Irma A. J. M. Bakker-Woudenberg

Continuous versus intermittent administration of ceftazidime in experimental Klebsiella pneumoniae pneumonia in normal and leukopenic rats.

Experimental Klebsiella pneumoniae pneumonia was used to study the influence of cyclophosphamide-induced leukopenia on the relative therapeutic efficacy of continuous and intermittent (6-h intervals) administration of ceftazidime. The antimicrobial response was evaluated with respect to the calculated daily dose that protected 50% of the animals from death (PD50) until 16 days after the termination of a 4-day treatment. When ceftazidime was administered intermittently to leukopenic rats, the PD50 was 24.37 mg/kg per day, 70 times (P less than 0.001) the PD50 of 0.35 mg/kg per day for normal rats. Continuous administration of ceftazidime to leukopenic rats resulted in a PD50 of 1.52 mg/kg per day, four times (P less than 0.001) the PD50 of 0.36 mg/kg per day for normal rats. Continuous administration of ceftazidime in daily doses that protected 100% of normal and leukopenic rats from death resulted in serum levels of 0.06 and 0.38 micrograms/ml, respectively, whereas the MIC for the infecting K. pneumoniae strain was 0.2 micrograms of ceftazidime per ml. The effect of the duration of ceftazidime treatment by continuous infusion on the therapeutic efficacy in relation to the persistence of leukopenia was then investigated in leukopenic rats. The administration of 3.75 mg of ceftazidime/kg per day for 4 days protected all leukopenic rats from death, provided the circulating leukocytes returned at the end of antibiotic treatment. When leukopenia persisted for 8 days this ceftazidime treatment schedule resulted in the mortality of rats (P less than 0.05). However, when ceftazidime treatment was continued for 8 days, until the return of the leukocytes, there was no significant mortality (P greater than 0.05).

Time–kill kinetics of anti-tuberculosis drugs, and emergence of resistance, in relation to metabolic activity of Mycobacterium tuberculosis

OBJECTIVES The pharmacodynamics of tuberculosis (TB) treatment should be further explored, to prevent emergence of resistance, treatment failure and relapse of infection. The diagnostic drug susceptibility tests guiding TB therapy investigate metabolically active Mycobacterium tuberculosis (Mtb) isolates under static conditions and as such are not informative with respect to the time-kill kinetics of anti-TB drugs and the emergence of resistance in metabolically lowly active or even dormant mycobacterial cells. METHODS In vitro, the killing capacity of rifampicin, isoniazid, ethambutol and amikacin regarding the degree of killing, killing rate and selection of resistant mutants was investigated in metabolically highly active versus metabolically lowly active Mtb cells. RESULTS Isoniazid showed rapid and high killing capacity towards highly active mycobacteria, but due to the emergence of resistance could not eliminate the Mtb. Efflux pump-mediated isoniazid resistance was predominant. Rifampicin revealed a relatively slow and time-dependent killing capacity, but achieved elimination of all mycobacteria. Ethambutol was not bactericidal. Amikacin showed a high and extremely rapid killing activity that was not time dependent and could eliminate all mycobacteria. Exposure of lowly active Mtb populations to isoniazid, rifampicin or amikacin led to the emergence of resistant mutants. Compared with the highly active mycobacteria, elimination of the susceptible lowly active mycobacteria required a 64-fold increased isoniazid concentration and a 4-fold increased rifampicin concentration, whereas amikacin was equally effective irrespective of the metabolic state of the mycobacteria. CONCLUSIONS The anti-TB drugs differ significantly regarding their time-kill kinetics. In addition, the metabolic state of Mtb significantly affects its susceptibility to antimicrobials, with the exception of amikacin. Optimization of dosage of anti-TB drugs is required to achieve maximum drug concentrations at the site of infection in order to maximize reduction in Mtb load and to minimize the emergence and selection of resistance.

Enhanced localization of liposomes with prolonged blood circulation time in infected lung tissue

In an experimental model of unilateral pneumonia caused by Klebsiella pneumoniae in rats we investigated whether intravenous administration of liposomes with prolonged blood circulation time resulted in significant localization of liposomes in infected lung tissue. Liposomes (100 nm) composed of hydrogenated phosphatidylinositol:hydrogenated phosphatidylcholine:cholesterol (molar ratio, 1:10:5) radiolabeled with gallium-67-deferoxamine showed relatively long blood circulation time. The degree of localization of these long circulating liposomes in the infected left lung was significantly higher compared to that of localization of 110 nm egg phosphatidylglycerol:egg phosphatidylcholine:cholesterol (molar ratio, 1:10:5) liposomes which exhibited relatively short blood circulation time. At 16 h after administration of the long circulating liposomes (when 10% of the injected dose was still present in the bloodstream) localization of liposomes in the infected left lung was increased up to 10-fold compared to the left lung of uninfected rats, and appeared to be highly correlated with the intensity of the infection. In the uninfected right lung the localization of long circulating liposomes was not increased. The degree of localization of liposomes in the infected tissue is dependent on the residence time of liposomes in the blood compartment. The extent of localization of long circulating liposomes in infected tissue appeared to be dependent on the liposomal dose administered.

