Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

Spatial transcriptome-guided multi-scale framework connects P. aeruginosa metabolic states to oxidative stress biofilm microenvironment.

With the generation of spatially resolved transcriptomics of microbial biofilms, computational tools can be used to integrate this data to elucidate the multi-scale mechanisms controlling heterogeneous biofilm metabolism. This work presents a Multi-scale model of Metabolism In Cellular Systems (MiMICS) which is a computational framework that couples a genome-scale metabolic network reconstruction (GENRE) with Hybrid Automata Library (HAL), an existing agent-based model and reaction-diffusion model platform. A key feature of MiMICS is the ability to incorporate multiple -omics-guided metabolic models, which can represent unique metabolic states that yield different metabolic parameter values passed to the extracellular models. We used MiMICS to simulate Pseudomonas aeruginosa regulation of denitrification and oxidative stress metabolism in hypoxic and nitric oxide (NO) biofilm microenvironments. Integration of P. aeruginosa PA14 biofilm spatial transcriptomic data into a P. aeruginosa PA14 GENRE generated four PA14 metabolic model states that were input into MiMICS. Characteristic of aerobic, denitrification, and oxidative stress metabolism, the four metabolic model states predicted different oxygen, nitrate, and NO exchange fluxes that were passed as inputs to update the agent's local metabolite concentrations in the extracellular reaction-diffusion model. Individual bacterial agents chose a PA14 metabolic model state based on a combination of stochastic rules, and agents sensing local oxygen and NO. Transcriptome-guided MiMICS predictions suggested microscale denitrification and oxidative stress metabolic heterogeneity emerged due to local variability in the NO biofilm microenvironment. MiMICS accurately predicted the biofilm's spatial relationships between denitrification, oxidative stress, and central carbon metabolism. As simulated cells responded to extracellular NO, MiMICS revealed dynamics of cell populations heterogeneously upregulating reactions in the denitrification pathway, which may function to maintain NO levels within non-toxic ranges. We demonstrated that MiMICS is a valuable computational tool to incorporate multiple -omics-guided metabolic models to mechanistically map heterogeneous microbial metabolic states to the biofilm microenvironment.

Identifying metabolic adaptations characteristic of cardiotoxicity using paired transcriptomics and metabolomics data integrated with a computational model of heart metabolism.

Improvements in the diagnosis and treatment of cancer have revealed long-term side effects of chemotherapeutics, particularly cardiotoxicity. Here, we present paired transcriptomics and metabolomics data characterizing in vitro cardiotoxicity to three compounds: 5-fluorouracil, acetaminophen, and doxorubicin. Standard gene enrichment and metabolomics approaches identify some commonly affected pathways and metabolites but are not able to readily identify metabolic adaptations in response to cardiotoxicity. The paired data was integrated with a genome-scale metabolic network reconstruction of the heart to identify shifted metabolic functions, unique metabolic reactions, and changes in flux in metabolic reactions in response to these compounds. Using this approach, we confirm previously seen changes in the p53 pathway by doxorubicin and RNA synthesis by 5-fluorouracil, we find evidence for an increase in phospholipid metabolism in response to acetaminophen, and we see a shift in central carbon metabolism suggesting an increase in metabolic demand after treatment with doxorubicin and 5-fluorouracil.

Reconstructor: a COBRApy compatible tool for automated genome-scale metabolic network reconstruction with parsimonious flux-based gap-filling.

