Blood Serum and Drainage Microbial and Mitochondrial Metabolites in Patients after Surgery for Pancreatic Cancer.

Pancreatic cancer (PC) has the highest mortality rate of all major cancers in the world despite improvements in clinical care and an understanding of the biology of pancreatic cancer. A study of 64 patients with verified pancreatic cancer who underwent surgery was included. Sampling was carried out at three points: before surgery and on days 1-3 after surgery and 5-7 days after surgery. Drainage fluid collection was taken from the drains installed intraoperatively one day after surgery. Tyrosine and phenylalanine metabolites and two mitochondrial metabolites, namely succinic and fumaric acids, were identified and quantified by GC-MS in the serum of healthy donors and patients. Differences in the metabolomic profile were found between the patients and healthy people. A statistically significant decrease in the level of p-hydroxyphenyllactic acid (p-HPhLA), the amount of sum 3 sepsis-associated metabolites (Σ 3AMM), as well as fumaric and succinic acids in patients was observed. It was also noted that p-hydroxyphenyllactic acid in the preoperative period may be considered as a predictor of complications and longer postoperative recovery.

Influence of Microbiota-Related Metabolites Associated with Inflammation and Sepsis on the Peroxidase Activity of Cyclooxygenase in Healthy Human Monocytes and Acute Monocytic Leukemia Cells.

The human microbiota produces metabolites that can enter the bloodstream and exert systemic effects on various functions in both healthy and pathological states. We have studied the participation of microbiota-related metabolites in bacterial infection by examining their influence on the activity of cyclooxygenase (COX) as a key enzyme of inflammation. The influence of aromatic microbial metabolites, derivatives of phenylalanine (phenylpropionic acid, PPA), tyrosine (4-hydroxyphenyllactic acid, HPLA), and tryptophan (indolacetic acids, IAA), the concentrations of which in the blood change notably during sepsis, was evaluated. Also, the effect of itaconic acid (ITA) was studied, which is formed in macrophages under the action of bacterial lipopolysaccharides (LPS) and appears in the blood in the early stages of infection. Metabiotic acetyl phosphate (AcP) as a strong acetylating agent was also tested. The activity of COX was measured via the TMPD oxidation colorimetric assay using the commercial pure enzyme, cultured healthy monocytes, and the human acute monocytic leukemia cell line THP-1. All metabolites in the concentration range of 100-500 µM lowered the activity of COX. The most pronounced inhibition was observed on the commercial pure enzyme, reaching up to 40% in the presence of AcP and 20-30% in the presence of the other metabolites. On cell lysates, the effect of metabolites was preserved, although it significantly decreased, probably due to their interaction with other targets subject to redox-dependent and acetylation processes. The possible contribution of the redox-dependent action of microbial metabolites was confirmed by assessing the activity of the enzyme in the presence of thiol reagents and in model conditions, when the COX-formed peroxy intermediate was replaced with tert-butyl hydroperoxide (TBH). The data show the involvement of the microbial metabolites in the regulation of COX activity, probably due to their influence on the peroxidase activity of the enzyme.

Analysis of 4-Hydroxyphenyllactic Acid and Other Diagnostically Important Metabolites of α-Amino Acids in Human Blood Serum Using a Validated and Sensitive Ultra-High-Pressure Liquid Chromatography-Tandem Mass Spectrometry Method.

The profile of and dynamic concentration changes in tyrosine, phenylalanine, and tryptophan metabolites in blood are of great interest since they could be considered potential biomarkers of different disorders. Some aromatic metabolites, such as 4-hydroxyphenyllactic, 4-hydroxyphenylacetic, phenyllactic, and 4-hydroxybenzoic acids have previously demonstrated their diagnostic significance in critically ill patients and patients with post-COVID-19 syndrome. In this study, a sensitive method, including serum protein precipitation with methanol and ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detection, was developed and validated for six phenyl- and five indole-containing acids in human serum. The liquid-liquid extraction was also examined, but it demonstrated unsatisfactory results based on analyte recoveries and the matrix effect. However, the recoveries for all analytes reached 100% and matrix effects were not observed using protein precipitation. This made it possible to use deionized water as a blank matrix. The lower limits of quantitation (LLOQs) were from 0.02 to 0.25 µmol/L. The validated method was used for the analysis of serum samples of healthy volunteers (n = 48) to reveal the reference values of the target analytes. The concentrations of the most clinically significant metabolite 4-hydroxyphenyllactic acid, which were revealed using UPLC-MS/MS and a previously developed gas chromatography-mass spectrometry method, were completely comparable. The proposed UPLC-MS/MS protocol can be used in the routine clinical practice of medical centers.

