A combination of metagenomic and cultivation approaches reveals hypermutator phenotypes within Vibrio cholerae infected patients

Abstract

Vibrio cholerae can cause a range of symptoms, ranging from severe diarrhea to asymptomatic infection. Previous studies using whole genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous in V. cholerae DNA repair genes in five out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome, and in one of three patients with isolate genomes sequenced. Second, mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, non-mutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCEPathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease, or asymptomatic infection. Here we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.