Transmission of Vibrio cholerae Is Antagonized by Lytic Phage and Entry into the Aquatic Environment

Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water—the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment.Author Summary: The biological factors that control the transmission of water-borne pathogens like Vibrio cholerae during outbreaks are ill defined. In this study, a molecular analysis of the active but non-culturable (ABNC) state of V. cholerae provides insights into the physiology of environmental adaptation. The ABNC state, lytic phage, and hyperinfectivity were concurrently followed as V. cholerae passaged from cholera patients to an aquatic reservoir. The relevance to transmission of each factor was weighed against the others. As the bacteria transitioned from the patient to pond water, there was a rapid decay into the ABNC state and a rise of lytic phage that compounded to block transmission in a mouse model. These two factors give reason for V. cholerae to make a quick transit through the environment and onto the next human host. Thus, in over-crowded locations with failed water infrastructure, the opportunity for fast transmission coupled with the increased infectivity and culturability of recently shed V. cholerae creates a charged setting for explosive cholera outbreaks.