Phylogenetically diverse bacterial species produce histamine.

Histamine is an important biogenic amine known to impact a variety of patho-physiological processes ranging from allergic reactions, gut-mediated anti-inflammatory responses, and neurotransmitter activity. Histamine is found both endogenously within specialized host cells and exogenously in microbes. Exogenous histamine is produced through the decarboxylation of the amino acid L-histidine by bacterial-derived histidine decarboxylase enzymes. To investigate how widespread histamine production is across bacterial species, we examined 102,018 annotated genomes in the Integrated Microbial Genomes Database and identified 3,679 bacterial genomes (3.6 %) which possess the enzymatic machinery to generate histamine. These bacteria belonged to 10 phyla: Bacillota, Bacteroidota, Actinomycetota, Pseudomonadota, Lentisphaerota, Fusobacteriota, Armatimonadota, Cyanobacteriota, Thermodesulfobacteriota, and Verrucomicrobiota. The majority of the identified bacteria were terrestrial or aquatic in origin, although several bacteria originated in the human gut microbiota. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics to confirm our genome discoveries correlated with L-histidine-to-histamine conversion in a chemically defined bacterial growth medium by a cohort of select environmental and human gut bacteria. We found that environmental microbes Vibrio harveyi, Pseudomonas fluorescens and Streptomyces griseus generated considerable levels of histamine (788 - 8,730 ng/mL). Interestingly, we found higher concentrations of histamine produced by gut-associated Fusobacterium varium, Clostridium perfringens, Limosilactobacillus reuteri and Morganella morganii (8,510--82,400 ng/mL). This work expands our knowledge of histamine production by diverse microbes.

Mapping the Geographical Distribution of the Mucosa-Associated Gut Microbiome in GI-Symptomatic Children with Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by cognitive, behavioral, and communication impairments. In the last few years, it has been proposed that alterations in the gut microbiota may contribute to an aberrant communication between the gut and brain in children with ASD. Consistent with this notion, several studies have demonstrated that children with ASD have an altered fecal microbiota compared to typically developing (TD) children. However, it is unclear where along the length of the gastrointestinal (GI) tract these alterations in microbial communities occur. Additionally, the variation between specific mucosa-associated communities remains unknown. To address this gap in knowledge of the microbiome associated with ASD, biopsies from the antrum, duodenum, ileum, ascending colon, and rectum of children with ASD and age- and sex-matched TD children were examined by 16s rRNA sequencing. We observed an overall elevated abundance of Bacillota and Bacteroidota and decreased abundance of Pseudomonadota in all GI tract regions of both male and female ASD children compared to TD children. Further analysis at the genera level revealed unique differences in the microbiome in the different regions of the GI tract in ASD children compared to TD children. We also observed sex-specific differences in the gut microbiota composition in children with ASD. These data indicate that the microbiota of ASD children is altered at multiple regions of the GI tract and that different anatomic locations have unique alterations in mucosa-associated bacterial genera.

Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts.

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

Repurposing dried blood spot device technology to examine bile acid profiles in human dried fecal spot samples.

Dried blood spot (DBS) analysis has existed for >50 years, but application of this technique to fecal analysis remains limited. To address whether dried fecal spots (DFS) could be used to measure fecal bile acids, we collected feces from five subjects for each of the following cohorts: 1) healthy individuals, 2) individuals with diarrhea, and 3) Clostridioides difficile-infected patients. Homogenized fecal extracts were loaded onto quantitative DBS (qDBS) devices, dried overnight, and shipped to the bioanalytical lab at ambient temperature. For comparison, source fecal extracts were shipped on dry ice and stored frozen. After 4 mo, frozen fecal extracts and ambient DFS samples were processed and subjected to targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics with stable isotope-labeled standards. We observed no differences in the bile acid levels measured between the traditional extraction and the qDBS-based DFS methods. This pilot data demonstrates that DFS-based analysis is feasible and warrants further development for fecal compounds and microbiome applications.NEW & NOTEWORTHY Stool analysis in remote settings can be challenging, as the samples must be stored at -80°C and transported on dry ice for downstream processing. Our work indicates that dried fecal spots (DFS) on Capitainer quantitative DBS (qDBS) devices can be stored and shipped at ambient temperature and yields the same bile acid profiles as traditional samples. This approach has broad applications for patient home testing and sample collection in rural communities or resource-limited countries.

The adolescent and young adult microbiome and its association with substance use: a scoping review.

