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The lungs, as vital organs in the human body, continuously engage in gas exchange with the external environment. The lung microbiota, a critical component in maintaining internal homeostasis, significantly influences the onset and progression of diseases. Beneficial interactions between the host and its microbial community are essential for preserving the host's health, whereas disease development is often linked to dysbiosis or alterations in the microbial community. Evidence has demonstrated that changes in lung microbiota contribute to the development of major chronic lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, and lung cancer. However, in-depth mechanistic studies are constrained by the small scale of the lung microbiota and its susceptibility to environmental pollutants and other factors, leaving many questions unanswered. This review examines recent research on the lung microbiota and lung diseases, as well as methodological advancements in studying lung microbiota, summarizing the ways in which lung microbiota impacts lung diseases and introducing research methods for investigating lung microbiota.

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An imbalanced microbiome is linked to several diseases, such as cancer, inflammatory bowel disease, obesity, and even neurological disorders. Bacteria and their by-products are used for various industrial and clinical purposes. The metabolites under discussion were chosen based on their biological impacts on host and gut microbiota interactions as established by metabolome research. The separation of bacterial metabolites by using statistics and machine learning analysis creates new opportunities for applications of bacteria and their metabolites in the environmental and medical sciences. Thus, the metabolite production strategies, methodologies, and importance of bacterial metabolites for human well-being are discussed in this review.

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BACKGROUND: Vitamins are essential micronutrients that play key roles in many biological pathways associated with sepsis. The gut microbiome plays a pivotal role in the progression of sepsis and may contribute to the onset of multi-organ dysfunction syndrome (MODS). The aim of this study was to investigate the changes in serum vitamins, and their correlation with intestinal flora and metabolomic profiles in patients with sepsis. METHODS: The serum levels of vitamins were determined by Ultra Performance Liquid Chromatography (UPLC). 16S rRNA gene sequencing and Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) targeted metabolomics were used for microbiome and metabolome analysis. RESULTS: In the training cohort: After univariate, multivariate (OPLS-DA) and Spearman analyses, it was concluded that vitamin levels of 25 (OH) VD3 and (VD2 + VD3), as well as vitamins A and B9, differed significantly among healthy controls (HC), non-septic critical patients (NS), and sepsis patients (SS) (P < 0.05). The validation cohort confirmed the differential vitamin findings from the training cohort. Moreover, analyses of gut flora and metabolites in septic patients and healthy individuals revealed differential flora, metabolites, and metabolic pathways that were linked to alterations in serum vitamin levels. We found for the first time that vitamin B9 was negatively correlated with g_Sellimonas. CONCLUSION: Sepsis patients exhibited significantly lower levels of 25 (OH) VD3 and (VD2 + VD3), vitamins A and B9, which hold potential as predictive markers for sepsis prognosis. The changes in these vitamins may be associated with inflammatory factors, oxidative stress, and changes in gut flora.

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The integrated photocatalysis and fluidized bed biofilm reactor (FBBR) is an attractive wastewater treatment technique for managing wastewater containing antibiotics. However, the fast recombination of photoinduced charge and low microbial activity limit the degradation and mineralization efficiency for antibiotics. To address this, we attempt to introduce magnetic field (MF) to the integrated system with B-doped Bi3O4Cl as the photocatalysts to effectively improve removal and mineralization of ciprofloxacin (CIP). As a consequence, the degradation rate reaches 96% after 40 d in integrated system with MF. The biofilm inside the integrated system with MF carrier can mineralize the photocatalytic products, thereby increasing the total organic carbon (TOC) degradation rate by more than 32%. The electrochemical experiment indicates the Lorentz force generated by MF can accelerate charge separation, increasing the electron concentration. Simultaneously, the increased amounts of electrons lead to the generation of more ·OH and ·O2-. MF addition also results in increased biomass, increased biological respiratory activity, microbial community evolution and accelerated microbial metabolism, enabling more members to biodegrade photocatalytic intermediates. Therefore, applied MF is an efficient method to enhance CIP degradation and mineralization by the integrated system.

