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% ofEscherichia coli, 85% ofSalmonella 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, nitrosoguanidineinduced mutants ofH. 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.We have reported that Haemophilus influenzae b produces a bactericidal factor that is active against Haemophilus species and certain members of the Enterobacteriaceae but not against gram-positive bacteria (13). Preliminary characterization of the bactericidal factor showed that it was a heat-stable protein of between 50,000 and 100,000 daltons in size, as determined by membrane filter retention. It was produced throughout the growth cycle ofH. influenzae when cultured aerobically. The bactericidal factor appeared to be different from "colicin-like" bacteriocins in that its production was not inducible by mitomycin C, it did not kill by "single-hit" kinetics and, as opposed to the strain specificity of bacteriocin production, we have observed that all H. influenzae b strains produced the factor. None of the clinical nontypable H. influenzae strains tested produced the bactericidal factor. It is interesting that H. influenzae Rb, i.e., type b strains that have lost the ability to produce a capsule, retained the capacity to produce the bactericidal factor. We have hypothesized that capsule production occurs independently of bactericidal factor ...
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