Phage predation and antibiotic exposure are inversely associated with disease severity and shape pathogen genetic diversity in cholera patients

Nearly a century has passed since d’Hérelle and colleagues discovered phages that infect Vibrio cholerae and influence the clinical and epidemiologic trajectories of cholera outbreaks. Despite an increasingly detailed picture of the molecular mechanisms of resistance and counter-resistance that shape phage and bacterial interactions, we lack an understanding of how these interactions play out during natural infection, how they are affected by antibiotic exposure, and how they relate to clinical outcomes. To fill these gaps, we conducted a nation-wide study of diarrheal disease patients in the cholera-endemic setting of Bangladesh. A total of 2574 stool samples were collected from enrolled patients at hospital admission and screened for V. cholerae and virulent phages (ICP1, 2, or 3). All 282 culture positive samples, and an additional 107 culture negative but PCR positive samples, were analyzed by shotgun metagenomic sequencing. From these metagenomes, we estimated the relative abundances of V. cholerae, phages, and members of the gut microbiome, while accounting for antibiotic exposure determined by quantitative mass spectrometry. Consistent with d'Hérelle's thesis, we observed higher phage to V. cholerae ratios in patients with mild dehydration, demonstrating in the modern era that phages are indicative of disease severity. Antibiotics were associated with less V. cholerae and with mild disease; ciprofloxacin in particular was associated with several known antibiotic resistance genes. Phage resistance genes in the V. cholerae integrative conjugative element (ICE) were associated with lower phage to V. cholerae ratios. In the absence of detectable ICEs, phages shaped genetic diversity, selecting for nonsynonymous point mutations in the V. cholerae genome. Together, our results point to both antibiotics and phages as inverse correlates of disease severity, while also selecting for resistance genes or mutations within cholera patients.