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) 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].
The resistome of uncontacted Amerindians. Pharmacologic-dose antibiotic exposure in humans is nearly ubiquitous worldwide, obscuring our knowledge of antibiotic resistance in commensal human microbiota in the pre-antibiotic era. To estimate the pre-modern human commensal resistome, we characterized the fecal and oral bacterial resistome of members of an isolated Yanomami Amerindian village with no documented previous contact with Western people and no known exposure to commercial antibiotics [Clemente, Pehrsson, et al. Science Advances 2015]. Surprisingly, we found that these antibiotic-naïve individuals harbored bacteria that carry functional resistance genes, including those that confer resistance to synthetic antibiotics and are syntenic with mobilization elements. These results suggest that antibiotic resistance is a natural feature of the human microbiome even in the absence of exposure to commercial antibiotics and that resistance genes are likely poised for mobilization and enrichment upon exposure to pharmacological levels of antibiotics. Our findings emphasize the need for extensive characterization of the commensal resistome of diverse people to inform the design and prudent deployment of antibiotics to minimize enrichment for preexisting resistance [Press].
- 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 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 resistomes is particularly relevant for investigating adaptive dynamics and genetic exchange in microbial populations. We focus our study on the gut 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, and (iii) antibiotic-induced changes in infant microbiota might persist into adulthood. 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]. This work has motivated our interest in illuminating why some host-associated microbial communities harbor clinically important resistance genes but others do not.
- Quantification of the impacts of antibiotic therapy 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 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] [Press]. 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].
- 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].
Additional ecology projects:
- Nonhuman primates as models for evolution of human gut microbiota
- Microbial community assembly within the roots of native vs. invasive plant species.
- Microbial community assembly and pathogen-resistance of transgenic model plants with boosted or suppressed immune systems
- Threat assessment of the human microbiota as a reservoir for human pathogens of the Enterobacteriaceae to acquire new antibiotic resistance genes
Novel antibiotic bioremediation activity in soil and gut microbiota
While a majority of microbes on our planet are beneficial (or at least benign) to humans, a small minority 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 these drugs in the 1930s, while the pipeline of new antibiotics coming to market has dramatically decreased over the past few decades. Some pathogens are now resistant to almost all of our current antibiotics, raising the dark prospect of a post-antibiotic era where we begin to succumb to common infectious agents. Drug resistant infections currently claim hundreds of thousands of lives worldwide, and are estimated to add over 35 billion dollars in healthcare costs annually in the US alone. We urgently require novel therapies to combat these threats, 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 states of the human microbiota, 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 a number of 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. A few of our recent and ongoing efforts in the realm of translational microbiology are described below:
- 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]
- 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]
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 tetracycline destructases, which confer high-level tetracycline resistance in model pathogens [Forsberg et al. Chemistry and 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.
Additional translational projects:
- Development of genomic-based antibiotic resistance clinical diagnostics
- Investigation of the effects of antibiotics on the gut microbiome for treatment of severe acute malnutrition in African children
- Investigation of the effects of long-term antibiotic therapy on the gut microbiome development of HIV-exposed infants in Africa
- High resolution profiling of microbial community structure in urinary tract infections.
- Microbiome and resistome response to autologous or allogeneic fecal microbiota transplants (FMTs) to treat C. difficile and reduce MDRO colonization
- Assessment of fitness landscapes in a novel bifunctional antibiotic resistance enzymes
- Identifying protective vs permissive microbiota states for symptomatic vs asymptomatic bacterial traveller's diarrhea
- Genomic, transcriptomic, and lipidomic characterization of in vitro and in vivo evolved high-level daptomycin in clinical pathogens
- Genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in hospital settings
- Comparative genomic epidemiology of surface transmission of MDROs in Intensive Care Units in Pakistan and the United States
As a complement to our ecological and translational focus on understanding microbial community functions, we aim to bioprospect for novel industrial and therapeutic applications. 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 other living hosts, is enabled by the incredible diversity of small molecules which they produce for modulating their intracellular interactions, both cooperative and offensive. Indeed, most antibiotics currently used in the clinic are natural products (or their semi-synthetic derivatives) of soil-dwelling bacteria. In addition to a source of therapeutic molecules, microbes also have the capacity to produce high-value industrial compounds (e.g. bioplastics, biofuels etc.) from renewable substrates, raising the prospect of biologically-derived alternatives to non-renewable, environmentally-unsustainable, fossil-based compounds (e.g. petroleum, plastics etc.). Accordingly, one of our major goals is to bioprospect and genetically engineer novel beneficial functions in microbes for biomedical and industrial applications. A few of our recent and ongoing efforts in the realm of Microbial Engineering are described below:
- Bioprospecting lignin-tolerance genes from soil microbiomes. The efficiency with which lignocellulose is converted into biofuel must be improved to make the industrial production of plant-based bioenergy economically sustainable. Presently, microbial catalysis of plant material to fuel is limited by the toxicity of many next-generation biofuels and lignocellulosic byproducts of biomass pretreatment. We have coupled functional selection with next generation sequencing based methods for interrogating resistance-conferring DNA fragments, and are applying this innovation towards the discovery of genes that confer tolerance against the many toxins associated with lignocellulosic biofuel production. We have captured many such tolerance genes from numerous soil microbiomes [Sommer et al. Molecular Systems BIology 2010] and are engineering hardier biofuel production hosts with broad-spectrum tolerance against key biofuel-associated inhibitors [Forsberg et al. Applied and Environmental Microbiology 2016]. Our work will allow improved yields and increased production efficiencies for biofuels.
- Improving biofuel-producing bacteria. Rhodococcus opacus naturally accumulates biodiesel precursors, and has the ability to tolerate and utilize phenolic compounds which are common degradation products from lignin (currently a wasted part of biomass). By understanding how phenol-adapted strains of R. opacus utilize higher concentrations of phenol, we aim to engineer R. opacus strains as a part of future biorefineries. [Yoneda, Henson et al. Nucleic Acids Research 2016]
- Improving probiotics for better colonization. Probiotics are typically cleared from the gut within weeks after treatment. Using our functional metagenomics approach, we aim to identify genes that make a probiotic strain of bacteria able to persist in the mammalian gut microbiota [Gibson, Pesesky et al. Journal of Molecular Biology 2014]
Additional engineering projects:
- 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
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]
- 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]
- ShortBRED facilitates inference of microbial community function from metagenomic sequencing data (in collaboration with Huttenhower lab), and is particularly useful for measuring the abundance of antibiotic resistance genes. [Kaminski, Gibson et al., PLOS Computational Biology 2015]
Additional technology development projects:
- 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