MOTIVATION: Genome-scale metabolic network reconstructions (GENREs) are valuable for understanding cellular metabolism in silico. Several tools exist for automatic GENRE generation. However, these tools frequently (i) do not readily integrate with some of the widely-used suites of packaged methods available for network analysis, (ii) lack effective network curation tools, (iii) are not sufficiently user-friendly, and (iv) often produce low-quality draft reconstructions. RESULTS: Here, we present Reconstructor, a user-friendly, COBRApy-compatible tool that produces high-quality draft reconstructions with reaction and metabolite naming conventions that are consistent with the ModelSEED biochemistry database and includes a gap-filling technique based on the principles of parsimony. Reconstructor can generate SBML GENREs from three input types: annotated protein .fasta sequences (Type 1 input), a BLASTp output (Type 2), or an existing SBML GENRE that can be further gap-filled (Type 3). While Reconstructor can be used to create GENREs of any species, we demonstrate the utility of Reconstructor with bacterial reconstructions. We demonstrate how Reconstructor readily generates high-quality GENRES that capture strain, species, and higher taxonomic differences in functional metabolism of bacteria and are useful for further biological discovery. AVAILABILITY AND IMPLEMENTATION: The Reconstructor Python package is freely available for download. Complete installation and usage instructions and benchmarking data are available at http://github.com/emmamglass/reconstructor.

Metabolic modeling of sex-specific liver tissue suggests mechanism of differences in toxicological responses.

Male subjects in animal and human studies are disproportionately used for toxicological testing. This discrepancy is evidenced in clinical medicine where females are more likely than males to experience liver-related adverse events in response to xenobiotics. While previous work has shown gene expression differences between the sexes, there is a lack of systems-level approaches to understand the direct clinical impact of these differences. Here, we integrate gene expression data with metabolic network models to characterize the impact of transcriptional changes of metabolic genes in the context of sex differences and drug treatment. We used Tasks Inferred from Differential Expression (TIDEs), a reaction-centric approach to analyzing differences in gene expression, to discover that several metabolic pathways exhibit sex differences including glycolysis, fatty acid metabolism, nucleotide metabolism, and xenobiotics metabolism. When TIDEs is used to compare expression differences in treated and untreated hepatocytes, we find several subsystems with differential expression overlap with the sex-altered pathways such as fatty acid metabolism, purine and pyrimidine metabolism, and xenobiotics metabolism. Finally, using sex-specific transcriptomic data, we create individual and averaged male and female liver models and find differences in the pentose phosphate pathway and other metabolic pathways. These results suggest potential sex differences in the contribution of the pentose phosphate pathway to oxidative stress, and we recommend further research into how these reactions respond to hepatotoxic pharmaceuticals.

Network analysis of toxin production in Clostridioides difficile identifies key metabolic dependencies.

Clostridioides difficile pathogenesis is mediated through its two toxin proteins, TcdA and TcdB, which induce intestinal epithelial cell death and inflammation. It is possible to alter C. difficile toxin production by changing various metabolite concentrations within the extracellular environment. However, it is unknown which intracellular metabolic pathways are involved and how they regulate toxin production. To investigate the response of intracellular metabolic pathways to diverse nutritional environments and toxin production states, we use previously published genome-scale metabolic models of C. difficile strains CD630 and CDR20291 (iCdG709 and iCdR703). We integrated publicly available transcriptomic data with the models using the RIPTiDe algorithm to create 16 unique contextualized C. difficile models representing a range of nutritional environments and toxin states. We used Random Forest with flux sampling and shadow pricing analyses to identify metabolic patterns correlated with toxin states and environment. Specifically, we found that arginine and ornithine uptake is particularly active in low toxin states. Additionally, uptake of arginine and ornithine is highly dependent on intracellular fatty acid and large polymer metabolite pools. We also applied the metabolic transformation algorithm (MTA) to identify model perturbations that shift metabolism from a high toxin state to a low toxin state. This analysis expands our understanding of toxin production in C. difficile and identifies metabolic dependencies that could be leveraged to mitigate disease severity.

A computational model of Pseudomonas syringae metabolism unveils a role for branched-chain amino acids in Arabidopsis leaf colonization.

