updated-protocol-for-assembly-use-minibioreactor-array

Updated Protocol for the Assembly and Use of the Minibioreactor Array (MBRA)

The human microbiome comprises diverse and dynamic microbial communities that play essential roles in host health. Understanding these communities and ...

Baylor College of Medicine

Hospital acquired Clostridioides (Clostridium) difficile infection is exacerbated by the continued evolution of C. difficile strains, a phenomenon studied by multiple laboratories using stock cultures specific to each laboratory. Intralaboratory evolution of strains contributes to interlaboratory variation in experimental results adding to the challenges of scientific rigor and reproducibility. To explore how microevolution of C. difficile within laboratories influences the metabolic capacity of an organism, three different laboratory stock isolates of the C. difficile 630 reference strain were whole-genome sequenced and profiled in over 180 nutrient environments using phenotypic microarrays. The results identified differences in growth dynamics for 32 carbon sources including trehalose, fructose, and mannose. An updated genome-scale model for C. difficile 630 was constructed and used to contextualize the 28 unique mutations observed between the stock cultures. The integration of phenotypic screens with model predictions identified pathways enabling catabolism of ethanolamine, salicin, arbutin, and N-acetyl-galactosamine that differentiated individual C. difficile 630 laboratory isolates. The reconstruction was used as a framework to analyze the core-genome of 415 publicly available C. difficile genomes and identify areas of metabolism prone to evolution within the species. Genes encoding enzymes and transporters involved in starch metabolism and iron acquisition were more variable while C. difficile distinct metabolic functions like Stickland fermentation were more consistent. A substitution in the trehalose PTS system was identified with potential implications in strain virulence. Thus, pairing genome-scale models with large-scale physiological and genomic data enables a mechanistic framework for studying the evolution of pathogens within microenvironments and will lead to predictive modeling to combat pathogen emergence.

Systems biology analysis of the Clostridioides difficile core-genome contextualizes microenvironmental evolutionary pressures leading to genotypic and phenotypic divergence.

Recent studies indicate that the human intestinal microbiota could impact the outcome of infection by  the etiological agent of the diarrheal disease cholera. A commensal bacterium,  was previously identified in high abundance in stool collected from individuals infected with  when compared to stool from uninfected persons. However, if and how  interacts with  has not been experimentally determined; moreover, whether any association between this bacterium alters the behaviors of  to affect the disease outcome is unclear. Here, we show that  and  together form dual-species biofilm structure at the air-liquid interface, with previously uncharacterized novel features. Importantly, the presence of  within the murine small intestine enhances  colonization in the same niche that is dependent on the  exopolysaccharide and other major components of mature  biofilm. These studies illustrate that multispecies biofilm formation is a plausible mechanism used by a gut microbe to increase the virulence of the pathogen, and this interaction may alter outcomes in enteric infections.

Impact of a human gut microbe on  host colonization through biofilm enhancement.

Sjögren syndrome (SS) is an autoimmune inflammatory disorder characterized by secretory dysfunction in the eye and mouth; in the eye, this results in tear film instability, reduced tear production, and corneal barrier disruption. A growing number of studies show that homeostasis of the ocular surface is impacted by the intestinal microbiome, and several 16S sequencing studies have demonstrated dysbiosis of the intestinal microbiota in SS patients. In this study, we utilized metagenomic sequencing to perform a deeper analysis of the intestinal microbiome using stools collected from sex- and age-matched healthy ( = 20), dry eye ( = 4) and SS ( = 7) subjects. The observed Operational Taxonomic Units (OTUs) and Shannon alpha diversity were significantly decreased in SS compared to healthy controls, and there was a significant inverse correlation between observed OTUs and ocular severity score. We also identified specific bacterial strains that are differentially modulated in SS vs. healthy subjects. To investigate if the differential composition of intestinal microbiome would have an impact on the immune and eye phenotype, we performed functional studies using germ-free mice colonized with human intestinal microbiota from SS patients and healthy controls. Flow cytometry analysis demonstrated reduced frequency of CD4 FOXP3 cells in ocular draining cervical lymph nodes (CLN) in mice colonized with SS patient intestinal microbiota 4 weeks post-colonization. We also found that offspring of SS-humanized mice also have fewer CD4FOXP3 cells in the CLN as well as spleen, demonstrating vertical transmission. SS-humanized mice subjected to desiccating stress exhibited greater corneal barrier disruption as compared to healthy control humanized mice under the same conditions. Taken together, these data support the hypothesis that the intestinal microbiota can modulate ocular surface health, possibly by influencing development of CD4 FOXP3 regulatory T cells (Tregs) in the ocular draining lymph nodes.

