Clostridium difficile cell attachment is modified by environmental factors

Abstract

Adherence of Clostridium difficile to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation. The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains. Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa.

FIG. 1

(A) Adherence of C. difficile to Vero cells under anaerobic conditions (part 1) or after a heat shock under aerobic (with CO₂) conditions (part 2). (B) Adherence to Vero cells of seven C. difficile strains as a function of time. Symbols for toxigenic strains: ⧫, 79-685; ■, VPI-10409. Symbols for nontoxigenic strains: ▴, 90-111; ×, 93-136; ○, 93-226; ●, 93-296; +, 93-379. (C) Effect of growth phase on the adherence of C. difficile to Vero cells. (D) Effect of iron in the culture medium on the adherence of C. difficile to Vero cells. The control was TGY, the concentration of FeCl₃ was 50 μM, and that of 2,2′-dipyridyl (22′D) was 200 μM. Nb, number.

FIG. 2

Effects of cations and pH on C. difficile adherence to Vero cells. (A) Part 1, NaCl concentrations in growth medium: ■ (control [TGY broth]), 50 mM; □, 150 mM; ▧, 500 mM. Part 2, NaCl concentrations in adhesion medium: ■, control; □, 50 mM; ▧, 150 mM; ▩, 500 mM. (B) Part 1, CaCl₂ concentrations in growth medium: ■ (control [TGY broth]), 1 mM; □, 12.5 mM; ▧, 25 mM. Part 2, CaCl₂ concentration in adhesion medium: ■, control; □, 1 mM; ▧, 12.5 mM; ▩, 25 mM. (C) Part 1 MgCl₂ concentrations in growth medium: ■ (control [TGY broth]), 1 mM; □, 22.5 mM; ▧, 45 mM. Part 2, MgCl₂ concentrations in adhesion medium: ■, control; □, 1 mM; ▧, 22.5 mM; ▩, 45 mM. (D) Treatment of C. difficile at pHs 4 to 10 before adhesion assay. Nb, number.

FIG. 3

Patterns of C. difficile adherence to Vero cells examined by light microscopy (magnification, ×1,000). Panels: A, control (C. difficile treated at pH 7.0 before adhesion assay); B, C. difficile treated at pH 4 before adhesion assay.

FIG. 4

Adhesins involved in cell attachment. (A) Surface proteins from C. difficile grown under various conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). (B) Proteins on membrane which bound Vero cells (autoradiography). (C) Optical density determined by densitometric scanning of autoradiograms for the 70-kDa (■), 50-kDa (□), and 40-kDa (▧) proteins (expressed in comparison with the control). Lanes: 1, non-heat-shocked C. difficile; 2, C. difficile heat shocked under partial anaerobic conditions; 3, C. difficile treated at pH 4; 4, C. difficile grown in the presence of 200 μM 2,2′-dipyridyl; 5, C. difficile grown in the presence of 500 mM NaCl; 6, negative control (Clostridium indolis proteins [nonadherent, normal-flora bacterial strain]).