Tissue-resident bacteria in metabolic diseases: emerging evidence and challenges.

Although the impact of the gut microbiome on health and disease is well established, there is controversy regarding the presence of microorganisms such as bacteria and their products in organs and tissues. However, recent contamination-aware findings of tissue-resident microbial signatures provide accumulating evidence in support of bacterial translocation in cardiometabolic disease. The latter provides a distinct paradigm for the link between microbial colonizers of mucosal surfaces and host metabolism. In this Perspective, we re-evaluate the concept of tissue-resident bacteria including their role in metabolic low-grade tissue and systemic inflammation. We examine the limitations and challenges associated with studying low bacterial biomass samples and propose experimental and analytical strategies to overcome these issues. Our Perspective aims to encourage further investigation of the mechanisms linking tissue-resident bacteria to host metabolism and their potentially actionable health implications for prevention and treatment.

Archaea oxidizing alkanes through alkyl-coenzyme M reductases.

This review synthesizes recent discoveries of novel archaea clades capable of oxidizing higher alkanes, from volatile ones like ethane to longer-chain alkanes like hexadecane. These archaea, termed anaerobic multicarbon alkane-oxidizing archaea (ANKA), initiate alkane oxidation using alkyl-coenzyme M reductases, enzymes similar to the methyl-coenzyme M reductases of methanogenic and anaerobic methanotrophic archaea (ANME). The polyphyletic alkane-oxidizing archaea group (ALOX), encompassing ANME and ANKA, harbors increasingly complex alkane degradation pathways, correlated with the alkane chain length. We discuss the evolutionary trajectory of these pathways emphasizing metabolic innovations and the acquisition of metabolic modules via lateral gene transfer. Additionally, we explore the mechanisms by which archaea couple alkane oxidation with the reduction of electron acceptors, including electron transfer to partner sulfate-reducing bacteria (SRB). The phylogenetic and functional constraints that shape ALOX-SRB associations are also discussed. We conclude by highlighting the research needs in this emerging research field and its potential applications in biotechnology.

Stable isotopes and nanoSIMS single-cell imaging reveals soil plastisphere colonizers able to assimilate sulfamethoxazole.

The presence and accumulation of both, plastics and antibiotics in soils may lead to the colonization, selection, and propagation of soil bacteria with certain metabolic traits, e.g., antibiotic resistance, in the plastisphere. However, the impact of plastic-antibiotic tandem on the soil ecosystem functioning, particularly on microbial function and metabolism remains currently unexplored. Herein, we investigated the competence of soil bacteria to colonize plastics and degrade 13C-labeled sulfamethoxazole (SMX). Using single-cell imaging, isotope tracers, soil respiration and SMX mineralization bulk measurements we show that microbial colonization of polyethylene (PE) and polystyrene (PS) surfaces takes place within the first 30 days of incubation. Morphologically diverse microorganisms were colonizing both plastic types, with a slight preference for PE substrate. CARD-FISH bacterial cell counts on PE and PS surfaces formed under SMX amendment ranged from 5.36 × 103 to 2.06 × 104, and 2.06 × 103 to 3.43 × 103 hybridized cells mm-2, respectively. Nano-scale Secondary Ion Mass Spectrometry measurements show that 13C enrichment was highest at 130 days with values up to 1.29 atom%, similar to those of the 13CO2 pool (up to 1.26 atom%, or 22.55 ‰). Independent Mann-Whitney U test showed a significant difference between the control plastisphere samples incubated without SMX and those in 13C-SMX incubations (P < 0.001). Our results provide direct evidence demonstrating, at single-cell level, the capacity of bacterial colonizers of plastics to assimilate 13C-SMX from contaminated soils. These findings expand our knowledge on the role of soil-seeded plastisphere microbiota in the ecological functioning of soils impacted by anthropogenic stressors.

Correction: High resolution microscopy to evaluate the efficiency of surface sterilization of Zea Mays seeds.

[This corrects the article DOI: 10.1371/journal.pone.0242247.].

Antibiotic resistance indicator genes in biofilm and planktonic microbial communities after wastewater discharge.

