Isolation and Characterization of Isogenic Pairs of Domed Hemolytic and Flat Nonhemolytic Colony Types of Bordetella pertussis

Abstract

Four diferent serotype strains of B. pertussis (3779BL2S4, Tohama I, 353/Z and 2753) were plated on Bordet-Gengou agar; they grew as domed, hemolytic (D+H+) wild-type colonies. Cloned D+H+ colony types of all 4 strains were passed onto modified Stainer-Scholte medium solidified with 1% Noble Agar. Colonies were selected from Stainer-Scholte agar; these subsequently grew as flat, nonhemolytic (D-H-) colonies when transferred back onto Bordet-Gengou agar. The frequency of D-H- organisms within a population of cloned D+H+ was determined to be between 5 .times. 10-5-5 .times. 10-6. The D-H- colony types maintained their flat, nonhemolytic characteristics for over 80 single-colony passages on Bordet-Gengou agar. The isogenic pairs of D+H+ and D-H- colony types from the 4 strains were compared for hemagglutination titer, lymphocytosis-promoting activity, adenylate cyclase activity and presence of agglutinogens by agglutination. In all cases, the D-H- colony types showed reduced activities or amounts of antigen compared with their D+H+ parents. Freely diffusible antigens were markedly different between the 2 phenotypes as noted by double diffusion of antisera added to plates on which colonies of the variants were growing. Antigens solubilized from the 2 colony types by Triton X-100 were also markedly different as judged by radial immunodiffusion with antifimbrial hemagglutinin, antilymphocytosis-promoting factor and anti-353/Z adsorbed with autoclaved 353/Z. Autoradiographs of 125I-surface-labeled whole cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed unique banding patterns for each colony type. Since all organisms, regardless of colony type, were grown on Bordet-Gengou agar, the differences reported could not be due to medium composition. Differences between phenotypes were also independent of passage number on Bordet-Gengou agar. By analogy to previous studies, the D-H- organisms appear to fulfill the criteria for phase III or phase IV in the system of Leslie and Gardner or phase III in the system of Kasuga.