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Highly selective and sensitive analytical techniques are necessary for microbial metabolomics due to the complexity of the microbial sample matrix. Hence, mass spectrometry (MS) has been successfully applied in microbial metabolomics due to its high precision, versatility, sensitivity, and wide dynamic range. The different analytical tools using MS have been employed in microbial metabolomics investigations and can contribute to the discovery or accelerate the search for bioactive substances. The coupling with chromatographic and electrophoretic separation techniques has resulted in more efficient technologies for the analysis of microbial compounds occurring in trace levels. This book chapter describes the current advances in the application of mass spectrometry-based metabolomics in the search for new biologically active agents from microbial sources; the development of new approaches for in silico annotation of natural products; the different technologies employing mass spectrometry imaging to deliver more comprehensive analysis and elucidate the metabolome involved in ecological interactions as they enable visualization of the spatial dispersion of small molecules. We also describe other ambient ionization techniques applied to the fingerprint of microbial natural products and modern techniques such as ion mobility mass spectrometry used to microbial metabolomic analyses and the dereplication of natural microbial products through MS.

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Confidently, nuclear magnetic resonance (NMR) is the most informative technique in analytical chemistry and its use as an analytical platform in metabolomics is well proven. This chapter aims to present NMR as a viable tool for microbial metabolomics discussing its fundamental aspects and applications in metabolomics using some chosen examples.

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Microbial metabolomics has gained significant interest as it reflects the physiological state of microorganisms. Due to the great variability of biological organisms, in terms of physicochemical characteristics and variable range of concentration of metabolites, the choice of sample preparation methods is a crucial step in the metabolomics workflow and will reflect on the quality and reliability of the results generated. The procedures applied to the preparation of microbial samples will vary according to the type of microorganism studied, the metabolomics approach (untargeted or targeted), and the analytical platform of choice. This chapter aims to provide an overview of the sample preparation workflow for microbial metabolomics, highlighting the pre-analytical factors associated with cultivation, harvesting, metabolic quenching, and extraction. Discussions focus on obtaining intracellular and extracellular metabolites. Finally, we introduced advanced sample preparation methods based on automated systems.

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Haemophilus influenzae is a gram-negative bacterium of relevant clinical interest. H. influenzae Rd KW20 was the first organism to be sequenced and for which a genome-scale metabolic model (GEM) was developed. However, current H. influenzae GEMs are unable to capture several aspects of metabolome nature related to metabolite pools. To directly and comprehensively characterize the endometabolome of H. influenzae Rd KW20, we performed a multiplatform MS-based metabolomics approach combining LC-MS, GC-MS and CE-MS. We obtained direct evidence of 15-20% of the endometabolome present in current H. influenzae GEMs and showed that polar metabolite pools are interconnected through correlating metabolite islands. Notably, we obtained high-quality evidence of 18 metabolites not previously included in H. influenzae GEMs, including the antimicrobial metabolite cyclo(Leu-Pro). Additionally, we comprehensively characterized and evaluated the quantitative composition of the phospholipidome of H. influenzae, revealing that the fatty acyl chain composition is largely independent of the lipid class, as well as that the probability distribution of phospholipids is mostly related to the conditional probability distribution of individual acyl chains. This finding enabled us to provide a rationale for the observed phospholipid profiles and estimate the abundance of low-level species, permitting the expansion of the phospholipidome characterization through predictive probabilistic modelling.