Bacterial pathogens adapt their metabolism to the plant environment to successfully colonize their hosts. In our efforts to uncover the metabolic pathways that contribute to the colonization of Arabidopsis thaliana leaves by Pseudomonas syringae pv tomato DC3000 (Pst DC3000), we created iPst19, an ensemble of 100 genome-scale network reconstructions of Pst DC3000 metabolism. We developed a novel approach for gene essentiality screens, leveraging the predictive power of iPst19 to identify core and ancillary condition-specific essential genes. Constraining the metabolic flux of iPst19 with Pst DC3000 gene expression data obtained from naïve-infected or pre-immunized-infected plants, revealed changes in bacterial metabolism imposed by plant immunity. Machine learning analysis revealed that among other amino acids, branched-chain amino acids (BCAAs) metabolism significantly contributed to the overall metabolic status of each gene-expression-contextualized iPst19 simulation. These predictions were tested and confirmed experimentally. Pst DC3000 growth and gene expression analysis showed that BCAAs suppress virulence gene expression in vitro without affecting bacterial growth. In planta, however, an excess of BCAAs suppress the expression of virulence genes at the early stages of infection and significantly impair the colonization of Arabidopsis leaves. Our findings suggesting that BCAAs catabolism is necessary to express virulence and colonize the host. Overall, this study provides valuable insights into how plant immunity impacts Pst DC3000 metabolism, and how bacterial metabolism impacts the expression of virulence.

Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms.

BACKGROUND & AIMS: The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. METHODS: We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. RESULTS: Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. CONCLUSIONS: Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Quantifying cumulative phenotypic and genomic evidence for procedural generation of metabolic network reconstructions.

Genome-scale metabolic network reconstructions (GENREs) are valuable tools for understanding microbial metabolism. The process of automatically generating GENREs includes identifying metabolic reactions supported by sufficient genomic evidence to generate a draft metabolic network. The draft GENRE is then gapfilled with additional reactions in order to recapitulate specific growth phenotypes as indicated with associated experimental data. Previous methods have implemented absolute mapping thresholds for the reactions automatically included in draft GENREs; however, there is growing evidence that integrating annotation evidence in a continuous form can improve model accuracy. There is a need for flexibility in the structure of GENREs to better account for uncertainty in biological data, unknown regulatory mechanisms, and context-specificity associated with data inputs. To address this issue, we present a novel method that provides a framework for quantifying combined genomic, biochemical, and phenotypic evidence for each biochemical reaction during automated GENRE construction. Our method, Constraint-based Analysis Yielding reaction Usage across metabolic Networks (CANYUNs), generates accurate GENREs with a quantitative metric for the cumulative evidence for each reaction included in the network. The structuring of CANYUNs allows for the simultaneous integration of three data inputs while maintaining all supporting evidence for biochemical reactions that may be active in an organism. CANYUNs is designed to maximize the utility of experimental and annotation datasets and to ultimately assist in the curation of the reference datasets used for the automatic construction of metabolic networks. We validated CANYUNs by generating an E. coli K-12 model and compared it to the manually curated reconstruction iML1515. Finally, we demonstrated the use of CANYUNs to build a model by generating an E. coli Nissle CANYUNs model using novel phenotypic data that we collected. This method may address key challenges for the procedural construction of metabolic networks by leveraging uncertainty and redundancy in biological data.

Comparative analyses of parasites with a comprehensive database of genome-scale metabolic models.

