Evidence of the proinflammatory role of Enterococcus faecalis in polymicrobial peritonitis in rats.

Although the role of members of the Enterobacteriaceae and anaerobes in the pathogenesis of intra-abdominal infections has been extensively demonstrated, the role played by enterococci in these infections remains controversial. The pathophysiological mechanisms induced by enterococci in intra-abdominal infection were studied in a nonfatal model of peritonitis in rats by implanting a gelatin capsule containing Escherichia coli and Bacteroides fragilis with or without increasing concentrations of Enterococcus faecalis or heat-inactivated enterococci. The ability of the rat peritoneal cavity to sterilize itself after bacterial challenge was evaluated by quantifying the inflammatory response in the peritoneal cavity, reflected by both phagocyte and cytokine responses. Effects were evaluated 6, 12, and 24 h and 3 and 6 days after inoculation. On day 6 after inoculation, the highest enterococcal concentration (10(8) CFU/ml) was accompanied by significantly increased concentrations of E. coli in peritoneal fluid and peritoneal phagocytes when compared to other groups. In the first 12 h after inoculation, tumor necrosis factor and interleukin-6 concentrations were significantly increased in the peritoneal fluid of the animals that had received the highest inoculum of enterococci or heat-inactivated enterococci. In the late period of the study (3 and 6 days), significantly increased leukocyte counts were observed in the peritoneal fluid of these animals. These results suggest that E. faecalis somehow inhibited phagocytosis and intracellular killing of the other pathogens and also played an inflammatory role, which might account for the bacterial synergy observed in this model.

Importance of penicillinase production for activity of penicillin alone or in combination with sulbactam in experimental endocarditis due to methicillin-resistant Staphylococcus aureus.

The activity of penicillin, alone and in combination with sulbactam, against a heterogeneously methicillin-resistant, penicillinase-producing clinical isolate of Staphylococcus aureus and its penicillinase-negative derivative was investigated in vitro and in a rabbit experimental endocarditis model. Penicillin was significantly more effective than vancomycin against the penicillinase-negative derivative in vivo (P < 0.001), and it sterilized 25% of the vegetations. The combination of penicillin and sulbactam exhibited an in vivo synergistic effect on the penicillinase-producing strain (P < 0.01) but did not produce any advantage over treatment with vancomycin, even when a high dose of sulbactam was used (100 mg/kg of body weight every 6 h). This combination was significantly less effective against the penicillinase-producing strain than was penicillin alone against the penicillinase-negative derivative (P < 0.03). In addition, the most resistant subpopulation of the surviving bacteria, which grew on agar containing 16 micrograms of methicillin per ml, was detected in 5 of 6 animals treated with penicillin and a high dose of sulbactam against the penicillinase-producing strain compared with only 1 of 12 animals treated with penicillin alone against the penicillinase-negative derivative (P < 0.01). We conclude that penicillin is highly effective against penicillinase-negative methicillin-resistant S. aureus and that penicillinase production, rather than methicillin resistance, appears to be the limiting factor for the activity of the penicillin-sulbactam combination against penicillinase-producing, methicillin-resistant S. aureus.

Comparison of fluconazole and amphotericin B for treatment of experimental Candida albicans endocarditis in rabbits.

Amphotericin B (AmB) and fluconazole, administered intraperitoneally for 7 days, were compared in a rabbit model for Candida albicans endocarditis. When given early, AmB was more effective than fluconazole for reducing CFU counts in vegetations (P < 0.01) and kidneys. Forty-eight hours after the last dose, AmB was still detected in all vegetations whereas fluconazole was detected in only one case.

Critical influence of resistance to streptogramin B-type antibiotics on activity of RP 59500 (quinupristin-dalfopristin) in experimental endocarditis due to Staphylococcus aureus.

