An experimental study of the population and evolutionary dynamics of Vibrio cholerae O1 and the bacteriophage JSF4

Abstract

Studies of Vibrio cholerae in the environment and infected patients suggest that the waning of cholera outbreaks is associated with rise in the density of lytic bacteriophage. In accordance with mathematical models, there are seemingly realistic conditions where phage predation could be responsible for declines in the incidence of cholera. Here, we present the results of experiments with the El Tor strain of V. cholerae (N16961) and a naturally occurring lytic phage (JSF4), exploring the validity of the main premise of this model: that phage predation limits the density of V. cholerae populations. At one level, the results of our experiments are inconsistent with this hypothesis. JSF4-resistant V. cholerae evolve within a short time following their confrontation with these viruses and their populations become limited by resources rather than phage predation. At a larger scale, however, the results of our experiments are not inconsistent with the hypothesis that bacteriophage modulate outbreaks of cholera. We postulate that the resistant bacteria that evolved play an insignificant role in the ecology or pathogenicity of V. cholerae. Relative to the phage-sensitive cells from whence they are derived, the evolved JSF4-resistant V. cholerae have fitness costs and other characters that are likely to impair their ability to compete with the sensitive cells in their natural habitat and may be avirulent in human hosts. The results of this in vitro study make predictions that can be tested in natural populations of V. cholerae and cholera-infected patients.

Figure 1.

Change in the density of bacteria and phage in continuous culture, 5% LB, where the solid lines with squares and broken lines with squares are, respectively, the densities of the bacteria and phage in mixed culture, and solid lines with diamonds are the density of bacteria in the phage-free control. All chemostats were inoculated with bacteria from an overnight LB culture for an initial density of approximately 10⁸ CFU ml⁻¹ and approximately 10⁶ PFU ml⁻¹ of phage. The dilution rates, w, are the fraction of the total volume of the chemostat change per hour. The lines with ‘% resistance’ denote the percentage of JSF4-resistant mutants out of 20 randomly chosen colonies at specific time points. (a) Chemostat inoculated with wild-type V. cholerae N16961, w = 0.07–0.12. (b) Chemostat inoculated with rugose variant of V. cholerae N16961, w = 0.24. (c) Chemostat inoculated with wild-type V. cholerae N16961; the phage JSF4 was introduced after the rugose variants in this chemostat reached a level of 9.4% of total bacteria, w = 0.21.

Figure 2.

(a) Relative Malthusian fitness of a rifampin-resistant JSF4-sensitive common competitor in pair-wise competition with rifampin-sensitive JSF4-sensitive wild-type N16961, rugose variant of N16961 and 16 rifampin-sensitive JSF4-resistant mutants (12 are smooth colony and four are rugose colony types). The horizontal line denotes the fitness of the common competitor, the rifampin-resistant mutant. The error bars are the standard deviations of the mean fitness for at least three independent pair-wise competition experiments. For clarity and aesthetic reasons, we have plotted only the negative standard error. RF, R1.1, R1.2, R2.1, R2.2, RR: JSF4-resistant mutants (see table 1 for the source). R3.1, R3.2, R3.3, R4.1, R4.2, R4.3, R4.4: smooth JSF4-resistant mutants. R5.1, R5.2, R5.3: rugose type JSF4-resistant mutants. All of them were isolated from three different chemostats. The source of these mutants is listed in the electronic supplementary material, table S2. (b) Relative fitness of different V. cholerae N16961 in competition with rifampin-resistant JSF4-sensitive common competitor in liquid and in surface culture. (S): competition on surface; (L): competition in liquid; R2.1, R2.2: non-motile JSF4-resistant mutants; NM: a non-motile JSF4-sensitive isolate.

Figure 3.

Colonization of wild-type V. cholerae N16961 and JSF4-resistant mutants on (a) D. pulex and (b) D. magna. RF, R1.1, R1.2, R2.1, R2.2: JSF4-resistant mutants. Error bar represents standard deviation of bacteria (CFU) attached to single Daphnia.

Figure 4.

Rate of mortality of C. elegans feeding on phage-sensitive and -resistant V. cholerae and an E. coli OP50 control. RF, R1.1, R1.2, R2.1, R2.2: smooth phage-resistant mutants; RR: rugose phage-resistant mutant. Triple asterisks (***) indicate a significant p-value of log-rank test for comparison of survival curves of JSF4-resistant relative to JSF4-sensitive.