Protozoan parasites cause diverse diseases with large global impacts. Research on the pathogenesis and biology of these organisms is limited by economic and experimental constraints. Accordingly, studies of one parasite are frequently extrapolated to infer knowledge about another parasite, across and within genera. Model in vitro or in vivo systems are frequently used to enhance experimental manipulability, but these systems generally use species related to, yet distinct from, the clinically relevant causal pathogen. Characterization of functional differences among parasite species is confined to post hoc or single target studies, limiting the utility of this extrapolation approach. To address this challenge and to accelerate parasitology research broadly, we present a functional comparative analysis of 192 genomes, representing every high-quality, publicly-available protozoan parasite genome including Plasmodium, Toxoplasma, Cryptosporidium, Entamoeba, Trypanosoma, Leishmania, Giardia, and other species. We generated an automated metabolic network reconstruction pipeline optimized for eukaryotic organisms. These metabolic network reconstructions serve as biochemical knowledgebases for each parasite, enabling qualitative and quantitative comparisons of metabolic behavior across parasites. We identified putative differences in gene essentiality and pathway utilization to facilitate the comparison of experimental findings and discovered that phylogeny is not the sole predictor of metabolic similarity. This knowledgebase represents the largest collection of genome-scale metabolic models for both pathogens and eukaryotes; with this resource, we can predict species-specific functions, contextualize experimental results, and optimize selection of experimental systems for fastidious species.

Megasphaera in the Stool Microbiota Is Negatively Associated With Diarrheal Cryptosporidiosis.

BACKGROUND: The protozoan parasites in the Cryptosporidium genus cause both acute diarrheal disease and subclinical (ie, nondiarrheal) disease. It is unclear if the microbiota can influence the manifestation of diarrhea during a Cryptosporidium infection. METHODS: To characterize the role of the gut microbiota in diarrheal cryptosporidiosis, the microbiome composition of both diarrheal and surveillance Cryptosporidium-positive fecal samples from 72 infants was evaluated using 16S ribosomal RNA gene sequencing. Additionally, the microbiome composition prior to infection was examined to test whether a preexisting microbiome profile could influence the Cryptosporidium infection phenotype. RESULTS: Fecal microbiome composition was associated with diarrheal symptoms at 2 timepoints. Megasphaera was significantly less abundant in diarrheal samples compared with subclinical samples at the time of Cryptosporidium detection (log2 [fold change] = -4.3; P = 10-10) and prior to infection (log2 [fold change] = -2.0; P = 10-4); this assigned sequence variant was detected in 8 children who had diarrhea and 30 children without diarrhea. Random forest classification also identified Megasphaera abundance in the pre- and postexposure microbiota as predictive of a subclinical infection. CONCLUSIONS: Microbiome composition broadly, and specifically low Megasphaera abundance, was associated with diarrheal symptoms prior to and at the time of Cryptosporidium detection. This observation suggests that the gut microenvironment may play a role in determining the severity of a Cryptosporidium infection. Clinical Trials Registration. NCT02764918.

Perinatal Outcomes of Asynchronous Influenza Vaccination, Ceará, Brazil, 2013-2018.

In Ceará, Brazil, seasonal influenza transmission begins before national annual vaccination campaigns commence. To assess the perinatal consequences of this misalignment, we tracked severe acute respiratory infection (SARI), influenza, and influenza immunizations during 2013-2018. Among 3,297 SARI cases, 145 (4.4%) occurred in pregnant women. Statewide vaccination coverage was >80%; however, national vaccination campaigns began during or after peak influenza season. Thirty to forty weeks after peak influenza season, birthweights decreased by 40 g, and rates of prematurity increased from 10.7% to 15.5%. We identified 61 children born to mothers with SARI during pregnancy; they weighed 10% less at birth and were more likely to be premature than 122 newborn controls. Mistiming of influenza vaccination campaigns adversely effects perinatal outcomes in Ceará. Because Ceará is the presumptive starting point for north-to-south seasonal influenza transmission in Brazil, earlier national immunization campaigns would provide greater protection for pregnant women and their fetuses in Ceará and beyond.

Community standards to facilitate development and address challenges in metabolic modeling.