AIMS: The microbiome is a critical factor in health throughout human development. The aims of this scoping review are to (i) elucidate the differences between the youth (post-natal day 21-65 for rodents, 2-7 years for non-human primates, and 10-25 years for humans) microbiome with other life stages and (ii) identify youth-specific microbial changes associated with substance use. METHODS: Peer-reviewed studies published up to May 2023 were identified in PubMed and SCOPUS and included gut and oral microbiome studies from rodents, non-human primates, and humans (N = 1733). Twenty-six articles were determined eligible based on inclusion criteria (aim 1: n = 19, aim 2: n = 7). RESULTS: The adolescent and young adult oral and gut microbiomes are distinct compared to other life stages, within both non-human and human models. While there is limited research in this area, the microbiome appears to be vulnerable to substance use exposure earlier in life, including substances commonly initiated and escalated during adolescence and young adulthood (i.e. alcohol, cannabis, and tobacco). CONCLUSIONS: Studies across the lifespan indicate that adolescence and young adulthood are distinct periods of development, where the microbiome is sensitive to exposures, including substance use. There is a need for more studies focused on the adolescent and young adult microbiome and substance use, as well as focused on the oral microbiome during this developmental period. Understanding the gut and oral microbiome during adolescence and young adulthood may provide insight into the pathophysiology of substance use disorders.

Bifidobacterium and the intestinal mucus layer.

Bifidobacterium species are integral members of the human gut microbiota and these microbes have significant interactions with the intestinal mucus layer. This review delves into Bifidobacterium-mucus dynamics, shedding light on the multifaceted nature of this relationship. We cover conserved features of Bifidobacterium-mucus interactions, such as mucus adhesion and positive regulation of goblet cell and mucus production, as well as species and strain-specific attributes of mucus degradation. For each interface, we explore the molecular mechanisms underlying these interactions and their potential implications for human health. Notably, we emphasize the ability of Bifidobacterium species to positively influence the mucus layer, shedding light on its potential as a mucin-builder and a therapeutic agent for diseases associated with disrupted mucus barriers. By elucidating the complex interplay between Bifidobacterium and intestinal mucus, we aim to contribute to a deeper understanding of the gut microbiota-host interface and pave the way for novel therapeutic strategies.

A high-throughput protocol for measuring solution pH of bacterial cultures using UV-Vis absorption spectrophotometry.

We present a protocol for measuring the pH of cell-free bacterial-conditioned media based on changes in the ultraviolet-visible (UV-Vis) absorbance spectrum using the pH indicator dye litmus. This protocol includes detailed procedures for performing bacterial culturing, examining bacterial growth, collecting cell-free supernatant, litmus dye addition, and pH-based calibration curve preparations. This assay has been designed for flexible formatting that can accommodate both high-volume and low-volume sample sets.

Identification of Differentiated Intestinal Epithelial Cells Using Immunostaining and Fluorescence Microscopy.

Immunofluorescence imaging enables visualization of a wide range of molecules in diverse cells and tissues. Determining the localization and endogenous protein levels in cells using immunostaining can be highly informative for researchers studying cell structure and function. The small intestinal epithelium is composed of numerous cell types including absorptive enterocytes, mucus-producing goblet cells, lysozyme positive Paneth cells, proliferative stem cells, chemosensing tuft cells, and hormone-producing enteroendocrine cells. Each cell type in the small intestine has unique functions and structures that are critical for maintaining intestinal homeostasis and identifiable by immunofluorescence labeling. In this chapter we provide a detailed protocol and representative images of immunostaining of paraffin-embedded mouse small intestinal tissue. The method highlights antibodies and micrographs that identify differentiated cell types. These details are important because quality immunofluorescence imaging can provide novel insights and a greater understanding of healthy and disease states.

Bioinformatics reveal elevated levels of Myosin Vb in uterine corpus endometrial carcinoma patients which correlates to increased cell metabolism and poor prognosis.

Carcinoma of the endometrium of the uterus is the most common female pelvic malignancy. Although uterine corpus endometrial cancer (UCEC) has a favorable prognosis if removed early, patients with advanced tumor stages have a low survival rate. These facts highlight the importance of understanding UCEC biology. Computational analysis of RNA-sequencing data from UCEC patients revealed that the molecular motor Myosin Vb (MYO5B) was elevated in the beginning stages of UCEC and occurred in all patients regardless of tumor stage, tumor type, age, menopause status or ethnicity. Although several mutations were identified in the MYO5B gene in UCEC patients, these mutations did not correlate with mRNA expression. Examination of MYO5B methylation revealed that UCEC patients had undermethylated MYO5B and undermethylation was positively correlated with increased mRNA and protein levels. Immunostaining confirmed elevated levels of apical MYO5B in UCEC patients compared to adjacent tissue. UCEC patients with high expressing MYO5B tumors had far worse prognosis than UCEC patients with low expressing MYO5B tumors, as reflected by survival curves. Metabolic pathway analysis revealed significant alterations in metabolism pathways in UCE patients and key metabolism genes were positively correlated with MYO5B mRNA. These data provide the first evidence that MYO5B may participate in UCEC tumor development.

