The molecular typing of 81 pretreatment Helicobacter pylori isolates and the comparison of 18 pretreatment-posttreatment pairs is described by restriction fragment length polymorphism (RFLP) of the ureC gene. The results of our study show the extreme genomic diversity of H. pylori and indicate that infection by H. pylori in the United States does not appear to be limited to a small number of RFLP types.

Identification of a 23S rRNA gene mutation in clarithromycin-resistant Helicobacter pylori.

BACKGROUND: Transition mutations (A-G) at residue 2143, cognate to position 2058 in the Escherichia coli 23S rRNA gene, have been shown to confer resistance to macrolides in Helicobacter pylori. This study reports the finding that transversion mutations (A-C) can occur at 2143 as well. MATERIALS AND METHODS: Three clarithromycin-resistant H. pylori isolated from three different patients after treatment with clarithromycin were analyzed for point mutations by cycle sequencing of a 163-bp amplified region surrounding residue 2143 within the conserved loop of the 23S rRNA gene. RESULTS: Nucleotide sequence comparisons of a 163-bp amplified product revealed that A-C transversion mutations occurred at position 2143. H. pylori isolated from the patients prior to treatment were susceptible to clarithromycin and displayed no polymorphism at 2143. CONCLUSION: This is the first report to show that A-C transversion mutations at position 2143 can confer resistance to clarithromycin in H. pylori and further support the role that mutations at position 2143 play in conferring macrolide resistance in H. pylori.

Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori.

Twelve clarithromycin-resistant Helicobacter pylori isolates (100% of resistant isolates examined) from seven different patients each contained an A-->G transition mutation within a conserved loop of 23S rRNA. A-->G transition mutations at positions cognate with Escherichia coli 23S rRNA positions 2058 and 2059 were identified. Clarithromycin-susceptible H. pylori isolates from 14 different patients displayed no polymorphisms in a conserved loop within domain V of 23S rRNA. The study is the first to report mutations in H. pylori associated with resistance to an antimicrobial agent used in established peptic ulcer treatment regimens.