Differentiation of Alkyl- and Plasmenyl-phosphatidylcholine by Endogenous Sphingomyelin RT-XLOGP3 Regression for Coronary Artery Disease Plasma Lipidomics Analysis.

Accurate identification between alkyl- and plasmenyl-phosphatidylcholine (PC(O-) and PC(P-)) isomers is a major analytical challenge in lipidomics studies due to a lack of structure-specific ions in conventional tandem mass spectrometry (MS/MS) methods and the absence of universal retention time (RT) references. Given the importance of PC(O-) and PC(P-), an easy-to-apply method for current research is urgently needed. In this study, we present a quadratic RT-XLOGP3SM regression model that uses endogenous sphingomyelin (SM) species in blood samples as retention time (RT) indicators to predict the RTs of PC(O-) and PC(P-) species by coupling their calculated partition coefficients based on XLOGP3. The prediction results were obtained with a root-mean-square error (RMSE) of 0.12 min (1.3%) for the RRHD (rapid resolution high definition) nonlinear LC condition. A lipidomic analysis with RT-XLOGP3SM regression was used to study lipid regulation in coronary artery disease (CAD) outpatient plasma samples, and we found that the types of exhibited regulation were highly dependent on the lipid subclasses in comparison to the healthy control group. In conclusion, given that the quadratic RT-XLOGP3SM regression model predicts the RTs of PC species based on the relative value of XLOGP3 and the RTs of endogenous SM species, it can be expected that most of the C18-based lipidomics analyses could apply this method to increase the identification ability of the PC(O-) and PC(P-) subclasses and to improve the understanding of their physiological functions.

Gut microbiome: A possible common therapeutic target for treatment of atherosclerosis and cancer.

Human gut microbiota is a dynamic and variable system that can change over time and in response to different diets and treatments. There is currently no doubt that gut microbiota can provide interesting therapeutic opportunities, since it can metabolize biologically active molecules, drugs, and their precursors, and control their bioavailability. Moreover, it can produce both beneficial and dangerous metabolites that influence host's health. In this review, we summarize the current knowledge on the involvement of gut microbiota in two chronic human pathologies that represent the greatest challenges of modern medicine: atherosclerosis and cancer. Interesting parallels are observed between the mechanisms and possible treatment approaches of these pathologies. Some of the common effects of therapeutic agents targeting both pathologies, such as anti-inflammatory activity, are partially mediated by the gut microbiota. We will discuss the effects of common drugs (metformin, statins and aspirin) and various nutraceuticals on gut microbiota and outline the pathways of microbial involvement in mediating the pleiotropic beneficial effects of these agents in atherosclerosis and cancer.

Development of an Efficient and Sensitive Chemical Derivatization-Based LC-MS/MS Method for Quantifying Gut Microbiota-Derived Metabolites in Human Plasma and Its Application in Studying Cardiovascular Disease.

Recently, the gut microbiota has been found to be associated with many diseases, such as inflammatory bowel disease, depression, Parkinson's disease, cancer, metabolic syndrome, and cardiovascular disease (CVD). Among various gut microbiota-derived metabolites (GMs), short-chain fatty acids (SCFAs), bile acids (BAs), and tryptophan (TRP) metabolites are the most frequently discussed metabolites. LC-MS/MS shows advantages in quantifying the levels of metabolites with good sensitivity and selectivity; however, the poor ionization efficiency and polar characteristics of SCFAs make their analysis challenging, especially when analyzing plasma samples with low SCFA concentrations. Moreover, without characteristic fragment ions for unconjugated BAs and different detection ion modes for TRP metabolites and BAs, GM analysis is complex and time-consuming. To overcome these problems, we developed a derivatization method combined with LC-MS/MS to enhance the sensitivity and LC retention of GMs. Through derivatization with 3-nitrophenylhydrazine (3-NPH), 7 SCFAs, 9 bile acids, and 6 tryptophan metabolites can be simultaneously analyzed via separation within 14 min on a reversed-phase C18 column. For accurate quantification, 13C6-3NPH-labeled standards were used as one-to-one internal standards. This derivatization approach was optimized and then validated. We further applied this method to investigate the targeted GM profile in patients with CVD. The results showed a significant reduction in plasma butyrate levels in CVD patients compared with healthy controls, suggesting its potentially protective role in CVD. In summary, this work provides a sensitive and effective LC-MS/MS method for simultaneously quantifying gut microbiota-related metabolites in human plasma, which could benefit various future gut microbiota-related studies.