Management of mycetoma: major challenge in tropical mycoses with limited international recognition

Purpose of review The present review highlights an orphan infectious disease in alarming need of international recognition. While money is being invested to develop new broad-spectrum antimicrobial drugs to treat infection in general, improvement in the management of complicated infections such as mycetoma receives little support. Recent findings Many case presentations describe single-center experience in the management of mycetoma. Unfortunately, randomized and blinded clinical studies into the efficacy of antimicrobial treatment are desperately lacking. Response to medical treatment is usually better in actinomycetoma than eumycetoma. Eumycetoma is difficult to treat using current therapies. Surgery in combination with azole treatment is the recommended regimen for small eumycetoma lesions in the extremities. Bone involvement complicates clinical management, leaving surgical amputation as the only treatment option. Although clinical management has not received major attention recently, laboratory technology has improved in areas of molecular diagnosis and epidemiology. Summary Management of mycetoma and laboratory diagnosis of its etiological agents need to be improved and better implemented in endemic regions. Optimized therapeutic approaches and more detailed epidemiological data are urgently needed. It is vital to initiate multicenter collaborations on national and international levels to develop consensus clinical score sheets and state-of-the-art treatment regimens for mycetoma patients.

Drug Susceptibility of Mycobacterium tuberculosis Beijing Genotype and Association with MDR TB

To determine differences in the ability of Mycobacterium tuberculosis strains to withstand antituberculosis drug treatment, we compared the activity of antituberculosis drugs against susceptible Beijing and East-African/Indian genotype M. tuberculosis strains. Beijing genotype strains showed high rates of mutation within a wide range of drug concentrations, possibly explaining this genotype’s association with multidrug-resistant tuberculosis.

Impact of the dosage schedule on the efficacy of ceftazidime, gentamicin and ciprofloxacin inKlebsiella pneumoniae pneumonia and septicemia in leukopenic rats

The impact of the dosage schedule on the therapeutic efficacy of antibiotics was investigated in experimentalKlebsiella pneumoniae pneumonia and septicemia in leukopenic rats. The daily doses (mg/kg) that protected 50 % of the animals from death, when calculated for administration at 6 h intervals or by continuous infusion, were as follows: ceftazidime 24.4 and 1.5 (p<0.001), gentamicin 2.8 and 3.8 (p>0.05), and ciprofloxacin 3.3 and 6.5 (p<0.05), respectively. This correlates with the observation that ceftazidime killedKlebsiella pneumoniae slowly but constantly, and relatively independently of concentration, whereas killing by gentamicin or ciprofloxacin was rapid, and markedly dependent on antibiotic concentration. Exposure of bacteria for 1 h to concentrations of fivefold the MBC did not give rise to a postantibiotic effect for any of the drugs. In our model ceftazidime was far more effective when given continuously than when administered at 6 h intervals. On the other hand, the activity of gentamicin was not influenced by the mode of administration, whereas ciprofloxacin was slightly more effective when given intermittently. However, to avoid misleading conclusions regarding the use of antibiotics in humans, the pharmacokinetic differences between rats and man must be considered when interpreting these results.

Localization of sterically stabilized liposomes in Klebsiella pneumoniae-infected rat lung tissue: influence of liposome characteristics.

Sterically stabilized liposomes are able to localize at sites of infection and could serve as carriers of antimicrobial agents. For a rational optimization of liposome localization, the blood clearance kinetics and biodistribution of liposomes differing in poly(ethylene glycol) (PEG) density, particle size, bilayer fluidity or surface charge were studied in a rat model of a unilateral pneumonia caused by Klebsiella pneumoniae. It is shown that all liposome preparations studied localize preferentially in the infected lung compared to the contralateral non-infected lung. A reduction of the PEG density or rise in particle size resulted in a higher uptake by the mononuclear phagocyte system, lower blood circulation time and lower infected lung localization. Differences in bilayer fluidity did not affect blood clearance kinetics or localization in the infected lung. Increasing the amount of negatively charged phospholipids in the liposome bilayer did not affect blood clearance kinetics, but did reduce localization of this liposome preparation at the site of lung infection. In conclusion, the degree of localization at the infected site is remarkably independent of the physicochemical characteristics of the PEG liposomes. Substantial selective liposome localization can be achieved provided that certain criteria regarding PEG density, size and inclusion of charged phospholipids are met. These properties seem to be a direct consequence of the presence of the polymer coating operating as a repulsive steric barrier opposing interactions with biological components.