Gut Microbiota From Sjögren syndrome Patients Causes Decreased T Regulatory Cells in the Lymphoid Organs and Desiccation-Induced Corneal Barrier Disruption in Mice.

Combatting Clostridioides difficile infections, a dominant cause of hospital-associated infections with incidence and resulting deaths increasing worldwide, is complicated by the frequent emergence of new virulent strains. Here, we employ whole-genome sequencing, high-throughput phenotypic screenings, and genome-scale models of metabolism to evaluate the genetic diversity of 451 strains of C. difficile. Constructing the C. difficile pangenome based on this set revealed 9,924 distinct gene clusters, of which 2,899 (29%) are defined as core, 2,968 (30%) are defined as unique, and the remaining 4,057 (41%) are defined as accessory. We develop a strain typing method, sequence typing by accessory genome (STAG), that identifies 176 genetically distinct groups of strains and allows for explicit interrogation of accessory gene content. Thirty-five strains representative of the overall set were experimentally profiled on 95 different nutrient sources, revealing 26 distinct growth profiles and unique nutrient preferences; 451 strain-specific genome scale models of metabolism were constructed, allowing us to computationally probe phenotypic diversity in 28,864 unique conditions. The models create a mechanistic link between the observed phenotypes and strain-specific genetic differences and exhibit an ability to correctly predict growth in 76% of measured cases. The typing and model predictions are used to identify and contextualize discriminating genetic features and phenotypes that may contribute to the emergence of new problematic strains.

Systems biology approach to functionally assess the  pangenome reveals genetic diversity with discriminatory power.

Cross feeding between microbes is ubiquitous, but its impact on the diversity and productivity of microbial communities is incompletely understood. A reductionist approach using simple microbial communities has the potential to detect cross feeding interactions and their impact on ecosystem properties. However, quantifying abundance of more than two microbes in a community in a high throughput fashion requires rapid, inexpensive assays. Here, we show that multicolor flow cytometry combined with a machine learning-based classifier can rapidly quantify species abundances in simple, synthetic microbial communities. Our approach measures community structure over time and detects the exchange of metabolites in a four-member community of fluorescent  species. Notably, we quantified species abundances in co-cultures and detected evidence of cooperation in polysaccharide processing and competition for monosaccharide utilization. We also observed that co-culturing on simple sugars, but not complex sugars, reduced microbial productivity, although less productive communities maintained higher community diversity. In summary, our multicolor flow cytometric approach presents an economical, tractable model system for microbial ecology using well-studied human bacteria. It can be extended to include additional species, evaluate more complex environments, and assay response of communities to a variety of disturbances.

Tracking defined microbial communities by multicolor flow cytometry reveals tradeoffs between productivity and diversity.

 is a gram-positive spore-forming pathogen that commonly causes diarrheal infections in the developed world. Although  is a genetically diverse species, certain ribotypes are overrepresented in human infections. It is unknown if metabolic adaptations are essential for the emergence of these epidemic ribotypes. Here, we tested carbon substrate utilization by 88  isolates and looked for differences in growth between 22 ribotypes. By profiling clinical isolates, we assert that  is a generalist species capable of growing on a variety of carbon substrates. Further,  strains clustered by phylogenetic relationship and displayed ribotype-specific and clade-specific metabolic capabilities. Surprisingly, we observed that two emerging lineages, ribotypes 023 and 255, have divergent metabolic phenotypes. In addition, although  Clade 5 is the most evolutionary distant clade and often detected in animals, it displayed more robust growth on simple dietary sugars than Clades 1-4. Altogether, our results corroborate the generalist metabolic strategy of  and demonstrate lineage-specific metabolic capabilities. In addition, our approach can be adapted to the study of additional pathogens to ascertain their metabolic niches in the gut.