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Non-alcoholic fatty liver disease (NAFLD) is a multifactorial metabolic disorder that poses health challenges worldwide and is expected to continue to rise dramatically. NAFLD is associated with metabolic syndrome, type 2 diabetes mellitus, and impaired gut health. Increased gut permeability, caused by disturbance of tight junction proteins, allows passage of damaging microbial components that, upon reaching the liver, have been proposed to trigger the release of inflammatory cytokines and generate cellular stress. A growing body of research has suggested the utilization of targeted probiotic supplements as a preventive therapy to improve gut barrier function and tight junctions. Furthermore, specific microbial interactions and metabolites induce the secretion of hormones such as GLP-1, resulting in beneficial effects on liver health. To increase the likelihood of finding beneficial probiotic strains, we set up a novel screening platform consisting of multiple in vitro and ex vivo assays for the screening of 42 bacterial strains. Analysis of transepithelial electrical resistance response via co-incubation of the 42 bacterial strains with human colonic cells (Caco-2) revealed improved barrier integrity. Then, strain-individual metabolome profiling was performed revealing species-specific clusters. GLP-1 secretion assay with intestinal secretin tumor cell line (STC-1) found at least seven of the strains tested capable of enhancing GLP-1 secretion in vitro. Gene expression profiling in human biopsy-derived intestinal organoids was performed using next generation sequencing transcriptomics post bacterial co-incubation. Here, different degrees of immunomodulation by the increase in certain cytokine and chemokine transcripts were found. Treatment of mouse primary hepatocytes with selected highly produced bacterial metabolites revealed that indole metabolites robustly inhibited de novo lipogenesis. Collectively, through our comprehensive bacterial screening pipeline, not previously ascribed strains from both Lactobacillus and Bifidobacterium genera were proposed as potential probiotics based on their ability to increase epithelial barrier integrity and immunity, promote GLP-1 secretion, and produce metabolites relevant to liver health.

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Bacteria can communicate through quorum sensing, allowing them to develop different survival or virulence traits that lead to increased bacterial resistance against conventional antibiotic therapy. Here, fifteen essential oils (EOs) were investigated for their antimicrobial and anti-quorum-sensing activities using Chromobacterium violaceum CV026 as a model. All EOs were isolated from plant material via hydrodistillation and analyzed using GC/MS. In vitro antimicrobial activity was determined using the microdilution technique. Subinhibitory concentrations were used to determine anti-quorum-sensing activity by inhibition of violacein production. Finally, a possible mechanism of action for most bioactive EOs was determined using a metabolomic approach. Among the EOs evaluated, the EO from Lippia origanoides exhibited antimicrobial and anti-quorum activities at 0.37 and 0.15 mg/mL, respectively. Based on the experimental results, the antibiofilm activity of EO can be attributed to the blockage of tryptophan metabolism in the metabolic pathway of violacein synthesis. The metabolomic analyses made it possible to see effects mainly at the levels of tryptophan metabolism, nucleotide biosynthesis, arginine metabolism and vitamin biosynthesis. This allows us to highlight the EO of L. origanoides as a promising candidate for further studies in the design of antimicrobial compounds against bacterial resistance.

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Passengers are at a higher risk of respiratory infections and chronic diseases due to microbial exposure in airline cabins. However, the presence of virulence factors (VFs), antimicrobial resistance genes (ARGs), metabolites, and chemicals are yet to be studied. To address this gap, we collected dust samples from the cabins of two airlines, one with textile seats (TSC) and one with leather seats (LSC), and analyzed the exposure using shotgun metagenomics and LC/MS. Results showed that the abundances of 17 VFs and 11 risk chemicals were significantly higher in TSC than LSC (p < 0.01). The predominant VFs in TSC were related to adherence, biofilm formation, and immune modulation, mainly derived from facultative pathogens such as Haemophilus parainfluenzae and Streptococcus pneumoniae. The predominant risk chemicals in TSC included pesticides/herbicides (carbofuran, bromacil, and propazine) and detergents (triethanolamine, diethanolamine, and diethyl phthalate). The abundances of these VFs and detergents followed the trend of TSC > LSC > school classrooms (p < 0.01), potentially explaining the higher incidence of infectious and chronic inflammatory diseases in aircraft. The level of ARGs in aircraft was similar to that in school environments. This is the first multi-omic survey in commercial aircraft, highlighting that surface material choice is a potential intervention strategy for improving passenger health.