Promising Markers of Inflammatory and Gut Dysbiosis in Patients with Post-COVID-19 Syndrome.

Post-COVID-19 syndrome is a complex of different symptoms, which results in a multisystemic impairment after the suffering from COVID-19 infection. The aim of the study was to reveal the clinical, laboratory, and gut disorders in patients with post-COVID-19 syndrome (n = 39) before and after taking part in the 14-day complex program of rehabilitation. A complete blood count, coagulation test, blood chemistry, biomarkers, and metabolites in serum samples, and gut dysbiosis were revealed in patients on the day of admission and after 14-day rehabilitation, in comparison with the variables of healthy volunteers (n = 48) or with reference ranges. On the day of discharge, patients noted an improvement in respiratory function, general well-being, and mood. At the same time, the levels of some metabolic (4-hydroxybenzoic, succinic, fumaric acids) and inflammatory (interleukin-6) variables, which were increased on admission, did not reach the level of healthy people during the rehabilitation program. Taxonomy disbalance was observed in patients' feces, namely, a high level of total bacterial mass, a decrease in the number of Lactobacillus spp., and an increase in pro-inflammatory microorganisms. The authors suggest that the post-COVID-19 rehabilitation program should be personalized, considering the patient's state together with not only the baseline levels of biomarkers, but also with the individual taxonomy of the gut microbiota.

Microbiota Metabolism Failure as a Risk Factor for Postoperative Complications after Aortic Prosthetics.

Postoperative complications in cardiovascular surgery remain an important unresolved problem, in particular in patients with aortic aneurysm. The role of the altered microbiota in such patients is of great interest. The aim of this pilot study was to determine whether the development of postoperative complications in patients with aortic aneurysm is related with initial or acquired disorders of microbiota metabolism by monitoring the level of some aromatic microbial metabolites (AMMs) circulating in the blood before the surgery and in the early postoperative period. The study comprised patients with aortic aneurysm (n = 79), including patients without complications (n = 36) and patients with all types of complications (n = 43). The serum samples from the patients were collected before and 6 h after the end of the surgery. The most significant results were obtained for the sum of three sepsis-associated AMMs. This level was higher before the surgery in comparison with that of healthy volunteers (n = 48), p < 0.001, and it was also higher in the early postoperative period in patients with all types of complications compared to those without complications, p = 0.001; the area under the ROC curve, the cut-off value, and the odds ratio were 0.7; 2.9 µmol/L, and 5.5, respectively. Impaired microbiota metabolism is important in the development of complications after complex reconstructive aortic surgery, which is the basis for the search for a new prevention strategy.

Bacteriophage Cocktails in the Post-COVID Rehabilitation.

Increasing evidence suggests that gut dysbiosis is associated with coronavirus disease 2019 (COVID-19) infection and may persist long after disease resolution. The excessive use of antimicrobials in patients with COVID-19 can lead to additional destruction of the microbiota, as well as to the growth and spread of antimicrobial resistance. The problem of bacterial resistance to antibiotics encourages the search for alternative methods of limiting bacterial growth and restoring the normal balance of the microbiota in the human body. Bacteriophages are promising candidates as potential regulators of the microbiota. In the present study, two complex phage cocktails targeting multiple bacterial species were used in the rehabilitation of thirty patients after COVID-19, and the effectiveness of the bacteriophages against the clinical strain of Klebsiella pneumoniae was evaluated for the first time using real-time visualization on a 3D Cell Explorer microscope. Application of phage cocktails for two weeks showed safety and the absence of adverse effects. An almost threefold statistically significant decrease in the anaerobic imbalance ratio, together with an erythrocyte sedimentation rate (ESR), was detected. This work will serve as a starting point for a broader and more detailed study of the use of phages and their effects on the microbiome.

Influence of Microbial Metabolites and Itaconic Acid Involved in Bacterial Inflammation on the Activity of Mitochondrial Enzymes and the Protective Role of Alkalization.

