MICROBIAL ECOLOGY

Microbes are the most ubiquitous lifeforms on Earth. They are found across all habitats studied to date, including the bodies of every living thing (including humans), as well terrestrial, subterranean, and aquatic environments. Microbes collectively represent one of the largest reservoirs of biomass, estimated to account for 350-550 Petagrams (1 Pg = 10^15 grams = 1 billion tons) of carbon, 85-130 Pg of nitrogen, and 9-14 Pg of phosphorous. Their diverse biochemical and metabolic activities impact and control nearly all aspects of biotic and abiotic processes on the planet. In virtually all cases, microbes live and work in complex ecosystems composed of incredibly diverse taxonomic lineages. We take a quantitative ecological perspective in our study of diverse microbial communities, with a focus on human associated microbiota and interconnected environmental habitats. Accordingly, one of our major goals is to understand and quantitatively predict the effects of anthropogenic interventions (e.g., antibiotics, diet, and hospitalization) on microbial community composition and function. A few of our recent and ongoing efforts in the realm of microbial ecology are described below:

  • Functional ecology of soil resistomes. The soil microbiome is an ancient and diverse reservoir of microbial antibiotic resistance genes. We apply a combination of functional metagenomic selections, 16S phylogenetic profiling, and whole metagenome shotgun sequencing to interrogate the reservoir of functional antibiotic resistance genes (the ‘resistome’) encoded in diverse soil metagenomes. Our studies on the soil resistome are designed to model the evolution of antibiotic resistance genes, and to quantifiably evaluate the impact of various anthropogenic practices on the exchange of resistance genes between the soil microbiota and other microbes, including human pathogens.  We provided the first genetic evidence for resistome exchange between benign cultured soil multidrug resistant (MDR) Proteobacteria [Dantas, Sommer et al. Science 2008] [Press] and MDR human pathogens [Forsberg, Reyes et al. Science 2012] [Press]. In contrast, we demonstrated that the uncultured majority of the soil resistome is structured by phylogeny and habitat and is not in recent exchange with human pathogens [Forsberg, Patel et al. Nature 2014] [Press].

Bacteria Subsisting on Antibiotics. Dantas, Sommer et al. Science 2008.

Bacteria Subsisting on Antibiotics. Dantas, Sommer et al. Science 2008.

The Shared Antibiotic Resistome of Soil Bacteria and Human Pathogens. Forsberg, Reyes et al. Science 2012

The Shared Antibiotic Resistome of Soil Bacteria and Human Pathogens. Forsberg, Reyes et al. Science 2012

Bacterial Phylogeny Structures Soil Resistomes Across Habitats. Forsberg, Patel et al. Nature 2014

Bacterial Phylogeny Structures Soil Resistomes Across Habitats. Forsberg, Patel et al. Nature 2014

  • Pediatric microbiome and resistome. Principles governing microbiota assembly and dynamics are best studied in infancy. Bacteria colonize infants’ sterile guts soon after birth. The composition of resulting bacterial communities fluctuates strikingly until about three years of age, when stable adult-like communities emerge. Disruption of normal community development during infancy can permanently alter its composition. Aberrant compositions of intestinal microbiota are implicated in various pathologies both in infancy (e.g., necrotizing enterocolitis and late onset sepsis) and in adulthood (e.g., asthma, allergies, and obesity). These clinical correlates of early dysfunctional microbial colonization illustrate the importance of understanding dynamics of infant gut microbial communities. Bacterial functions governing response to environmental perturbations are intrinsic to understanding their community dynamics. Hence, a focus on community function and resistomes is particularly relevant for investigating adaptive dynamics and genetic exchange in microbial populations. We focus our study on the gut microbiome and resistome during the first years of human life because (i) antibiotics are among the most frequently (and often inappropriately) prescribed medications in children, (ii) antibiotics significantly perturb microbiota diversity and function, (iii) antibiotic-induced changes in infant microbiota might persist into adulthood, and (iv) infancy accompanies numerous transitions in environment and diet during a time of microbiome dynamicity. Early research from our lab revealed that gut microbiota in infants harbor novel and diverse antibiotic resistance genes [Moore et al. PLOS One 2013] [Press]. We expanded this work to a cohort of HIV exposed, but uninfected infants who are treated with prophylactic cotrimoxazole during the first year of life. We showed that resistance gene prevalence and diversity increased with cotrimoxazole treatment versus placebo control [D’Souza et al. Clinical Infectious Diseases 2019]. This work has motivated our interest in illuminating why certain antibiotic treatments have greater effects on the microbiome and resistome. 

