Mechanisms involved in governing adherence of Vibrio cholerae to granular starch

Abstract

Vibrio cholerae has been shown to adhere to cornstarch granules. The present work explored the mechanisms involved in this adhesion and the possibility of its occurrence in vivo. The findings suggest that both specific and nonspecific interactions are involved in the adhesion. Nonspecific hydrophobic interactions may play a role, since both V. cholerae and cornstarch granules exhibited hydrophobic properties when they were tested using a xylene-water system. In addition, the presence of bile acids reduced the adhesion. The adhesion also involves some specific carbohydrate-binding moieties on the cell surface, as reflected by reduced adhesion following pretreatment of the bacteria with proteinase K and sodium m-periodate. Further investigations supported these observations and showed that media containing low-molecular-weight carbohydrates had a significant inhibitory effect. Binding cell lysate to starch granules and removing the adhered proteins using either glucose or bile acids led to identification (by liquid chromatography-tandem mass spectrometry analysis) of several candidate V. cholerae outer membrane-associated starch-binding proteins. Different sets of proteins were isolated by removal in a glucose solution or bile acids. When the upper gastrointestinal tract conditions were simulated in vitro, both bile salts and the amylolytic activity of the pancreatic juices were found to have an inhibitory effect on the adherence of V. cholerae to starch. However, during acute diarrhea, this inhibitory effect may be significantly reduced due to dilution, suggesting that adhesion does occur in vivo. Such adhesion may contribute to the beneficial effects observed following administration of granular starch-based oral rehydration solutions to cholera patients.

FIG. 1.

Possible factors affecting adhesion of V. cholerae to starch granules: levels of adherence of V. cholerae under different conditions and with different treatments that may affect adhesion (see Table 1). The light gray bars indicate results obtained with various medium compositions or bacterial pretreatments. The dark gray bars indicate results obtained with starch granule pretreatments. Each experiment was repeated at least three times. The error bars indicate standard errors. Different letters above the bars indicate that the levels of adhesion are significantly different (α = 0.05).

FIG. 2.

Adhesion under conditions simulating the upper gastrointestinal tract. The open stippled bars indicate results obtained with different adhesion conditions, and the gray stippled bars indicate results obtained with starch granule pretreatments (see Table 2). Each experiment was repeated at least three times. The error bars indicate standard errors. Different letters above the bars indicate that the levels of adhesion are significantly different (α = 0.05).

FIG. 3.

Correlation between adhesion to starch granules and cell surface hydrophobicity. The cell surface hydrophobicity of the strains was measured by using the test for bacterial adhesion to hydrocarbon. The hydrocarbons used were xylene (filled symbols) and hexadecane (open symbols). A linear fit was found only with xylene (R² = 0.769, P = 0.0217). Each experiment was repeated at least three times. The error bars indicate standard errors.

FIG. 4.

Effect of growth phase and carbon source on adhesion. (A) Levels of adhesion of V. cholerae to cornstarch granules determined with three carbon sources, glucose, maltose, and DE19. The adhesion was examined at three points along the growth curve, two points in the log phase (90 min and 150 min) and one point in the early stationary phase (270 min). The error bars indicate standard errors. (B) Growth curves for V. cholerae in the presence of three carbon sources. Each experiment was repeated at least three times, and the results shown are averages.

FIG. 5.

Removal of adhered bacteria. Adhered bacteria were resuspended and incubated in several solutions containing agents that might facilitate their removal from the starch granules. The percentage of bacteria released from the granules was estimated after 2 min and 15 min of incubation and compared to the values for two controls composed of oral rehydration solution (ORS) or phosphate buffer (PB) with no added reagent. Each experiment was repeated at least three times. The error bars indicate standard errors. Different letters above the bars indicate that the levels are significantly different (α = 0.05).