Attenuated mutants of Staphylococcus aureus with two temperature-sensitive lesions: isolation and characterization.

The feasibility of constructing attenuated mutants of Staphylococcus aureus with two temperature-sensitive (ts) lesions for ultimate development of a live-attenuated strain was investigated. Temperature-sensitive S. aureus strain G/1/2, which grows well at 31 degrees C but does not replicate at 37 degrees C, was subjected to chemical mutagenesis. After two enrichment cycles, fifteen mutants able to grow at 25 degrees C but unable to grow at 31 degrees C, were identified. Growth curves with temperature shifts from 25 to 31 degrees C, and from 31 to 37 degrees C confirmed that these were mutants with two lesions (dts), each with a different cut-off temperature. The reversion frequency of mutant G/1/2 at 37 degrees C was 2 x 10(-6) whereas those of several dts mutants were much lower (dts7: 7 x 10(-9) and dts12: 1 x 10(-9)). There was no increase in ts mutation reversion rate in response to prolonged incubation at 37 degrees C. The data support the further development of these mutants for use as a stable attenuated vaccine.

Specific pulmonary defences against Pseudomonas aeruginosa after local immunization with temperature-sensitive mutants.

The specificity of the enhancement in lung defences after local immunization of mice with three temperature-sensitive (ts) mutants of Pseudomonas aeruginosa was investigated. The three selected mutants display altered growth characteristics when transferred from 29 degrees C to mammalian body temperature. Mice immunized with the live ts mutants by aerosol exposure or multiple intranasal inoculations were challenged with aerosols containing wild-type (wt) P. aeruginosa. Aerosol immunization with ts mutant A/10/25 significantly enhanced the lung clearance of the wt but did not enhance the clearance of either Klebsiella pneumoniae or Staphylococcus aureus. Aerosol immunization with ts mutants D/1/8 or E/9/9 enhanced the lung defences against the parental wt (of identical immunotype 1) but not against immunotype 4; similarly, intranasal immunization enhanced the lung defences against the parental wt but not against immunotypes 4 or 5. We conclude that local immunization with ts mutants of P. aeruginosa enhances lung defences against the wt in a genus- and immunotype-specific fashion. It is suggested that local immunity may play a central role in immunoprophylaxis against P. aeruginosa lung infection.

Differential growth characteristics and immunogenicity of tight and coasting temperature-sensitive mutants of Pseudomonas aeruginosa.

Genetically attenuated vaccines capable of limited replication in the vaccinate may elicit stronger, longer-lasting immunity than that induced by component, killed whole-cell, or nonreplicating live vaccines. We have isolated and partially characterized temperature-sensitive Pseudomonas aeruginosa mutants of two different phenotypes: a tight mutant, which ceases all growth immediately after its transfer to 36 degrees C, and a coaster, which continues to replicate for five generations at 36 degrees C. The growth profiles of the two temperature-sensitive phenotypes were compared both in vitro and in vivo; maintenance of the coasting phenotype in vivo was confirmed. The immunogenicity of the two phenotypes was compared in two models. In model 1, ICR mice were immunized intraperitoneally (i.p.) with graded doses of either mutant and challenged 3 weeks later i.p. with lethal doses of the wild-type strain. In model 2, DBA/2J mice were immunized intranasally with either mutant and subsequently challenged with an aerosolized inoculum of the wild-type strain, and lung clearance was measured over 4 h. In both models, the coaster demonstrated slightly higher immunogenic potential and, in addition, induced significantly higher levels of immunotype-specific serum immunoglobulin G after i.p. immunization.

Analysis of macrophage bactericidal function in genetically resistant and susceptible mice by using the temperature-sensitive mutant of Listeria monocytogenes.