The spread of bacteria with antibiotic resistance genes (ARGs) in aquatic ecosystems is of growing concern as this can pose a risk of transmission to humans and animals. While the impact of wastewater treatment plant (WWTP) effluent on ARG abundance in surface waters has been studied extensively, less is known about the fate of ARGs in biofilms. The proximity and dense growth of microorganisms in combination with the accumulation of higher antibiotic concentrations in biofilms might render biofilms a reservoir for ARGs. Seasonal parameters such as water temperature, precipitation, and antibiotic concentrations should be considered as well, as they may further influence the fate of ARGs in aquatic ecosystems. Here we investigated the effect of WWTP effluent on the abundance of the sulfonamide resistance genes sul1 and sul2, and the integrase gene intI1 in biofilm and surface water compartments of a river in Germany with a gradient of anthropogenic impact using quantitative PCR. Furthermore, we analyzed the bacterial community structure in both compartments via 16S rRNA gene amplicon sequencing, following the river downstream. Additionally, conventional water parameters and sulfonamide concentrations were measured, and seasonal aspects were considered by comparing the fate of ARGs and bacterial community diversity in the surface water compartment between the summer and winter season. Our results show that biofilm compartments near the WWTP had a higher relative abundance of ARGs (up to 4.7%) than surface waters (<2.8%). Sulfonamide resistance genes were more persistent further downstream (>10 km) of the WWTP in the hot and dry summer season than in winter. This finding is likely a consequence of the higher proportion of wastewater and thus wastewater-derived microorganisms in the river during summer periods. We observed distinct bacterial communities and ARG abundance between the biofilm and surface water compartment, but even greater variations when considering seasonal and spatiotemporal parameters. This underscores the need to consider seasonal aspects when studying the fate of ARGs in aquatic ecosystems.

Tracking deuterium uptake in hydroponically grown maize roots using correlative helium ion microscopy and Raman micro-spectroscopy.

BACKGROUND: Investigations into the growth and self-organization of plant roots is subject to fundamental and applied research in various areas such as botany, agriculture, and soil science. The growth activity of the plant tissue can be investigated by isotope labeling experiments with heavy water and subsequent detection of the deuterium in non-exchangeable positions incorporated into the plant biomass. Commonly used analytical methods to detect deuterium in plants are based on mass-spectrometry or neutron-scattering and they either suffer from elaborated sample preparation, destruction of the sample during analysis, or low spatial resolution. Confocal Raman micro-spectroscopy (CRM) can be considered a promising method to overcome the aforementioned challenges. The substitution of hydrogen with deuterium results in the measurable shift of the CH-related Raman bands. By employing correlative approaches with a high-resolution technique, such as helium ion microscopy (HIM), additional structural information can be added to CRM isotope maps and spatial resolution can be further increased. For that, it is necessary to develop a comprehensive workflow from sample preparation to data processing. RESULTS: A workflow to prepare and analyze roots of hydroponically grown and deuterium labeled Zea mays by correlative HIM-CRM micro-analysis was developed. The accuracy and linearity of deuterium detection by CRM were tested and confirmed with samples of deuterated glucose. A set of root samples taken from deuterated Zea mays in a time-series experiment was used to test the entire workflow. The deuterium content in the roots measured by CRM was close to the values obtained by isotope-ratio mass spectrometry. As expected, root tips being the most actively growing root zone had incorporated the highest amount of deuterium which increased with increasing time of labeling. Furthermore, correlative HIM-CRM analysis allowed for obtaining the spatial distribution pattern of deuterium and lignin in root cross-sections. Here, more active root zones with higher deuterium incorporation showed less lignification. CONCLUSIONS: We demonstrated that CRM in combination with deuterium labeling can be an alternative and reliable tool for the analysis of plant growth. This approach together with the developed workflow has the potential to be extended to complex systems such as plant roots grown in soil.

Corrigendum: The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

[This corrects the article DOI: 10.3389/fmicb.2023.1058350.].

Spatial organization and proteome of a dual-species cyanobacterial biofilm alter among N2-fixing and non-fixing conditions.

