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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.
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Treatment with antibiotics is a major risk factor for Clostridioides difficile infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit C. difficile colonization include conversion of conjugated primary bile salts into secondary bile salts toxic to C. difficile growth, and competition between the microbiota and C. difficile for limiting nutrients. Using a continuous flow model of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility to C. difficile infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with C. difficile susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate, and decreasing levels of the secondary bile salt, deoxycholate. However, decreased levels of deoxycholate did not correlate with increased C. difficile susceptibility. Further, bile salts were not required to inhibit C. difficile colonization. We tested whether amino acid fermentation contributed to persistence of C. difficile in antibiotic-treated communities. C. difficile mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (ΔprdB) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization of C. difficile.
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Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.
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Bacteriocinas , Humanos , Bacteriocinas/farmacología , Bacteriocinas/química , Bacterias , Antibacterianos/farmacología , Antibacterianos/química , PéptidosRESUMEN
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.
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Eje Cerebro-Intestino , Espectrometría de Masas en Tándem , Animales , Ratones , Cromatografía Liquida , Vida Libre de Gérmenes , Metabolómica/métodos , Bacterias , Mamíferos , OrganoidesRESUMEN
Background: Atovaquone has traditionally been used as an antiparasitic and antifungal agent, but recent studies have shown its potential as an anticancer agent. The high variability in atovaquone bioavailability highlights the need for therapeutic drug monitoring, especially in pediatric patients. The goal of our study was to develop and validate the performance of an assay to quantify atovaquone plasma concentrations collected from pediatric cancer patients using LC-MS/MS. Methods: Atovaquone was extracted from a 10 µL volume of K2-EDTA human plasma using a solution consisting of ACN: EtOH: DMF (8:1:1 v:v:v), separated using reverse-phase chromatography, and detected using a SCIEX 5500 QTrap MS system. LC-MS/MS assay performance was evaluated for precision, accuracy, carryover, sensitivity, specificity, linearity, and interferences. Results: Atovaquone and its deuterated internal standard were analyzed using a gradient chromatographic method that had an overall cycle-time of 7.4 min per injection, and retention times of 4.3 min. Atovaquone was measured over a dynamic concentration range of 0.63 - 80 µM with a deviation within ≤ ± 5.1 % of the target value. Intra- and inter-assay precision were ≤ 2.7 % and ≤ 8.4 %, respectively. Dilutional, carryover, and interference studies were also within acceptable limits. Conclusions: Our studies have shown that our LC-MS/MS-based method is both reliable and robust for the quantification of plasma atovaquone concentrations and can be used to determine the effective dose of atovaquone for pediatric patients treated for AML.
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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.
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Subgrupos de Linfocitos B , Esclerosis Múltiple , Humanos , Interleucina-4 , Clorhidrato de FingolimodRESUMEN
BACKGROUND: Peanut oral immunotherapy has emerged as a novel, active management approach for peanut-allergic sufferers, but limited data exist currently on the role of the microbiome in successful desensitization. OBJECTIVE: We examined the oral and gut microbiome in a cohort of 17 children undergoing peanut oral immunotherapy with the aim to identify the microbiome signatures associated with successful desensitization. We also set out to characterize their fecal metabolic profiles after successful therapy. METHODS: Participants gradually built up their daily dose from 2 mg (starting dose) to 300 mg (maintenance dose) within approximately 40 weeks. We collected a buccal and stool specimen from each subject at two different time points: at baseline and post-therapy (1 month after reaching maintenance). The oral (buccal) and gut (fecal) microbiome was characterized based on sequencing of 16S rRNA gene amplicons with Illumina MiSeq. Fecal short chain fatty acid levels were measured using liquid chromatography-tandem mass spectrometry. RESULTS: We report increased alpha diversity of the oral microbiome post-therapy and have also identified a significant increase in the relative abundance of oral Actinobacteria, associated with the desensitized state. However, the baseline gut microbiome did not differ from the post-therapy. Additionally, fecal short chain fatty acids increased after therapy, but not significantly. CONCLUSION: Our research adds to the limited current knowledge on microbiome and metabolic signatures in pediatric patients completing oral immunotherapy. Post-therapy increased trends of fecal fatty acid levels support a role in modulating the allergic response and potentially exerting protective and anti-inflammatory effects alongside successful desensitization. A better understanding of the microbiome-related mechanisms underlying desensitization may allow development of smarter therapeutic approaches in the near future. CLINICAL IMPLICATION: The oral microbiome composition is altered following successful peanut oral immunotherapy, with a significant increase in alpha diversity and the relative abundance of phylum Actinobacteria. CAPSULE SUMMARY: Significant microbiome changes in children completing peanut immunotherapy include increase in alpha-diversity and overrepresentation of Actinobacteria in the oral microbiome, and increased trends for fecal short chain fatty acids, suggesting a protective effect against the allergic response.
