In vitro inhibition of lymphocyte proliferation by Pseudomonas aeruginosa phenazine pigments.

Human lymphocyte proliferation is inhibited in vitro in the presence of killed Pseudomonas aeruginosa or cell-free P. aeruginosa culture supernatants. A comparison of culture supernatants obtained under similar conditions from Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Pseudomonas cepacia strains demonstrated that all P. aeruginosa supernatants were strongly inhibitory, whereas supernatants from other bacteria were mildly inhibitory or not inhibitory at all. These P. aeruginosa inhibitors prevent proliferative responses of resting cells upon mitogen activation and decrease [3H]thymidine uptake when added to human lymphocytes undergoing active proliferation in culture. The inhibitory effect is reversible and not due to cytotoxicity. Most of the inhibitory activity present in crude supernatants was detected in ultrafiltrates of molecular weights below 2,000. Purified P. aeruginosa pyocyanine, a low-molecular-weight phenazine pigment present in culture supernatant, was strongly inhibitory for lymphocyte proliferation. Extraction of pyocyanine and phenazine pigments from inhibitory P. aeruginosa supernatants eliminated their inhibitory activity. Inhibitors were recovered from reverse-phase chromatographic cartridges by both chloroform and methanol elution, indicating that pyocyanine and other phenazine pigments present in P. aeruginosa supernatants are responsible for the inhibition of lymphocyte proliferation. In addition to the identification of phenazine pigments as lymphocyte proliferation inhibitors, several criteria ruled out major contributions of P. aeruginosa polysaccharide, exotoxin A, and proteases to this phenomenon. P. aeruginosa strains selected for very low protease production or for very low exotoxin A production produced supernatants as inhibitory for lymphocyte proliferation as supernatants obtained from clinical P. aeruginosa isolates. Purified P. aeruginosa lipopolysaccharide and protease preparations failed to induce reversible lymphocyte proliferation inhibition. Finally, heat inactivation of P. aeruginosa supernatants at 100 degrees C for 60 min inactivates exotoxin A and proteases but produced only a moderate decrease of the inhibitory activity for lymphocyte proliferation.

Once-daily vs. continuous aminoglycoside dosing: efficacy and toxicity in animal and clinical studies of gentamicin, netilmicin, and tobramycin.

The dosing frequency of aminoglycoside antibiotics may alter efficacy and toxicity independent of total daily dose. Once-daily tobramycin dosing was compared with continuous infusion in three models of efficacy. Acute pneumonia due to Pseudomonas aeruginosa in guinea pigs responded better to once-daily dosing, and chronic pneumonia in rats and endocarditis in rabbits responded equally to both regimens. Dogs given gentamicin, tobramycin, or netilmicin once daily, with maximum serum concentrations of greater than 100 mg/liter, had less nephrotoxicity than dogs given continuous infusions. Tobramycin was given once daily or continuously to 52 patients with cystic fibrosis who in 10 days had no change in creatinine clearance or hearing despite maximum serum tobramycin concentrations of 40 mg/liter. Intermittent dosing of aminoglycosides, causing infrequent large maximum serum concentrations, may be less toxic and equally efficacious as frequent dosing.

Pseudomonas aeruginosa exoproducts as pulmonary virulence factors.

An experimental animal model of chronic pseudomonas pneumonia was used to document the production of potential virulence factors by Pseudomonas aeruginosa during the infection. The production of exotoxin A, proteolytic enzymes, and the serotype-specific lipopolysaccharide and slime-layer antigens during the infection was examined by solid-phase radioimmunoassay of serum from infected rats and by indirect immunofluorescence tests of their lung tissue. Rats inoculated intratracheally with purified bacterial exoproducts, delivered alone or in combination, developed pulmonary histopathology similar to that induced by the experimental infection. The results indicate that these exoproducts are produced during the course of the pulmonary infection and suggest that they are involved in the observed lung pathology.

Protective immunization against chronic Pseudomonas aeruginosa pulmonary infection in rats.

Rats were immunized systemically with various doses of the polyvalent Pseudomonas aeruginosa vaccine PEV-01. After a series of two or three doses (25 to 50 micrograms each) at 8- to 11-day intervals, animals were challenged intratracheally by the agarose bead technique with a serotype 5 P. aeruginosa strain at periods of 9 to 42 days. Immunized animals developed circulating antibodies (primarily immunoglobulin M) against vaccine components at levels significantly higher than challenged, nonimmunized controls (P less than 0.005). Eight to ten days postinfection, histological sections of lungs from immunized animals showed only minimal inflammation associated with infectious foci (agarose beads) as compared with the extensive pathological changes of airways and parenchyma seen in infected nonimmunized control animals. However, no significant reduction in bacterial numbers was observed. Such protection lasted at least 6 weeks after the final immunization. It is speculated that the vaccine may contain components of cell surface proteins and virulence exoproducts.

A rat model of chronic respiratory infection with Pseudomonas aeruginosa.

Chronic, nonlethal, pulmonary infection of rats by Pseudomonas aeruginosa can be initiated by intratracheal inoculation of 10(4) bacteria enmeshed in agar beads. The number of bacteria recoverable from the lung increased to approximately 10(6) within 3 days and remained at that number during 35 days of observation. Histologic examination of the infected lungs revealed lesions resembling those seen in lung tissue of humans with acute or chronic nonbacteremic, Pseudomonas aeruginosa pneumonia, including the presence of goblet-cell hyperplasia, focal areas of necrosis, and acute and chronic inflammatory infiltrate. This model should be useful for investigating the interactions between microbial virulence factors and host defense mechanisms.