Many disciplines have become increasingly interested in cyanobacteria, due to their ability to fix CO2 while using water and sunlight as electron and energy sources. Further, several species of cyanobacteria are also capable of fixing molecular nitrogen, making them independent of the addition of nitrate or ammonia. Thereby they hold huge potential as sustainable biocatalysts. Here, we look into a dual-species biofilm consisting of filamentous diazotrophic cyanobacteria Tolypothrix sp. PCC 7712 and heterotrophic bacteria Pseudomonas taiwanensis VLB 120 growing in a capillary biofilm reactor. Such systems have been reported to enable high cell densities continuous process operation. By combining confocal laser scanning and helium-ion microscopy with a proteomics approach, we examined these organisms' interactions under two nitrogen-feeding strategies: N2-fixing and nitrate assimilation. Not only did Pseudomonas facilitate the biofilm formation by forming a carpet layer on the surface area but also did N2-fixing biofilms show greater attachment to the surface. Pseudomonas proteins related to surface and cell attachments were observed in N2-fixing biofilms in particular. Furthermore, co-localized biofilm cells displayed a resilient response to extra shear forces induced by segmented media/air flows. This study highlights the role of Pseudomonas in the initial attachment process, as well as the effects of different nitrogen-feeding strategies and operation regimes on biofilm composition and growth. IMPORTANCE Cyanobacteria are highly interesting microorganisms due to their ability to synthesize sugars from CO2 while using water and sunlight as electron and energy sources. Further, many species are also capable of utilizing molecular nitrogen, making them independent of artificial fertilizers. In this study, such organisms are cultivated in a technical system, which enables them to attach to the reactor surface, and form three-dimensional structures termed biofilms. Biofilms achieve extraordinarily high cell densities. Furthermore, this growth format allows for continuous processing, both being essential features in biotechnological process development. Understanding biofilm growth and the influence technical settings and media composition have on biofilm maturation and stability are crucial for reaction and reactor design. These findings will help to open up these fascinating organisms for applications as sustainable, resource-efficient industrial workhorses.

Live tracking metabolic networks and physiological responses within microbial assemblages at single-cell level.

Microbial interactions impact the functioning of both natural and engineered systems, yet our ability to directly monitor these highly dynamic and spatially resolved interactions in living cells is very limited. Here, we developed a synergistic approach coupling single-cell Raman microspectroscopy with 15N2 and 13CO2 stable isotope probing in a microfluidic culture system (RMCS-SIP) for live tracking of the occurrence, rate, and physiological shift of metabolic interactions in active microbial assemblages. Quantitative and robust Raman biomarkers specific for N2 and CO2 fixation in both model and bloom-forming diazotrophic cyanobacteria were established and cross-validated. By designing a prototype microfluidic chip allowing simultaneous microbial cultivation and single-cell Raman acquisition, we achieved temporal tracking of both intercellular (between heterocyst and vegetative cells of cyanobacteria) and interspecies N and C metabolite exchange (from diazotroph to heterotroph). Moreover, single-cell N and C fixation and bidirectional transfer rate in living cells were quantified via SIP-induced characteristic Raman shifts. Remarkably, RMCS captured physiological responses of metabolically active cells to nutrient stimuli through comprehensive metabolic profiling, providing multimodal information on the evolution of microbial interactions and functions under fluctuating conditions. This noninvasive RMCS-SIP is an advantageous approach for live-cell imaging and represents an important advancement in the single-cell microbiology field. This platform can be extended for real-time tracking of a wide range of microbial interactions with single-cell resolution and advances the understanding and manipulation of microbial interactions for societal benefit.

The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river stream.

Introduction: Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2. Methods: Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ). Results: Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication. Discussion: We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics.

Efficient carbon and nitrogen transfer from marine diatom aggregates to colonizing bacterial groups.

Bacterial degradation of sinking diatom aggregates is key for the availability of organic matter in the deep-ocean. Yet, little is known about the impact of aggregate colonization by different bacterial taxa on organic carbon and nutrient cycling within aggregates. Here, we tracked the carbon (C) and nitrogen (N) transfer from the diatom Leptocylindrus danicus to different environmental bacterial groups using a combination of 13C and 15N isotope incubation (incubated for 72 h), CARD-FISH and nanoSIMS single-cell analysis. Pseudoalteromonas bacterial group was the first colonizing diatom-aggregates, succeeded by the Alteromonas group. Within aggregates, diatom-attached bacteria were considerably more enriched in 13C and 15N than non-attached bacteria. Isotopic mass balance budget indicates that both groups showed comparable levels of diatom C in their biomass, accounting for 19 ± 7% and 15 ± 11%, respectively. In contrast to C, bacteria of the Alteromonas groups showed significantly higher levels of N derived from diatoms (77 ± 28%) than Pseudoalteromonas (47 ± 17%), suggesting a competitive advantage for Alteromonas in the N-limiting environments of the deep-sea. Our results imply that bacterial succession within diatom aggregates may largely impact taxa-specific C and N uptake, which may have important consequences for the quantity and quality of organic matter exported to the deep ocean.

