Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages

Abstract

The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh. Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V. cholerae strain than both (P < 0.00001). The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V. cholerae strains in water samples that otherwise lacked detectable cholera phages. Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria. Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V. cholerae pandemic serogroups or clones.

Fig. 1.

Mean concentration of lytic vibriophages in the aquatic environment of Dhaka, Bangladesh, and the estimated number of cholera cases reporting to the International Centre for Diarrhoeal Disease Research hospital in Dhaka from 2001 to 2003. Number of cholera cases is extrapolated from a 2% surveillance sample of all patients presenting for treatment. pfu, plaque-forming units.

Fig. 2.

Electron micrograph of vibriophages isolated from environmental waters in Bangladesh. (A–F) Morphology of phages JSF1–JSF6. (Scale bar: 50 nm.)

Fig. 3.

Plaque morphology of different vibriophages on lawns of susceptible V. cholerae strains. Description of phages and host strains are as follows: JSF1 phage plated on a V. cholerae O1 clinical strain isolated from the recent epidemic (A), JSF2 phage isolated from water (B) and from a lysogenic non-O1/non-O139 strain (G) plated on an epidemic V. cholerae O1 strain, JSF3 phage plated on a V. cholerae O139 clinical strain (C), JSF6 phage plated on a V. cholerae O1 strain (F) and an environmental non-O1/non-O139 strain (H), JSF6 phage produced from an environmental non-O1/non-O139 strain and plated on a clinical V. cholerae O1 strain (E), and JSF4 phage plated on a V. cholerae O1 strain (D).

Fig. 4.

Schematic model for the influence of phage on cholera seasonality and serogroup emergence. Seasonal cholera epidemics (increased cases over time) occur in waves, with different serogroups (e.g., O1 or O139) dominating. Corresponding conditions that exist in the aquatic environment are diagramed in the circles shown. The absence of yellow phage specific for yellow oval cells provides an opportunity for that serogroup to begin the seasonal epidemic and transmit efficiently. However, yellow phages eventually amplify in the environment and attack this serogroup, ending that epidemic. A different serogroup (red oval cells) is resistant to the yellow phage. In the case shown, red cells actually carry a prophage (yellow intracellular circle) and thus shed yellow phage. A second epidemic wave due to the red serogroup follows and runs its course until red phages bloom and prevent environmental transmission of this serogroup. The interepidemic period is dominated by sporadic disease due to other serogroups (blue cells) resistant to both phages. These strains usually lack typical virulence factors but are more environmentally adapted than virulent strains. However, blue cells may harbor prophages that kill virulent serogroups and may acquire virulence determinants by horizontal gene transfer. Thus, blue cells may eventually emerge to become a new epidemic serogroup.