Pediatric Fecal Microbiota Harbor Diverse and Novel Antibiotic Resistance Genes. Moore et al. PLOS ONE 2013

Pediatric Fecal Microbiota Harbor Diverse and Novel Antibiotic Resistance Genes. Moore et al. PLOS ONE 2013

  • Quantification of the impacts of antibiotic therapy and diet on pediatric microbiome diversity and function. We are currently studying the impact of various early childhood exposures on the developing gut microbiota and associated antibiotic resistance genes using a combination of microbiological and omics techniques [Gibson, Crofts et al. Current Opinion in Microbiology 2015]. We have correlated breastfeeding, formula composition, and maternal weight with distinct microbiome profiles. Furthermore, we identified key functional pathways for short-chain fatty acid utilization enriched in the microbiome when infants consume soy formula instead of milk-based formula [Baumann-Dudenhoeffer et al. Nature Medicine 2018]. These findings suggest host-metabilic mutualism during early life that we reasoned could be interrupted by early life antibiotics. Accordingly, we have analyzed longitudinal samples and clinical data from both preterm infants and healthy infants to compare resistomes with their microbial community structures [Moore et al. Microbiome 2015]. We found that the abundant use of antibiotics in the premature infants results in acute perturbations of the premature infant gut microbiota, including decreases in species richness and enrichment of antibiotic resistance genes and multidrug resistant Escherichia, Klebsiella, and Enterobacter genera [Gibson et al. Nature Microbiology 2016] [Press]. Additionally, antibiotic treatment during time in the neonatal intensive care unit leads to persistent enrichment of antibiotic resistance genes and prolonged carriage of multidrug resistant organisms over one year after discharge [Gasparrini et al. Nature Microbiology 2019].  

Developmental dynamics of the preterm infant gut microbiota and antibiotic resistome. Gibson et al. Nature Microbiology 2016

Developmental dynamics of the preterm infant gut microbiota and antibiotic resistome. Gibson et al. Nature Microbiology 2016

Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant gut microbiota and resistome. Gasparrini et al. Nature Microbiology 2019.

Persistent metagenomic signatures of early-life hospitalization and antibiotic treatment in the infant gut microbiota and resistome. Gasparrini et al. Nature Microbiology 2019.

  • Transmission dynamics of antibiotic resistance genes in rural and urban communities in Latin America. We are studying the composition and exchange of microbial communities and their resistomes between humans, animals, and the broader environment in rural and urban shanty town communities in El Salvador and Peru. This work is motivated by the recognition that antibiotic resistance is one of most urgent global public health challenges, and yet most studies to date on the commensal human microbiota and resistome have focused on either industrialized or remote hunter-gatherer communities, which represent the extremes of the global human population. In contrast, our Salvadoran and Peruvian study sites represent resource-limited, low-income settings in which over two-thirds of the world’s population lives. Because of marked differences in lifestyle and environmental conditions compared to industrialized nations and remote hunter-gatherer communities, as well as the high potential for microbial exchange between individuals and their environment, these communities provide an ideal setting in which to study the exchange and transmission dynamics of microbial communities and their associated resistomes. Our first publication on these two large-scale projects was recently featured on the cover of Nature [Pehrsson, Tsukayama et al. Nature 2016]. We characterized the bacterial community structure and resistance exchange networks of 263 fecal samples from 115 individuals in 27 houses over two years from our Salvador village and Peruvian slum study sites, as well as 209 environmental samples from donor households and surrounding environments, including the local sewage treatment systems. We found that resistomes across habitats are generally structured by bacterial phylogeny along ecological gradients, but identified key antibiotic resistance genes that cross habitat boundaries. We also identified chicken coops in the Salvadoran village and the waste-water sewage treatment plant outside the Peruvian slum as hotspots for antibiotic resistance gene enrichment and transmission between humans and the environment. This work lays the foundation for real-time molecular surveillance of drug-resistant microbes and their resistance genes, and informs the design of public health interventions to decrease their global enrichment and dissemination [Press].