Gastrodia elata Blume water extract modulates neurotransmitters and alters the gut microbiota in a mild social defeat stress-induced depression mouse model.

Gastrodia elata Blume has multiple bioactive functions, such as antioxidant and antidepressant activities, immune modulation, neuroplasticity, and neuroprotection. We previously found that the water extract of G. elata exerts antidepressant-like effects in unpredictable chronic mild stress models and animals exposed to the forced swimming test. We aimed to investigate the mechanisms by which the water extract of G. elata protects against subchronic- and mild-social defeat-stress-induced dysbiosis. After a 10-day subchronic and mild-social-defeat-stress program, oral treatment with the water extract of G. elata (500 mg/kg bw) resulted in reversal of depression-like behavior. In addition, monoamine analyses showed that the water extract of G. elata normalized the 5-hydroxyindoleacetic acid:5-HT ratio in the prefrontal cortex and colon and reduced the defeat-stress-induced kynurenine:tryptophan ratio in the colon. After the 10-day subchronic and mild social-defeat-stress program, the water extract of G. elata altered the intestinal microbiome by increasing Actinobacteria levels, modulating intestinal inflammation, and shifting the relative abundances of multiple bacterial groups in the gut. Our results suggest that the water extract of G. elata exhibits a potent antidepressant-like effect via the regulation of monoaminergic neurotransmission and alteration of gut microbiota composition and function, and that it may be an effective prevention for depression.

Measurement of gut microbial metabolites in cardiometabolic health and translational research.

The human gut microbiota is a functioning endocrine organ and stands at the intersection between dietary components and health or disease. There are very many microbial metabolites with numerous structures and functions arising from the gut microbial fermentation of foods and become signals for biological communication in the human body. These small molecules can be absorbed and delivered to distant organs through the circulatory system to build the gut-systemic axis. The gut microbial metabolomes are thus believed to play important roles in regulating cardiometabolic health and provide opportunities in mechanistic research and new drug discovery. Measurement of these novel microbial metabolites in clinical samples may serve as a tool for investigating disease biomarkers. In the past decade, the development of untargeted and targeted metabolomics approaches using NMR, LC/MS, and GC/MS has contributed to the exploration of gut microbial metabolomes in cardiometabolic health and disease. Some important targets are currently being translated into clinical applications. In this review article, we introduce an oral carnitine challenge test developed as an example to demonstrate the potential applications in personalized nutrition based on the function of gut microbiota. It is a method taking the gut microbiota as a bioreactor and provides fermentable materials as inputs and measures the outputs of targeted microbial byproducts in the blood or urine. This challenge test may be extended to measure metabolites from microbial fermentation related to other endocrinological or inflammatory diseases. We review current gut metabolome research approaches and propose a gut microbial functional measurement using a challenge test. We suggest that the maturation in measuring gut microbial metabolites may provide an important piece to complete the puzzle of precision medicine.

Comparison of DNA stabilizers and storage conditions on preserving fecal microbiota profiles.

BACKGROUND/PURPOSE: Appropriate storage of fecal samples is a critical step for the unbiased analysis of microbial communities in metagenomic studies. Rapid freezing at -80 °C is usually considered to be best practice, but this approach is challenging. DNA stabilizing kits may provide a more convenient method to preserve and store clinical samples. We evaluated the reliability of two collection kits (Stratec stool collection tube with stabilizer, #1038111200 and OMNIgene.GUT OMR-200) on preserving fecal microbiota. METHODS: Samples were collected from two locations of the fecal specimen, in four healthy volunteers. The samples were sub-aliquoted and stored in a -80 °C freezer, in Stratec and OMNIgene.GUT (incubation at ambient temperature for 0, 3, or 7 days). The fecal microbial composition was assessed by 16S rRNA sequencing. RESULTS: We found that alpha diversity was not significantly affected by storage conditions. Samples stored in DNA stabilizers were still representative of the original microbial community after 7 days at ambient temperature. Individual differences were found to have a greater contribution to the differences in microbial community composition than storage conditions or sampling location. Samples subjected to stabilizers displayed microbial community shifts compared with immediately frozen samples. A linear discriminant analysis effect size (LEfSe) analysis showed that the relative abundances of Faecalibacterium were significantly higher in samples stored in Stratec kits. CONCLUSION: Our study reveals that both Stratec and OMNIgene.GUT kits provide good microbiome preservation for up to 7 days in ambient temperature and would represent good options for fecal sample collection in large scale, population-based studies.

