Vibrio choleraegenomic diversity within and between patients
Authors: Levade Inès , Terrat Yves , Leducq Jean-Baptiste , Weil Ana A. , Mayo-Smith Leslie M. , Chowdhury Fahima , Khan Ashraful I. , Boncy Jacques , Buteau Josiane , Ivers Louise C. , Ryan Edward T. , Charles Richelle C. , Calderwood Stephen B. , Qadri Firdausi , Harris Jason B. , LaRocque Regina C. , Shapiro B. Jesse 
Published: July 2017

Cholera is a severe, waterborne diarrheal disease caused by toxin-producing strains of the bacteriumVibrio cholerae. Comparative genomics has revealed “waves” of cholera transmission and evolution, in which clones are successively replaced over decades and centuries. However, the extent ofV. choleraegenetic diversity within an epidemic or even within an individual patient is poorly understood. Here, we characterizedV. choleraegenomic diversity at a micro-epidemiological level within and between individual patients from Bangladesh and Haiti. To capture within-patient diversity, we isolated multiple (8 to 20)V. choleraecolonies from each of eight patients, sequenced their genomes and identified point mutations and gene gain/loss events. We found limited but detectable diversity at the level of point mutations within hosts (zero to three single nucleotide variants within each patient), and comparatively higher gene content variation within hosts (at least one gain/loss event per patient, and up to 103 events in one patient). Much of the gene content variation appeared to be due to gain and loss of phage and plasmids within theV. choleraepopulation, with occasional exchanges betweenV. choleraeand other members of the gut microbiota. We also show that certain intra-host variants have phenotypic consequences. For example, the acquisition of aBacteroidesplasmid and nonsynonymous mutations in a sensor histidine kinase gene both reduced biofilm formation, an important trait for environmental survival. Together, our results show thatV. choleraeis measurably evolving within patients, with possible implications for disease outcomes and transmission dynamics.

Author Summary

Vibrio choleraeis the etiological agent of cholera, a severe diarrheal disease endemic to Bangladesh and responsible for global outbreaks, including one ongoing in Haiti. Certain bacterial pathogens can evolve and diversify within the human host, often altering virulence and antibiotic resistance. However, most examples of within-host evolution have come from chronic infections, in which the pathogen has sufficient time to mutate and diversify, and little attention has been paid to more acute infections such as the one caused byV. cholerae. The goal of this study was to measure the extent of within-host evolution ofV. choleraewithin individual infected patients. By sequencing multiple bacterial isolates_from each of eight patients from Bangladesh and Haiti, we found that cholera patients can harbor a diverse population ofV. cholerae. As expected for an acute infection, this diversity is limited, ranging from zero to three point mutations (single nucleotide variants) per patient. However, gene gain/loss events are more prevalent than point mutations, occurring in every single patient, and sometimes involving the transfer of dozens of genes on plasmids. Even if rare, point mutations and gene gain/loss events may be maintained by natural selection, and can alter clinically-and environmentally-relevant phenotypes such as biofilm formation. Therefore, within-patient evolution has the potential to impact clinical and epidemiological outcomes. Together, our results demonstrate that within-patient evolution may be a general feature of both acute and chronic infections, and that gene gain/loss may be an important but under-appreciated feature of within-host evolution.