Interconnected microbiomes and resistomes in low-income human habitats. Pehrsson EC, Tsukayama P et al. Nature 2016

Interconnected microbiomes and resistomes in low-income human habitats. Pehrsson EC, Tsukayama P et al. Nature 2016

Additional ecology projects:

  • Functional metagenomics and microbial characterization of dairy farm soils, manure, dairy cows and farm workers to asses transmission of antibiotic resistance genes

  • Nonhuman primates as models for evolution of human gut microbiota [Tsukayama, Boolchandani et al. mSystems 2018]

  • Competitive selection of near-isogenic strains expressing different resistance mechanisms to the same antibiotic

  • Microbial community assembly and pathogen-resistance of root and soil samples from the model plant Arabidopsis thaliana

  • Threat assessment of the human microbiota as a reservoir for human pathogens of the Enterobacteriaceae to acquire new antibiotic resistance genes

  • Characterizing the role of local skin microbiota in post-operative infections following breast reconstructive surgery

  • Examining the impact of travel to regions with high infection and resistance burdens on individuals’ resistomes

  • Novel antibiotic bioremediation activity in soil and gut microbiota

  • Transmission dynamics of Staphylococcus intermedi group (SIG) between humans and companion animals

TRANSLATIONAL MICROBIOLOGY

While many microbes are beneficial (or at least benign) to humans, some microbes are pathogenic, and contribute to human suffering and mortality. Antibiotics are our primary therapeutics against the infectious diseases caused by these pathogens. Unfortunately, resistance to these life-saving drugs has steadily increased in pathogens since the first wide-scale discovery and deployment of antibiotics in the 1930s. Concurrently, the pipeline of new antibiotics coming to market has slowed. Some pathogens are now resistant to almost all clinically used antibiotics, raising specter of a post-antibiotic era where treatment options for common infectious agents are severely curtailed or non-existent. Drug resistant infections currently claim hundreds of thousands of lives worldwide, and they are estimated to add over 35 billion dollars in healthcare costs annually in the US alone. We urgently require novel therapies for drug resistant infections, as well as an improved understanding of ways to detect and curb the evolution of new resistance. A parallel motivation for novel microbial therapeutics is the maintenance of healthy human microbiota states and rescue from perturbed states (for instance, those caused by overuse of antibiotics in agriculture and the clinic). The thousands of years of co-evolution of humans and their resident microbiota has resulted in a delicate ecological balance which provides benefits to both the host and the trillions of microbes that live in and on the body. Disruption of this balance has been hypothesized to lead to several human pathologies which may parallel the burden of infectious diseases, including aberrant immune responses (e.g., asthma and allergies), gastrointestinal disorders (e.g., inflammatory bowel disease), and cancer. Accordingly, one of our major goals is to evaluate and design novel diagnostics and therapies for maintaining healthy human commensal microbial communities and defeating pathogenic microbes. Several of our recent and ongoing efforts in translational microbiology are described below:

  • Shared strategies for β-lactam catabolism in the soil microbiome. The soil microbiome can produce, resist, or degrade antibiotics and even catabolize them. While resistance genes are widely distributed in the soil, there is a dearth of knowledge concerning antibiotic catabolism. We found a pathway for penicillin catabolism in bacteria. Genomic and transcriptomic sequencing revealed β-lactamase, amidase, and phenylacetic acid catabolon upregulation. Knocking out part of the phenylacetic acid catabolon or an apparent penicillin utilization operon (put) resulted in loss of penicillin catabolism in one bacterial isolate. A hydrolase from the put operon was found to degrade in vitro benzylpenicilloic acid, the β-lactamase penicillin product. To test the generality of this strategy, an Escherichia coli strain was engineered to co-express a β-lactamase and a penicillin amidase or the put operon, enabling it to grow using penicillin or benzylpenicilloic acid, respectively. Elucidation of additional pathways may allow bioremediation of antibiotic-contaminated soils and discovery of antibiotic-remodeling enzymes with industrial and therapeutic utility [Crofts et al. Nature Chemical Biology 2018].

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  • Spatiotemporal dynamics of multidrug resistant bacteria. Bacterial pathogens that infect patients also contaminate hospital surfaces. These contaminants impact hospital infection control and epidemiology, prompting quantitative examination of their transmission dynamics. We investigated the spatiotemporal and phylogenetic relationships of multidrug resistant (MDR) bacteria on intensive care unit surfaces from two hospitals in the United States (US) and Pakistan collected over one year. While the US surfaces were relatively, clean, the Pakistani surfaces almost all yielded MDR bacteria, including common nosocomial pathogens, rare opportunistic pathogens, and novel taxa. Many resistance genes (e.g., blaNDM, blaOXA carbapenamases), were shared by multiple species and flanked by mobilization elements. We identified Acinetobacter baumannii and Enterococcus faecium co-association on multiple surfaces, and demonstrated these species establish synergistic biofilms in vitro. Our results highlighted substantial MDR pathogen burdens in hospital built-environments, provided evidence for spatiotemporal-dependent transmission, and demonstrated potential mechanisms for multispecies surface persistence [D’Souza, Potter et al. Nature Communications 2019].

Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces. D’Souza, Potter et al. Nature Communications 2019.

Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces. D’Souza, Potter et al. Nature Communications 2019.

  • In silico analysis of Gardnerella genomospecies detected in the setting of bacterial vaginosis. Gardnerella vaginalis is implicated as 1 of the causative agents of bacterial vaginosis, but it can also be isolated from the vagina of healthy women. This discrepancy may be due to genomic variation. In fact, G. vaginalis may be multiple species. We performed comprehensive taxonomic and phylogenomic analysis to quantify the number of species called G. vaginalis, the similarity of their core genes, and their burden of their accessory genes. We additionally analyzed publicly available metatranscriptomic data sets of bacterial vaginosis to determine whether the newly delineated genomospecies are present and to identify putative conserved features of Gardnerella pathogenesis. Our consensus classification identified 9 different Gardnerella genomospecies. The genomospecies could be readily distinguished by the phylogeny of their shared genes and burden of accessory genes. We identified all of these genomospecies in metatranscriptomes of bacterial vaginosis. Multiple Gardnerella genomospecies operating in isolation or in concert with one another may be responsible for bacterial vaginosis. These results have important implications for future efforts to understand the evolution of the Gardnerella genomospecies, host– pathogen interactions of the genomospecies during bacterial vaginosis, diagnostic assay development for bacterial vaginosis, and metagenomic investigations of the vaginal microbiota [Potter et al. Clinical Chemistry 2019].

In silico analysis of Gardnerella genomospecies detected in the setting of bacterial vaginosis. Potter et al. Clinical Chemistry 2019.

In silico analysis of Gardnerella genomospecies detected in the setting of bacterial vaginosis. Potter et al. Clinical Chemistry 2019.