Microbiota-Associated Therapy for Non-Alcoholic Steatohepatitis-Induced Liver Cancer: A Review.

Even though advancement in medicine has contributed to the control of many diseases to date, cancer therapy continues to pose several challenges. Hepatocellular carcinoma (HCC) etiology is multifactorial. Recently, non-alcoholic fatty liver disease (NAFLD) has been considered as an important risk factor of HCC. NAFLD can be divided into non-alcoholic simple fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) based on histopathological features. Recently, studies have indicated that the gut microbiota is associated with NAFLD and HCC. Therefore, in this review, we have discussed the effects of gut microbiota-related mechanisms, including dysbiosis and gut barrier function, and gut microbiota-derived metabolites on NAFLD and HCC pathogenesis and the potential therapeutic strategies for NAFLD and HCC. With a better understanding of the gut microbiota composition and function, new and improved diagnostic, prognostic, and therapeutic strategies for common liver diseases can be developed.

Mutual Interplay of Host Immune System and Gut Microbiota in the Immunopathology of Atherosclerosis.

Inflammation is the key for the initiation and progression of atherosclerosis. Accumulating evidence has revealed that an altered gut microbiome (dysbiosis) triggers both local and systemic inflammation to cause chronic inflammatory diseases, including atherosclerosis. There have been some microbiome-relevant pro-inflammatory mechanisms proposed to link the relationships between dysbiosis and atherosclerosis such as gut permeability disruption, trigger of innate immunity from lipopolysaccharide (LPS), and generation of proatherogenic metabolites, such as trimethylamine N-oxide (TMAO). Meanwhile, immune responses, such as inflammasome activation and cytokine production, could reshape both composition and function of the microbiota. In fact, the immune system delicately modulates the interplay between microbiota and atherogenesis. Recent clinical trials have suggested the potential of immunomodulation as a treatment strategy of atherosclerosis. Here in this review, we present current knowledge regarding to the roles of microbiota in contributing atherosclerotic pathogenesis and highlight translational perspectives by discussing the mutual interplay between microbiota and immune system on atherogenesis.

Sialylated Immunoglobulins for the Treatment of Immuno-Inflammatory Diseases.

Immunoglobulins are the potent effector proteins of the humoral immune response. In the course of evolution, immunoglobulins have formed extremely diverse types of molecular structures with antigen-recognizing, antigen-binding, and effector functions embedded in a single molecule. Polysaccharide moiety of immunoglobulins plays the essential role in immunoglobulin functioning. There is growing evidence that the carbohydrate composition of immunoglobulin-linked glycans, and especially their terminal sialic acid residues, provide a key effect on the effector functions of immunoglobulins. Possibly, sialylation of Fc glycan is a common mechanism of IgG anti-inflammatory action in vivo. Thus, the post-translational modification (glycosylation) of immunoglobulins opens up significant possibilities in the diagnosis of both immunological and inflammatory disorders and in their therapies. This review is focused on the analysis of glycosylation of immunoglobulins, which can be a promising addition to improve existing strategies for the diagnosis and treatment of various immuno-inflammatory diseases.

Signaling Pathways Potentially Responsible for Foam Cell Formation: Cholesterol Accumulation or Inflammatory Response-What is First?

