Cultivation of Bordetella pertussis Strains

B. pertussis strains 18323 (85 (link)) and Tohama (86 (link)) were maintained in our laboratory. The B. pertussis clinical strains BP140, BP141, BP142, and BP144 were provided by K. Kamachi (National Institute of Infectious Diseases). B. pertussis was grown on Bordet-Gengou agar (Becton, Dickinson) plates containing 1% Hipolypepton (Nihon Pharmaceutical), 1% glycerol, 15% defibrinated horse blood, and 10 μg/mL ceftibuten (BG plate). The bacteria recovered from colonies on BG plates were suspended in Stainer-Scholte (SS) medium (87 (link)) to yield an optical density at 650 nm (OD₆₅₀) value of 0.2 and incubated at 37°C for 12 to 14 h with shaking. The obtained bacteria were used as Bvg⁺-phase bacteria. Unless otherwise specified, B. pertussis in the Bvg⁻ phase was obtained by cultivation in the presence of 40 mM MgSO₄. The number of CFU was estimated from the OD₆₅₀ values of fresh cultures according to the following equation: 1 OD₆₅₀ unit = 3.3 × 10⁹ CFU/mL. Escherichia coli was grown with Luria-Bertani (LB) agar or broth. E. coli strains DH5α λpir and HB101 harboring pRK2013 (88 (link)) were provided by K. Minamisawa (Tohoku University). The growth media were supplemented with antibiotics when necessary at the following concentrations: ampicillin, 50 μg/mL; gentamicin 10 μg/mL; kanamycin 50 μg/mL.