  • Genomic analysis of the evolution and spread of Carbapenem-Resistant Enterobacteriaceae (CREs) across distinct geographies. The similarities in the spread and resistance spectra of KPC and NDM-1 (both provide resistance to nearly all β-lactam antimicrobial drugs) leads to the hypothesis that similar mobile elements will make both genes available to similar pathogen populations. We tested this hypothesis by sequencing 78 clinical Enterobacteriaceae isolates from Pakistan and the United States encoding NDM-1, KPC, or no carbapenemase. High similarities of the results indicate rapid spread of carbapenem resistance between strains, including globally disseminated pathogens. [Pesesky, Hussain et al. Emerging Infectious Diseases 2015] [Press].

KPC and NDM-1 Genes in Related Enterobacteriaceae Strains and Plasmids from Pakistan and the United States. Pesesky, Hussain et al. Emerging Infectious Diseases 2015.

KPC and NDM-1 Genes in Related Enterobacteriaceae Strains and Plasmids from Pakistan and the United States. Pesesky, Hussain et al. Emerging Infectious Diseases 2015.

  • Mechanistic design of antibiotic combinations that suppress development of resistance. We propose that effective therapies for defeating multi-drug resistant (MDR) pathogens can derive from combinations of drugs that target essential nodes in distinct biological processes. Synergistic drug combinations might overcome single agent problems with toxicity, spectrum, potency, and emerging resistance. By combining synergy with collateral sensitivity, in which resistance to one drug increases sensitivity to another, combinations can be found that also combat development of antibiotic resistance. We are using high-throughput robotic assays to identify novel drug combinations, and utilize forward and reverse genetic screens and whole-genome sequencing to elucidate the genetic and biochemical basis for the observed drug interactions. In our first implementation of this strategy, we found that the triple β-lactam combination of meropenem-piperacillin-tazobactam acts synergistically to kill methicillin-resistant Staphylococcus aureus (MRSA) both in vitro and in a murine model of aggressive MRSA infection, while also suppressing the evolution of new resistance [Gonzales et al. Nature Chemical Biology 2016] [Press].

Synergistic, collaterally sensitive β-lactam combinations suppress resistance in MRSA. Gonzales et al. Nature Chemical Biology 2016.

Synergistic, collaterally sensitive β-lactam combinations suppress resistance in MRSA. Gonzales et al. Nature Chemical Biology 2016.

  • Biochemical and structural characterization of novel antibiotic resistance and catabolic genes. We discovered a family of flavoenzymes in tetracycline selections of soil functional metagenomic libraries, termed the tetracycline destructases, which confer high-level tetracycline resistance in model pathogens [Forsberg et al. Chemistry & Biology 2015]. We showed that these genes, previously unrecognizable as resistance determinants, function by enzymatically inactivating tetracycline. Because this mechanism is distinct from the dominant tetracycline resistance mechanisms of efflux and ribosomal protection, we believe these genes may fill an empty niche in pathogens and are ripe for dissemination to the clinic. We are currently employing a multipronged structural, biochemical, and microbiologic approach to better understand and inhibit the mechanism of action of these enzymes. We recently found a small-molecule inhibitor that prevents degradation of tetracycline in vitro and restores the efficacy of tetracycline against bacteria expressing a tetracycline destructase. Furthermore, we established the structural and molecular basis for this observed inhibition [Park, Gasparrini et al. Nature Chemical Biology 2017] [Press] [News and Views]. Such inhibitors could be co-administered with tetracyclines in the future to restore tetracycline activity in the face of widespread resistance. In addition to designing and testing new inhibitors, we are also currently working to better understand the environmental evolutionary origins of the tetracycline destructases, and modeling their prevalence and risk of dissemination across diverse habitats. 

The Tetracycline Destructases: A Novel Family of Tetracycline-Inactivating Enzymes. Forsberg et al. Chemistry & Biology 2015.

The Tetracycline Destructases: A Novel Family of Tetracycline-Inactivating Enzymes. Forsberg et al. Chemistry & Biology 2015.