Interrogation of the mammalian gut-brain axis using LC-MS/MS-based targeted metabolomics with in vitro bacterial and organoid cultures and in vivo gnotobiotic mouse models.

Interest in the communication between the gastrointestinal tract and central nervous system, known as the gut-brain axis, has prompted the development of quantitative analytical platforms to analyze microbe- and host-derived signals. This protocol enables investigations into connections between microbial colonization and intestinal and brain neurotransmitters and contains strategies for the comprehensive evaluation of metabolites in in vitro (organoids) and in vivo mouse model systems. Here we present an optimized workflow that includes procedures for preparing these gut-brain axis model systems: (stage 1) growth of microbes in defined media; (stage 2) microinjection of intestinal organoids; and (stage 3) generation of animal models including germ-free (no microbes), specific-pathogen-free (complete gut microbiota) and specific-pathogen-free re-conventionalized (germ-free mice associated with a complete gut microbiota from a specific-pathogen-free mouse), and Bifidobacterium dentium and Bacteroides ovatus mono-associated mice (germ-free mice colonized with a single gut microbe). We describe targeted liquid chromatography-tandem mass spectrometry-based metabolomics methods for analyzing microbially derived short-chain fatty acids and neurotransmitters from these samples. Unlike other protocols that commonly examine only stool samples, this protocol includes bacterial cultures, organoid cultures and in vivo samples, in addition to monitoring the metabolite content of stool samples. The incorporation of three experimental models (microbes, organoids and animals) enhances the impact of this protocol. The protocol requires 3 weeks of murine colonization with microbes and ~1-2 weeks for liquid chromatography-tandem mass spectrometry-based instrumental and quantitative analysis, and sample post-processing and normalization.

Serum 3-phenyllactic acid level is reduced in benign multiple sclerosis and is associated with effector B cell ratios.

BACKGROUND: 3-phenyllactic acid (PLA) is produced by both intestinal bacteria and the human host. PLA exists in its D- and L- chiral forms. It modulates human immune functions, thereby acting as a mediator of bacterial-host interactions. We aim to determine the amount and potential influence of PLA on clinical and immunological features of MS. METHODS: We measured D- and L-PLA levels in bacterial supernatants and in sera of 60 MS patients and 25 healthy controls. We investigated potential associations between PLA levels, clinical features of MS, serum cytokine levels and ratios of peripheral blood lymphocyte subsets. RESULTS: Multiple gut commensal bacteria possessed the capacity to generate D- and L-PLA. MS patients with benign phenotype showed markedly lower PLA levels than healthy controls or other MS patients. Fingolimod resistant patients had higher PLA levels at baseline. Furthermore, MS patients with higher PLA levels tended to display increased memory B and plasma cell ratios, elevated IL-4 levels and increased ratios of IL-4 and IL-10 producing T cell subsets. CONCLUSION: Collectively, our work indicates that reduced serum levels of PLA could be associated with a favorable clinical course in MS and possibly be used as a biomarker.

Profiling Antibiotic Resistance in Acinetobacter calcoaceticus.

Background: Acinetobacter spp. have emerged as troublesome pathogens due to their multi-drug resistance. The majority of the work to date has focused on the antibiotic resistance profile of Acinetobacter baumannii. Although A. calcoaceticus strains are isolated in the hospital setting, limited information is available on these closely related species. Methods & Results: The computational analysis of antibiotic resistance genes in 1441 Acinetobacter genomes revealed that A. calcoaceticus harbored a similar repertoire of multi-drug efflux pump and beta-lactam resistance genes as A. baumannii, leading us to speculate that A. calcoaceticus would have a similar antibiotic resistance profile to A. baumannii. To profile the resistance patterns of A. calcoaceticus, strains were examined by Kirby−Bauer disk diffusion and phenotypic microarrays. We found that Acinetobacter strains were moderately to highly resistant to certain antibiotics within fluoroquinolones, aminoglycosides, tetracyclines, and other antibiotic classes. These data indicate that A. calcoaceticus has a similar antibiotic resistance profile as A. baumannii ATCC 19606. We also identified that all Acinetobacter species were sensitive to 5-fluoroorotic acid, novobiocin, and benzethonium chloride. Conclusion: Collectively, these data provide new insights into the antibiotic resistance in A. calcoaceticus and identify several antibiotics that could be beneficial in treating Acinetobacter infections.

