Biofilm formation and phenotypic variation enhance predation-driven persistence of Vibrio cholerae

Abstract

Persistence of the opportunistic bacterial pathogen Vibrio cholerae in aquatic environments is the principal cause for seasonal occurrence of cholera epidemics. This causality has been explained by postulating that V. cholerae forms biofilms in association with animate and inanimate surfaces. Alternatively, it has been proposed that bacterial pathogens are an integral part of the natural microbial food web and thus their survival is constrained by protozoan predation. Here, we report that both explanations are interrelated. Our data show that biofilms are the protective agent enabling V. cholerae to survive protozoan grazing while their planktonic counterparts are eliminated. Grazing on planktonic V. cholerae was found to select for the biofilm-enhancing rugose phase variant, which is adapted to the surface-associated niche by the production of exopolymers. Interestingly, grazing resistance in V. cholerae biofilms was not attained by exopolymer production alone but was accomplished by the secretion of an antiprotozoal factor that inhibits protozoan feeding activity. We identified that the cell density-dependent regulator hapR controls the production of this factor in biofilms. The inhibitory effect of V. cholerae biofilms was found to be widespread among toxigenic and nontoxigenic isolates. Our results provide a mechanistic explanation for the adaptive advantage of surface-associated growth in the environmental persistence of V. cholerae and suggest an important contribution of protozoan predation in the selective enrichment of biofilm-forming strains in the out-of-host environment.

Fig. 1.

Persistence of V. cholerae biofilms as opposed to planktonic cells during flagellate grazing. Planktonic (A) and biofilm (B) subpopulations of the smooth and rugose phase variant of V. cholerae A1552 were examined in the absence (-GRAZ) and in the presence (+GRAZ) of flagellate grazers. Planktonic bacteria were exposed to the suspension feeder C. roenbergensis and biofilms to the surface feeder R. nasuta for 72 h. Error bars indicate standard deviations of four replicates.

Fig. 2.

Inhibition of flagellate growth by V. cholerae biofilms. Growth rates were determined for the suspension feeder C. roenbergensis on planktonic cells (A) and for the surface-feeder R. nasuta on biofilms (B) of V. cholerae A1552 over 4 days. Error bars indicate standard deviations of four replicates.

Fig. 3.

Stimulation of biofilm formation by planktonic grazers. Planktonic subpopulations of the smooth and rugose phase variant of V. cholerae A1552 were cultivated without (-GRAZ) and with (+GRAZ) the suspension feeder C. roenbergensis. Biofilm formation was quantified by a crystal violet staining assay. Error bars indicate standard deviations of four replicates.

Fig. 4.

Inhibition of protozoan grazers in V. cholerae biofilms is regulated by hapR. Growth of the surface-feeding flagellate R. nasuta was evaluated on biofilms of the rugose wild-type A1552 and the isogenic mutants vpsR, vpsA, vpsL, vpsT, hapR, and luxT. Biofilms without (-GRAZ) and with (+GRAZ) grazer were examined by staining with SYTO 9 and by using confocal laser scanning microscopy. (Magnification, ×200; scale bar, 50 μm.) Note the elimination of the hapR^- biofilm. Error bars indicate standard deviations of four replicates.

Fig. 5.

Widespread resistance of V. cholerae biofilms to protozoan grazing among toxigenic and nontoxigenic isolates. Growth of the surface-feeding flagellate R. nasuta was followed on biofilms of eight environmental, two sixth pandemic, and six seventh pandemic strains over 4 days. Error bars indicate standard deviations of three replicates.