Accumulation of lipid-laden (foam) cells in the arterial wall is known to be the earliest step in the pathogenesis of atherosclerosis. There is almost no doubt that atherogenic modified low-density lipoproteins (LDL) are the main sources of accumulating lipids in foam cells. Atherogenic modified LDL are taken up by arterial cells, such as macrophages, pericytes, and smooth muscle cells in an unregulated manner bypassing the LDL receptor. The present study was conducted to reveal possible common mechanisms in the interaction of macrophages with associates of modified LDL and non-lipid latex particles of a similar size. To determine regulatory pathways that are potentially responsible for cholesterol accumulation in human macrophages after the exposure to naturally occurring atherogenic or artificially modified LDL, we used transcriptome analysis. Previous studies of our group demonstrated that any type of LDL modification facilitates the self-association of lipoprotein particles. The size of such self-associates hinders their interaction with a specific LDL receptor. As a result, self-associates are taken up by nonspecific phagocytosis bypassing the LDL receptor. That is why we used latex beads as a stimulator of macrophage phagocytotic activity. We revealed at least 12 signaling pathways that were regulated by the interaction of macrophages with the multiple-modified atherogenic naturally occurring LDL and with latex beads in a similar manner. Therefore, modified LDL was shown to stimulate phagocytosis through the upregulation of certain genes. We have identified at least three genes (F2RL1, EIF2AK3, and IL15) encoding inflammatory molecules and associated with signaling pathways that were upregulated in response to the interaction of modified LDL with macrophages. Knockdown of two of these genes, EIF2AK3 and IL15, completely suppressed cholesterol accumulation in macrophages. Correspondingly, the upregulation of EIF2AK3 and IL15 promoted cholesterol accumulation. These data confirmed our hypothesis of the following chain of events in atherosclerosis: LDL particles undergo atherogenic modification; this is accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. This chain of events may explain the relationship between cholesterol accumulation and inflammation. The primary sequence of events in this chain is related to inflammatory response rather than cholesterol accumulation.

Role of Phagocytosis in the Pro-Inflammatory Response in LDL-Induced Foam Cell Formation; a Transcriptome Analysis.

Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell formation) caused by modified low-density lipoprotein (LDL) is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. Recently, we have identified the top 10 master regulators that were involved in the accumulation of cholesterol in cultured macrophages induced by the incubation with modified LDL. It was found that most of the identified master regulators were related to the regulation of the inflammatory immune response, but not to lipid metabolism. A possible explanation for this unexpected result is a stimulation of the phagocytic activity of macrophages by modified LDL particle associates that have a relatively large size. In the current study, we investigated gene regulation in macrophages using transcriptome analysis to test the hypothesis that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. We identified genes that were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads (inert phagocytosis stimulators). Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. The obtained results indicated that pro-inflammatory response to phagocytosis stimulation precedes the accumulation of intracellular lipids and possibly contributes to the formation of foam cells. In this way, the currently recognized hypothesis that the accumulation of lipids triggers the pro-inflammatory response was not confirmed. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. Additionally, the knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked down these genes in primary macrophages derived from human monocytes. The addition of atherogenic naturally occurring LDL caused a significant accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages. The knock-down of the ANXA1 gene caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to explain in which way the inflammatory response and the accumulation of cholesterol are related confirming our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response.

Identification of TMAO-producer phenotype and host-diet-gut dysbiosis by carnitine challenge test in human and germ-free mice.

OBJECTIVE: The gut microbiota-derived metabolite, trimethylamine N-oxide (TMAO) plays an important role in cardiovascular disease (CVD). The fasting plasma TMAO was shown as a prognostic indicator of CVD incident in patients and raised the interest of intervention targeting gut microbiota. Here we develop a clinically applicable method called oral carnitine challenge test (OCCT) for TMAO-related therapeutic drug efforts assessment and personalising dietary guidance. DESIGN: A pharmacokinetic study was performed to verify the design of OCCT protocol. The OCCT was conducted in 23 vegetarians and 34 omnivores to validate gut microbiota TMAO production capacity. The OCCT survey was integrated with gut microbiome, host genotypes, dietary records and serum biochemistry. A humanised gnotobiotic mice study was performed for translational validation. RESULTS: The OCCT showed better efficacy than fasting plasma TMAO to identify TMAO producer phenotype. The omnivores exhibited a 10-fold higher OR to be high TMAO producer than vegetarians. The TMAO-associated taxa found by OCCT in this study were consistent with previous animal studies. The TMAO producer phenotypes were also reproduced in humanised gnotobiotic mice model. Besides, we found the faecal CntA gene was not associated with TMAO production; therefore, other key relevant microbial genes might be involved. Finally, we demonstrated the urine TMAO exhibited a strong positive correlation with plasma TMAO (r=0.92, p<0.0001) and improved the feasibility of OCCT. CONCLUSION: The OCCT can be used to identify TMAO-producer phenotype of gut microbiota and may serve as a personal guidance in CVD prevention and treatment. TRIAL REGISTRATION NUMBER: NCT02838732; Results.