Myosins and membrane trafficking in intestinal brush border assembly.

Myosins are a class of motors that participate in a wide variety of cellular functions including organelle transport, cell adhesion, endocytosis and exocytosis, movement of RNA, and cell motility. Among the emerging roles for myosins is regulation of the assembly, morphology, and function of actin protrusions such as microvilli. The intestine harbors an elaborate apical membrane composed of highly organized microvilli. Microvilli assembly and function are intricately tied to several myosins including Myosin 1a, non-muscle Myosin 2c, Myosin 5b, Myosin 6, and Myosin 7b. Here, we review the research progress made in our understanding of myosin mediated apical assembly.

Acinetobacter calcoaceticus is Well Adapted to Withstand Intestinal Stressors and Modulate the Gut Epithelium.

Background: The gastrointestinal tract has been speculated to serve as a reservoir for Acinetobacter, however little is known about the ecological fitness of Acinetobacter strains in the gut. Likewise, not much is known about the ability of Acinetobacter to consume dietary, or host derived nutrients or their capacity to modulate host gene expression. Given the increasing prevalence of Acinetobacter in the clinical setting, we sought to characterize how A. calcoaceticus responds to gut-related stressors and identify potential microbe-host interactions. Materials and Methods: To accomplish these aims, we grew clinical isolates and commercially available strains of A. calcoaceticus in minimal media with different levels of pH, osmolarity, ethanol and hydrogen peroxide. Utilization of nutrients was examined using Biolog phenotypic microarrays. To examine the interactions of A. calcoaceticus with the host, inverted murine organoids where the apical membrane is exposed to bacteria, were incubated with live A. calcoaceticus, and gene expression was examined by qPCR. Results: All strains grew modestly at pH 6, 5 and 4; indicating that these strains could tolerate passage through the gastrointestinal tract. All strains had robust growth in 0.1 and 0.5 M NaCl concentrations which mirror the small intestine, but differences were observed between strains in response to 1 M NaCl. Additionally, all strains tolerated up to 5% ethanol and 0.1% hydrogen peroxide. Biolog phenotypic microarrays revealed that A. calcoaceticus strains could use a range of nutrient sources, including monosaccharides, disaccharides, polymers, glycosides, acids, and amino acids. Interestingly, the commercially available A. calcoaceticus strains and one clinical isolate stimulated the pro-inflammatory cytokines Tnf, Kc, and Mcp-1 while all strains suppressed Muc13 and Muc2. Conclusion: Collectively, these data demonstrate that A. calcoaceticus is well adapted to dealing with environmental stressors of the gastrointestinal system. This data also points to the potential for Acinetobacter to influence the gut epithelium.

Characterizing the mucin-degrading capacity of the human gut microbiota.

Mucin-degrading microbes are known to harbor glycosyl hydrolases (GHs) which cleave specific glycan linkages. Although several microbial species have been identified as mucin degraders, there are likely many other members of the healthy gut community with the capacity to degrade mucins. The aim of the present study was to systematically examine the CAZyme mucin-degrading profiles of the human gut microbiota. Within the Verrucomicrobia phylum, all Akkermansia glycaniphila and muciniphila genomes harbored multiple gene copies of mucin-degrading GHs. The only representative of the Lentisphaerae phylum, Victivallales, harbored a GH profile that closely mirrored Akkermansia. In the Actinobacteria phylum, we found several Actinomadura, Actinomyces, Bifidobacterium, Streptacidiphilus and Streptomyces species with mucin-degrading GHs. Within the Bacteroidetes phylum, Alistipes, Alloprevotella, Bacteroides, Fermenitomonas Parabacteroides, Prevotella and Phocaeicola species had mucin degrading GHs. Firmicutes contained Abiotrophia, Blautia, Enterococcus, Paenibacillus, Ruminococcus, Streptococcus, and Viridibacillus species with mucin-degrading GHs. Interestingly, far fewer mucin-degrading GHs were observed in the Proteobacteria phylum and were found in Klebsiella, Mixta, Serratia and Enterobacter species. We confirmed the mucin-degrading capability of 23 representative gut microbes using a chemically defined media lacking glucose supplemented with porcine intestinal mucus. These data greatly expand our knowledge of microbial-mediated mucin degradation within the human gut microbiota.

