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

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Abstract
Four different serotype strains of Bordetella pertussis , 3779BL 2 S 4 , Tohama I, 353/Z, and 2753, were plated on Bordet-Gengou agar, where they grew as domed, hemolytic (D + H + ) wild-type colonies. Cloned D + H + colony types of all four strains were passed onto modified Stainer-Scholte medium solidified with 1% Noble Agar. Colonies were selected from Stainer-Scholte agar, and 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 × 10 −5 and 5 × 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 four 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 two phenotypes as noted by double diffusion of antisera added to plates on which colonies of the variants were growing. Antigens solubilized from the two 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. In addition, autoradiographs of 125 I-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 (P. H. Leslie and A. D. Gardner, J. Hyg. 31:423-434, 1931) or phase III in the system of Kasuga et al. (T. Kasuga, Y. Nakase, K. Ukishima, and K. Takatsu, Kitasato Arch. Exp. Med. 26:121-134, 1954).