Kiran Raosaheb Patil

Commensal gut bacteria are key contributors to the resilience against pathogen invasion. This is exemplified by the success of faecal microbiota transplantation in treating recurrent Clostridioides difficile infection. Yet, characteristics of communities that can confer colonisation resistance and the underlying mechanisms remain largely unknown. Here we use a synthetic community of 14 commensal gut bacteria to uncover inter-species interactions and metabolic pathways underpinning the emergent resilience against C. difficile invasion. We challenged this synthetic community as well as faecal-matter-derived communities with antibiotic treatment and C. difficile in a continuous flow bioreactor. Using generalised Lotka-Volterra and genome-scale metabolic modelling, we identified interactions between Escherichia coli and Bacteroides/Phocaeicola sp. as key to the pathogen’s suppression. Metabolomics and targeted validation experiments revealed that fructan-derived fructooligosaccharides, vitamin B3 biosynthesis, and competition for Stickland metabolism precursors causally contribute to suppression across multiple C. difficile strains and donor backgrounds. Analysis of metagenomics data from patient cohorts and clinical trials attested the in vivo relevance of the identified metabolic pathways and the ratio between Bacteroides and Escherichia in successful colonisation resistance. The latter was found to be a stronger discriminator than commonly used alpha diversity metrics. Together, our study uncovers emergent microbial interactions in pathogen resistance with implications for rational design of bacteriotherapies.

Ecological theory predicts that obligate cross-feeding of metabolites is common in microbial communities. However, systematic studies testing this prediction in natural microbial communities such as soil are missing, thus hindering the understanding of these ecologically important ecosystems. Here we address this gap by analysing 6,931 bacterial isolates from 27 soil microbial communities sampled in Germany. We find that the growth of up to 50% of community members depended on supplementation with amino acids, vitamins or nucleobases. For 73% of isolates, supplementation with multiple amino acids was necessary. Genomic analysis of 62 strains revealed that accumulation of insertion sequences and gene loss was associated with the observed auxotrophic phenotypes. Genome-scale metabolic models, computational analyses and cocultivation experiments suggest other co-occurring genotypes complement the metabolic needs of auxotrophs, thus enabling their growth and survival. Together, our results indicate that soil bacteria probably exist within integrated metabolic networks rather than as metabolically autonomous units.

Many small-molecule drugs accumulate in both host and gut microbial cells. Yet, the molecular consequences of intracellular drug accumulation are largely unknown. Here we show that the antidepressant duloxetine broadly disrupts RNA-protein interactions revealing a previously unrecognized mode of drug effect. Across phylogenetically diverse bacteria and human intestinal cells, duloxetine consistently reduced RNA-binding capacity, with approximately 80% of RNA-binding proteins responding to duloxetine in bacteria ( E. coli IAI1) and human (Caco-2) cells. As a mechanistic example, we show how duloxetine disrupts interactions between the pyrimidine biosynthesis enzyme PyrB and transcripts encoding metabolically linked functions. Biochemical and structural analyses show that duloxetine competes with aspartate, the natural substrate of PyrB, weakening the enzyme’s binding with 3′ UTR-localized stem-loop structures in its RNA partners. Overall, our results uncover the off-target disruption of RNA-protein interactions due to drug accumulation and have implications for understanding molecular basis of variation in drug efficacy and toxicity.

Yeasts are key microbial members of several ecosystems. Yet, the impact of widespread chemical pollutants on yeasts is only sparsely studied. Here we report the effect of >1000 chemical pollutants on fourteen diverse yeast species spanning the Saccharomycotina subphylum. Starmerella bombicola , a symbiont of various bee species, was the most sensitive and inhibited by several fungicides as well as by non-fungicides. To identify the molecular basis of this ultra-sensitivity, we selected resistant lineages against nine chemicals using adaptive laboratory evolution. Whole-genome-sequencing uncovered convergent evolution on YBP1 , a key regulator of oxidative stress. Proteomic analysis confirmed the protective role of oxidative stress response pathways, including proteins encoded by horizontally transferred bacterial genes. We find that the evolved S. bombicola stably colonized the bee gut and ameliorated the negative effect of paclobutrazol, a plant hormone regulator, on gut microbes, sucrose responsiveness, and learning. Our findings demonstrate how laboratory evolution can be used to mitigate the negative impact of chemical pollutants on pollinators.