Standardization of data and models facilitates effective communication, especially in computational systems biology. However, both the development and consistent use of standards and resources remain challenging. As a result, the amount, quality, and format of the information contained within systems biology models are not consistent and therefore present challenges for widespread use and communication. Here, we focused on these standards, resources, and challenges in the field of constraint-based metabolic modeling by conducting a community-wide survey. We used this feedback to (i) outline the major challenges that our field faces and to propose solutions and (ii) identify a set of features that defines what a "gold standard" metabolic network reconstruction looks like concerning content, annotation, and simulation capabilities. We anticipate that this community-driven outline will help the long-term development of community-inspired resources as well as produce high-quality, accessible models within our field. More broadly, we hope that these efforts can serve as blueprints for other computational modeling communities to ensure the continued development of both practical, usable standards and reproducible, knowledge-rich models.

Predicting changes in renal metabolism after compound exposure with a genome-scale metabolic model.

The kidneys are metabolically active organs with importance in several physiological tasks such as the secretion of soluble wastes into the urine and synthesizing glucose and oxidizing fatty acids for energy in fasting (non-fed) conditions. Once damaged, the metabolic capability of the kidneys becomes altered. Here, we define metabolic tasks in a computational modeling framework to capture kidney function in an update to the iRno network reconstruction of rat metabolism using literature-based evidence. To demonstrate the utility of iRno for predicting kidney function, we exposed primary rat renal proximal tubule epithelial cells to four compounds with varying levels of nephrotoxicity (acetaminophen, gentamicin, 2,3,7,8-tetrachlorodibenzodioxin, and trichloroethylene) for six and twenty-four hours, and collected transcriptomics and metabolomics data to measure the metabolic effects of compound exposure. For the transcriptomics data, we observed changes in fatty acid metabolism and amino acid metabolism, as well as changes in existing markers of kidney function such as Clu (clusterin). The iRno metabolic network reconstruction was used to predict alterations in these same pathways after integrating transcriptomics data and was able to distinguish between select compound-specific effects on the proximal tubule epithelial cells. Genome-scale metabolic network reconstructions with coupled omics data can be used to predict changes in metabolism as a step towards identifying novel metabolic biomarkers of kidney function and dysfunction.

Medusa: Software to build and analyze ensembles of genome-scale metabolic network reconstructions.

Uncertainty in the structure and parameters of networks is ubiquitous across computational biology. In constraint-based reconstruction and analysis of metabolic networks, this uncertainty is present both during the reconstruction of networks and in simulations performed with them. Here, we present Medusa, a Python package for the generation and analysis of ensembles of genome-scale metabolic network reconstructions. Medusa builds on the COBRApy package for constraint-based reconstruction and analysis by compressing a set of models into a compact ensemble object, providing functions for the generation of ensembles using experimental data, and extending constraint-based analyses to ensemble scale. We demonstrate how Medusa can be used to generate ensembles and perform ensemble simulations, and how machine learning can be used in conjunction with Medusa to guide the curation of genome-scale metabolic network reconstructions. Medusa is available under the permissive MIT license from the Python Packaging Index (https://pypi.org) and from github (https://github.com/opencobra/Medusa), and comprehensive documentation is available at https://medusa.readthedocs.io/en/latest.

Transcriptome-guided parsimonious flux analysis improves predictions with metabolic networks in complex environments.

The metabolic responses of bacteria to dynamic extracellular conditions drives not only the behavior of single species, but also entire communities of microbes. Over the last decade, genome-scale metabolic network reconstructions have assisted in our appreciation of important metabolic determinants of bacterial physiology. These network models have been a powerful force in understanding the metabolic capacity that species may utilize in order to succeed in an environment. Increasingly, an understanding of context-specific metabolism is critical for elucidating metabolic drivers of larger phenotypes and disease. However, previous approaches to use network models in concert with omics data to better characterize experimental systems have met challenges due to assumptions necessary by the various integration platforms or due to large input data requirements. With these challenges in mind, we developed RIPTiDe (Reaction Inclusion by Parsimony and Transcript Distribution) which uses both transcriptomic abundances and parsimony of overall flux to identify the most cost-effective usage of metabolism that also best reflects the cell's investments into transcription. Additionally, in biological samples where it is difficult to quantify specific growth conditions, it becomes critical to develop methods that require lower amounts of user intervention in order to generate accurate metabolic predictions. Utilizing a metabolic network reconstruction for the model organism Escherichia coli str. K-12 substr. MG1655 (iJO1366), we found that RIPTiDe correctly identifies context-specific metabolic pathway activity without supervision or knowledge of specific media conditions. We also assessed the application of RIPTiDe to in vivo metatranscriptomic data where E. coli was present at high abundances, and found that our approach also effectively predicts metabolic behaviors of host-associated bacteria. In the setting of human health, understanding metabolic changes within bacteria in environments where growth substrate availability is difficult to quantify can have large downstream impacts on our ability to elucidate molecular drivers of disease-associated dysbiosis across the microbiota. Our results indicate that RIPTiDe may have potential to provide understanding of context-specific metabolism of bacteria within complex communities.

