The impact of phage predation on bacterial pathogens in the context of human disease is not currently appreciated. Here, we show that predatory interactions of a phage with an important environmentally transmitted pathogen, Vibrio cholerae, can modulate the evolutionary trajectory of this pathogen during the natural course of infection within individual patients. We analyzed geographically and temporally disparate cholera patient stool samples from Haiti and Bangladesh and found that phage predation can drive the genomic diversity of intra-patient V. cholerae populations. Intra-patient phage-sensitive and phage-resistant isolates were isogenic except for mutations conferring phage resistance, and moreover, phage-resistant V. cholerae populations were composed of a heterogeneous mix of many unique mutants. We also observed that phage predation can significantly alter the virulence potential of V. cholerae shed from cholera patients. We provide the first molecular evidence for predatory phage shaping microbial community structure during the natural course of infection in humans.
Cholera epidemics occur seasonally in areas such as Bangladesh, and outbreaks can also strike in vulnerable regions, as has occurred recently in Haiti. The disease is caused by Vibrio cholerae, a water-borne bacterium that colonizes the small intestine, and its symptoms include severe diarrhea and vomiting which can lead to death if the patient is not treated promptly.
Lytic phages are viruses that specifically attack and kill bacteria. After replicating many times inside the bacterial cell, the phages break open and destroy the cell. Over time a bacterial population can evolve to resist this phage ‘predation’; however, it is not known if bacterial pathogens need to defend themselves against phage attack when they infect humans. It had been suggested that phages might affect the progress of cholera infections in people, but molecular evidence that supports this hypothesis was lacking.
When testing stool samples from Haitian cholera patients, Seed et al. found one sample contained a lot of lytic phage relative to the amount of V. cholerae present. This phage was very similar to—but distinct from—a phage found in Bangladeshi patients.
The V. cholerae bacteria isolated from the stool sample were resistant to attack by the phage. Sequencing the genome of individual bacteria from this sample revealed that each had a mutation that made them resistant to the phage; and while many types of these mutations were found, these were the only differences between all the V. cholerae bacteria in this patient sample. This suggests that this resistance developed independently many different times within the patient due to strong selective pressure from phage predation.
When Seed et al. looked at a phage-positive stool sample from a Bangladeshi patient, more mutations that made the bacteria resistant to this phage were found; however, these mutations were different again from the ones in the Haitian bacteria. Because of the nature of these mutations the bacteria from this patient were rendered unable to cause disease and non-transmissible.
This work shows that phages can indeed have access to pathogenic bacteria during human infection. It also indicates that the pressure imposed by phage predation can, in some cases, be so strong that the bacteria lose their virulence and ability to spread to other humans in order to become resistant to the phage.