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Microbial metabolomics allows understanding and to comprehensively analyse metabolites, and their related cellular and metabolic processes, that are produced and released to the extracellular environment under specific conditions. In that regard, the main objective of this research is to understand the impact of culture media changes in the metabolic profile of Pedobacter lusitanus NL19 (NL19) and Pedobacter himalayensis MTCC 6384 (MTCC6384) and respective influence on the production of biotechnologically relevant compounds. Solid-phase microextraction combined with comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry with time-of-flight analyser (GC × GC-ToFMS) was applied to comprehensively study the metabolites produced by NL19 and MTCC6384 both in tryptic soy broth 100% (TSB100) and tryptic soy broth with 25% casein peptone (PC25). A total of 320 metabolites were putatively identified, which belong to different chemical families: alcohols, aldehydes, esters, ethers, hydrocarbons, ketones, nitrogen compounds, sulphur compounds, monoterpenes, and sesquiterpenes. Metabolites that were statistically different from the control (sterile medium) were selected allowing for the construction of the metabolic profile of both strains. A set of 80 metabolites was tentatively associated to the metabolic pathways such as the metabolism of fatty acids, branched-chain aminoacids, phenylalanine, methionine, aromatic compounds, and monoterpene and sesquiterpene biosynthesis. This study allowed to better understand how slight changes of the culture media and thus the composition of nutrients impair the metabolic profile of bacteria, which may be further explored for metabolomics pipeline construction or biotechnological applications.

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Glyphosate (GLY) contamination widely occurred in aquatic environments including aquaculture systems and raised hazard to aquatic organisms such as fish. Probiotics have been reported to alleviate contaminants-induced toxicity. However, whether probiotics could reduce the health risk of GLY to fish remain unknown. Here we investigated the impacts of GLY on crucian carp (Carassius auratus) by focusing on the protective roles of two commonly used aquaculture probiotics, Bacillus coagulans (BC) and Clostridium butyricum (CB). Exposure to GLY significantly caused growth retardation and reduced visceral fat and intestinal lipase activity in crucian carp. 16S rRNA sequencing indicated that dysbiosis of Bacteroidetes at phylum level and Flavobacterium at genus level might be primarily responsible for GLY-induced negative growth performance. High throughput targeted quantification for metabolites revealed that GLY changed intestinal metabolites profiles, especially the reduced bile acids and short-chain fatty acids. However, the addition of BC or CB effectively attenuated the adverse effects above by remodeling the gut microbiota composition and improving microbial metabolism. The present study provides novel evidence for ameliorating the harmful effects of GLY on fish species by adding probiotics, which highlights the potential application of probiotics in reducing the health risks of GLY in aquatic environment.

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Objectives: We aimed to study the effect of antibiotic-induced disruption of gut microbiome on host metabolomes and inflammatory responses after long-term use of antibiotics. Methods: A total of three groups of 3-week-old female C57BL/6 mice (n = 44) were continuously treated with vancomycin (VAN), polymyxin B (PMB), or water, respectively, for up to 28 weeks. Fecal samples collected at different time points were analyzed by bacterial 16S rRNA gene sequencing and untargeted metabolomics by ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS). Serum cytokines (IFN-γ, IL-2, IL-10, IL-13, IL-17A, and TNF-α) were determined by multiplex immunoassay. Results: Treatment by VAN or PMB did not affect the average body weight of mice. However, a heavier caecum observed in VAN-treated mice. Compared with PMB-treated and control mice, VAN treatment induced more rapid dysbiosis of gut microbiota and dysmetabolism. Instead of Bacteroides, VAN-treated mice had a compositional shift to Proteobacteria and its species Escherichia coli and Verrucomicrobia and its species Akkermansia muciniphila. The shift was accompanied by decreased richness and diversity in microbiota. PMB-treated mice had an increased Firmicutes, and the diversity was shortly increased and further decreased to the baseline. Decreased levels of short-chain and long-chain fatty acids, bile acids, L-arginine, dopamine, L-tyrosine, and phosphatidylcholine (all p < 0.05) were observed in VAN-treated mice. In contrast, significantly increased levels of amino acids including L-aspartic acid, beta-alanine, 5-hydroxy-L-tryptophan, L-glutamic acid, and lysophosphatidylcholines (all p < 0.05) were found. These changes occurred after 3-week treatment and remained unchanged up to 28 weeks. For PMB-treated mice, metabolites involved in the metabolic pathway of vitamin B6 were decreased, whereas glycocholic acid and chenodeoxycholic acid were increased (all p < 0.05). After 8-week treatment, VAN-treated mice had significantly higher levels of serum IFN-γ, IL-13, and IL-17A, and PMB-treated mice had higher levels of IL-13 and IL-17 compared to control mice. At 28-week treatment, only IL-17A remained high in PMB-treated mice. Conclusion: This study showed that the antibiotic-induced alterations in gut microbiota contribute to host inflammatory responses through the change in metabolic status, which are likely related to the type, rather than timing of antibiotic used.

