Attacking the Global Problem of Antimicrobial Resistance

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Project Summary Besides the impact on health care, the economic impact of antimicrobial resistance is significant. More than two million infections a year are caused by bacteria that are resistant to at least first-line antibiotics, costing the US health care system 20 billion dollars each year. Pseudomonas aeruginosa (PA) causes over 51,000 health care associated infections per year in the US, of which 13% are multi-drug resistant (MDR). Non-antibiotic approaches to prevent and treat bacterial infections provide attractive alternatives/adjuncts to antibiotics no longer effective against MDR PA isolates. One of these, glycyrrhizin (GLY), an extract from the licorice root (Glycyrrhiza glabra), is effective in treatment of animal models of sepsis, colitis, lung and brain injury; and is used in the clinical management of chronic hepatitis. After infection, GLY reduces extracellularly released high mobility group box 1 (HMGB1), a prototypic alarmin that activates cell surface innate immune receptors affecting host inflammatory responses. GLY has direct antimicrobial effects in PA experimental keratitis induced by non-MDR clinical (keratitis) isolates (e.g., KEI 1025) and by MDR9, a non-ocular isolate. Preliminary/recently published data support that in MDR9 induced PA keratitis, GLY: a) permeabilizes bacterial membranes, b) reduces efflux pump activity, increases bacterial killing and d) combined with Ciprofloxacin in vivo, reduces the neutrophil infiltrate and plate count optimally. Therefore, our overarching hypothesis is that GLY is non-toxic to the cornea and ocular adnexa and after PA infection, binds to HMGB1 preventing activation of innate immune receptors. To test this hypothesis two Specific aims are proposed. Specific Aim 1: Tests the hypothesis that GLY is well-tolerated and does not alter the cytoarchitectue nor the physiological parameters of the normal cornea, conjunctiva and ocular adnexa. In this aim, we will test the effects of GLY given topically on the uninfected, normal cornea, and if intraocular pressure (IOP), tear volume, corneal nerve pattern, corneal sensitivity, resident immune cells and/or conjunctiva are altered. Specific Aim 2: Tests the hypothesis that GLY's protective effect on PA keratitis is mediated by inhibiting HMGB1 amplification of TLR4/RAGE signaling pathways in myeloid cells. This aim will test if GLY binding to HMGB1, inhibits HMGB1-TLR4 (-RAGE) interactions and signaling pathways, with downstream effects on immature myeloid dendritic cell (DC) maturation, macrophage production of proinflammatory cytokines and chemokines and neutrophil infiltration and function. Aim 2a will test this hypothesis in GLY treated mice after infection with KEI1025, a non-MDR PA keratitis isolate using TLR4 and RAGE KO mice and myeloid specific HMGB1 KO mice. Aim 2b is translational and will test this hypothesis in mice in which GLY treatment is combined with Moxifloxacin and initiated 18h after infection with MDR PA.