Evaluation and Optimization of Sample Handling Methods for Quantification of Short-Chain Fatty Acids in Human Fecal Samples by GC-MS.

The gut microbiota has attracted a great deal of interest in recent years due to its association with many diseases. Short-chain fatty acids (SCFAs), the end products of dietary fiber fermentation by the intestinal microbiota, are among the most frequently discussed gut metabolites. As the sample handling method greatly affects the integrity of data, this study investigated the most important parameters that affect the bias of SCFA comparisons in human fecal studies. An accurate gas chromatography-mass spectrometry (GC-MS) method was first established and validated for quantifying six SCFAs, including acetic, propionic, butyric, isobutyric, isovaleric, and valeric acids. To remove interfering species, we used butanol to extract SCFAs from acidified fecal suspensions. The validated quantification method was then applied to evaluate fecal sample handling protocols. We found that lyophilization of fecal samples can not only minimize bias due to the water content but also provide better stability of SCFAs. Six SCFAs were stable and that their recoveries were higher than 90% after lyophilization. Lyophilization of a large fecal sample is extremely time-consuming, and 1 g of fecal sample is suggested for lyophilization to minimize sampling bias. The interindividual difference was significantly higher than the intra-individual difference when using 1 g of fecal sample to study SCFAs. Finally, an effective protocol from sample collection to GC-MS analysis was proposed. As SCFAs have been shown to play an important role in health maintenance and disease development, the proposed protocol is anticipated to be applicable to clinical studies to delineate the biological functions of each SCFA.

Optimization of fecal sample processing for microbiome study - The journey from bathroom to bench.

Although great interest has been displayed by researchers in the contribution of gut microbiota to human health, there is still no standard protocol with consensus to guarantee the sample quality of metagenomic analysis. Here we reviewed existing methodology studies and present suggestions for optimizing research pipeline from fecal sample collection to DNA extraction. First, we discuss strategies of clinical metadata collection as common confounders for microbiome research. Second, we propose general principles for freshly collected fecal sample and its storage and share a DIY stool collection kit protocol based on the manual procedure of Human Microbiome Project (HMP). Third, we provide a useful information of collection kit with DNA stabilization buffers and compare their pros and cons for multi-omic study. Fourth, we offer technical strategies as well as information of novel tools for sample aliquoting before long-term storage. Fifth, we discuss the substantial impact of different DNA extraction protocols on technical variations of metagenomic analysis. And lastly, we point out the limitation of current methods and the unmet needs for better quality control of metagenomic analysis. We hope the information provided here will help investigators in this exciting field to advance their studies while avoiding experimental artifacts.

Healthy diet intervention reverses the progression of NASH through gut microbiota modulation.

IMPORTANCE: The link between gut microbiota and diet is crucial in the development of non-alcoholic steatohepatitis (NASH). This study underscores the essential role of a healthy diet in preventing and treating NASH by reversing obesity, lipidemia, and gut microbiota dysbiosis. Moreover, the supplementation of functional food or drug to the diet can provide additional advantages by inhibiting hepatic inflammation through the modulation of the hepatic inflammasome signaling pathway and partially mediating the gut microbiota and lipopolysaccharide signaling pathway. This study highlights the importance of adopting healthy dietary habits in treating NASH and proposes that supplementing with ginger essential oil or obeticholic acid may offer additional benefits. Nonetheless, further clinical studies are necessary to validate these findings.

Safety assessment of a proprietary fermented soybean solution, Symbiota®, as an ingredient for use in foods and dietary supplements: Non-clinical studies and a randomized trial.