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Soil nitrogen (N) transformations constrain terrestrial net primary productivity and are driven by the activity of soil microorganisms. Free-living N fixation (FLNF) is an important soil N transformation and key N input to terrestrial systems, but the forms of N contributed to soil by FLNF are poorly understood. To address this knowledge gap, a focus on microorganisms and microbial scale processes is needed that links N-fixing bacteria and their contributed N sources to FLNF process rates. However, studying the activity of soil microorganisms in situ poses inherent challenges, including differences in sampling scale between microorganism and process rates, which can be addressed with culture-based studies and an emphasis on microbial-scale measurements. Culture conditions can differ significantly from soil conditions, so it also important that such studies include multiple culture conditions like liquid and solid media as proxies for soil environments like soil pore water and soil aggregate surfaces. Here we characterized extracellular N-containing metabolites produced by two common, diazotrophic soil bacteria in liquid and solid media, with or without N, across two sampling scales (bulk via GC-MS and spatially resolved via MALDI mass spec imaging). We found extracellular production of inorganic and organic N during FLNF, indicating terrestrial N contributions from FLNF occur in multiple forms not only as ammonium as previously thought. Extracellular metabolite profiles differed between liquid and solid media supporting previous work indicating environmental structure influences microbial function. Metabolite profiles also differed between sampling scales underscoring the need to quantify microbial scale conditions to accurately interpret microbial function. IMPORTANCE Free-living nitrogen-fixing bacteria contribute significantly to terrestrial nitrogen availability; however, the forms of nitrogen contributed by this process are poorly understood. This is in part because of inherent challenges to studying soil microorganisms in situ, such as vast differences in scale between microorganism and ecosystem and complexities of the soil system (e.g., opacity, chemical complexity). Thus, upscaling important ecosystem processes driven by soil microorganisms, like free-living nitrogen fixation, requires microbial-scale measurements in controlled systems. Our work generated bulk and spatially resolved measurements of nitrogen released during free-living nitrogen fixation under two contrasting growth conditions analogous to soil pores and aggregates. This work allowed us to determine that diverse forms of nitrogen are likely contributed to terrestrial systems by free-living nitrogen bacteria. We also demonstrated that microbial habitat (e.g., liquid versus solid media) alters microbial activity and that measurement of microbial activity is altered by sampling scale (e.g., bulk versus spatially resolved) highlighting the critical importance of quantifying microbial-scale processes to upscaling of ecosystem function.

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The gut microbiome and microbial metabolomic influences on liver diseases and their diagnosis, prognosis, and treatment are still controversial. Research studies have provocatively claimed that the gut microbiome, metabolomics understanding, and microbial metabolite screening are key approaches to understanding liver cancer and liver diseases. An advance of logical innovations in metabolomics profiling, the metabolome inclusion, challenges, and the reproducibility of the investigations at every stage are devoted to this domain to link the common molecules across multiple liver diseases, such as fatty liver, hepatitis, and cirrhosis. These molecules are not immediately recognizable because of the huge underlying and synthetic variety present inside the liver cellular metabolome. This review focuses on microenvironmental metabolic stimuli in the gut-liver axis. Microbial small-molecule profiling (i.e., semiquantitative monitoring, metabolic discrimination, target profiling, and untargeted profiling) in biological fluids has been incompletely addressed. Here, we have reviewed the differential expression of the metabolome of short-chain fatty acids (SCFAs), tryptophan, one-carbon metabolism and bile acid, and the gut microbiota effects are summarized and discussed. We further present proof-of-evidence for gut microbiota-based metabolomics that manipulates the host's gut or liver microbes, mechanosensitive metabolite reactions and potential metabolic pathways. We conclude with a forward-looking perspective on future attention to the "dark matter" of the gut microbiota and microbial metabolomics.