History of antibiotic adaptation influences microbial evolutionary dynamics during subsequent treatment.

Antibiotic regimens often include the sequential changing of drugs to limit the development and evolution of resistance of bacterial pathogens. It remains unclear how history of adaptation to one antibiotic can influence the resistance profiles when bacteria subsequently adapt to a different antibiotic. Here, we experimentally evolved Pseudomonas aeruginosa to six 2-drug sequences. We observed drug order-specific effects, whereby adaptation to the first drug can limit the rate of subsequent adaptation to the second drug, adaptation to the second drug can restore susceptibility to the first drug, or final resistance levels depend on the order of the 2-drug sequence. These findings demonstrate how resistance not only depends on the current drug regimen but also the history of past regimens. These order-specific effects may allow for rational forecasting of the evolutionary dynamics of bacteria given knowledge of past adaptations and provide support for the need to consider the history of past drug exposure when designing strategies to mitigate resistance and combat bacterial infections.

Reconciling high-throughput gene essentiality data with metabolic network reconstructions.

The identification of genes essential for bacterial growth and survival represents a promising strategy for the discovery of antimicrobial targets. Essential genes can be identified on a genome-scale using transposon mutagenesis approaches; however, variability between screens and challenges with interpretation of essentiality data hinder the identification of both condition-independent and condition-dependent essential genes. To illustrate the scope of these challenges, we perform a large-scale comparison of multiple published Pseudomonas aeruginosa gene essentiality datasets, revealing substantial differences between the screens. We then contextualize essentiality using genome-scale metabolic network reconstructions and demonstrate the utility of this approach in providing functional explanations for essentiality and reconciling differences between screens. Genome-scale metabolic network reconstructions also enable a high-throughput, quantitative analysis to assess the impact of media conditions on the identification of condition-independent essential genes. Our computational model-driven analysis provides mechanistic insight into essentiality and contributes novel insights for design of future gene essentiality screens and the identification of core metabolic processes.

Gram-negative Microbiota Blooms in Premature Twins Discordant for Parenteral Nutrition-associated Cholestasis.

Parenteral nutrition-associated cholestasis (PNAC) causes serious morbidity in the neonatal intensive care unit. Infection with gut-associated bacteria is associated with cholestasis, but the role of intestinal microbiota in PNAC is poorly understood. We examined the composition of stool microbiota from premature twins discordant for PNAC as a strategy to reduce confounding from variables associated with both microbiota and cholestasis. Eighty-four serial stool samples were included from 4 twin sets discordant for PNAC. Random Forests was utilized to determine genera most discriminatory in classifying samples from infants with and without PNAC. In infants with PNAC, we detected a significant increase in the relative abundance of Klebsiella, Veillonella, Enterobacter, and Enterococcus (P < 0.05). Bray-Curtis dissimilarities in infants with PNAC were significantly different (P < 0.05) from infants without PNAC. Our findings warrant further exploration in larger cohorts and experimental models of PNAC to determine if a microbiota signature predicts PNAC, as a basis for future interventions to mitigate liver injury.