Isolation and characterization of Bordetella pertussis phenotype variants capable of growing on nutrient agar: comparison with phases III and IV.

Unsupplemented nutrient agar (NA) was used to select spontaneous phenotype variants (PVs) of Bordetella pertussis Tohama I and 3779 which, by their growth on NA, could possibly be considered equivalent to 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, 1953). NA growers (Gna+) were selected from the flat, nonhemolytic, non-NA grower (Dom- Hly- Gna-) PV of both strains at a rate of between 10(-7) to 10(-8) per cell per generation. When cultured on Bordet-Gengou agar (BGA), more than one colony type was observed in strain 3779; these all retained the Gna+ characteristic during 10 to 30 passages on BGA. Analysis of 125I-surface-labeled whole cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no major changes between the Dom- Hly- Gna+ PV and their Dom- Hly- Gna- PV parents in polypeptide profile (by Coomassie stain), in surface exposure of proteins (by autoradiography), or in lipopolysaccharide profile (by silver stain). Increased resistance to oleic acid, tetracycline, erythromycin, rifampin, and penicillin G, however, was characteristic for the Dom- Hly- Gna+ PV. Five phase IV strains and a phase III B. pertussis strain had similar antibiotic and oleic acid sensitivity profiles as the Dom- Hly- Gna+ isolates and plated with similar efficiency on NA, despite heterogeneity in BGA colonial morphology and lipopolysaccharide profile.