Toxicity of doxorubicin entrapped within long-circulating liposomes

We studied the effect of doxorubicin entrapped within long-circulating liposomes (Dox-LCL) on the phagocytic capacity and bacterial blood clearance capacity of rat liver macrophages. Dox-LCL (125 nm in diameter) were composed of egg phosphatidylcholine (PC), cholesterol (CH) and poly(ethyleneglycol)distearoylphosphatidylethanolamine (PEG-PE) (55:45:5 molar ratio; MW PEG 1900), and loaded with doxorubicin by means of a trans-membrane pH gradient. The doxorubicin/lipid ratio was 0.36:1 (mol/mol). At different time-points after one, two or three intravenous injections of Dox-LCL, radiolabeled negatively charged test liposomes (egg PC, CH, and phosphatidylserine in a 4:5:1 molar ratio) were injected. After 2 h, liver macrophages were isolated and the amount of macrophage-associated radioactivity was determined. Twenty-four hours after a single injection of 5 mg/kg Dox-LCL, no significant effect was observed. However, 48 h after injection, phagocytic activity was reduced significantly (49%). Recovery of phagocytic capacity of the liver macrophages took 8 days after two injections of Dox-LCL (2 x 5 mg/kg). Seventy-two hours after the last of two injections of Dox-LCL, bacterial blood clearance was significantly reduced as compared to clearance in control rats and in rats injected twice with doxorubicin combined with placebo liposomes. When comparing these Dox-LCL data with previous data on the effects of Dox-L, Dox-LCL appears less toxic than Dox-L for the liver macrophage population following i.v. administration both with respect to specific phagocytic activity and cell numbers. Due to the delay in onset of toxic effects and the faster recovery from Dox-LCL treatment as compared to Dox-L treatment, it is conceivable that therapeutic protocols can be designed with Dox-LCL that circumvent long-term impairment of the liver macrophage population.

Testing of the In Vitro Susceptibilities of Madurella mycetomatis to Six Antifungal Agents by Using the Sensititre System in Comparison with a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5- [(Phenylamino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) Assay and a Modified NCCLS Method

ABSTRACT The in vitro susceptibilities of 36 clinical isolates of Madurella mycetomatis, the prime agent of eumycetoma in Africa, to ketoconazole, itraconazole, fluconazole, voriconazole, amphotericin B, and flucytosine were determined by the Sensititre YeastOne system. This system appeared to be a rapid and easy test, and by use of hyphal suspensions it generated results comparable to those of a modified NCCLS method. After 10 days of incubation, the antifungal activities of ketoconazole (MIC at which 90% of isolates were inhibited [MIC90], 0.125 μg/ml), itraconazole (MIC90, 0.064 μg/ml), and voriconazole (MIC90, 0.125 μg/ml) appeared superior to those of fluconazole (MIC90, 128 μg/ml) and amphotericin B (MIC90, 1 μg/ml), with MICs in the clinically relevant range. All isolates were resistant to flucytosine (all MICs above 64 μg/ml). Based on the relatively broad range of MICs obtained for the antifungal agents, routine testing of M. mycetomatis isolates for susceptibility to antifungal agents seems to be relevant to adequate therapeutic management.

Apolipoprotein CI stimulates the response to lipopolysaccharide and reduces mortality in Gram-negative sepsis

Gram‐negative sepsis is a major death cause in intensive care units. Accumulating evidence indicates the protective role of plasma lipoproteins such as high‐density lipoprotein (HDL) in sepsis. It has recently been shown that septic HDL is almost depleted from apolipoprotein CI (apoCI), suggesting that apoCI may be a protective factor in sepsis. Sequence analysis revealed that apoCI possesses a highly conserved consensus KVKEKLK binding motif for lipopolysaccharide (LPS), an outer‐membrane component of Gram‐negative bacteria. Through avid binding to LPS involving this motif, apoCI improved the presentation of LPS to macrophages in vitro and in mice, thereby stimulating the inflammatory response to LPS. Moreover, apoCI dose‐dependently increased the early inflammatory response to Klebsiella pneumoniae‐induced pneumonia, reduced the number of circulating bacteria, and protected mice against fatal sepsis. Our data support the hypothesis that apoCI is a physiological protector against infection by enhancing the early inflammatory response to LPS and suggest that timely increase of apoCI levels could be used to efficiently prevent and treat early sepsis.—Berbée, J. F. P., van der Hoogt, C. C., Kleemann, R., Schippers, E. F., Kitchens, R. L., van Dissel, J. T., Bakker‐Woudenberg, I. A. J. M., Havekes, L. M., Rensen, P. C. N. Apolipoprotein CI stimulates the response to lipopolysaccharide and reduces mortality in Gram‐negative sepsis. FASEB J. 20, E1560 –E1569 (2006)