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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.
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Mathematical models have many applications in infectious diseases: epidemiologists use them to forecast outbreaks and design containment strategies; systems biologists use them to study complex processes sustaining pathogens, from the metabolic networks empowering microbial cells to ecological networks in the microbiome that protects its host. Here, we (1) review important models relevant to infectious diseases, (2) draw parallels among models ranging widely in scale. We end by discussing a minimal set of information for a model to promote its use by others and to enable predictions that help us better fight pathogens and the diseases they cause.
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Background: Bacteroidetes are the most common bacterial phylum in the mammalian intestine and the effects of several Bacteroides spp. on multiple facets of host physiology have been previously described. Of the Bacteroides spp., Bacteroides ovatus has recently garnered attention due to its beneficial effects in the context of intestinal inflammation. In this study, we aimed to examine model host intestinal physiological conditions and dietary modifications to characterize their effects on B. ovatus growth. Methods and Results: Using Biolog phenotypic microarrays, we evaluated 62 primary carbon sources and determined that B. ovatus ATCC 8384 can use the following carbohydrates as primary carbon sources: 10 disaccharides, 4 trisaccharides, 4 polysaccharides, 4 polymers, 3 L-linked sugars, 6 D-linked sugars, 5 amino-sugars, 6 alcohol sugars, and 15 organic acids. Proteomic profiling of B. ovatus bacteria revealed that a significant portion of the B. ovatus proteome contains proteins important for metabolism. Among the proteins, we found glycosyl hydrolase (GH) familes GH2, GH5, GH20, GH 43, GH88, GH92, and GH95. We also identified multiple proteins with antioxidant properties and reasoned that these proteins may support B. ovatus growth in the GI tract. Upon further testing, we showed that B. ovatus grew robustly in various pH, osmolarity, bile, ethanol, and H2O2 concentrations; indicating that B. ovatus is a well-adapted gut microbe. Conclusion: Taken together, we have demonstrated that key host and diet-derived changes in the intestinal environment influence B. ovatus growth. These data provide the framework for future work toward understanding how diet and lifestyle interventions may promote a beneficial environment for B. ovatus growth.
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BACKGROUND: Accumulating evidence indicates that the gut microbiota can synthesize neurotransmitters as well as impact host-derived neurotransmitter levels. In the past, it has been challenging to decipher which microbes influence neurotransmitters due to the complexity of the gut microbiota. METHODS: To address whether a single microbe, Bifidobacterium dentium, could regulate important neurotransmitters, we examined Bifidobacteria genomes and explored neurotransmitter pathways in secreted cell-free supernatant using LC-MS/MS. To determine if B. dentium could impact neurotransmitters in vivo, we mono-associated germ-free mice with B. dentium ATCC 27678 and examined fecal and brain neurotransmitter concentrations. RESULTS: We found that B. dentium possessed the enzymatic machinery to generate γ-aminobutyric acid (GABA) from glutamate, glutamine, and succinate. Consistent with the genome analysis, we found that B. dentium secreted GABA in a fully defined microbial media and elevated fecal GABA in B. dentium mono-associated mice compared to germ-free controls. We also examined the tyrosine/dopamine pathway and found that B. dentium could synthesize tyrosine, but could not generate L-dopa, dopamine, norepinephrine, or epinephrine. In vivo, we found that B. dentium mono-associated mice had elevated levels of tyrosine in the feces and brain. CONCLUSIONS: These data indicate that B. dentium can contribute to in vivo neurotransmitter regulation.