Emerging  ribotypes have divergent metabolic phenotypes.

Characterizing the dynamics of microbial community succession in the infant gut microbiome is crucial for understanding child health and development, but no normative model currently exists. Here, we estimate child age using gut microbial taxonomic relative abundances from metagenomes, with high temporal resolution (±3 months) for the first 1.5 years of life. Using 3154 samples from 1827 infants across 12 countries, we trained a random forest model, achieving a root mean square error of 2.56 months. We identified key taxonomic predictors of age, including declines in Bifidobacterium spp. and increases in Faecalibacterium prausnitzii and Lachnospiraceae. Microbial succession patterns are conserved across infants from diverse human populations, suggesting universal developmental trajectories. Functional analysis confirmed trends in key microbial genes involved in feeding transitions and dietary exposures. This model provides a normative benchmark of "microbiome age" for assessing early gut maturation that may be used alongside other measures of child development.

Early life microbial succession in the gut follows common patterns in humans across the globe.

 is a gram-positive spore-forming pathogen that commonly causes diarrheal infections in the developed world. Although  is a genetically diverse species, certain ribotypes are overrepresented in human infections, and it is unclear if metabolic adaptations are essential for the emergence of these epidemic ribotypes. To identify ribotype-specific metabolic differences, we therefore tested carbon substrate utilization by 88 .  isolates and looked for differences in growth between 22 ribotypes. As expected,  was capable of growing on a variety of carbon substrates. Further,  strains clustered by phylogenetic relationship and displayed ribotype-specific and clade-specific metabolic capabilities. Surprisingly, we observed that two emerging lineages, ribotypes 023 and 255, have divergent metabolic phenotypes. In addition, although  Clade 5 is the most evolutionary distant clade and often detected in animals, it displayed robust growth on simple sugars similar to Clades 1-4. Altogether, our results corroborate the generalist metabolic strategy of  but also demonstrate lineage-specific metabolic capabilities.IMPORTANCEThe gut pathogen  utilizes a wide range of carbon sources. Microbial communities can be rationally designed to combat  by depleting its preferred nutrients in the gut. However,  is genetically diverse with hundreds of identified ribotypes, and most of its metabolic studies were performed with lab-adapted strains. To identify ribotype-specific metabolic differences, we profiled carbon metabolism by a myriad of  clinical isolates. While the metabolic capabilities of these isolates clustered by their genetic lineage, we observed surprising metabolic divergence between two emerging lineages. We also found that genetically newer and older clades grew to a similar level on simple sugars, which contrasts with recent findings that newer clades experienced positive selection on genes involved in simple sugar metabolism. Altogether, our results underscore the importance of considering the metabolic diversity of pathogens in the study of their evolution and the rational design of therapeutic interventions.

Article	Year
Systems biology analysis of the Clostridioides difficile core-genome contextualizes microenvironmental evolutionary pressures leading to genotypic and phenotypic divergence. NPJ systems biology and applications\| PubMed ID: 33082337	2020
Impact of a human gut microbe on host colonization through biofilm enhancement. eLife\| PubMed ID: 35343438	2022
Gut Microbiota From Sjögren syndrome Patients Causes Decreased T Regulatory Cells in the Lymphoid Organs and Desiccation-Induced Corneal Barrier Disruption in Mice. Frontiers in medicine\| PubMed ID: 35355610	2022
Systems biology approach to functionally assess the pangenome reveals genetic diversity with discriminatory power. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 35476524	2022
Tracking defined microbial communities by multicolor flow cytometry reveals tradeoffs between productivity and diversity. Frontiers in microbiology\| PubMed ID: 36687598	2022
Emerging ribotypes have divergent metabolic phenotypes. bioRxiv : the preprint server for biology\| PubMed ID: 39185189	2024
Early life microbial succession in the gut follows common patterns in humans across the globe. Nature communications\| PubMed ID: 39809768	2025
Emerging ribotypes have divergent metabolic phenotypes. mSystems\| PubMed ID: 40013784	2025

Firas S. Midani

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