New Paradigms for Familiar Diseases: Lessons Learned on Circulatory Bacterial Signatures in Cardiometabolic Diseases.

Despite the strongly accumulating evidence for microbial signatures in metabolic tissues, including the blood, suggesting a novel paradigm for metabolic disease development, the notion of a core blood bacterial signature in health and disease remains a contentious concept. Recent studies clearly demonstrate that under a strict contamination-free environment, methods such as 16 S rRNA gene sequencing, fluorescence in-situ hybridization, transmission electron microscopy, and several more, allied with advanced bioinformatics tools, allow unambiguous detection and quantification of bacteria and bacterial DNA in human tissues. Bacterial load and compositional changes in the blood have been reported for numerous disease states, suggesting that bacteria and their components may partially induce systemic inflammation in cardiometabolic disease. This concept has been so far primarily based on measurements of surrogate parameters. It is now highly desirable to translate the current knowledge into diagnostic, prognostic, and therapeutic approaches.This review addresses the potential clinical relevance of a blood bacterial signature pertinent to cardiometabolic diseases and outcomes and new avenues for translational approaches. It discusses pitfalls related to research in low bacterial biomass while proposing mitigation strategies for future research and application approaches.

Iron corrosion by methanogenic archaea characterized by stable isotope effects and crust mineralogy.

Carbon and hydrogen stable isotope effects associated with methane formation by the corrosive archaeon Methanobacterium strain IM1 were determined during growth with hydrogen and iron. Isotope analyses were complemented by structural, elemental and molecular composition analyses of corrosion crusts. During growth with H2 , strain IM1 formed methane with average δ13 C of -43.5‰ and δ2 H of -370‰. Corrosive growth led to methane more depleted in 13 C, with average δ13 C ranging from -56‰ to -64‰ during the early and the late growth phase respectively. The corresponding δ2 H were less impacted by the growth phase, with average values ranging from -316 to -329‰. The stable isotope fractionation factors, α 13 C CO 2 / CH 4 , were 1.026 and 1.042 for hydrogenotrophic and corrosive growth respectively. Corrosion crusts formed by strain IM1 have a domed structure, appeared electrically conductive and were composed of siderite, calcite and iron sulfide, the latter formed by precipitation of sulfide (from culture medium) with ferrous iron generated during corrosion. Strain IM1 cells were found attached to crust surfaces and encrusted deep inside crust domes. Our results may assist to diagnose methanogens-induced corrosion in the field and suggest that intrusion of sulfide in anoxic settings may stimulate corrosion by methanogenic archaea via formation of semiconductive crusts.

Corrigendum: Microbial identification, high-resolution microscopy and spectrometry of the rhizosphere in its native spatial context.

[This corrects the article DOI: 10.3389/fpls.2021.668929.].

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae.

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low 13 C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.

Metabolic history and metabolic fitness as drivers of anabolic heterogeneity in isogenic microbial populations.

Microbial populations often display different degrees of heterogeneity in their substrate assimilation, that is, anabolic heterogeneity. It has been shown that nutrient limitations are a relevant trigger for this behaviour. Here we explore the dynamics of anabolic heterogeneity under nutrient replete conditions. We applied time-resolved stable isotope probing and nanoscale secondary ion mass spectrometry to quantify substrate assimilation by individual cells of Pseudomonas putida, P. stutzeri and Thauera aromatica. Acetate and benzoate at different concentrations were used as substrates. Anabolic heterogeneity was quantified by the cumulative differentiation tendency index. We observed two major, opposing trends of anabolic heterogeneity over time. Most often, microbial populations started as highly heterogeneous, with heterogeneity decreasing by various degrees over time. The second, less frequently observed trend, saw microbial populations starting at low or very low heterogeneity, and remaining largely stable over time. We explain these trends as an interplay of metabolic history (e.g. former growth substrate or other nutrient limitations) and metabolic fitness (i.e. the fine-tuning of metabolic pathways to process a defined growth substrate). Our results offer a new viewpoint on the intra-population functional diversification often encountered in the environment, and suggests that some microbial populations may be intrinsically heterogeneous.