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Bifidobacterium/metabolismo , Neurotransmisores/metabolismo , Animales , Infecciones por Bifidobacteriales/metabolismo , Encéfalo/metabolismo , Calibración , Cromatografía Liquida , Microbioma Gastrointestinal , Genoma , Intestinos/patología , Masculino , Ratones , Microbiota , Espectrometría de Masas en Tándem , Tirosina/metabolismoRESUMEN
BACKGROUND: Bifidobacteria are commensal microbes of the mammalian gastrointestinal tract. In this study, we aimed to identify the intestinal colonization mechanisms and key metabolic pathways implemented by Bifidobacterium dentium. RESULTS: B. dentium displayed acid resistance, with high viability over a pH range from 4 to 7; findings that correlated to the expression of Na+/H+ antiporters within the B. dentium genome. B. dentium was found to adhere to human MUC2+ mucus and harbor mucin-binding proteins. Using microbial phenotyping microarrays and fully-defined media, we demonstrated that in the absence of glucose, B. dentium could metabolize a variety of nutrient sources. Many of these nutrient sources were plant-based, suggesting that B. dentium can consume dietary substances. In contrast to other bifidobacteria, B. dentium was largely unable to grow on compounds found in human mucus; a finding that was supported by its glycosyl hydrolase (GH) profile. Of the proteins identified in B. dentium by proteomic analysis, a large cohort of proteins were associated with diverse metabolic pathways, indicating metabolic plasticity which supports colonization of the dynamic gastrointestinal environment. CONCLUSIONS: Taken together, we conclude that B. dentium is well adapted for commensalism in the gastrointestinal tract.
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Bifidobacterium/metabolismo , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Ácidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bifidobacterium/genética , Bifidobacterium/crecimiento & desarrollo , Tracto Gastrointestinal/fisiología , Genoma Bacteriano , Glucosa/metabolismo , Humanos , SimbiosisRESUMEN
Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although Bifidobacterium can beneficially modulate mucin production, little work has been done investigating the effects of Bifidobacterium on goblet cell ER stress. We hypothesized that secreted factors from Bifidobacterium dentium downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that B. dentium secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. B. dentium cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, B. dentium supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that B. dentium supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress in vivo, we first examined mono-association of B. dentium in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that B. dentium can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live B. dentium or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in B. dentium- and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both B. dentium- and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by B. dentium-secreted compounds.
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Bifidobacterium/metabolismo , Colitis/microbiología , Colitis/fisiopatología , Colon/inmunología , Dipéptidos/metabolismo , Estrés del Retículo Endoplásmico , Células Caliciformes/inmunología , Animales , Colitis/inducido químicamente , Colitis/inmunología , Colon/microbiología , Colon/fisiopatología , Chaperón BiP del Retículo Endoplásmico , Microbioma Gastrointestinal , Humanos , Masculino , Ratones , Mucina 2/genética , Mucina 2/inmunología , Ácido Trinitrobencenosulfónico/efectos adversosRESUMEN
The intestinal microbiota influences the development and function of the mucosal immune system. However, the exact mechanisms by which commensal microbes modulate immunity is not clear. We previously demonstrated that commensal Bacteroides ovatus ATCC 8384 reduces mucosal inflammation. Herein, we aimed to identify immunomodulatory pathways employed by B. ovatus. In germ-free mice, mono-association with B. ovatus shifted the CD11b+/CD11c+ and CD103+/CD11c+ dendritic cell populations. Because indole compounds are known to modulate dendritic cells, B. ovatus cell-free supernatant was screened for tryptophan metabolites by liquid chromatography-tandem mass spectrometry and larger quantities of indole-3-acetic acid were detected. Analysis of cecal and fecal samples from germ-free and B. ovatus mono-associated mice confirmed that B. ovatus could elevate indole-3-acetic acid concentrations in vivo. Indole metabolites have previously been shown to stimulate immune cells to secrete the reparative cytokine IL-22. Addition of B. ovatus cell-free supernatant to immature bone marrow-derived dendritic cells stimulated IL-22 secretion. The ability of IL-22 to drive repair in the intestinal epithelium was confirmed using a physiologically relevant human intestinal enteroid model. Finally, B. ovatus shifted the immune cell populations in trinitrobenzene sulfonic acid-treated mice and up-regulated colonic IL-22 expression, effects that correlated with decreased inflammation. Our data suggest that B. ovatus-produced indole-3-acetic acid promotes IL-22 production by immune cells, yielding beneficial effects on colitis.
