Phage Predation as a Determinant of Diarrheal Disease Severity and Antimicrobial Resistance Emergence

SUMMARY The interaction between virulent bacteriophage (predators), bacterial pathogens (prey) and antibiotics is complex and governs disease severity and antibiotic resistance emergence in known and unknown ways. As antimicrobial resistance (AMR) expands, there is a growing need to better understand these interactions to effectively combat this public health crisis while not compromising care at the level of individual patients. This challenge is daunting among diarrheal diseases because inappropriate use of antibiotics is high, global annual case incidence is in the billions, and mobile genetic elements with multi-antibiotic resistance gene cassettes are common. Using cholera as a model, we discovered that 'effective' phage predation was associated with decreased disease severity and increased pathogen genetic diversity. 'Ineffective' phage predation was associated with increased disease severity and increased phage genetic diversity. These effects were modulated by phage resistance factors that are genetically linked with antibiotic resistance genes on a mobile element, a situation likely common among enteric pathogens. In this proposal, our objectives are to validate this discovery in a new cohort of cholera patients, test our underlying theory on effective phage predation for generalization among high-priority enteric pathogens (Shigella spp., Enterotoxigenic Escherichia coli (ETEC)), and determine if phage predation is also associated with AMR emergence within patients. To pursue these objectives, and associated hypotheses, we will leverage two clinical and laboratory resources we gathered through recent NIH and Wellcome Trust funded research: (i) a cohort study of over 2000 patients in Bangladesh who were assessed through highly accurate and standardized clinical measures of disease severity, and (ii) a time-series study of 500 patients pre/post antibiotic exposure in Bangladesh who were clinically assessed through similar means. In these collections now housed in the USA, the targets (Vibrio cholerae, Shigella spp., ETEC) are at rates amenable to testing our hypotheses. Samples will be analyzed in a high-throughput analytic pipeline that we developed for pathogen detection, mass spectrometry for antibiotic detection, and microbial characterization via metagenomic and single-cell sequencing. In Aim 1, we will identify and characterize dominant bacteriophages associated with each infection. In Aim 2, we will test if phage and antibiotic exposures are determinants of disease severity, pathogen genetic diversity and AMR emergence. In Aim 3, we will derive and validate predictive models for disease severity and AMR emergence that are inclusive to antibiotic and phage exposures. This proposal will generate a new framework to evaluate diarrheal disease severity and antimicrobial resistance emergence in a way that is inclusive to the complex interactions between phage, pathogens and antibiotics. The framework, and predictive models, will have applied value for clinicians, scientists and policy makers as they navigate the intended and unintended consequences of antibiotic therapy and prophylaxis today, and possibly phage therapy and prophylaxis tomorrow.