HIGH-THROUGHPUT FUNCTIONAL CHARACTERIZATION OF SOIL MICROBIOTA

We are applying functional metagenomic selections to interrogate diverse soil metagenomes for (i) their complement of antibiotic resistance genes and (ii) functional elements that confer resistance to key toxins involved in biofuel production. 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. Our work on shotgun capture of biomass toxin remediation genes enables our goal of engineering hardier microbial biofuel production hosts, allowing improved yields and increased production efficiencies.

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 associated functions between humans, animals, and the broader environment in rural and urban shanty town communities in El Salvador and Peru. Using 16S ribosomal DNA sequencing and high-throughput functional metagenomic assays we are investigating sources of resistance genes and various factors, both internal and external, that influence dissemination patterns into the human gut microbiota. Because of marked differences in lifestyle and environmental conditions compared to industrialized nations, 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.

QUANTIFY THE IMPACTS OF ANTIBIOTIC THERAPY ON PEDIATRIC MICROBIOME DIVERSITY AND FUNCTION

Although the bacteria that live in the gut are known to be an important reservoir of antibiotic resistance genes, little is known about why some host-associated microbial communities harbor clinically important resistance genes but others do not. 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 genomics techniques including 16S rRNA sequencing, shotgun metagenome sequencing, and high throughput functional metagenomic assays.

SYSTEMS MODELING OF EVOLUTIONARY FITNESS LANDSCAPES USING BI-FUNCTIONAL ANTIBIOTIC RESISTANCE GENES

Adaptive evolution of antibiotic resistance genes under stringent selections provide an ideal system for modeling evolutionary fitness landscapes at a population level. We are studying such fitness landscapes by measuring the relative fitness of mutant libraries of bi-functional antibiotic resistance genes, with combination drug gradients and massively-parallel, indexed, next-generation sequencing.

MECHANISTIC DESIGN OF SYNERGISTIC ANTIBIOTIC COMBINATIONS

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. 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.

COMPUTATIONAL TOOLS FOR NGS ANALYSIS OF FUNCTIONAL METAGENOMICS

To improve our understanding of microbial community function, we are developing several computational tools tailored to the large datasets generated in the lab experimentally. One such tool is PARFuMS, a pipeline for the de-novo assembly and annotation of functionally-selected metagenomic DNA inserts. PARFuMS enables multiplexed processing of functional metagenomic selections at less than 1/100th the cost of traditional methods. Once assembled, metagenomic DNA inserts selected for antibiotic resitance funciton are annotated using the Resfams database of profile HMMs, confirmed for antibiotic resistance function and organized by ontology.

NEXT GENERATION SEQUENCING CLINICAL DIAGNOSTICS

Next Generation Sequencing (NGS) technologies show great potential to continue increasing in speed and decreasing in cost to the point that they will become viable options for diagnosing infectious disease. Leveraging our extensive experience in identifying novel as well as known antibiotic resistance genes, we are developing new software, Diagnostics for Resistance Utilizing Genome Sequences (DRUGS), which can predict antibiotic susceptibility profiles for multidrug resistant pathogens from draft genome sequences. Our software will be implemented in improving diagnostics and standard clinical treatment of urinary tract infections (UTI) by providing a holistic bacterial community approach. We plan to develop and validate an innovative, culture-independent, UTI diagnostic platform through integration of NGS methods, DRUGS, and microbial ecological and evolutionary analyses.

ENGINEERING MICROORGANISMS TO IMPROVE BIOFUEL-PRODUCING PROPERTIES

To make biofuels and value-added chemicals sustainably, lignocellulosic biomass must be utilized efficiently. Working with teams of engineers and chemists at Washington University, we are bringing together systems biology, thermochemistry, metabolic engineering and genomics to understand how a biofuel-producing bacterium metabolizes toxic compounds derived from biomass. We are investigating systems level changes during directed evolution of this non-model organism using whole genome sequencing and transcriptomic analysis. We are also working toward developing a functional selection in E. coli to "mine" novel enzymes from hard-to-culture bacteria and to engineer terpene synthases to generate a wide variety of useful bioactive compounds and biofuels.

ENGINEERING ENHANCED PROBIOTICS TO INCREASE GUT COLONIZATION

Clinical trials of probiotics have demonstrated positive outcomes when administered continuously, however after treatment is terminated, probiotics are cleared from the gut microbiota in less than three weeks in the majority of patients. The established adult microbiota excludes news species through a variety of mechanisms, and has therefore limited probiotic efficacy to short-term use and reduced their ability to treat chronic gastrointestinal diseases. We aim to engineer enhanced probiotics capable of colonizing the adult gut microbiota by using in-vivo functional metagenomics to identify genes that allow probiotic species to persist in the gut microbiota.