Characterization of flagella of Clostridium difficile and their role in serogrouping reactions

Abstract

Slide agglutination with rabbit antisera allows the differentiation of 10 serogroups of Clostridium difficile, namely, A, B, C, D, F, G, H, I, K, and X. Each serogroup displays a specific protein profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for A, which displays 12 different protein profiles (A1 to A12). In the present work, electron microscopy revealed the presence of uniformly distributed flagella in the reference strains of serogroups G and K and in all strains representative of the 12 subgroups within serogroup A. No flagella were observed in the other reference strains. Flagella sheared from the cell surface were partially purified by differential centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these preparations revealed one distinct band with an apparent molecular mass of approximately 39 kilodaltons. Antiserum was prepared by immunizing a rabbit with the serogroup A flagellin, which had been eluted from the gel. In immunoblotting, this antiserum cross-reacted with the flagellin of the other strains. When the cells were deflagellated by a short sonication, the cross-reactions observed by slide agglutination with A, G, and K antisera were suppressed. Similarly, shearing of flagella allowed specific slide agglutination of the 12 subgroups of serogroup A.