In order to determine the microbiological and pharmacokinetic parameters that best predicted the in vivo antistaphylococcal activity of the streptogramin RP 59500 (quinupristin-dalfopristin), we evaluated the activity in rabbit aortic endocarditis of three regimens of quinupristin-dalfopristin against five strains of Staphylococcus aureus with various streptogramin B-type antibiotic resistance phenotypes and susceptible to streptogramin A-type antibiotics. Quinupristin-dalfopristin was as active as vancomycin against three strains that were susceptible to its streptogramin B component quinupristin, including one strain that was inducibly resistant to erythromycin, but had a significantly decreased activity against two strains that were resistant to quinupristin, for all quinupristin-dalfopristin regimens tested (P < 0.05). The area under the concentration-time curve for quinupristin-dalfopristin in plasma divided by the MIC of quinupristin was the only parameter retained by multilinear regression that predicted the in vivo activity of quinupristin-dalfopristin (P = 0.0001), emphasizing the importance of determining the susceptibility to quinupristin in order to predict the in vivo activity of quinupristin-dalfopristin against S. aureus.

In vivo activity of interferon-gamma in combination with amphotericin B in the treatment of experimental cryptococcosis.

The effect of recombinant interferon-gamma (rIFN-gamma) on Cryptococcus neoformans infection was investigated in vivo. BALB/c mice were injected intravenously with 2 x 10(6) C. neoformans. rIFN-gamma alone (10 micrograms intraperitoneally 18 h before, at, 24 h after infection) significantly increased the survival and decreased the colony-forming unit counts in the lungs compared with untreated mice. rIFN-gamma association significantly enhanced the effect of a single dose of amphotericin B (0.25 mg/kg 24 h after infection) to prolong mouse survival and to reduce colony counts in the brain. Tumor necrosis factor-alpha levels, measured in spleen 3, 6, and 24 h after infection, were not increased by rIFN-gamma. These results suggest that exogenous rIFN-gamma might improve the effect of antifungal therapy during cryptococcosis.

In vitro renal toxicity and in vivo therapeutic efficacy in experimental murine cryptococcosis of amphotericin B (Fungizone) associated with Intralipid.

We compared the experimental toxicities and activities of deoxycholate amphotericin B (d-AmB) dissolved in glucose (Dd-AmB) or mixed with 20% Intralipid (ILd-AmB). In vitro, ILd-AmB against renal tubular cells in primary culture. In vivo, the toxicities and activities of Dd-AmB and ILd-AmB were studied in DBA2 mice with cryptococcosis. The maximum tolerated dose of intravenously administered d-AmB, i.e., the dose that induced less than 15% mortality because of toxicity, was 1.7 to 2.5 times higher when it was administered as ILd-AmB than when it was administered as Dd-AmB. Both treatments given intravenously at the same dose were equivalent for improving the survival of mice and reducing CFU counts in infected tissue, but at maximum tolerated doses, ILd-AmB (2 mg/kg of body weight) was more effective than Dd-AmB (0.8 to 1.2 mg/kg). AmB concentrations in spleen, liver, lung, and kidney were measured by high-pressure liquid chromatography 4 and 24 h after a single injection of 1.2 mg of Dd-AmB per kg, 1.2 mg of ILd-AmB per kg, or 2 mg of ILd-AmB per kg. In a given organ, AmB levels were similar after administration of 1.2 mg of Dd-AmB or ILd-AmB per kg but were significantly higher after administration of 2 mg of ILd-AmB per kg. The lower level of toxicity of ILd-AmB might be explained by circular dichroism experiments, showing that ILd-AmB contained 10-fold less soluble oligomeric AmB, which is believed to be the toxic form of the drug, than Dd-AmB. We conclude that ILd-AmB is as efficient as Dd-AmB and is better tolerated than Dd-AmB in mice with experimental cryptococcosis. By allowing higher doses of AmB to be infused, Intralipid enhances AmB concentrations in infected sites, and thus the therapeutic activity of the drug.

Activity of MS-8209, a nonester amphotericin B derivative, in treatment of experimental systemic mycoses.