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Bacteroides/efectos de los fármacos , Colon/metabolismo , Inflamación/tratamiento farmacológico , Interleucinas/metabolismo , Ácido Trinitrobencenosulfónico/farmacología , Animales , Colitis/tratamiento farmacológico , Colitis/metabolismo , Colon/efectos de los fármacos , Citocinas/metabolismo , Sulfato de Dextran/metabolismo , Humanos , Inflamación/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Ratones , Interleucina-22RESUMEN
BACKGROUND & AIMS: The human gut microbiota can regulate production of serotonin (5-hydroxytryptamine [5-HT]) from enterochromaffin cells. However, the mechanisms underlying microbial-induced serotonin signaling are not well understood. METHODS: Adult germ-free mice were treated with sterile media, live Bifidobacterium dentium, heat-killed B dentium, or live Bacteroides ovatus. Mouse and human enteroids were used to assess the effects of B dentium metabolites on 5-HT release from enterochromaffin cells. In vitro and in vivo short-chain fatty acids and 5-HT levels were assessed by mass spectrometry. Expression of tryptophan hydroxylase, short-chain fatty acid receptor free fatty acid receptor 2, 5-HT receptors, and the 5-HT re-uptake transporter (serotonin transporter) were assessed by quantitative polymerase chain reaction and immunostaining. RNA in situ hybridization assessed 5-HT-receptor expression in the brain, and 5-HT-receptor-dependent behavior was evaluated using the marble burying test. RESULTS: B dentium mono-associated mice showed increased fecal acetate. This finding corresponded with increased intestinal 5-HT concentrations and increased expression of 5-HT receptors 2a, 4, and serotonin transporter. These effects were absent in B ovatus-treated mice. Application of acetate and B dentium-secreted products stimulated 5-HT release in mouse and human enteroids. In situ hybridization of brain tissue also showed significantly increased hippocampal expression of 5-HT-receptor 2a in B dentium-treated mice relative to germ-free controls. Functionally, B dentium colonization normalized species-typical repetitive and anxiety-like behaviors previously shown to be linked to 5-HT-receptor 2a. CONCLUSIONS: These data suggest that B dentium, and the bacterial metabolite acetate, are capable of regulating key components of the serotonergic system in multiple host tissues, and are associated with a functional change in adult behavior.
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Bifidobacterium/metabolismo , Eje Cerebro-Intestino/fisiología , Microbioma Gastrointestinal/fisiología , Interacciones Microbiota-Huesped/fisiología , Serotonina/metabolismo , Acetatos/metabolismo , Animales , Conducta Animal/fisiología , Bifidobacterium/aislamiento & purificación , Técnicas de Cultivo de Célula , Células Enterocromafines/metabolismo , Vida Libre de Gérmenes , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Modelos Animales , Organoides , Receptores de Serotonina/metabolismoRESUMEN
Recently, an opportunity to perform a broad ruggedness assessment of our liquid chromatography-tandem mass spectrometry (LC-MS/MS) system presented itself during the analytical planning phase of a large-scale human fecal microbiome study. The specific aim of this project was to study the microbial-mediated metabolism of a targeted set of bile acids/salts by mixed bacterial communities cultured from the feces of 12 healthy volunteers when grown in a custom growth medium and following exposure to different clinically-relevant antibiotics. The magnitude of this study offered a rare opportunity to significantly stress procedures and LC-MS/MS system components comprised in our bile acid/salt targeted metabolomics method. With this second specific aim in mind, we modified the sample analysis plan to include a series of figure-of-merit (FoM)-based tests that are commonly used in regulated bioanalytical labs to assess LC and MS system ruggedness for a specific assay - these FoM-based testing parameters were monitored continuously over the course of sample analysis and the results are presented in this report. In total, the assessment included 1206 sequential injections (180 calibration standards, 136 blank-internal standard samples, and 890 diluted medium samples) that took place over 8-days. Completion of the 8-days of non-stop sample analysis revealed no critical hardware or software failures, and the analysis of the FoM-based tests indicated no observable degradation of system performance over the number of samples and time tested. The FoM-based test metrics presented may be used as a template to assess the ruggedness of any LC-MS/MS-based targeted metabolomics workflow.