Cytoklepty in the plankton: A host strategy to optimize the bioenergetic machinery of endosymbiotic algae.

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition.

Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context.

During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro- to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (µ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance.

Circulating bacterial signature is linked to metabolic disease and shifts with metabolic alleviation after bariatric surgery.

BACKGROUND: The microbiome has emerged as an environmental factor contributing to obesity and type 2 diabetes (T2D). Increasing evidence suggests links between circulating bacterial components (i.e., bacterial DNA), cardiometabolic disease, and blunted response to metabolic interventions. In this aspect, thorough next-generation sequencing-based and contaminant-aware approaches are lacking. To address this, we tested whether bacterial DNA could be amplified in the blood of subjects with obesity and high metabolic risk under strict experimental and analytical control and whether a putative bacterial signature is related to metabolic improvement after bariatric surgery. METHODS: Subjects undergoing bariatric surgery were recruited into sex- and BMI-matched subgroups with (n = 24) or without T2D (n = 24). Bacterial DNA in the blood was quantified and prokaryotic 16S rRNA gene amplicons were sequenced. A contaminant-aware approach was applied to derive a compositional microbial signature from bacterial sequences in all subjects at baseline and at 3 and 12 months after surgery. We modeled associations between bacterial load and composition with host metabolic and anthropometric markers. We further tested whether compositional shifts were related to weight loss response and T2D remission. Lastly, bacteria were visualized in blood samples using catalyzed reporter deposition (CARD)-fluorescence in situ hybridization (FISH). RESULTS: The contaminant-aware blood bacterial signature was associated with metabolic health. Based on bacterial phyla and genera detected in the blood samples, a metabolic syndrome classification index score was derived and shown to robustly classify subjects along their actual clinical group. T2D was characterized by decreased bacterial richness and loss of genera associated with improved metabolic health. Weight loss and metabolic improvement following bariatric surgery were associated with an early and stable increase of these genera in parallel with improvements in key cardiometabolic risk parameters. CARD-FISH allowed the detection of living bacteria in blood samples in obesity. CONCLUSIONS: We show that the circulating bacterial signature reflects metabolic disease and its improvement after bariatric surgery. Our work provides contaminant-aware evidence for the presence of living bacteria in the blood and suggests a putative crosstalk between components of the blood and metabolism in metabolic health regulation.

Novel clades of soil biphenyl degraders revealed by integrating isotope probing, multi-omics, and single-cell analyses.

Most microorganisms in the biosphere remain uncultured and poorly characterized. Although the surge in genome sequences has enabled insights into the genetic and metabolic properties of uncultured microorganisms, their physiology and ecological roles cannot be determined without direct probing of their activities in natural habitats. Here we employed an experimental framework coupling genome reconstruction and activity assays to characterize the largely uncultured microorganisms responsible for aerobic biodegradation of biphenyl as a proxy for a large class of environmental pollutants, polychlorinated biphenyls. We used 13C-labeled biphenyl in contaminated soils and traced the flow of pollutant-derived carbon into active cells using single-cell analyses and protein-stable isotope probing. The detection of 13C-enriched proteins linked biphenyl biodegradation to the uncultured Alphaproteobacteria clade UBA11222, which we found to host a distinctive biphenyl dioxygenase gene widely retrieved from contaminated environments. The same approach indicated the capacity of Azoarcus species to oxidize biphenyl and suggested similar metabolic abilities for species of Rugosibacter. Biphenyl oxidation would thus represent formerly unrecognized ecological functions of both genera. The quantitative role of these microorganisms in pollutant degradation was resolved using single-cell-based uptake measurements. Our strategy advances our understanding of microbially mediated biodegradation processes and has general application potential for elucidating the ecological roles of uncultured microorganisms in their natural habitats.