The in vitro and in vivo toxicities and activities of MS-8209, a new hydrosoluble amphotericin B (deoxycholate-amphotericin B [D-AmB]; Fungizone) derivative, were studied. In vitro, MS-8209 was less toxic than AmB against renal tubular cells in primary culture and less active against Candida albicans and Cryptococcus neoformans. However, at 10-fold the AmB concentration, MS-8209 in vitro antifungal activity paralleled that of AmB. Fifty-percent lethal doses of MS-8209 and D-AmB in OF1 noninfected mice were 26 and 2.3 mg/kg, respectively. Therapeutic efficacy of MS-8209 was assessed in murine candidiasis, cryptococcosis, and aspergillosis. In each model of infection, we determined the maximum tolerated dosages of MS-8209 and D-AmB, i.e., the dosage inducing less than 15% mortality due to toxicity; the efficacies of MS-8209 and D-AmB at their respective maximum tolerated dosages were compared. In candidiasis, MS-8209 (15 mg/kg) significantly increased the survival time compared with D-AmB (0.5 mg/kg). Both compounds were equally effective at reducing CFU counts in the kidney. MS-8209 was the most effective agent for increasing the survival time in cryptococcal meningoencephalitis and for reducing CFU counts in spleen, brain, and lung during both cryptococcal pneumonia and meningoencephalitis. In aspergillosis, MS-8209 and D-AmB similarly prolonged the survival of treated mice compared with controls. These results show that when MS-8209 and D-AmB were used at the maximum tolerated dosage, MS-8209 was as effective as or more effective than D-AmB for the treatment of systemic mycoses. These findings warrant further experiments to study the pharmacokinetic properties and toxicity of MS-8209 under conditions of chronic administration.

Tissue distribution and antifungal effect of liposomal itraconazole in experimental cryptococcosis and pulmonary aspergillosis.

We studied the tissue distribution and in vivo antifungal effect of itraconazole, incorporated into pure dipalmitoylphosphatidylcholine (DPPC) multilamellar liposomes and administered intravenously. Eighty percent of the itraconazole was associated with DPPC. Drug levels in lung, brain, and liver, obtained after intravenous administration of tritiated itraconazole, were higher when the drug was administered intravenously as liposomal than when it was dissolved in cyclodextrin. Administration of the liposomal formulation also led to higher and sustained levels of intact itraconazole in serum. Efficacy was assessed in DBA/2 mice infected intravenously with 3 x 10(6) Cryptococcus neoformans, an inoculum responsible for early fatal pneumonia, or 3 x 10(5) C. neoformans, leading to delayed meningitis. In pneumonia, 20 mg/kg of liposomal itraconazole was more effective on survival than the same dose given intravenously in cyclodextrin or twice the dose administered orally dissolved in polyethylene glycol 200. In meningitis, liposomal itraconazole was also more efficient than the drug dissolved in cyclodextrin. These results were confirmed by colony counts in the brain and lung of infected mice. In immunosuppressed OF1 mice infected after inhalation of Aspergillus fumigatus spores, liposomal itraconazole (20 mg/kg x 3) was the only effective treatment. We conclude that intravenous liposomal delivery of itraconazole enhances both concentrations in infected tissues and the in vivo efficacy of the drug. Such passive targeting of antifungal agents other than amphotericin B might be helpful in the treatment of severe systemic mycoses, especially in the case of lung or brain involvement.

Influence of phospholipid/amphotericin B ratio and phospholipid type on in vitro renal cell toxicities and fungicidal activities of lipid-associated amphotericin B formulations.

