Efficient treatment of murine systemic infection with Candida albicans using amphotericin B incorporated in nanosize range particles (emulsomes).

The effects of emulsome nanosize range lipid particles containing amphotericin B (EAmB) were compared with the reference formulation containing deoxycholate (Fungizone; Bristol-Myers Squibb, Munich, Germany) and with the commercial amphotericin lipid complex preparation (AmBisome; Nexstar, San Dimas, CA, USA). The minimal inhibitory concentrations of Fungizone and EAmB were identical although killing of Candida albicans was delayed when EAmB was used. In a tissue culture model and in mice, the incorporation of AmB into emulsomes resulted in a considerable reduction of toxicity in comparison with Fungizone. For comparison of the in vivo effect of the preparations a mouse model of systemic infection with C. albicans was used. All preparations were able to reduce the fungal burden in the liver and kidneys in comparison with control animals treated with isotonic saline. AmBisome was more efficient in the reduction of the fungal burden of the liver than EAmB and Fungizone, even when applied in a similar dosage of 1 mg kg(-1). In the kidneys, EAmB and Fungizone was slightly more effective than AmBisome. Therefore, in our models, the incorporation of AmB into nanosize particles was able to reduce toxicity without loss of efficiency. EAmB may be considered a candidate preparation for the treatment of infections with C. albicans in humans.

Systemic and intracerebral infections of mice with Listeria monocytogenes successfully treated with linezolid.

Linezolid is an oxazolidinone derivative which is active mostly against Gram-positive bacteria. In this work its activity against the facultatively intracellular bacterium Listeria monocytogenes was examined in vitro, in tissue culture and in animal models of systemic and intracerebral infection and compared with ampicillin which is the antibiotic of choice for treatment of listeriosis. All strains of L. monocytogenes were susceptible to the substance, with minimal inhibitory concentrations (MICs) determined by E-test ranging from 0.38 to 1.5 mg/l which is below the preliminary breakpoint of this substance. Linezolid was bacteriostatic against L. monocytogenes since up to 64 times the MIC did not kill the bacteria in 24 hours. Linezolid was also bacteriostatic on L monocytogenes in infected tissue culture cells. In animal models of systemic and intracerebral infection, linezolid was able to inhibit bacterial growth but was clearly less effective than ampicillin. In conclusion, linezolid might be useful for the treatment of infections with L monocytogenes in humans when ampicillin may not be used.

The ketolide antibiotic HMR 3647, a candidate substance for the treatment of systemic and intracerebral infections with Listeria monocytogenes.

HMR 3647 is a novel macrolide derivative with a broad spectrum of activity against grampositive bacteria and some fastiduous gramnegative bacteria, anaerobes and Toxoplasma gondii. In this work, its activity against the facultatively intracellular bacterium, Listeria monocytogenes, was examined in vitro, in tissue culture and in animal models of systemic and intracerebral infection and compared with that of erythromycin. All strains of L. monocytogenes were susceptible to the substance, with minimal inhibitory concentrations (MICs) that were consistently lower than the MICs of erythromycin. HMR 3647 was bacteriostatic against L. monocytogenes since concentrations of up to 64 times the MIC did not kill the bacteria within 24 hours. HMR 3647 produced a pronounced postantibiotic effect (PAE) and was bacteriostatic in tissue culture cells infected with L. monocytogenes. In animal models of systemic and intracerebral infection, HMR 3647 was slightly more effective than erythromycin in the livers and spleens and comparably effective in the brains when given in the same dosage. In conclusion, HMR 3647 is a candidate substance for the treatment of infections with L. monocytogenes in immunocompetent subjects.

Clinafloxacin (CL 960) is superior to standard therapeutics in the treatment of murine listeriosis and salmonellosis.

