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At least 523 records · Page 29

The Martini 3 Lipidome: Expanded and Refined Parameters Improve Lipid Phase Behavior

Lipid membranes are central to cellular life. Complementing experiments, computational modeling has been essential in unraveling complex lipid-biomolecule interactions, crucial in both academia and industry. The Martini model, a coarse-grained force field for efficient molecular dynamics simulations, is widely used to study membrane phenomena but has faced limitations, particularly in capturing realistic lipid phase behavior. Here, we present refined Martini 3 lipid models with a mapping scheme that distinguishes lipid tails that differ by just two carbon atoms, enhancing the structural resolution and thermodynamic accuracy of model membrane systems including ternary mixtures. The expanded Martini lipid library includes thousands of models, enabling simulations of complex and biologically relevant systems. These advancements establish Martini as a robust platform for lipid-based simulations across diverse fields.

Lipids↗

Spaceflight Microbiology: Benefits for Long Duration Spaceflight and Our Understanding of Microorganisms on Earth

Spaceflight microbiology is composed of both operational and experimental components that complement each other in our understanding of microbial interactions and their responses in the microgravity of spaceflight. Operationally, efforts to mitigate microbiological risk to the crew and the spacecraft have historically focused on minimizing the number of detectable organisms, relying heavily on preventative measures, including appropriate vehicle design, crew quarantine prior to flight, and stringent microbial monitoring. Preflight monitoring targets have included the astronauts, spaceflight foods, potable water systems, the vehicle air and surfaces, and the cargo carried aboard the spacecraft. This approach has been very successful for earlier missions; however, the construction and long-term habitation of the International Space Station (ISS) has created the need for additional inflight monitoring of the environment and potable water systems using hardware designed for both in-flight microbial enumeration and sample collection and return to Earth. In addition to operational activities, the ISS is providing a research platform to advance our understanding of microbiomes in the built environment. Adding to the research possibilities of this system are multiple reports of unique changes in microbial gene expression and phenotypic responses, including virulence and biofilm formation, in response to spaceflight culture. The tremendous potential of the ISS research platform led the National Research Council to recommend that NASA utilize the ISS as a microbial observatory. Collectively, the findings from operational and research activities on the ISS are expected to both enable future space exploration and translate to basic and applied research on Earth.

Ott, C. Mark↗

Microbial polyphenol metabolism is part of the thawing permafrost carbon cycle

Abstract With rising global temperatures, permafrost carbon stores are vulnerable to microbial degradation. The enzyme latch theory states that polyphenols should accumulate in saturated peatlands due to diminished phenol oxidase activity, inhibiting resident microbes and promoting carbon stabilization. Pairing microbiome and geochemical measurements along a permafrost thaw-induced saturation gradient in Stordalen Mire, a model Arctic peatland, we confirmed a negative relationship between phenol oxidase expression and saturation but failed to support other trends predicted by the enzyme latch. To inventory alternative polyphenol removal strategies, we built CAMPER, a gene annotation tool leveraging polyphenol enzyme knowledge gleaned across microbial ecosystems. Applying CAMPER to genome-resolved metatranscriptomes, we identified genes for diverse polyphenol-active enzymes expressed by various microbial lineages under a range of redox conditions. This shifts the paradigm that polyphenols stabilize carbon in saturated soils and highlights the need to consider both oxic and anoxic polyphenol metabolisms to understand carbon cycling in changing ecosystems.

54 ENVIRONMENTAL SCIENCES↗

An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity

Plasticity in gene expression allows bacteria to adapt to diverse environments. This is particularly relevant in the dynamic niche of the human intestinal tract; however, transcriptional networks remain largely unknown for gut-resident bacteria. Here we apply differential RNA sequencing (RNA-seq) and conventional RNA-seq to the model gut bacterium Bacteroides thetaiotaomicron to map transcriptional units and profile their expression levels across 15 in vivo-relevant growth conditions. We infer stress- and carbon source-specific transcriptional regulons and expand the annotation of small RNAs (sRNAs). Integrating this expression atlas with published transposon mutant fitness data, we predict conditionally important sRNAs. These include MasB, which downregulates tetracycline tolerance. Using MS2 affinity purification and RNA-seq, we identify a putative MasB target and assess its role in the context of the MasB-associated phenotype. These data—publicly available through the Theta-Base web browser (http://micromix.helmholtz-hiri.de/bacteroides/)—constitute a valuable resource for the microbiome community.