Innate resistance to infection by Listeria monocytogenes is genetically controlled and is critically dependent on prompt macrophage recruitment to the sites of infection. Experiments reported here were designed to examine whether there was an additional, qualitative difference between the intrinsic bactericidal activity of the inflammatory macrophages of genetically resistant (C57BL/6J) and susceptible (A/J) hosts. To critically evaluate the bactericidal (rather than bacteriostatic) function of the macrophage, a temperature-sensitive (ts) mutant of L. monocytogenes was developed. Mutagenesis was induced with nitrosoguanidine, and the ts mutants were isolated following enrichment with penicillin-gentamicin combinations. The ts mutants were found to carry the cell surface and biochemical characteristics of the original wild-type strain of L. monocytogenes. Inflammatory peritoneal macrophages from resistant C57BL/6J mice were found to have enhanced listericidal activity when compared with inflammatory macrophages from susceptible A/J mice. However, further analysis of the macrophage populations revealed that this seemingly qualitative advantage was due to the relatively greater proportion of inflammatory macrophages present in the inflammatory exudates of resistant C57BL/6J mice. When homogeneous populations of pure inflammatory macrophages were compared, no interstrain differences in their listericidal activity in vitro were seen. These results suggest that the susceptibility of A/J strain mice to L. monocytogenes is not due to an intrinsic deficiency of the listericidal activity of the inflammatory macrophage. The slight increase in bactericidal activity of macrophages from resistant mice that was reported by others (C. J. Czuprynski, B. P. Canono, P. M. Henson, and P. A. Campbell, Immunology 55:511-518, 1985) is caused by the difference in the relative percentage of resident cells present in the peritoneal exudates from resistant and susceptible mice.

Effect of chemotactins released by Staphylococcus aureus and Pseudomonas aeruginosa on the murine respiratory tract.

Staphylococcus aureus, the Pseudomonas aeruginosa temperature-sensitive (ts) mutant A/10/25, and the P. aeruginosa parental wild type were aerosolized to C5-deficient mice, and the total number of polymorphonuclear leukocytes (PMN) recovered by lung lavage was determined 4 h after aerosol exposure. S. aureus induced a slight but significant recruitment of PMN, as compared with the effect of a saline aerosol. Both wild-type P. aeruginosa and the ts mutant induced a significant PMN recruitment of a magnitude ca. 180 times higher than that produced by S. aureus. Gentamicin-killed ts P. aeruginosa induced a PMN recruitment of a magnitude similar to that produced by live ts P. aeruginosa. Thorough washing of the bacteria, however, removed ca. 90% of the chemotactic activity. Exposure of the animals to a ts P. aeruginosa culture supernatant aerosol induced significant PMN recruitment into the lower airways. The same culture supernatants were chemotactic for mouse PMN in a dose-dependent fashion when tested in vitro in the absence of serum. Culture supernatants of S. aureus exhibited weak chemotactic activity in vitro and did not induce PMN recruitment in the lungs when aerosolized to DBA/2J mice. The results suggest that chemotactins released by P. aeruginosa may be an important virulence factor and play a significant role in lung tissue damage.

Quantitative determination of bacterial replication in vivo.

A new methodology which permits the quantitative measurement of absolute bacterial replication in vivo is proposed. Mice were inoculated with mixtures of temperature-sensitive mutants and parental wild types, and the changes in the ratios of the two strains were measured. The number of wild-type generations was calculated from the declining ratios over time with the formula n = log (r0/rt)/log 2; n is the number of generations, and r0 and rt are the ratio of temperature-sensitive mutants to the parental wild type at time zero and at the times sampled throughout the experiment. The replication rate was determined by regression analysis. A mathematical argument for the formula is presented. Using this technique, we determined the mean generation times of Escherichia coli (33 min) and Pseudomonas aeruginosa (20 min) in the peritoneal cavities of mice, in the face of host clearance mechanisms during the first stages of infection.

Inflammatory responses to Pseudomonas aeruginosa and Staphylococcus aureus in the murine lung.

The changes in pulmonary cell population in response to aerosols containing either Pseudomonas aeruginosa or Staphylococcus aureus were studied in a murine model. The lungs of inbred DBA/2J mice received an inoculum of 2 X 10(5) colony-forming units of the microorganism and lung lavages were performed at various time intervals thereafter. P. aeruginosa aerosols produced an immediate decrease in the number of resident alveolar macrophages (AM), followed by a two-waved recruitment of cells into the respiratory tract; the first wave was composed of polymorphonuclear leukocytes (PMN) and the second of monocyte-like peroxidase-positive AM. The change in cell populations was transient and returned to baseline values within a week after aerosolization. In contrast, aerosolized S. aureus initially induced a slight increase in mononuclear cells, and by 60 min after aerosol exposure, the cell population was not different from that of control animals.