Human microbiota produces metabolites that may enter the bloodstream and exert systemic influence on various functions including mitochondrial. Mitochondria are not only a target for microbial metabolites, but also themselves, due to the inhibition of several enzymes, produce metabolites involved in infectious processes and immune response. The influence of indolic acids, microbial derivatives of tryptophan, as well as itaconic acid, formed in the tricarboxylic acid cycle under the action of bacterial lipopolysaccharides, on the activity of mitochondrial enzymes was studied by methyl thiazolyl tetrazolium (MTT), dichlorophenolindophenol (DCPIP) and pyridine nucleotide fluorescence assays. Thus, it was found that indolic acids suppressed succinate and glutamate oxidation, shifting the redox potential of pyridine nucleotides to a more oxidized state. Itaconic acid, in addition to the well-known inhibition of succinate oxidation, also decreased NAD reduction in reactions with glutamate as a substrate. Unlike itaconic acid, indolic acids are not direct inhibitors of succinate dehydrogenase and glutamate dehydrogenase as their effects could be partially eliminated by the thiol antioxidant dithiothreitol (DTT) and the scavenger of lipid radicals butyl-hydroxytoluene (BHT). Alkalization turned out to be the most effective means to decrease the action of these metabolites, including itaconic acid, which is due to the protective influence on redox-dependent processes. Thus, among mitochondrial oxidative enzymes, the most accessible targets of these microbial-related metabolites are succinate dehydrogenase and glutamate dehydrogenase. These are important in the context of the shifting of metabolic pathways involved in bacterial inflammation and sepsis as well as the detection of new markers of these pathologies.

Multivariate Prognostic Model for Predicting the Outcome of Critically Ill Patients Using the Aromatic Metabolites Detected by Gas Chromatography-Mass Spectrometry.

A number of aromatic metabolites of tyrosine and phenylalanine have been investigated as new perspective markers of infectious complications in the critically ill patients of intensive care units (ICUs). The goal of our research was to build a multivariate model for predicting the outcome of critically ill patients regardless of the main pathology on the day of admission to the ICU. Eight aromatic metabolites were detected in serum using gas chromatography-mass spectrometry. The samples were obtained from the critically ill patients (n = 79), including survivors (n = 44) and non-survivors (n = 35), and healthy volunteers (n = 52). The concentrations of aromatic metabolites were statistically different in the critically ill patients and healthy volunteers. A univariate model for predicting the outcome of the critically ill patients was based on 3-(4-hydroxyphenyl)lactic acid (p-HPhLA). Two multivariate classification models were built based on aromatic metabolites using SIMCA method. The predictive models were compared with the clinical APACHE II scale using ROC analysis. For all of the predictive models the areas under the ROC curve were close to one. The aromatic metabolites (one or a number of them) can be used in clinical practice for the prognosis of the outcome of critically ill patients on the day of admission to the ICU.

4-Hydroxyphenyllactic Acid in Cerebrospinal Fluid as a Possible Marker of Post-Neurosurgical Meningitis: Retrospective Study.

The search for new potential biomarkers for the diagnostics of post-neurosurgical bacterial meningitis is required because of the difficulties in its early verification using results of the routine laboratory and biochemical analyses of the cerebrospinal fluid (CSF). The goal of the study was to determine the contents of the aromatic metabolites and biomarkers in the CSF samples of the post-neurosurgical patients (n = 82) and their potential diagnostical significance for the evaluation of the risk of post-neurosurgical meningitis. Patients with signs of post-neurosurgical meningitis (n = 30) had lower median values of glucose and higher values of cell count, neutrophils, lactate, protein, 3-(4-hydroxyphenyl)lactic acid (p-HPhLA), and interleukin-6 (IL-6) than patients without signs of post-neurosurgical meningitis (n = 52). ROC analysis for IL-6 and p-HPhLA resulted in 0.785 and 0.734 values of the area under the ROC curve, with sensitivity 96.30 and 66.67%; specificity 54.17 and 82.69%, respectively. IL-6 should be considered as a non-specific biomarker, in contrast to the microbial metabolite p-HPhLA. If the concentration of p-HPhLA was more or equal to 0.9 µmol/L, the risk of bacterial complications was 9.6 times higher. p-HPhLA is a promising marker for the prognosis of post-neurosurgical meningitis, and its determination on a larger group of post-neurosurgical patients can subsequently prove its diagnostic significance for the verification of CNS infections.

Gut Microbiota as Early Predictor of Infectious Complications before Cardiac Surgery: A Prospective Pilot Study.