59 BASIC BIOLOGICAL SCIENCES↗

Extreme environments and exobiology

Ecological research on extreme environments can be applied to exobiological problems such as the question of life on Mars. If life forms (fossil or extant) are found on Mars, their study will help to solve fundamental questions about the nature of life on Earth. Extreme environments that are beyond the range of adaptability of their inhabitants are defined as "absolute extreme". Such environments can serve as terrestrial models for the last stages of life in the history of Mars, when the surface cooled down and atmosphere and water disappeared. The cryptoendolithic microbial community in porous rocks of the Ross Desert in Antarctica and the microbial mats at the bottom of frozen Antarctic lakes are such examples. The microbial communities of Siberian permafrost show that, in frozen but stable communities, long-term survival is possible. In the context of terraforming Mars, selected microorganisms isolated from absolute extreme environments are considered for use in creation of a biological carbon cycle.

NASA Discipline Exobiology↗

The ecological assembly of bacterial communities in Antarctic wetlands varies across levels of phylogenetic resolution

Summary As functional traits are conserved at different phylogenetic depths, the ability to detect community assembly processes can be conditional on the phylogenetic resolution; yet most previous work quantifying their influence has focused on a single level of phylogenetic resolution. Here, we have studied the ecological assembly of bacterial communities from an Antarctic wetland complex, applying null models across different levels of phylogenetic resolution (i.e. clustering ASVs into OTUs with decreasing sequence identity thresholds). We found that the relative influence of the community assembly processes varies with phylogenetic resolution. More specifically, selection processes seem to impose stronger influence at finer (100% sequence similarity ASV) than at coarser (99%–97% sequence similarity OTUs) resolution. We identified environmental features related with the ecological processes and propose a conceptual model for the bacterial community assembly in this Antarctic ecosystem. Briefly, eco‐evolutionary processes appear to be leading to different but very closely related ASVs in lotic, lentic and terrestrial environments. In all, this study shows that assessing community assembly processes at different phylogenetic resolutions is key to improve our understanding of microbial ecology. More importantly, a failure to detect selection processes at coarser phylogenetic resolution does not imply the absence of such processes at finer resolutions.

59 BASIC BIOLOGICAL SCIENCES↗

Machine Learning of All Mycobacterium tuberculosis H37Rv RNA-seq Data Reveals a Structured Interplay between Metabolism, Stress Response, and Infection

Mycobacterium tuberculosis is one of the most consequential human bacterial pathogens, posing a serious challenge to 21st century medicine. A key feature of its pathogenicity is its ability to adapt its transcriptional response to environmental stresses through its transcriptional regulatory network (TRN). While many studies have sought to characterize specific portions of the M. tuberculosis TRN, and some studies have performed system-level analysis, few have been able to provide a network-based model of the TRN that also provides the relative shifts in transcriptional regulator activity triggered by changing environments. Here, we compiled a compendium of nearly 650 publicly available, high quality M. tuberculosis RNA-sequencing data sets and applied an unsupervised machine learning method to obtain a quantitative, top-down TRN. It consists of 80 independently modulated gene sets known as “iModulons,” 41 of which correspond to known regulons. These iModulons explain 61% of the variance in the organism’s transcriptional response. We show that iModulons (i) reveal the function of poorly characterized regulons, (ii) describe the transcriptional shifts that occur during environmental changes such as shifting carbon sources, oxidative stress, and infection events, and (iii) identify intrinsic clusters of regulons that link several important metabolic systems, including lipid, cholesterol, and sulfur metabolism. This transcriptome-wide analysis of the M. tuberculosis TRN informs future research on effective ways to study and manipulate its transcriptional regulation and presents a knowledge-enhanced database of all published high-quality RNA-seq data for this organism to date.

59 BASIC BIOLOGICAL SCIENCES↗

Property space mapping of Pseudomonas aeruginosa permeability to small molecules

Two membrane cell envelopes act as selective permeability barriers in Gram-negative bacteria, protecting cells against antibiotics and other small molecules. Significant efforts are being directed toward understanding how small molecules permeate these barriers. In this study, we developed an approach to analyze the permeation of compounds into Gram-negative bacteria and applied it to Pseudomonas aeruginosa, an important human pathogen notorious for resistance to multiple antibiotics. The approach uses mass spectrometric measurements of accumulation of a library of structurally diverse compounds in four isogenic strains of P. aeruginosa with varied permeability barriers. We further developed a machine learning algorithm that generates a deterministic classification model with minimal synonymity between the descriptors. This model predicted good permeators into P. aeruginosa with an accuracy of 89% and precision above 58%. The good permeators are broadly distributed in the property space and can be mapped to six distinct regions representing diverse chemical scaffolds. We posit that this approach can be used for more detailed mapping of the property space and for rational design of compounds with high Gram-negative permeability.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Mapping microhabitats of lignocellulose decomposition by a microbial consortium