Bacteroides ovatus colonization influences the abundance of intestinal short chain fatty acids and neurotransmitters.

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.

Identifying single-strain growth patterns of human gut microbes in response to preterm human milk and formula.

The intestinal microbiota of the preterm neonate has become a major research focus, with evidence emerging that the microbiota influences both short and long-term health outcomes, in the neonatal intensive care unit and beyond. Similar to the term microbiome, the preterm gut microbiome is highly influenced by diet, specifically formula and human milk use. This study aims to analyze next-generation products including preterm formula, human milk-oligosaccharide term formula, and preterm breastmilk. We used a culture-based model to differentially compare the growth patterns of individual bacterial strains found in the human intestine. This model probed 24 strains of commensal bacteria and 8 pathobiont species which have previously been found to cause sepsis in preterm neonates. Remarkable differences between strain growth and culture pH were noted after comparing models of formulas and between human milk and formula. Both formula and human milk supported the growth of commensal bacteria; however, the formula products, but not human milk, supported the growth of several specific pathogenic strains. Computational analysis revealed potential connections between long-chain fatty acid and iron uptake from formula in pathobiont organisms. These findings indicate that there is a unique profile of growth in response to human milk and formula and shed light into how the infant gut microbiota could be influenced.

Maternal Lactobacillus reuteri supplementation shifts the intestinal microbiome in mice and provides protection from experimental colitis in female offspring.

The purpose of our experiment was to explore how stochastic (inter-individual variation) gut microbiome composition may link to inflammatory bowel disease (IBD) susceptibility and guide the development of a perinatal preventative probiotic. Dextran sodium sulfate (DSS) was introduced to C57BL/BJ mice to induce acute colitis as a model of IBD. Potentially protective bacteria were identified using a discovery-validation cohort approach toward stochastic DSS susceptibility. Lactobacilli (two different cocktails of L. reuteri and L. johnsonii strains) or control media were supplemented by mouth to dams prior to delivery and during lactation (i.e., perinatal probiotic). The pups were evaluated for DSS susceptibility at young adulthood. Fecal Lactobacillus was increased in the DSS-resistant mice in both the discovery and validation cohorts. Maternal supplementation of female offspring with an L. reuteri cocktail (strains 6798-1, 6798-jm, and 6798-cm) induced progressive microbiome separation and protection against colitis by young adulthood. Maternal supplementation of L. reuteri could confer protection against DSS colitis in young adult female mice. This work is the first to exploit stochastic mammalian microbiome variation to guide microbial therapeutic identification. Our findings underscore neonatal microbiome plasticity and set the stage for the potential development of perinatally deliverable protective probiotics against human IBD.

Loss of H2R Signaling Disrupts Neutrophil Homeostasis and Promotes Inflammation-Associated Colonic Tumorigenesis in Mice.

BACKGROUND & AIMS: We previously showed that histamine suppressed inflammation-associated colonic tumorigenesis through histamine type 2 receptor (H2R) signaling in mice. This study aimed to precisely elucidate the downstream effects of H2R activation in innate immune cells. METHODS: Analyses using online databases of single-cell RNA sequencing of intestinal epithelial cells in mice and RNA sequencing of mouse immune cells were performed to determine the relative abundances of 4 histamine receptors among different cell types. Mouse neutrophils, which expressed greater amounts of H2R, were collected from the peritoneum of wild-type and H2R-deficient mice, of which low-density and high-density neutrophils were extracted by centrifugation and were subjected to RNA sequencing. The effects of H2R activation on neutrophil differentiation and its functions in colitis and inflammation-associated colon tumors were investigated in a mouse model of dextran sulfate sodium-induced colitis. RESULTS: Data analysis of RNA sequencing and quantitative reverse-transcription polymerase chain reaction showed that Hrh2 is highly expressed in neutrophils, but barely detectable in intestinal epithelial cells. In mice, the absence of H2R activation promoted infiltration of neutrophils into both sites of inflammation and colonic tumors. H2R-deficient high-density neutrophils yielded proinflammatory features via nuclear factor-κB and mitogen-activated protein kinase signaling pathways, and suppressed T-cell proliferation. On the other hand, low-density neutrophils, which totally lack H2R activation, showed an immature phenotype compared with wild-type low-density neutrophils, with enhanced MYC pathway signaling and reduced expression of the maturation marker Toll-like receptor 4. CONCLUSIONS: Blocking H2R signaling enhanced proinflammatory responses of mature neutrophils and suppressed neutrophil maturation, leading to accelerated progression of inflammation-associated colonic tumorigenesis.