Cardiac surgery remains a field of medicine with a high percentage of postoperative complications, including infectious ones. Modern data indicate a close relationship of infectious disorders with pathological changes in the composition of the gut microbiome; however, the extent of such changes in cardiac surgery patients is not fully clarified. In this prospective, observational, single center, pilot study, 72 patients were included, 12 among them with the infectious complications. We analyzed the features of the fecal microbiota before and in the early postoperative period, as one of the markers for predicting the occurrence of bacterial infection. We also discovered the significant change in microbial composition in the group of patients with infectious complications compared to the non-infectious group before and after cardiac surgery, despite the intra-individual variation in composition of gut microbiome. Our study demonstrated that the group of patients that had a bacterial infection in the early postoperative period already had an altered microbial composition even before the surgery. Further studies will evaluate the clinical significance of the identified proportions of individual taxa of the intestinal microbiota and consider the microbiota as a novel target for reducing the risk of infectious complications.

Influence of Microbial Metabolites on the Nonspecific Permeability of Mitochondrial Membranes under Conditions of Acidosis and Loading with Calcium and Iron Ions.

Mitochondrial dysfunction is currently considered one of the main causes of multiple organ failure in chronic inflammation and sepsis. The participation of microbial metabolites in disorders of bioenergetic processes in mitochondria has been revealed, but their influence on the mitochondrial membrane permeability has not yet been studied. We tested the influence of various groups of microbial metabolites, including indolic and phenolic acids, trimethylamine-N-oxide (TMAO) and acetyl phosphate (AcP), on the nonspecific permeability of mitochondrial membranes under conditions of acidosis, imbalance of calcium ions and excess free iron, which are inherent in sepsis. Changes in the parameters of the calcium-induced opening of the mitochondrial permeability transition pore (MPTP) and iron-activated swelling of rat liver mitochondria were evaluated. The most active metabolites were indole-3-carboxylic acid (ICA) and benzoic acid (BA), which activated MPTP opening and swelling under all conditions. AcP showed the opposite effect on the induction of MPTP opening, increasing the threshold concentration of calcium by 1.5 times, while TMAO activated swelling only under acidification. All the redox-dependent effects of metabolites were suppressed by the lipid radical scavenger butyl-hydroxytoluene (BHT), which indicates the participation of these microbial metabolites in the activation of membrane lipid peroxidation. Thus, microbial metabolites can directly affect the nonspecific permeability of mitochondrial membranes, if conditions of acidosis, an imbalance of calcium ions and an excess of free iron are created in the pathological state.

Applicability of modified carbon sorbent for removing potentially toxic biologically active molecules of aromatic structure from blood plasma.

The modification of the mesoporous carbon sorbent with 3-phenylpropanoic acid was carried out in order to create preparations of complex, prolonged action, exhibiting detoxifying, antibacterial, and antifungal properties due to the applied modifier, which is capable of migrating into the solution and exhibiting its own biospecific properties. A technique was developed for fixing 3-phenylpropionic acid (PhPA) on a carbon support by its adsorption from solution. Three types of sorbents with various content of the modifier (PhPA) and the sorbent without modifier were studied. The sorption activity of new sorbents was studied using liquid-liquid extraction and gas chromatography-mass spectrometry methods on model experiments with plasma and aqueous additives of hydroxylated phenyl-containing acids (PhCAs) in various concentrations. The specific surface area was significantly changed for sorbent, modified with 1 × 10-3 mol/L of PhPA solution, and was 25% less than the area of unmodified sorbent. Potentially toxic biologically active hydroxylated PhCAs were used to create model solutions. The degrees of sorption of these compounds were close to 100%, except phenyllactic acid (over 80%). The sorbent without modifier and two sorbents with the lowest content of the modifier are considered to be more effective for the purification of the plasma from the hydroxylated PhCAs than the sorbent with the highest concentration of the modifier. Simultaneous adsorption of toxic metabolites from the bloodstream and desorption of beneficial ones can be used for a more subtle correction of the patient's condition.

Host-Microbiome Interactions Mediated by Phenolic Metabolites in Chronically Critically Ill Patients.

The community structure and metabolic potential of gut microbiome is not well investigated, especially in chronically critically ill patients with prolonged dependence on support systems after severe brain disorders. Microbial phenolic metabolites can target the brain function by the direct and indirect modulation of inflammation. The aim of this study was to investigate the features of the gut microbiota and profile of certain metabolites in the progression and reversibility of neurological disorders in chronically critically ill patients. Fecal samples were collected in dynamics from such patients (n = 44) and analyzed using 16S rRNA sequencing. Serum microbial and mitochondrial metabolites were measured by GC-MS and compared with the biomarkers and clinical neurological scores. The identified associations between specific bacterial taxa in fecal samples, neurological status and serum levels of metabolites suggest that impacts on specific members of the gut microbiota and their metabolism might be a promising tool for regulating brain function in future.