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Identification of carbohydrate gene clusters obtained from in vitro fermentations as predictive biomarkers of prebiotic responses

Prebiotic fibers are non-digestible substrates that modulate the gut microbiome by promoting expansion of microbes having the genetic and physiological potential to utilize those molecules. Although several prebiotic substrates have been consistently shown to provide health benefits in human clinical trials, responder and non-responder phenotypes are often reported. These observations had led to interest in identifying, a priori, prebiotic responders and non-responders as a basis for personalized nutrition. In this study, we conducted in vitro fecal enrichments and applied shotgun metagenomics and machine learning tools to identify microbial gene signatures from adult subjects that could be used to predict prebiotic responders and non-responders. Using short chain fatty acids as a targeted response, we identified genetic features, consisting of carbohydrate active enzymes, transcription factors and sugar transporters, from metagenomic sequencing of in vitro fermentations for three prebiotic substrates: xylooligosacharides, fructooligosacharides, and inulin. A machine learning approach was then used to select substrate-specific gene signatures as predictive features. These features were found to be predictive for XOS responders with respect to SCFA production in an in vivo trial. Our results confirm the bifidogenic effect of commonly used prebiotic substrates along with inter-individual microbial responses towards these substrates. We successfully trained classifiers for the prediction of prebiotic responders towards XOS and inulin with robust accuracy (≥ AUC 0.9) and demonstrated its utility in a human feeding trial. Overall, the findings from this study highlight the practical implementation of pre-intervention targeted profiling of individual microbiomes to stratify responders and non-responders.

59 BASIC BIOLOGICAL SCIENCES↗

Rapid Design and Engineering of Smart and Secure Microbiological Systems (Final Report)

The design and application of successfully engineered biosystems requires an understanding of how engineered microbes will interact with other organisms – either as one-on-one competitors or in the context of microbial consortia. Engineering microorganisms from first principles for non-laboratory, environmental applications is inherently challenging because: (1) engineered systems tend to quickly revert back to their wild-type behaviors; and (2) these systems typically pay a price in reduced fitness, making them uncompetitive against invasive contaminating species (i.e., metabolic burden). For this project, we used a synthetic biology-based strategy to investigate the organization, control, stabilization, and destabilization of natural and engineered microbes. This approach enabled development of (1) single-strain systems capable of detecting and responding to target organisms in the environment; (2) a pipeline for refining and engineering biological constructs in new, non-model host organisms; and (3) improved systems for rapidly designing, engineering, and assaying new biological modules. This coupled approach to safeguard system design is predictable and portable across bacterial species and is focused on microbes that are part of the beneficial plant microbiome. A long-term goal beyond the proposed research is to enable the rational engineering of microbial communities based on first principles of biological design that mimic the smart performance of microorganisms observed in natural systems.

59 BASIC BIOLOGICAL SCIENCES↗

Exploration of a Potential Desirability of Outcome Ranking Endpoint for Complicated Intra-Abdominal Infections Using 9 Registrational Trials for Antibacterial Drugs

Abstract Background Desirability of outcome ranking (DOOR) is a novel approach to clinical trial design that incorporates safety and efficacy assessments into an ordinal ranking system to evaluate overall outcomes of clinical trial participants. Here, we derived and applied a disease-specific DOOR endpoint to registrational trials for complicated intra-abdominal infection (cIAI). Methods Initially, we applied an a priori DOOR prototype to electronic patient-level data from 9 phase 3 noninferiority trials for cIAI submitted to the US Food and Drug Administration between 2005 and 2019. We derived a cIAI-specific DOOR endpoint based on clinically meaningful events that trial participants experienced. Next, we applied the cIAI-specific DOOR endpoint to the same datasets and, for each trial, estimated the probability that a participant assigned to the study treatment would have a more desirable DOOR or component outcome than if assigned to the comparator. Results Three key findings informed the cIAI-specific DOOR endpoint: (1) a significant proportion of participants underwent additional surgical procedures related to their baseline infection; (2) infectious complications of cIAI were diverse; and (3) participants with worse outcomes experienced more infectious complications, more serious adverse events, and underwent more procedures. DOOR distributions between treatment arms were similar in all trials. DOOR probability estimates ranged from 47.4% to 50.3% and were not significantly different. Component analyses depicted risk-benefit assessments of study treatment versus comparator. Conclusions We designed and evaluated a potential DOOR endpoint for cIAI trials to further characterize overall clinical experiences of participants. Similar data-driven approaches can be utilized to create other infectious disease–specific DOOR endpoints.