Additional translational projects:

  • Identifying gut microbiota states which are protective against or permissive to travelers’ diarrhea

  • Host-microbiome profiling to identify precursors for neonatal sepsis, and genomic analysis of suspected Borderline Oxacillin-Resistant Staphylococcus aureus (BORSA) isolates in a Neonatal Intensive Care Unit (NICU)

  • Transmission dynamics and host-microbial determinants of Acinetobacter disease

  • Assessing the fitness landscape of a novel family of tetracycline-inactivating enzymes

  • Characterization of long-term within-host evolution of Nontuberculous Mycobacteria (NTM) in chronically infected patients

  • Antibiotic resistance gene profiling of Bacteroides fragilis Group (BFG) clinical isolates

  • Gut microbial determinants of C. difficile infection (CDI) and transmission

  • Multi-omic characterization of host and microbial etiologies of necrotizing enterocolitis (NEC)

  • Genetic epidemiology of resistance-conferring mobile genetic elements in home and hospital environments

  • Characterization of the urinary tract microbiome of Men Who Have Sex with Men (MSM)—a unique population at increased risk for genitourinary diseases

  • Transcriptional analysis of highly-related methicillin-resistant Staphylococcus aureus (MRSA) strains with wide-ranging susceptibilities to antibiotic combination therapies

  • Genomic, transcriptomic, and lipidomic characterization of in vitro and in vivo evolved high-level daptomycin in clinical pathogens

MICROBIAL ENGINEERING

As a complement to our ecological and translational work on understanding microbial community functions, we aim to engineer microbial platforms for novel industrial and therapeutic applications. We focus specifically on (i) improving bacteria-mediated biofuel production, and (ii) developing engineered microbes for diagnosis and delivery of biologics to the gut against disease. One of the reasons that microbes are ubiquitous on our planet is their ability to metabolize and biochemically modify virtually all available organic substrates. Their capacity to exist in complex communities, often in association with living hosts, is enabled by the incredible diversity of small molecules they produce that modulate intercellular interactions, both cooperative and offensive. Indeed, most antibiotics used in the clinic are natural products, or corresponding semi-synthetic derivatives, of soil-dwelling bacteria. Microbes also have the capacity to produce high-value industrial compounds such as bioplastics and biofuels from renewable substrates, raising the prospect of biologically derived alternatives to non-renewable, unsustainable, fossil-based compounds like plastics and petroleum. In a separate vein, engineered commensal microbes are exciting platforms for in situ drug synthesis and delivery, as well as diagnostics. As living therapeutics, they can interact with and respond to the human microbiome in ways that traditional drugs cannot, but as such they replicate in the host and are therefore subject to natural selection.  With this in mind our work with engineered therapeutic microbes has two aims. First, we aim to develop commensal yeast and bacterial strains that can detect and respond to infectious and metabolic disease. In parallel, we aim to understand the selective pressures that affect the fitness and drive adaptation of these commensal microbes in the host, so to better predict their behavior and safety profiles in the contexts of health and disease.  A few of our recent and ongoing efforts in the realm of Microbial Engineering are described below: 

