Bactericidal factor produced by Haemophilus influenzae b: partial purification of the factor and transfer of its...

When aerobically grown on complex media, Haemophilus influenzae b and unencapsulated variants, Rb strains, produced a bactericidal factor that was active against other Haemophilus species and certain genera of the Enterobacteriaceae. A total of 341 clinical isolates of Haemophilus were tested for susceptibility to the factor. Ninety-three percent of H. influenzae (nontypable), 75% of H. haemolyticus, 71% of H. parainfluenzae, and 22% of H. parahaemolyticus were susceptible. H. influenaze b strains were resistant producers of the bactericidal factor and H. influenzae f strains were susceptible nonproducers. Only one strain each of H. aegyptius and H. aphrophilus was isolated and each was susceptible and resistant, respectively. 143 clinical isolates of the Enterobacteriaceae were tested and of those 82% of Escherichia coli, 85% of Salmonella sp., and all Citrobacter sp., Shigella sp., and Yersinia sp. were sensitive to the bactericidal factor produced by H. influenzae b. Attempts to isolate the bactericidal activity from mechanically disrupted, solubilized, or osmotically shocked cells failed to release active bactericidal factor. However, we partially purified the bactericidal factor from the spent culture medium of aerobically grown H. influenzae b by a series of extractions. The ability to produce the bactericidal factor was transferable to nonproducer strains without also genetically transforming for type b encapsulation. The converse was also true in that type b capsules were produced by transformed H. influenzae Rd strains but no bactericidal factor was detected from these strains. Additionally, nitrosoguanidine-induced mutants of H. influenzae b lost the ability to produce bactericidal factor without loss of their type-specific capsule, demonstrating that production of the bactericidal factor was genetically separable from production of the type capsule of H. influenzae b.