During bacteriophage studies on Haemophilus influenzer, it was observed that encapsulated type b and unencapsulated Rb strains released a bactericidal substance acitve against types a, c, d, e, and f H. influenzae, non-typable H. influenzae strains, other Haemophilus species, and certain members of the Enterobacteriaceae. The bactericidal activity was assayed by a plaque test utilizing an Rd strain as an indicator lawn and was also demonstrated in mixed broth cultures of a producer strain and an indicator strain. Immediately lysis of sensitive bacteria by the factor was not evident. The factor is sensitive to trypsin but resistant to deoxyribonuclease, treatment with 2-mercaptoethanol, lipase, alpha-amylase, and heating in a 100 degrees C water bath for 20 min. The activity is not dependent upon increased Ca2+ or Mg2+ concentration as is necessary for HP1C1 and S2 phage propagation. The bactericidal factor is not pelleted by high-speed centrifugation at 150,000 X g for 6 h. Treatment with ultraviolet light or mitomycin C does not result in observable phage, phage-like particles, or increased bactericidal activity. T-HE BACTERICIDAL FACTOR IS NOT A TYPICAL SMALL MOLECULAR WEIGHT "COLICIN-LIKE" BACTERiocin in that it is not inducible, has a wider range of activity, and does not kill by "single-hit" kinetics. On preliminary characterization, it is a thermostable protein toxic to certain bacterial strains.
To reduce the incubation time requirement in the Bauer-Kirby antibiotic susceptibility test, comparisons were made of the test results at 18 to 20 h (standard) and 7 to 8 h (rapid) utilizing 100 recent clinical isolates. The zone diameters for 664 disks were monitored by using the standard classification: resistant, intermediate, or susceptible. The susceptibility determination was unchanged in 558 out of 664 instances (84.0%). An analysis of the remaining 106 sets revealed that an initial interpretation of intermediate in zone size, subsequently determined resistant or susceptible, accounted for 49 of the observed differences. The reverse changes, initial resistant or susceptible subsequently classified as intermediate, accounted for 20 of the changes. In five instances the interpretation changed from susceptible to resistant; in two cases the interpretation changed from resistant to susceptible. The remaining 30 determinations were classified as indeterminant due to (i) insufficient growth at the early (7 to 8 h) determination, and to (ii) zones which were so large that they could not be measured accurately. The data indicate that zone sizes when measured to the nearest 0.1 mm can be interpreted with reasonable accuracy and the results can be available 10 to 14 h sooner.
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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