  • Improving biofuel-producing bacteria: Accordingly, one of our major goals is to optimize bacteria-mediated biofuel production. Towards this end the efficiency with which lignocellulose is converted into biofuel must be improved to make the industrial production of plant-based energy economically viable. Presently, microbial catalysis of plant material to fuel is limited by the toxicity of lignocellulosic byproducts of biomass pretreatment and the biofuel products themselves. In previous work, we coupled functional selection with next-generation sequencing to discover genes from soil microbiomes that confer tolerance against many of the toxins associated with lignocellulosic biofuel production [Forsberg et al. Applied and Environmental Microbiology 2016], which can be engineered into lignocellulosic strains to improve yields and increase production efficiencies for biofuels. Our current work focuses on Rhodococcus opacus, a soil bacterium that natively catabolizes aromatics such as phenolic compounds found in depolymerized lignin to accumulate biofuel precursors. These aromatics are common degradation products from lignin but to date are considered a waste byproduct of biofuel production instead of useable substrate due to their inherent heterogeneity and toxicity. The co-consumption of mixtures of lignin-derived aromatic monomers is an underexplored area of study that is important towards the valorization of lignin in biofuel production and therefore its overall economic profitability. We adaptively evolved R. opacus on 32 combinations of lignin model compounds and identified an optimal strain (PVHG6) that achieved a 225% increase in lipid production when grown on combination of aromatics as the sole carbon source [Henson, Campbell et al. Metabolic Engineering 2018]. Using comparative genomics and transcriptomics, we identified mutations in pathways involved in redox reactions and upregulation of aromatic funneling pathways, e.g to the β-ketoadipate pathway, in response to specific aromatic compounds, and confirmed the role of the β-ketoadipate pathway in gene knockout experiments. By understanding how adapted strains of R. opacus utilize higher concentrations of lignin model compounds, we aim to further develop R. opacus strains as a part of future biorefineries. 

Identification of genes conferring tolerance to lignocellulose-derived inhibitors by functional selections in soil metagenomes. Forsberg KJ et al. Applied and Environmental Microbiology 2015.

Identification of genes conferring tolerance to lignocellulose-derived inhibitors by functional selections in soil metagenomes. Forsberg KJ et al. Applied and Environmental Microbiology 2015.

Activation of β-ketoadipate pathway gene clusters by lignin model compounds in R. opacus. Henson, Campbell et al. Metabolic Engineering 2018.

Activation of β-ketoadipate pathway gene clusters by lignin model compounds in R. opacus. Henson, Campbell et al. Metabolic Engineering 2018.

  • Genetically engineered probiotics. Escherichia coli Nissle 1917 (EcN) is a commensal bacterium that has been clinically evaluated as a probiotic and is an attractive platform for engineered therapies due to its genetic tractability and demonstrated safety. We have engineered and continue to optimize EcN for the treatment of phenylketonuria, a metabolic disease in which mutations in the phenylalanine hydroxylase gene hinders degradation of the amino acid phenylalanine, leading to neurocognitive, and we have demonstrated efficacy in a murine model of phenylketonuria. Additional applications we are developing for EcN include delivery of biologics against helminthiasis, immunotherapies against colorectal cancer, and bacteriophages against infectious disease, as well as various biocontainment (‘killswitch’) modules. We are also interested in the adaptive strategies of EcN in the mammalian gut impairment [Crook et al. Cell Host and Microbe 2019]. We used in vivo functional selections as well as whole genome sequencing of in situ adapted EcN isolates to understand the selective forces that drive fitness in the gut for this promising therapeutic platform. We identified improved utilization of mucin-derived carbon sources as an important driver of fitness in the context of low microbiome diversity, a pressure that is relieved in more complex gut microbiomes that include species that degrade dietary polysaccharides to constituent mono- and disaccharides, EcN’s preferred carbon sources. Currently, we are assaying the fitness of EcN against clinically important pathogens and using transposon-mutagenesis and sequencing techniques to further understand the fitness of EcN in health and disease. We are also developing therapeutic applications in the commensal yeast Saccharomyces boulardii (Sb). As a yeast, Sb has the capacity to produce high titers of biologics, including those with more complex post-translational modifications, and platforms for protein display and secretion using Sb are well-studied. Applications we are currently pursuing in Sb include display or secretion of short chain variable fragments for detection or clearance of pathogens, delivery of immune checkpoint inhibitors, and delivery of such biologics in response to gut-relevant chemical signals such as oxygen concentration, pH, or concentration of bile salts or short chain fatty acids. Robust sensitivity to such signals will not only enable the targeted delivery of therapeutics but also open the door for a new type of living therapeutic that actually responds to the host and its environment. 