Hi-C Metagenomics in the ICU: Exploring Clinically Relevant Features of Gut Microbiome in Chronically Critically Ill Patients.

Gut microbiome in critically ill patients shows profound dysbiosis. The most vulnerable is the subgroup of chronically critically ill (CCI) patients - those suffering from long-term dependence on support systems in intensive care units. It is important to investigate their microbiome as a potential reservoir of opportunistic taxa causing co-infections and a morbidity factor. We explored dynamics of microbiome composition in the CCI patients by combining "shotgun" metagenomics with chromosome conformation capture (Hi-C). Stool samples were collected at 2 time points from 2 patients with severe brain injury with different outcomes within a 1-2-week interval. The metagenome-assembled genomes (MAGs) were reconstructed based on the Hi-C data using a novel hicSPAdes method (along with the bin3c method for comparison), as well as independently of the Hi-C using MetaBAT2. The resistomes of the samples were derived using a novel assembly graph-based approach. Links of bacteria to antibiotic resistance genes, plasmids and viruses were analyzed using Hi-C-based networks. The gut community structure was enriched in opportunistic microorganisms. The binning using hicSPAdes was superior to the conventional WGS-based binning as well as to the bin3c in terms of the number, completeness and contamination of the reconstructed MAGs. Using Klebsiella pneumoniae as an example, we showed how chromosome conformation capture can aid comparative genomic analysis of clinically important pathogens. Diverse associations of resistome with antimicrobial therapy from the level of assembly graphs to gene content were discovered. Analysis of Hi-C networks suggested multiple "host-plasmid" and "host-phage" links. Hi-C metagenomics is a promising technique for investigating clinical microbiome samples. It provides a community composition profile with increased details on bacterial gene content and mobile genetic elements compared to conventional metagenomics. The ability of Hi-C binning to encompass the MAG's plasmid content facilitates metagenomic evaluation of virulence and drug resistance dynamics in clinically relevant opportunistic pathogens. These findings will help to identify the targets for developing cost-effective and rapid tests for assessing microbiome-related health risks.

Metabolic profiling of aromatic compounds in cerebrospinal fluid of neurosurgical patients using microextraction by packed sorbent and liquid-liquid extraction with gas chromatography-mass spectrometry analysis.

A new approach to the quantitative analysis of aromatic metabolites in cerebrospinal fluid samples of neurosurgical patients based on microextraction by packed sorbent coupled with derivatization and GC-MS was developed. Analytical characteristics such as recoveries (40-90%), limit of detection (0.1-0.3 µm) and limit of quantitation (0.4-0.7 µm) values, accuracy (<±20%), precision (<20%) and linear correlations (R2 ≥ 0.99) over a 0.4-10 µm range of concentrations demonstrated that microextraction by packed sorbent provides results for the quantitative analysis of target compounds comparable with those for liquid-liquid extraction. Similar results were achieved using 40 µl of sample for microextraction by packed sorbent instead of 200 µl for liquid-liquid extraction. Benzoic, 3-phenylpropionic, 3-phenyllactic, 4-hydroxybenzoic, 2-(4-hydroxyphenyl)acetic, homovanillic and 3-(4-hydroxyphenyl)lactic acids were found in cerebrospinal fluid samples (n = 138) of neurosurgical patients in lower concentrations than in serum samples (n = 110) of critically ill patients. Analysis of the cerebrospinal fluid and serum samples taken at the same time from neurosurgical patients (n = 5) revealed similar results for patients without infection and multidirectional results for patients with central nervous system infection. Our preliminary results demonstrate the necessity of further evaluating the aromatic compound profile in cerebrospinal fluid for its subsequent verification for potential diagnostic markers.

Indolic Structure Metabolites as Potential Biomarkers of Non-infectious Diseases.