Immunology↗

Hyperactive nanobacteria with host-dependent traits pervade Omnitrophota

Abstract Candidate bacterial phylum Omnitrophota has not been isolated and is poorly understood. We analysed 72 newly sequenced and 349 existing Omnitrophota genomes representing 6 classes and 276 species, along with Earth Microbiome Project data to evaluate habitat, metabolic traits and lifestyles. We applied fluorescence-activated cell sorting and differential size filtration, and showed that most Omnitrophota are ultra-small (~0.2 μm) cells that are found in water, sediments and soils. Omnitrophota genomes in 6 classes are reduced, but maintain major biosynthetic and energy conservation pathways, including acetogenesis (with or without the Wood-Ljungdahl pathway) and diverse respirations. At least 64% of Omnitrophota genomes encode gene clusters typical of bacterial symbionts, suggesting host-associated lifestyles. We repurposed quantitative stable-isotope probing data from soils dominated by andesite, basalt or granite weathering and identified 3 families with high isotope uptake consistent with obligate bacterial predators. We propose that most Omnitrophota inhabit various ecosystems as predators or parasites.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic dependencies govern microbial syntrophies during methanogenesis in an anaerobic digestion ecosystem

Methanogenesis, a biological process mediated by complex microbial communities, has attracted great attention due to its contribution to global warming and potential in biotechnological applications. The current study unveiled the core microbial methanogenic metabolisms in anaerobic vessel ecosystems by applying combined genome-centric metagenomics and metatranscriptomics. Here, we demonstrate that an enriched natural system, fueled only with acetate, could support a bacteria-dominated microbiota employing a multi-trophic methanogenic process. Moreover, significant changes, in terms of microbial structure and function, were recorded after the system was supplemented with additional H 2 . Methanosarcina thermophila, the predominant methanogen prior to H 2 addition, simultaneously performed acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis. The methanogenic pattern changed after the addition of H 2 , which immediately stimulated Methanomicrobia-activity and was followed by a slow enrichment of Methanobacteria members. Interestingly, the essential genes involved in the Wood-Ljungdahl pathway were not expressed in bacterial members. The high expression of a glycine cleavage system indicated the activation of alternative metabolic pathways for acetate metabolism, which were reconstructed in the most abundant bacterial genomes. Moreover, as evidenced by predicted auxotrophies, we propose that specific microbes of the community were forming symbiotic relationships, thus reducing the biosynthetic burden of individual members. These results provide new information that will facilitate future microbial ecology studies of interspecies competition and symbiosis in methanogenic niches.

59 BASIC BIOLOGICAL SCIENCES↗

The 6-minute walk test predicts mortality in a pulmonary nontuberculous mycobacteria-predominant bronchiectasis cohort

Background: Bronchiectasis is a chronic lung condition frequently associated with nontuberculous mycobacteria pulmonary (NTM) disease. Persons with these conditions are at increased risk of mortality. Patient reported outcome (PRO) instruments and the 6-minute walk test (6MWT) have been shown to predict mortality for several lung conditions, but these measures have not been fully evaluated for bronchiectasis and NTM. Methods: We conducted a retrospective cohort study among adult patients enrolled in a natural history study of bronchiectasis at the National Heart, Lung, and Blood Institute. Electronic medical records were queried for demographic, clinical, microbiologic, radiographic, and PRO instrument data: St. George’s Respiratory Questionnaire (SGRQ), Medical Research Council Dyspnea Scale, and the Pulmonary Symptom Severity Score (PSSS). The study baseline date was defined as the patient’s first visit after January 1st, 2015 with a SGRQ or 6MWT completed. Follow-up was defined as the interval between the study baseline visit and date of death or December 31st, 2019. Sex-stratified Cox proportional-hazards regression was conducted to identify predictors of mortality. Separate models were run for each PRO and 6MWT measure, controlling for age, body mass index (BMI), fibrocavitary disease status, and M. abscessus infection. Results: In multivariable Cox proportional-hazards regression models, the PSSS-severity (aHR 1.29, 95% CI 1.04–1.59), the 6MWT total distance walked (aHR 0.938, 95% CI 0.896–0.981) and distance saturation product (aHR 0.930, 95% CI 0.887–0.974) independently predicted mortality. In addition, BMI was significantly predictive of mortality in all models. Conclusions: The 6MWT and a PRO instrument capturing symptom severity are independently predictive of mortality in our cohort of bronchiectasis patients.