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Técnicas Bacteriológicas/métodos , Cromatografía Liquida/métodos , Microbiota , Espectrometría de Masas en Tándem/métodos , Bacterias , Ácidos y Sales Biliares , Calibración , Heces , Humanos , Metabolómica , Sensibilidad y EspecificidadRESUMEN
Antibiotic resistance is one of the world's greatest public health challenges and adjunct probiotic therapies are strategies that could lessen this burden. Clostridioides difficile infection (CDI) is a prime example where adjunct probiotic therapies could decrease disease incidence through prevention. Human-derived Lactobacillus reuteri is a probiotic that produces the antimicrobial compound reuterin known to prevent C. difficile colonization of antibiotic-treated fecal microbial communities. However, the mechanism of inhibition is unclear. We show that reuterin inhibits C. difficile outgrowth from spores and vegetative cell growth, however, no effect on C. difficile germination or sporulation was observed. Consistent with published studies, we found that exposure to reuterin stimulated reactive oxygen species (ROS) in C. difficile, resulting in a concentration-dependent reduction in cell viability that was rescued by the antioxidant glutathione. Sublethal concentrations of reuterin enhanced the susceptibility of vegetative C. difficile to vancomycin and metronidazole treatment and reduced toxin synthesis by C. difficile. We also demonstrate that reuterin is protective against C. difficile toxin-mediated cellular damage in the human intestinal enteroid model. Overall, our results indicate that ROS are essential mediators of reuterin activity and show that reuterin production by L. reuteri is compatible as a therapeutic in a clinically relevant model.
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Clostridioides difficile/efectos de los fármacos , Gliceraldehído/análogos & derivados , Propano/farmacología , Especies Reactivas de Oxígeno/metabolismo , Antibacterianos/farmacología , Clostridioides difficile/crecimiento & desarrollo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidad , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Gliceraldehído/metabolismo , Gliceraldehído/farmacología , Humanos , Limosilactobacillus reuteri/metabolismo , Organoides/efectos de los fármacos , Organoides/microbiología , Estrés Oxidativo/efectos de los fármacos , Probióticos/metabolismo , Propano/metabolismo , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
Due to the physicochemical properties of bile acids/salts (i.e., hydrophobic and ionizable), the application of reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods are ideally suited for the measurement of these compounds in a host of microbiologically-relevant matrices. Here, we provide a detailed bioanalytical protocol that contains several modifications of a method previously described by Wegner et al. [1]. Briefly, this modified method exhibits the following advantages for the measurement of cholic acid (CA), taurocholic acid (TCA), and deoxycholic acid (DCA) in microbiome-relevant sample matrices: i) fecal sample processing has been streamlined by the elimination of lyophilization and manual homogenization steps; ii) the Sciex 6500 QTRAP hybrid triple-quadrupole/linear ion trap mass spectrometer has sufficient sensitivity to perform the measurement of bile acids/salts in negative ion mode - ammonium adducts of bile acids/salts are not required for detection; and, iii) assay throughput has been boosted by more than 5-fold by shortening the chromatographic duty cycle of a single sample injection from 45 min to 8.4 min. Recently, the method was used to perform 508 sequential injections (72 calibration standards, 52 blank-internal standard sample, and 368 MiniBioReactor Array (MBRA)-derived samples) from four separate batches over a 4-day time period.
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Herein we report that the Thomson Standard and eXtreme/FV® filter vials (0.2⯵m polyvinylidene difluoride filter membrane) are as effective as gold standard microporous membrane-based syringe filters at removing bacteria, such as Klebsiella pneumonia, from media samples produced in the microbiological laboratory.
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Filtración/métodos , Técnicas Microbiológicas/métodos , Esterilización/métodos , Jeringas , Bacterias , Equipos y Suministros , Filtración/instrumentación , Ensayos Analíticos de Alto Rendimiento , Técnicas Microbiológicas/instrumentaciónRESUMEN
Mass spectrometers are comprised of three main components: an ion source, a mass analyzer, and a detector. Ionization of the analyte occurs in the ion source and the resulting ions are counted at the detector. However, it is the mass analyzer that is responsible for determing the mass-to-charge ratio (m/z) of the ions (Jennings KR, Dolnikowski GG, Method Enzymol 193:37-61, 1990). Therefore, it is primarily the analyzer that allows the mass spectrometer to serve its primary goal - determining the mass of the analytes being measured. This becomes important in the field of molecular biology, where biomolecules may be of low molecular weight or often take on multiple charges (z) after ionization (Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM, Science 246:64-71, 1989). For this reason, the choice of analyzer is dependant on the properties of the analyte after ionization and the requirements of the experiment being performed.