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Synthetic Biology to Discover New ESBL-PE-Eradicating Microbiome-Based Therapeutics

Medical complications related to bacteria resistant to multiple antibiotics are a major issue in modern healthcare due to the increased morbidity, mortality, length of hospitalization, and related healthcare costs. Infections caused by multidrug-resistant bacteria from the family Enterobacteriaceae, such as extended-spectrum ß-lactamase producing Escherichia coli (ESBL-PE) are of critical concern for military personnel deployed in developing countries as colonization rates are high, and they may result in severe complicated wound and systemic infections as well as refractory urinary tract infections in female Service members. Infections caused by ESBL-producing organisms have, in fact, been associated with poor clinical outcomes, reduced rates of clinical and microbiological responses, longer hospital stays, and greater hospital expenses. Multiple outbreaks of ESBL-PE and increased rates of illness and death, especially in intensive care units (ICUs) have also been reported. Because ESBL-PE asymptomatically colonize the gastrointestinal (GI) tract after which they opportunistically lead to potentially life-threatening and treatment-challenging infections, development of interventions for reliable eradication of ESBL-PE from the GI compartment would be of high value for maintaining Warfighter health and minimizing transmission and risk of infection in military hospitals. While rational supplementation of the GI microbiota with defined sets of probiotics aimed at ensuring robust ESBL-PE decolonization could revolutionize how we manage multidrug-resistant infections, it is still unknown what bacteria and encoded functions are necessary to achieve it.

In recent work, investigators at the University of Massachusetts Dartmouth pioneered the first microbiome dynamical modeling toolset allowing to in silico discover and rationally optimize microbiome assemblies maximizing a desired clinical outcome (e.g., GI decolonization of a specific enteric pathogen). In addition to this computer-guided approach to microbiome engineering, they also pioneered the first set of synthetically engineered, single-strain probiotics that are capable of killing a number of drug-resistant Enterobacteriaceae in vitro, through the overproduction of an array of cathecol IIb microcins, small antimicrobial peptides that bacteria evolved to use as chemical weapons during conditions of stress such as nutrient starvation.

Leveraging these preliminary data and a collaborative effort with world-renown infectious disease scientists at University of Nevada Reno and at the Naval Research Laboratory, in the studies of this proposal, the team will develop and use novel computational modelling and synthetic biology approaches to identify and prototype in animal models combinations of bacteria and underlying mechanisms enabling robust and precise decolonization of ESBL-PE from the GI tract. Specifically, in the first aim of the project the team will use microbiota abundance DNA sequencing data from experiments with mice administered with ESBL-PE first and then dosed with different combinations of GI bacteria from healthy human donors to train a computational simulator that accurately captures this system’s temporal dynamics. The team will then use this simulator to predict what subsets of these human-derived bacteria are optimal in achieving ESBL-PE GI eradication. The team will then validate these findings back using the same animal model of ESBL-PE colonization and identify what microbial functions underlie potent decolonization ability by developing new genome-based, community model of these communities.

In the second aim of the project, the team will use a complementary approach and leverage their published and preliminary data showing the ability of single-strain probiotics that have been genetically engineered to overproduce anti-ESBL-PE molecules in killing ESBL-PE in vitro. Specifically, after optimizing the genetic architecture for overproduction of these molecules and after integrating it in the genome of U.S. Food and Drug Administration (FDA)-approved probiotic strains, the team will validate the ability of these engineered bacteria in decolonizing ESBL-PE in vivo.

As one of the motivations of this work is to develop the next-generation of GI microbiome engineering strategies that are more targeted compared to traditional broad-spectrum antibiotics, the team will perform animal experiments where they will evaluate the effect of the discovered bacteriotherapeutics on the host resident microbiota and immunity. The anticipated outcome is that these bacterial therapeutics will be capable of effective pathogen eradication and produce minimal effects on the host.

These studies  will leverage and extend the cutting-edge work that distinguishes our research in analytics and synthetic biology for probiotic discovery and optimization. Upon completion of this project, we will have: (1) discovered and validated in preclinical settings different combinations of healthy human-derived bacterial consortia that optimally prevent colonization by and eradication of ESBL-PE from the GI tract and (2) developed and validated in preclinical settings new single-strain, microcin-overproducing, genetically engineered bacteria for prevention and eradication of ESBL-PE. These studies will represent the first assessment of how host and resident microbiota respond to the developed bacteriotherapeutics. The results will give the Department of Defense (DoD) and industry partners a strong positive signal to encourage further microbiome bacteriotherapeutics development in support of FDA licensure and acquisition for use in Force health protection strategies in the DoD. Furthermore, these studies will provide new strategies to tackle multidrug-resistant bacteria threat to complement develop vaccines and best management practices.