Interest in indolic structure metabolites, including a number of products of microbial biotransformation of the aromatic amino acid tryptophan, is increasingly growing. The review prepared by a team of authors is based on in-depthscrutiny of data available in PubMed, Scopus, Cyberleninka, Clinical Trials, and Cochrane Library, eventually narrowing the search to a set of keywords such as tryptophan metabolites; plasma metabolomics profiling; metabolomics fingerprinting; gas-, liquid chromatography mass spectrometry; serotonin; melatonin; tryptamine; indoxyl sulfate; indole-3-acetic acid; indole-3-propionic acid; 5-hydroxyindole-3-acetic acid; gut microbiota and microbial metabolites. It provides a summary that outlines the pattern of changes in the level of indolic structure metabolites in a number of diseases and deals with the data from the field of human microbiota metabolites. In modern experimental studies, including the use of gnotobiological (germ-free) animals, it has been convincingly proved that the formation of tryptophan metabolites such as indole-3-acetic acid, indole-3-propionic acid, tryptamine, and indoxyl sulfate is associated with gut bacteria. Attention to some concentration changes of indolic compounds is due to the fact that pronounced deviations and a significant decrease of these metabolites in the blood were found in a number of serious cardiovascular, brain or gastrointestinal diseases. The literature-based analysis allowed the authors to conclude that a constant (normal) level of the main metabolites of the indolic structure in the human body is maintained by a few strict anaerobic bacteria from the gut of a healthy body belonging to the species of Clostridium, Bacteroides, Peptostreptococcus, Eubacteria, etc. The authors focus on several metabolites of the indolic structure that can be called clinically significant in certain diseases, such as schizophrenia, depression, atherosclerosis, colorectal cancer, etc. Determining the level of indole metabolites in the blood can be used to diagnose and monitor the effectiveness of a comprehensive treatment approach.

Determination of Tryptophan Metabolites in Serum and Cerebrospinal Fluid Samples Using Microextraction by Packed Sorbent, Silylation and GC-MS Detection.

Indole-containing acids-tryptophan metabolites-found in serum and cerebrospinal fluid (CSF) samples of patients with diseases of the central nervous system (CNS) were determined with the use of microextraction by packed sorbent (MEPS) followed by silylation and gas chromatography-mass spectrometry (GC-MS) analysis. MEPS with the following silylation led to the reproducible formation of derivatives with an unsubstituted hydrogen ion in the indole ring, the chromatographic peaks of which are symmetric and can be used for GC-MS analysis without additional derivatization. The recoveries of analytes at the limit of quantitation (LOQ) levels were 40-80% for pooled CSF and 40-60% for serum. The limit of detection (LOD) and LOQ values were 0.2-0.4 and 0.4-0.5 µM, respectively, for both CSF and serum. The precision (the reproducibility, RSD) value of less than 20% and the accuracy (the relative error, RE) value of less than ±20% at the LOQ concentrations meet the Food and Drug Administration (FDA) recommendations. Linear correlations for all analytes were determined over a potentially clinically significant range of concentrations (0.4-10 µM for serum, R2 ≥ 0.9942, and 0.4-7 µM for CSF, R2 ≥ 0.9949). Moreover, MEPS significantly reduced the matrix effect of serum compared to liquid-liquid extraction (LLE), which was revealed in the example of reducing the amount of cholesterol and its relative compounds.

Serum and fecal profiles of aromatic microbial metabolites reflect gut microbiota disruption in critically ill patients: a prospective observational pilot study.

BACKGROUND: High serum levels of certain aromatic microbial metabolites (AMM) are associated with severity and mortality in critically ill patients. Omics-based studies suggest gut dysbiosis and reduced microbiome diversity in critical conditions. However, the landscape of gut microbial metabolites is still to be outlined, not to mention the interplay correlation between the metabolome and gut microbiome in critically ill patients. The aim of this study was to analyze the association between serum and fecal levels of AMM and compare them with the composition of gut microbiota in critically ill patients in the acute and chronic stages. METHODS: In this prospective observational pilot study, we analyzed the temporal dynamics of the gut microbiome and the AMM spectrum across two distinct subgroups-acute critical ill (ACI) patients with nosocomial pneumonia and chronically critically ill (CCI) patients (9 subjects each group)-as well as performed comparison with 23 healthy volunteers. The AMM levels for each patient were measured using GC-MS in simultaneously taken serum and fecal samples (SFS). These parameters were compared with 16S rRNA fecal microbiome profiles. RESULTS: The observed proportions of bacterial taxa suggest a significant gut dysbiosis in the ACI and the CCI patients. Stronger imbalance in microbiome composition and dynamics observed in the ACI patients compared to the CCI ones resonates with a higher severity in the former group. The total levels of AMM in serum samples were higher for the ACI patients than for the CCI patients (3.7 (1.4-6.3) and 1.1 (1.0-1.6) µM, respectively; p = 0.0003). The qualitative composition of the SFS was also altered. We discovered significant associations between gut microbial taxa levels and metabolite concentrations in blood serum as well as in feces in each of the ACI and the CCI patients. CONCLUSIONS: Aromatic microbial metabolite profiles in the gut and the serum are interlinked and reflect a disruption of the gut microbial community in critically ill patients.

