Engineering Antimicrobial Probiotics for the Treatment of Vancomycin-Resistant Enterococcus

University of Minnesota Ph.D. dissertation.December 2016. Major: Chemical Engineering. Advisor: Yiannis Kaznessis. 1 computer file (PDF); ix, 134 pages.The rise of antibiotic resistance in bacteria has become an urgent concern in global healthcare. There is now a strong drive for the preservation of our current antibiotics as well as for the rapid development of new antibacterial therapies. Antimicrobial peptides (AMPs) are a vast collection of proteins naturally produced by living organisms as a defense mechanism against invading microbes. Unfortunately, though society has been aware of the therapeutic potential of AMPs for many years, their utility has been limited to topical applications because of toxicity and degradation in the body. Moreover, many bacterial infections originate in the gastrointestinal (GI) tract, which is largely unreachable for most AMPs by oral administration. To overcome this delivery challenge, we are engineering probiotic bacteria that can actively produce and deliver AMPs inside the GI tract. Vancomycin-resistant enterococci (VRE) are among the most difficult to treat pathogens in hospital environments. These bacteria frequently reside in the GI tract, often in low counts because of competition from the surrounding microbiota. When patients are given broad-spectrum antibiotics, the competition is reduced and VRE dominate in numbers. The pathogen can then spread to other host organs or to the surrounding hospital environment. Delivery of AMPs targeting VRE by probiotics may provide an alternative treatment or prevention option against these deadly pathogens. Importantly, the elimination of VRE from the GI tract using VRE-specific AMPs may allow removal of VRE while minimizing disruption of the surrounding bacteria. In this thesis, we describe the development of two different probiotic delivery systems for the reduction of the two major causative species of VRE infections, Enterococcus faecium and Enterococcus faecalis. The first probiotic platform employs the Gram-positive species, Lactococcus lactis, for the production of three class IIa bacteriocins, AMPs endogenously produced by bacteria. We have developed a chloride-inducible expression vector for AMP delivery from L. lactis, which we show to be activated by physiologically-relevant chloride concentrations. Herein, we demonstrate the ability of this system to inhibit VRE, first in in vitro cultures. VRE intestinal colonization models in mice were then developed and used to test the efficacy of our engineered L. lactis in the GI tract. Multiple trials showed statistically significant reduction of Enterococcus faecium in L. lactis treated mice compared to untreated mice. The second probiotic delivery system uses probiotic Escherichia coli Nissle 1917 (EcN). Currently, no anti-enterococcal peptides are known to be naturally produced from E. coli. In this project, we developed a modular AMP expression system that can be used in E. coli to express and secrete a variety of AMPs derived from a wide range of producer strains. With this system, we are able to produce AMPs targeting not only Gram-positive pathogens like VRE, but also Gram-negative pathogens including Salmonella and diarrheagenic E. coli. We show this system can be used to simultaneously express multiple anti-enterococcal peptides in vitro. Lastly, we demonstrate the efficacy of Nissle producing Enterocin B, Enterocin A, and Hiracin JM79 in reducing VRE colonization in mice. The final section of this thesis addresses the concern of bacterial resistance development to our antimicrobial probiotics. Class IIa bacteriocins are currently the most thoroughly-studied class of AMPs targeting enterococci. Though the mechanism of resistance to these peptides has been studied in E. faecalis, it has never been examined in E. faecium. In this project, we identified a mannose phosphotransferase in E. faecium that appears to be directly involved in E. faecium susceptibility to class IIa bacteriocins. We show that resistant mutants exhibit either downregulation or direct mutation of this transporter and that heterologous expression of this transporter in L. lactis confers susceptibility to the otherwise unsusceptible strain. We then include a brief discussion of the implications of this mode of resistance and potential methods for preventing resistance development in the future