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Scientific
Publications - Work Done by Microbiology Reader
A. Georgopoulos, W. Graninger, P. Bezecny, A. Buxbaum, In vitro activity of fosfomycin combined with glycopeptides, moxifloxacin, gentamicin, and ampicillin against Enterococci, assessed by the modified spectrophotometric method, Poster, Wien, Austria, 2001 ABSTRACT Fifty vancomycin-susceptible and six vancomycin-resistant clinical strains of enterococci were tested on their susceptibility to fosfomycin, gentamicin, teicoplanin, vancomycin, moxifloxacin, ampicillin, piperacillin-tazobactam, ceftazidime, cefpirome, cefepime, ceftriaxone, meropenem, and imipenem in vitro by the broth microdilution method. Fosfomycin MICs ranged from 16 to 128 µg/mL and were not significantly influenced by the antibiotic susceptibility patterns of the strains. The activity of fosfomycin, a phosphonic acid derivative with a broad spectrum of bactericidal activity against Gram-positive cocci and several Gram-negative bacteria, combined with vancomycin, teicoplanin, moxifloxacin, gentamicin, and ampicillin was studied in kinetic spectrophotometric assays using the automatic turbidometer Bioscreen-C-Analyzer which permittes continuous monitoring of bacterial growth. All five antibiotics provided an enhanced (synergistic or additive) bacteriostatic effect in combination with fosfomycin against most enterococcal strains. While a weak or moderate beneficial effect was observed with vancomycin, gentamicin and moxifloxacin, a strong effect was demonstrated if fosfomycin was combined with teicoplanin and ampicillin. Fosfomycin and teicoplanin appeared to be the most effective combination and exhibited synergistic activity against 44 of 50 vancomycin-susceptible and 4 of 6 vancomycin-resistant strains, respectively. To compare the Bioscreen results with a conventionally used technique, evaluation of antibiotic combinations was performed also by checkerboard titration. Good overall correlation between the spectrophotometric assay and the checkerboard technique could be observed. We conclude that the new approach of spectrophotometric assessment of antimicrobial combinations using the Bioscreen analyzer could be a valuable alternative for routine investigations.
INTRODUCTION The synergistic combination of a cell wall active antibiotic and an aminoglycoside remains the treatment of choice for serious enterococcal infections. However, such regimens are sometimes not feasible because of adverse reactions or high levels of resistance. Increasing resistance of enterococci to cell wall active agents, such as ßlactams and glycopeptides, stresses the need for new agents or combinations of agents that exhibit antienterococcal activity. This study was designed to evaluate the in vitro activity of fosfomycin alone and in combination with other antibiotics against clinical strains of Enterococcus faecalis and Enterococcus faecium with various resistance patterns, using a kinetic spectrophotometric method.
MATERIAL AND METHODS Bacterial strains and media. 56 clinical strains of enterococci (41 vancomycinsusceptible E. faecalis, six vancomycin-resistant E. faecalis and nine E. faecium) isolated from urin, blood, sputum and BAL in 1998/99 were used as test strains. The reference strain used in the study was E. faecalis ATCC 29212. Mueller-Hinton broth (Oxoid) was used as growth medium, glucose-6-phosphate (25 µg/ml) being added for tests with fosfomycin.
Experiments performed with the Bioscreen-C-Analyzer. The Bioscreen-C Analyzer (Lab Systems, Helsinki, Finland) is an automatic device designed for the kinetic measurement of turbidity in microbial samples. Dilutions of antimicrobials in Mueller-Hinton broth were made automatically in the Bioscreen honeycomb -type plates containing 400 µl wells. To the wells with 100 µl volumes of antimicrobials with a concentration of 0.5 × MIC were added samples (100 µl each) of the cultures to give a bacterial inoculum of 5 × 105 CFU/ml, glucose-6-phosphate concentration of 25 µg/ml and the antimicrobial concentration of 0.25 × MIC. The final volume in each well was 200 µl. Each well was recorded continuously every 10 minutes during the experiment, using a wavelength of 540 nm. The lowest detectable level of opti cal density for enterococci corresponded to approximately 5 × 105 CFU/ml. Each strain was exposed to an antibiotic A alone, antibiotic B alone and to the combination of the both A and B in the same concentration (0.25 × MIC). Drug free controls of each strain were included with each run.The results were processed by the Bioscreen C and reported as growth curves. Growth parameters, such as growth rate and the area under the concentration-time curve (AUC) were calculatedSynergism, antagonism, additivity and indifference were determined according to following definitions (modified from King et al. and Eliopoulos and Moellering):
RESULTS As shown in Table I, all five antibiotics provided a beneficial - synergistic or additiv - effect in combination with fosfomycin against most enterococcal strains. While a weak or moderate beneficial effect was observed with vancomycin, gentamicin and moxifloxacin, a strong effect was demonstrated with teicoplanin and ampicillin. Teicoplanin combined with fosfomycin appeared to be the most efficient combination and provided a synergistic effect against 44 of the 50 vancomycinsusceptible strains tested. Among vancomycin-resistant enterococci teicoplanin/fosfomycin synergism could be found in four strains and additivity in two strains (Table II).
CONCLUSIONS The strongest beneficial effects were observed with the combinations of fosfomycin with teicoplanin and with ampicillin, regardless of the resistance pattern of enterococcal strains. Fosfomycin combined with other cell wall active agents seems to act stronger than when combined with moxifloxacin or with gentamicin. The in-vitro synergistic combinations of fosfomycin with teicoplanin and ß-lactams as possible therapeutic alternatives for serious enterococcal infections should be evaluated in in-vivo trials.
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