Clinafloxacin is a novel fluoroquinolone with a broad spectrum of antibacterial activity against both gramnegative and grampositive bacteria. In this work, the activity against the facultatively intracellular bacteria Listeria monocytogenes and Salmonella typhimurium was examined in vitro, in tissue culture and in animal models of infection. All strains of L. monocytogenes and S. enterica were highly susceptible against clinafloxacin, with minimal inhibitory concentrations (MICs) that were consistently lower than those for ampicillin and ciprofloxacin, respectively. Clinafloxacin was rapidly bactericidal against L. monocytogenes and S. typhimurium since 8 times the MIC killed the bacteria within 2 hours. In contrast to ampicillin and ciprofloxacin, there was a postantibiotic effect (PAE) of 2 hours with 8 x MIC on L. monocytogenes. Clinafloxacin was more effective than ampicillin and ciprofloxacin in tissue culture cells infected with S. typhimurium or L. monocytogenes. In animal models of infection, clinafloxacin was also more potent than the reference substances. In conclusion, clinafloxacin is an excellent candidate substance for the treatment of infections caused by facultatively intracellular grampositive and gramnegative bacteria.

Management of listeriosis.

Determination of the MIC in vitro is often used as the basis for predicting the clinical efficacy of antibiotics. Listeriae are uniformly susceptible in vitro to most common antibiotics except cephalosporins and fosfomycin. However, the clinical outcome is poor. This is partially because listeriae are refractory to the bactericidal mechanisms of many antibiotics, especially to ampicillin-amoxicillin, which still is regarded as the drug of choice. A true synergism can be achieved by adding gentamicin. Another point is that listeriae are able to reside and multiply within host cells, e.g., macrophages, hepatocytes, and neurons, where they are protected from antibiotics in the extracellular fluid. Only a few agents penetrate, accumulate, and reach the cytosol of host cells, where the listeriae are found. Furthermore, certain host cells may exclude antibiotics from any intracellular compartment. Thus, determination of the antibacterial efficacy of a drug against listeriae in cell cultures may be a better approximation of potential therapeutic value. Certain host cells may have acquired the property of excluding certain antibiotics, for example macrolides, from intracellular spaces, which might explain therapeutic failures of antibiotic therapy in spite of low MICs. Animal models do not completely imitate human listeriosis, which is characterized by meningitis, encephalitis, soft tissue and parenchymal infections, and bacteremia. Meningitis produced in rabbits is a hyperacute disease, whereby most listeriae lie extracellularly, fairly accessible to antibiotics that can cross the blood-cerebrospinal fluid barrier. In the murine model of systemic infection, Listeria monocytogenes is located mainly within macrophages and parenchymal cells of the spleen and liver, hardly accessible to certain drugs, such as ampicillin and gentimicin. The therapeutic efficacy of drugs clearly depends on the model used. Thus, for example, the combination of ampicillin with gentamicin acts synergistically in the rabbit meningitis model but not in the mouse model. Since conventional antimicrobial therapy with antibiotics is not satisfactory, particularly in the immunocompromised host (about 30% of patients with listeriosis die in spite of a rational choice of antibiotics), other possibilities must be considered for therapy as well as prevention. Indeed, listeriae are highly susceptible to several endogenous antibiotics, such as defensins. Bacteriocins produced by related bacterial species, e.g., lactobacilli and enterococci, are rapidly bactericidal. However, unfortunately, the use of such alternative measures along with immunization and immunmodulation is not yet feasible.

Excellent activity of newer quinolones on Legionella pneumophila in J774 macrophages.

The activity of six antibiotics directed against intracellularly multiplying Legionella pneumophilia was examined in tissue cultures with J774 macrophages. The drugs tested were the new quinolones, BAY Y 3118 and clinafloxacin, and ciprofloxacin, erythromycin, gentamicin and ampicillin served as reference drugs. Additionally, the MICs of these drugs against L. pneumophila were determined in vitro by broth microdilution. Despite their low MIC values, ampicillin and gentamicin did not inhibit intracellular multiplication of L. pneumophila in J774 macrophages. In contrast, an inhibition of intracellular growth could be demonstrated for the four other antibiotics. The new quinolones BAY Y 3118 and clinafloxacin showed the highest activity against intracellular L. pneumophila. At a concentration of 0.00078 mg/L already, a marked reduction in bacterial counts was seen for both drugs in comparison to the growth control without antibiotics. The corresponding effective concentrations were 0.0125 mg/L for ciprofloxacin and 0.2 mg/L for erythromycin. It may be concluded that new quinolone derivatives might become an alternative to erythromycin and rifampicin which at present are the drugs of primary choice for the treatment of legionnaires' disease.