Phylogenies and phenotypic effects of nagC and gnat mutations. Crook et al. Cell Host and Microbe 2019.

Phylogenies and phenotypic effects of nagC and gnat mutations. Crook et al. Cell Host and Microbe 2019.

Additional engineering projects:

  • Using the probiotic yeast, Saccharomyces boulardii, to construct biosensors capable of detecting and responding in vivo biological stimuli

  • Construction of antimicrobial probiotics that target designated pathogens with high specificity

  • Engineering of probiotic bacteria and yeast to produce and target delivery of recombinant immunotherapy drugs to gastrointestinal tumor microenvironment

  • Identification of genes responsible for chlorophyll d synthesis to improve light harvesting ability of photosynthetic organisms.

  • Engineering improved biological conversion of lignin to next-generation biofuels.

  • Development of a terpene synthase selection system to mine useful terpene synthases from metagenomic sources

TECHNOLOGY DEVELOPMENT

Central to our diverse biological goals to understand and harness microbial community functions is a strong focus on technology development. We are particularly interested in technologies for microbial systems-level analyses and engineering, requiring the development and application of meta-omics methods and complementary computational tools to both analyze multi-scale data and build predictive models. Our published efforts have focused on methods for studying (meta)genomes and (meta)transcriptomes, and we are currently expanding our capacities in lipidomics, metabolic analyses, mechanistic protein biochemical and structural analyses, and conventional and gnotobiotic mouse husbandry and manipulation. One of our major goals is to develop novel high-throughput experimental and computational tools to study and modulate microbial communities. A few of our recent and ongoing efforts in the realm of Technology Development are described below:

  • We develop and apply a suite of complementary metagenomic methods to understand and engineer bacterial community composition and functional responses (e.g. antibiotic resistance) to xenobiotic perturbation (e.g. antibiotics) including 16S ribosomal gene sequencing, whole genome shotgun sequencing, and functional metagenomics.

  • Functional metagenomic selections identify novel resistance genes in uncultured microbiota. Metagenomic fragments are randomly ligated into plasmids and transformed into a culturable host, which is then subjected to selection from antibiotics. PARFuMS takes short-read sequence data from functional selections and applies a bioinformatics pipeline to annotate the functional resistance genes in metagenomic fragments [Forsberg, Reyes et al. Science 2012].

The Shared Antibiotic Resistome of Soil Bacteria and Human Pathogens. Forsberg, Reyes et al. Science 2012.

The Shared Antibiotic Resistome of Soil Bacteria and Human Pathogens. Forsberg, Reyes et al. Science 2012.

  • Resfams provides high-throughput annotation of sequence-novel antibiotic resistance determinants. It is a curated database of protein families and associated highly precise, accurate profile hidden Markov models (pHMMs), confirmed by the above methods for antibiotic resistance function and organized by ontology. Resfams is the most comprehensive and accurate resistance gene annotator to date. [Gibson et al. ISME Journal 2014].

Improved Annotation of Antibiotic Resistance Reveals Microbial Communities Cluster by Ecology. Gibson et al. ISME J 2015.

Improved Annotation of Antibiotic Resistance Reveals Microbial Communities Cluster by Ecology. Gibson et al. ISME J 2015.

High-specificity targeted functional profiling in microbial communities with ShortBRED. Kaminski et al. PLOS Computational Biology 2015.

High-specificity targeted functional profiling in microbial communities with ShortBRED. Kaminski et al. PLOS Computational Biology 2015.

Additional technology development projects:

  • Using functional metagenomics to mine novel terpene syntheses from soil metagenomes

  • Curating a database of functionally-selected and curated antibiotic resistance genes for isolate and metagenomic analyses

  • Development of engineering technologies for the gut microbiome

  • Development of novel methods to study and detect antifungal resistance

  • Development of improved annotators for virulence factor genes

  • Development of statistical models for predicting microbial community responses to xenobiotic perturbations