We studied the influence of the lipid/amphotericin B (AMB) ratio and the phospholipid type on the in vitro renal cell toxicity and antifungal efficacy of lipid-associated AMB (L-AMB). L-AMB was prepared at one of two different lipid/AMB ratios (1 and 40) by incubating AMB with empty small unilamellar vesicles, made from one of three different phospholipids: dipalmitoyl-, dimirystoyl-, and distearoylphosphatidylcholine (DPPC, DMPC, and DSPC, respectively). Renal cell toxicity, investigated through an assessment of the Na-dependent uptake of phosphate by proximal tubular cells, and fungicidal effect against Candida albicans were studied after 1 h of treatment at 37 degrees C. The amount of unbound AMB present in each L-AMB formulation was studied by use of circular dichroism. At a lipid/AMB ratio of 40, the three lipidic formulations were not toxic for renal cells but were less effective against C. albicans than AMB; however, DSPC-AMB, which contained 50% unbound AMB, was more effective against C. albicans than DPCC-AMB or DMPC-AMB, containing 0 and 13% unbound AMB, respectively. At a lipid/AMB ratio of 1, the antifungal effects of L-AMB and AMB were similar, whatever the phospholipid used, but only DMPC-AMB remained highly protective against AMB renal cell toxicity, despite the presence of the same amount of unbound AMB (50%) in DMPC-AMB and DPPC-AMB. We conclude that the in vitro activities and renal cell toxicities of different L-AMB formulations are influenced by the phospholipid type and the lipid/AMB ratio. The optimal ratio depends on the phospholipid itself. At a lipid/AMB ratio of 40, the antifungal activity depends mainly on the amount of unbound AMB in the formulation. At a lipid/AMB ratio of 1, the renal cell toxicity also depends on the fluidity of the phospholipid.

Inhibiting cholesterol synthesis reduces the binding and toxicity of amphotericin B against rabbit renal tubular cells in primary culture.

Renal tubular cells are a target of amphotericin B (AmB) toxicity, but the mechanisms involved in the tubular cell-AmB interactions are unknown. Ketoconazole was selected to lower the cholesterol content of rabbit renal tubular cells in primary culture. The consequences of cholesterol depletion on AmB nephrotoxicity was investigated in vitro as the inhibition of Na(+)-dependent phosphate uptake. After 1 h of exposure, AmB decreased phosphate uptake (49%, 77%, and 82% inhibition with 5, 10, and 20 microM of AmB, respectively). Pretreatment of cells with ketoconazole (10 microM for 24 h) reduced by 50% (P less than .01) the phosphate uptake inhibition induced by AmB, decreased cellular cholesterol synthesis (greater than 80% inhibition), and decreased AmB binding to cell membrane by 50%, as measured by the fluorescence extinction of a probe bound to tubular cell membrane. Incubation with exogenous exchangeable cholesterol again increased AmB binding to plasma membrane and restored AmB toxicity. These results demonstrate that the first step of AmB renal tubular toxicity is mediated by cellular cholesterol content and is parallel to the binding of AmB to cell membrane.

Interactions of free and liposomal amphotericin B with renal proximal tubular cells in primary culture.

We studied in vitro the renal toxicity of amphotericin B (AMB) and liposomal AMB using primary cultures of rabbit proximal tubular cells grown to confluence in serum-free medium. Toxicity was assessed by changes 1) in the Na(+)-dependent uptakes of P1 and alpha-methylglucopyranoside (MGP), characteristic functions of proximal tubular cells; 2) K+ release into the supernatant, dependent upon membrane permeability; and 3) lactic dehydrogenase release as a marker for cellular death. Cells were exposed for 1 hr to AMB, alone or intercalated in small unilamellar vesicles prepared with one of the following phospholipids: dipalmitoylphosphatidyl choline, distearoylphosphatidyl choline or dimyristoylphosphatidyl choline. Although AMB concentrations of 20 microM or less did not increase lactic dehydrogenase release, P1 and MGP uptakes were significantly reduced (50% inhibition) by 2.5 and 5 microM AMB, respectively. AMB toxicity was dose-dependent, up to 20 microM. Analysis of P1 and MGP uptake kinetics, after treatment of the cells with 10 microM AMB, showed that inhibition occurred through a decrease in Vmax (66 and 57% inhibition for P1 and MGP, respectively) without affecting Km value. K+ release appeared for 2.5 microM AMB and increased with higher concentrations. Alteration of Na(+)-dependent uptakes by AMB, which parallels K+ release, may result from an alteration of the sodium gradient. Na(+)-dependent uptakes and K+ release were unaffected by liposomal AMB, even at the highest concentration tested (80 microM). The protective effect of liposomes was the same regardless of the phospholipid used.(ABSTRACT TRUNCATED AT 250 WORDS)