Effects of amphotericin B incorporated into liposomes and in lipid suspensions in the treatment of murine candidiasis.

The effects of similar amounts of amphotericin B (CAS 1397-89-3, AmB) in different preparations either as conventional amphotericin B (des-AmB), or liposomal AmB (lipos-AmB), or des-AmB dissolved in a lipid emulsion (lipid-AmB) on Candida albicans and other Candida species were compared in several in vitro and in vivo models. The minimal inhibitory concentration (MIC) of des-AmB was equal to the MIC of lipid-AmB when determined after 24 h. In contrast, the MIC of lipos-AmB was 4-8 times the MIC of des-AmB. When tested at 4 times the MIC of the respective preparations suspension of lipid-AmB led to a reduced ability to kill the fungi whereas des-AmB reduced the inoculum by 99% within 6 h. Four times the MIC of lipos-AmB failed completely to kill the fungi in the same time, but was only fungistatic. At 24 h all preparations had killed the yeasts at concentrations 4 times the MIC. In contrast to the in vitro data, lipos-AmB was more active in the treatment of murine candidiasis than lipid-AmB and des-AmB. Lipos-AmB but not lipid-AmB or des-AmB was able to significantly reduce the amount of Candida albicans in the liver when given in the same dosage. Concomitantly, AmB measured by HPLC was highly concentrated in the livers of the mice treated with lipos-AmB. It is concluded that even when given in the same dosage as des-AmB and lipid-AmB, lipos-AmB is more effective in the treatment of murine candidiasis, although it is less effective in vitro. Lipid-AmB is no alternative to lipos-AmB in this model of systemic infection of mice with Candida albicans.

Bay Y 3118, a new quinolone derivative, rapidly eradicates Listeria monocytogenes from infected mice and L929 cells.

Bay Y 3118 is a new quinolone derivative with pronounced activity against gram-positive bacteria including the facultatively intracellular bacterium Listeria monocytogenes. Bay Y 3118 was tested in vitro and in animal models of listeriosis. All strains of L. monocytogenes and other Listeria spp. were highly susceptible in vitro; the MICs for these organisms ranged from 0.062 to 0.25 micrograms/ml. Bay Y 3118 was rapidly bactericidal in vitro, with a postantibiotic effect occurring for 3 h after removal of the antibiotic. L. monocytogenes was eliminated from infected L929 cells treated with Bay Y 3118, suggesting a bactericidal effect on the listeriae in these cells. Immunocompetent mice were rapidly cured by treatment with 4 mg every 12 h. Concomitantly, the levels of interleukin 6 and gamma interferon in mouse sera declined rapidly. In immunocompetent mice, treatment with 2 mg of Bay Y 3118 every 12 h resulted in a greater initial reduction in the listerial counts in the organs than treatment with 2 mg of ampicillin every 12 h. Bay Y 3118 completely eliminated L. monocytogenes from the livers and spleens of chronically infected nude mice. However, some of the bacteria reappeared after the cessation of therapy. In conclusion, Bay Y 3118 is an excellent candidate substance for the therapy of infections caused by facultatively intracellular gram-positive bacteria such as L. monocytogenes.

Effect of various antibiotics on Listeria monocytogenes multiplying in L 929 cells.

Various antibiotics were evaluated as to their effect on Listeria monocytogenes SLCC 4013 multiplying within L 929 mouse fibroblast cells. Antibiotics were employed in concentrations above the MIC value. However, there was no measurable effect of some drugs on intracellular listeriae (azlocillin, mezlocillin, cephalothin, ciprofloxacin, chloramphenicol). With other drugs an inhibition of intracellular growth was observed (penicillin, ampicillin, rifampicin, rifapentine, erythromycin, doxycycline, co-trimoxazole, coumermycin). Notably, with none of the antibiotics a complete eradication of the listeriae was achieved. There is a good correlation of these results with animal experiments. Therefore, the cell culture system might be useful for the screening of new antibiotics.