A safety evaluation was performed of Symbiota®, which is made by a proprietary anaerobic fermentation process of soybean with multistrains of probiotics and a yeast. The battery of genotoxicity studies showed that Symbiota® has no genotoxic effects. Safety and tolerability were further assessed by acute or repeated dose 28- and 90-day rodent studies, and no alterations in clinical observations, ophthalmological examination, blood chemistry, urinalysis, or hematology were observed between the control group and the different dosing groups (1.5, 5, and 15 mL/kg/day). There were no adverse effects on specific tissues or organs in terms of weight and histopathology. Importantly, the Symbiota® treatment did not perturb hormones and other endocrine-related endpoints. Of note, the No-Observed-Adverse-Effect-Level was determined to be 15 mL/kg/day in rats. Moreover, a randomized, double-blind, placebo-controlled clinical trial was recently conducted with healthy volunteers who consumed 8 mL/day of placebo or Symbiota® for 8 weeks. Only mild adverse events were reported in both groups, and the blood chemistry and blood cell profiles were also similar between the two groups. In summary, this study concluded that the oral consumption of Symbiota® at 8 mL/day by the general population does not pose any human health concerns.

Bifidobacterium alleviate metabolic disorders via converting methionine to 5'-methylthioadenosine.

Dietary patterns and corresponding gut microbiota profiles are associated with various health conditions. A diet rich in polyphenols, primarily plant-based, has been shown to promote the growth of probiotic bacteria in the gastrointestinal tract, subsequently reducing the risk of metabolic disorders in the host. The beneficial effects of these bacteria are largely due to the specific metabolites they produce, such as short-chain fatty acids and membrane proteins. In this study, we employed a metabolomics-guided bioactive metabolite identification platform that included bioactivity testing using in vitro and in vivo assays to discover a bioactive metabolite produced from probiotic bacteria. Through this approach, we identified 5'-methylthioadenosine (MTA) as a probiotic bacterial-derived metabolite with anti-obesity properties. Furthermore, our findings indicate that MTA administration has several regulatory impacts on liver functions, including modulating fatty acid synthesis and glucose metabolism. The present study elucidates the intricate interplay between dietary habits, gut microbiota, and their resultant metabolites.

Impact of Esophageal Motility on Microbiome Alterations in Symptomatic Gastroesophageal Reflux Disease Patients With Negative Endoscopy: Exploring the Role of Ineffective Esophageal Motility and Contraction Reserve.

Background/Aims: Ineffective esophageal motility (IEM) is common in patients with gastroesophageal reflux disease (GERD) and can be associated with poor esophageal contraction reserve on multiple rapid swallows. Alterations in the esophageal microbiome have been reported in GERD, but the relationship to presence or absence of contraction reserve in IEM patients has not been evaluated. We aim to investigate whether contraction reserve influences esophageal microbiome alterations in patients with GERD and IEM. Methods: We prospectively enrolled GERD patients with normal endoscopy and evaluated esophageal motility and contraction reserve with multiple rapid swallows during high-resolution manometry. The esophageal mucosa was biopsied for DNA extraction and 16S ribosomal RNA gene V3-V4 (Illumina)/full-length (Pacbio) amplicon sequencing analysis. Results: Among the 56 recruited patients, 20 had normal motility (NM), 19 had IEM with contraction reserve (IEM-R), and 17 had IEM without contraction reserve (IEM-NR). Esophageal microbiome analysis showed a significant decrease in microbial richness in patients with IEM-NR when compared to NM. The beta diversity revealed different microbiome profiles between patients with NM or IEM-R and IEM-NR (P = 0.037). Several esophageal bacterial taxa were characteristic in patients with IEM-NR, including reduced Prevotella spp. and Veillonella dispar, and enriched Fusobacterium nucleatum. In a microbiome-based random forest model for predicting IEM-NR, an area under the receiver operating characteristic curve of 0.81 was yielded. Conclusions: In symptomatic GERD patients with normal endoscopic findings, the esophageal microbiome differs based on contraction reserve among IEM. Absent contraction reserve appears to alter the physiology and microbiota of the esophagus.