D J Bibel’s scientific contributions

Scanning electron microscopy was used to search for local or limited synthesis of antibiotics by a strain of Trichophyton mentagrophytes. Micrococcus luteus, which was susceptible to penicillin and to the dermatophyte when both were cultured on agar media, displayed morphological alterations of flattened walls, invagination, enlargement, and collapse in skin cultures with the fungus. Penicillinase did not neutralize these effects. A control strain of Staphylococcus spp with poor sensitivity to penicillin was resistant to the dermatophyte in all test systems. T. mentagrophytes showed positive tropism to the bacteria. Regional variation in fungal germination and antibiotic production, the limited yield of antibiotics, and the pattern of dermatophyte growth and alteration of bacterial morphology demonstrated the significant influence of microenvironment on antibiosis.

The colonization and survival of Bacillus species, members of the cutaneous microbial community of humans, were investigated by applying spores of Bacillus licheniformis to the forearms of volunteers. Four strains were tested, including the bacitracin producer ATCC 10716 and its bacitracin-negative mutant. Germination occurred within 24 h. Significant differences in survival population and duration were found among the test strains; however, ATCC 10716 and its mutant produced statistically similar survival curves. In general, an inoculum density of 10(4) colony-forming units per cm2 allowed survival for at least 2 weeks. Individual variation was extreme, for one subject harbored bacilli for over 2 months and another eliminated the microorganism within 3 days. Individuals could be differentiated into long-term (greater than 21 days) and short-term (less than 14 days) carriers. Eight of the 11 volunteers (73%) inoculated with ATCC 10716 carried it for 2 weeks, and 5 subjects (45%) continued to support the bacilli for 3 weeks. Spreading of the organism to other regions of the body occurred, but bacilli were not detected in these areas beyond 6 days.

To determine whether antibiotic production might be ecologically advantageous in the survival of Bacillus species on human skin, we applied spores of a bacitracin-producing strain of Bacillus licheniformis (ATCC 10716) to the forearms of 11 volunteers. Three additional strains of B. licheniformis which did not synthesize antibiotics, including a mutant of ATCC 10716, were used in subsequent control trials. Samples of flora were taken from inoculated and control (opposite forearm) sites during the colonization period, generally 3 weeks. Although population densities were unaltered, changes in the carriage, composition, and bacitracin sensitivity of resident flora were related with the presence of ATCC 10716 only, which suggests that microbial interactions are important in bacillus colonization and in maintenance of normal flora. Interactions were examined in vitro by comparing growth curves of representative skin bacteria, including isolates of Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus luteus, and a large-colony diphtheroid, grown individually, in mixed culture with each other, and together in presence of each test strain of B. licheniformis. We observed some diminution of growth of M. luteus and the diphtheroid in the first mixed culture, and the diphtheroid was completely retarded in common culture with ATCC 10716. Lesser antibiotic effects were seen on the cocci, whose rank of sensitivity was similar to that in vivo. The growth of the diphtheroid was enhanced in mixed culture with those strains of bacilli which lack antibiotic activity.

Micrococcus luteus produced a diffusible, esterase inhibitory factor (EIF) which inhibited the activity of cutaneous diphtheroid esterases on Tween 80-CaCl2 agar media. Esterases of Staphylococcus, Micrococcus, Bacillus, and Serratia were not susceptible. EIF did not appear to combine with the substrate or to prevent enzyme synthesis; it was unable to reverse the precipitation of calcium oleate. The composition of the medium, especially peptones, influenced the production of EIF. EIF was synthesized in the absence of diphtheroids, but production required the presence of Tween. The interaction was observed on agar medium of pH 5.5-8.5, at 25-43 degrees C, under an atmosphere of 10-20% CO2, in the presence of urea, but not after the addition of NaCl or dextrose. Supernatants of broth cultures had to be concentrated to detect EIF. Crude dialyzed and concentrated preparations of EIF withstood 60 degrees C for 60 min but were inactivated after 100 degrees C for 10 min. EIF may possibly be associated with a lipoid substance, since it did not precipitate in ethanol.

Two surveys were conducted to ascertain the effect which Staphylococcus aureus has on resident flora and on skin of patients with eczema. Forty paired sites, normal and lesions, were sampled from 32 patients with chronic lichenified atopic dermatitis, and 162 sites covering the entire body of a patient with an acute flare of chronic atopic dermatitis were examined. In the first study, 34 lesions (85%) carried S. aureus, and 85% of these positive samples supported greater than 10(4) colony-forming units (CFU) of total aerobic bacteria/cm2. About 55% of normal sites carried S. aureus with 18% of such samples supporting greater than 10(4) CFU/cm2 total aerobic bacteria. Normal sites carried fewer but more diverse flora than lesions. The composition of flora on lesion sites was mainly S. aureus or S. epidermidis biotype 1, or both. At densities greater than 10(7) CFU/cm2, S. aureus constituted almost 100% of the total aerobic bacterial flora. The total body survey of the single eczema patient yielded similar results. Evidence supports the hypotheses that (1) S. aureus is able to exploit the environment of eczematous lesions and to influence the composition of cohabiting aerobic skin bacteria, and (2) its inhabitation of normal appearing sites is transitory.

Arthrosporogenesis of the dermatophyte Trichophyton mentagrophytes was examined by light and by scanning and transmission electron microscopy. Sabouraud dextrose agar plates were inoculated with microconidia and incubated in an atmosphere of 8% CO2. Typical germination and hyphal branching continued to day 4, when hyphae began to be increasingly coated with granular-fibrillar material. Multiple replication of nuclei and formation of segregating septa followed. By day 6 the thick surface mesh sometimes was restricted to protruding rings, probably over septa. Between days 6 and 7, after thickening of outer and septal walls, units began to round and separate. Triangular gaps, which developed at the junction of septa and outer wall layers, enlarged so that spores were held together at their poles and along a tangential ring. With elongation of the spore to its barrel shape, the halves of the septum separated and the ring pulled apart, leaving a jagged, circular flange originating from the outer layer of cell wall extended toward the poles, covering the apparently exposed inner wall layer. Newly formed arthrospores, which measured 2.0 to 3.3 by 2.9 to 3.8 micronm and possessed walls of about 0.33-micronm thickness, has smooth sides but somewhat rough poles.

The purpose of the study was to ascertain whether, among the Enterobacteriaceae, Tween 80 hydrolysis is specific for the genus Serratia. Free fatty acid is released upon the hydrolysis of Tween 80. In the presence of Ca2+, calcium oleate is precipitated, forming an opaque zone under and around the colony. The composition of our test medium, a modification of that of Sierra is as follows: 3.3% (wt/vol) tryptose blood agar base (Difco Laboratories), 0.4% (vol/vol) Teen 80 (J.T. Baker Chemical Co.), and 0.015% (wt/vol) CaCl2. Unless used shortly after preparation, plates are stored in sealed containers to prevent desiccation and remain satisfactory at least for 2 weeks. Pates are examined 24 and 48 h after incubation at 37°C. A positive reaction, normally observed within 24 h after inoculation, is a distinct precipitate in the agar. The test also can be carried out successfully at lower temperatures. Studies were limited to Enterobacteriaceae. Among members of this family, only Serratia yielded positive reactions. The use of Tween 80/CaC12 agar medium can greatly simplify the identification of Serratia.