Genome-scale metabolic models (GSMMs) are important aids towards system-level understanding of the metabolic physiology of the gut microbes and for rational microbiome engineering. While large-scale repositories of GSMMs for gut-associated bacteria are available, strain-level variability and the continuous discovery of novel taxa through metagenomics and culturomics underscore the need for scalable, ab initio reconstruction tools. Here, we present CarveMe-GutMicrobes, a client-side framework for rapid reconstruction of metabolic models directly from (meta)genomic input. Building upon the original CarveMe framework, CarveMe-GutMicrobes incorporates an expanded, gut-microbe-centric biochemical database that includes reactions, metabolites, and gene-protein-reaction (GPR) associations curated specifically for Bacteria and Archaea inhabiting the human gut. The tool supports taxonomic restriction of the reference database to improve context-specific accuracy. To test the CarveMe-GutMicrobes and to address the paucity of experimental data for non-model gut taxa, we generated new experimental datasets on metabolite secretion profiles and gene essentiality. CarveMe-GutMicrobes models demonstrated high predictive performance performance against these as well as previously available datasets. By integrating curated resources, extending reaction coverage, and offering new empirical datasets, CarveMe-GutMicrobes provides a scalable platform for high-resolution metabolic reconstruction towards broader adoption of GSMMs in gut microbiome research.

The gut microbiota is implicated in adverse effects associated with low-calorie sweeteners. Yet, the direct impact of sweeteners on gut bacteria remains largely uncharacterized. Here, we report interactions between 25 phylogenetically diverse gut bacterial strains and 39 commercially used sweeteners. We tested these sweeteners individually and in combination with four commonly co-consumed compounds, viz., advantame, caffeine, vanillin, and duloxetine. Three-quarters of the tested sweeteners individually impacted the growth of at least one tested bacterial strain. Further, over 100 interactions were found between sweeteners and the four co-consumed compounds. Isosteviol, a commonly used sweetener-component, and duloxetine, an antidepressant, synergistically inhibited Roseburia intestinalis , a bacterium previously linked to glucose homeostasis, and Parabacteroides merdae , a prevalent commensal linked to healthy microbiota. Proteomic, metabolomic, and genetic analyses indicate altered small molecule transport underpinning this sweetener–drug synergy. The isosteviol-duloxetine combination also modulated metabolism of a synthetic gut bacterial community, leading to increased toxicity to HeLa cells and altered secretion of inflammation-modulatory cytokines IL-6 and IL-8 by Caco-2 cells. Our data warrant further studies on interactions between low-calorie sweeteners and common xenobiotics.

Microbial interactions unfold within environments structured by physical transport and chemical gradients. Yet most mechanistic studies rely on well-mixed systems that mask the reciprocal influences of environmental heterogeneity on metabolism and ecology. Here, we investigate how the physical environment modulates the interaction between the gut commensal Bacteroides thetaiotaomicron and Escherichia coli . In anoxic liquid culture, cell-resolved isotope imaging and genetic perturbations reveal exploitative cross-feeding, where E. coli consumes diffusible sugars released by B . thetaiotaomicron during starch degradation. When exposed to intestinal-like oxygen gradients in microfluidics, the interaction is restructured by spatial organization. The species self-organize into complementary niches: E. coli locally depletes sugars and oxygen, thereby expanding the anoxic niche required by B. thetaiotaomicron . A reactive transport model confirms that this organization arises from coupled feedback between physical transport and metabolic reaction rates. Together, our results reveal how physical structure and chemical gradients convert an exploitative cross-feeding interaction into a dynamic niche-construction process that generates emergent spatial organization and stabilizes coexistence.

Species in microbial communities need to stave off competition and capitalize on new resources that become available because of metabolic activities of others. However, intra-cellular molecular changes that underpin these responses are understudied, preventing mechanistic insights into community function and dynamics. Here we analyse proteomic and metabolomic responses in 104 pairwise co-cultures of 15 gut bacteria, spanning a diversity of ecological interactions from competition to mutualism. We find that molecular responses to co-culturing are substantial, with typically 50% of the quantified proteome changing in at least one co-culture, jointly influenced by genome size, species abundance and pH. Even closely related species and orthologue proteins show different expression profiles in response to the same partner, indicating functional diversification at both protein and species level. Small-molecule transport and carbon metabolism are among the most responsive processes, indicating pervasive metabolic interactions. Using metabolomics, we identify likely cross-fed metabolites, emergent polyamine metabolism and niche partitioning in amino acid utilization. Overall, our study uncovers how bacteria respond to the presence of other species through extensive remodelling of their proteome and metabolome.