Serum Levels of Mitochondrial and Microbial Metabolites Reflect Mitochondrial Dysfunction in Different Stages of Sepsis.

Mechanisms of mitochondrial dysfunction in sepsis are being extensively studied in recent years. During our study, concentrations of microbial phenolic acids and mitochondrial metabolites (succinic, α-ketoglutaric, fumaric, itaconic acids) as indicators of sepsis and mitochondrial dysfunction, respectively, are measured by gas chromatography-mass spectrometry (GC-MS) in the blood of critically ill patients at the early and late stages of documented sepsis. The increase in levels of some phenylcarboxylic (phenyllactic (PhLA), p-hydroxyphenylacetic (p-HPhAA), p-hydroxyphenyllactic (p-HPhAA)) acids (PhCAs), simultaneously with a rise in levels of mitochondrial dicarboxylic acids, are mainly detected during the late stage of sepsis, especially succinic acid (up to 100-1000 µM). Itaconic acid is found in low concentrations (0.5-2.3 µM) only at early-stage sepsis. PhCAs in vitro inhibits succinate dehydrogenase (SDH) in isolated mitochondria but, unlike itaconic acid which acts as a competitive inhibitor of SDH, microbial metabolites most likely act on the ubiquinone binding site of the respiratory chain. A close correlation of the level of succinic acid in serum and sepsis-induced organ dysfunction is revealed, moreover the most significant correlation is observed at high concentrations of phenolic microbial metabolites (PhCAs) in late-stage sepsis. These data indicate the promise of such an approach for early detection, monitoring the progression of organ dysfunction and predicting the risk of non-survival in sepsis.

Metabolomic findings in sepsis as a damage of host-microbial metabolism integration.

Metabolomics globally evaluates the totality of the endogenous metabolites in patient's body, at the same time reflecting gene function, enzyme activity and degree of organ dysfunction in sepsis. The authors performed the analysis of the main chemical classes of low molecular weight compounds (amino acids, polyols, fatty acids, hydroxy acids, amines, nucleotides and their derivatives) that quantitatively distinguish patients with sepsis from healthy ones. The following keywords were used to find papers published in the Scopus and Web of Science databases from 2008 to 2015: (marker OR biomarker) AND (sepsis OR critical ill OR pneumonia OR hypoxia). Key words for the search were the following: metabolomics, metabolic profiling, sepsis, metabolism, biomarkers, critically ill patients, multiple organ failure. Several metabolomic findings in sepsis are still waiting for an explanation. When assessing metabolomic analysis results in patients with sepsis we should take into account the intervention of microbial metabolism. Among the low molecular weight compounds detected in septic patient blood, a special attention should be paid to the molecules which could be attributed to "common metabolites" of man and bacteria. The genomic region overlap and the production of enzymes which are similar in function and final products could be a possible reason for this phenomenon. For example, microbial biodegradation products of aromatic compounds are increased many times in blood of patients with sepsis. On the one hand, it shows a high metabolic activity of the bacteria. On the other hand, these molecules are intermediates in the metabolism of aromatic amino acids such as tyrosine and phenylalanine in human body. It is important that there are many clinical studies, which confirmed the diagnostic and prognostic significance of series of aromatic metabolites, including those with intrinsic biological activity. We can't exclude the presence of signaling pathways, cell receptors, transmembrane transporters and others which are common for a human and bacteria and their direct participation in mechanisms of organ dysfunction and hypotension in sepsis. Thus, today, we should not limit ourselves studying eukaryotic cells while searching for new molecular mechanisms of sepsis-associated organ failure and septic shock. We should take into account and simulate in the experiments the changes of a human internal environment, which occur during the radical microbiome "restructuring" in critically ill patients. This approach opens up new prospects for an objective monitoring of diseases, carrying out an assessment of the integral metabolic profile in a given time on common metabolites (particularly aromatic), and in future will provide new targets for therapeutic effects.