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Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally, a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample. An issue which has often been overlooked in microbial metabolomics is the fact that many intracellular metabolites are also present in significant amounts outside the cells and may interfere with the quantification of the endo metabolome. Attempts to remove the extracellular metabolites with dedicated quenching methods often induce release of intracellular metabolites into the quenching solution. For eukaryotic microorganisms, this release can be minimized by adaptation of the quenching method. For prokaryotic cells, this has not yet been accomplished, so the application of a differential method whereby metabolites are measured in the culture supernatant as well as in total broth samples, to calculate the intracellular levels by subtraction, seems to be the most suitable approach. Here we present an overview of different sampling, quenching, and extraction methods developed for microbial metabolomics, described in the literature. Detailed protocols are provided for rapid sampling, quenching, and extraction, for measurement of metabolites in total broth samples, washed cell samples, and supernatant, to be applied for quantitative metabolomics of both eukaryotic and prokaryotic microorganisms.

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Untargeted metabolomic profiling provides the opportunity to comprehensively explore metabolites of interest. Herein, we investigated the metabolic pathways associated with Jhp0106, a glycosyltransferase enzyme in Helicobacter pylori. Through untargeted exometabolomic and metabolomic profiling, we identified 9 and 10 features with significant differences in the culture media and pellets of the wild-type (WT) J99 and jhp0106 mutant (Δjhp0106). After tentative identification, several phosphatidylethanolamines (PEs) were identified in the culture medium, the levels of which were significantly higher in WT J99 than in Δjhp0106. Moreover, the reduced lysophosphatidic acid absorption from the culture medium and the reduced intrinsic diacylglycerol levels observed in Δjhp0106 indicate the possibility of reduced PE synthesis in Δjhp0106. The results suggest an association of the PE synthesis pathway with flagellar formation in H. pylori. Further investigations should be conducted to confirm this finding and the roles of the PE synthesis pathway in flagellar formation. This study successfully demonstrates the feasibility of the proposed extraction procedure and untargeted exometabolomic and metabolomic profiling strategies for microbial metabolomics. They may also extend our understanding of metabolic pathways associated with flagellar formation in H. pylori.

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Food safety is veryimportant in our daily life. In food processing or disinfection, microorganisms are commonly exposed to oxidative stress perturbations. However, microorganisms can adapt and respond to physicochemical interventions, leading to difficulty and complexity for food safety assurance. Therefore, understanding the response mechanisms of microbes and providing an overview of the responses under oxidative stress conditions are beneficial for ensuring food safety for the industry. The current review takes the metabolomics approach to reveal small metabolite signatures and key pathway alterations during oxidative stress at the molecular and technical levels. These alterations are involved in primary oxidative stress responses due to inactivation treatments such as using hypochlorite (HOCl), hydrogen peroxide (H2 O2 ), electrolyzed water (EW), irradiation, pulsed light (PL), electron beam (EB), and secondary oxidative stress responses due to exposures to excessive conditions such as heat, pressure, acid, and alkaline. Details on the putative origin of exogenous or endogenous reactive oxygen species (ROS) are discussed, with particular attention paid to their effects on lipid, amino acid, nucleotide, and carbohydrate metabolism. In addition, mechanisms on counteracting oxidative stresses, stabilization of cell osmolality as well as energy provision for microbes to survive are also discussed.

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