6-minute walk test↗

From Earth to Space: Application of Biological Treatment for the Removal of Ammonia from Water

Managing ammonia is often a challenge in both drinking water and wastewater treatment facilities. Ammonia is unregulated in drinking water, but its presence may result in numerous water quality issues in the distribution system such as loss of residual disinfectant, nitrification, and corrosion. Ammonia concentrations need to be managed in wastewater effluent to sustain the health of receiving water bodies. Biological treatment involves the microbiological oxidation of ammonia to nitrate through a two‐step process. While nitrification is common in the environment, and nitrifying bacteria can grow rapidly on filtration media, appropriate conditions, such as the presence of dissolved oxygen and required nutrients, need to be established. This presentation will highlight results from two ongoing research programs - one at NASA's Johnson Space Center, and the other at a drinking water facility in California. Both programs are designed to demonstrate nitrification through biological treatment. The objective of NASA's research is to be able to recycle wastewater to potable water for spaceflight mission. To this end, a biological water processor (BWP) has been integrated with a forward osmosis secondary treatment system (FOST). Bacteria mineralize organic carbon to carbon dioxide as well as ammonia‐nitrogen present in the wastewater to nitrogen gas, through a combination of nitrification and denitrification. The effluent from the BWP system is low in organic contaminants, but high in total dissolved solids. The FOST system, integrated downstream of the BWP, removes dissolved solids through a combination of concentration‐driven forward osmosis and pressure driven reverse osmosis. The integrated system testing planned for this year is expected to produce water that requires only a polishing step to meet potable water requirements for spaceflight. The pilot study in California is being conducted on Golden State Water Company's Yukon wellsthat have hydrogen sulfide odor, color, total organic carbon, bromide, iron and manganese in addition to ammonia. A treatment evaluation, conducted in 2011, recommended the testing of biological oxidation filtration for the removal of ammonia and production of biologically stable water. A 8‐month pilot testing program was conducted to develop and optimize key design and operational variables. Steadystate operational data was collected to demonstrate long‐term performance and inform California Department of Public Health permitting of the full‐scale process. As ammonia continues to present challenges to water and wastewater systems, innovative strategies such as biological treatment can be applied to successfully manage it. This presentation will discuss application of cutting‐age research being conducted by NASA that will bridge existing information gaps, and benefit municipal utilities.

Ghosh, Amlan↗

From Earth to Space: Application of Biological Treatment for the Removal of Ammonia from Water

Managing ammonia is often a challenge in both drinking water and wastewater treatment facilities. Ammonia is unregulated in drinking water, but its presence may result in numerous water quality issues in the distribution system such as loss of residual disinfectant, nitrification, and corrosion. Ammonia concentrations need to be managed in wastewater effluent to sustain the health of receiving water bodies. Biological treatment involves the microbiological oxidation of ammonia to nitrate through a two‐step process. While nitrification is common in the environment, and nitrifying bacteria can grow rapidly on filtration media, appropriate conditions, such as the presence of dissolved oxygen and required nutrients, need to be established. This presentation will highlight results from two ongoing research programs - one at NASA's Johnson Space Center, and the other at a drinking water facility in California. Both programs are designed to demonstrate nitrification through biological treatment. The objective of NASA's research is to be able to recycle wastewater to potable water for spaceflight missions. To this end, a biological water processor (BWP) has been integrated with a forward osmosis secondary treatment system (FOST). Bacteria mineralize organic carbon to carbon dioxide as well as ammonia‐nitrogen present in the wastewater to nitrogen gas, through a combination of nitrification and denitrification. The effluent from the BWP system is low in organic contaminants, but high in total dissolved solids. The FOST system, integrated downstream of the BWP, removes dissolved solids through a combination of concentration‐driven forward osmosis and pressure driven reverse osmosis. The integrated system testing planned for this year is expected to produce water that requires only a polishing step to meet potable water requirements for spaceflight. The pilot study in California is being conducted on Golden State Water Company's Yukon wells that have hydrogen sulfide odor, color, total organic carbon, bromide, iron and manganese in addition to ammonia. A treatment evaluation, conducted in 2011, recommended the testing of biological oxidation filtration for the removal of ammonia and production of biologically stable water. An 8‐month pilot testing program was conducted to develop and optimize key design and operational variables. Steadystate operational data was collected to demonstrate long‐term performance and inform California Department of Public Health permitting of the full‐scale process. As ammonia continues to present challenges to water and wastewater systems, innovative strategies such as biological treatment can be applied to successfully manage it. This presentation will discuss application of cutting‐age research being conducted by NASA that will bridge existing information gaps, and benefit municipal utilities.

Pickering, Karen↗

Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

59 BASIC BIOLOGICAL SCIENCES↗