Department of Clinical Microbiology, Albert Szent-Györgyi Medical University, H-6720 Szeged, PO Box 482, Hungary
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Abstract |
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Introduction |
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In the present study, three methods (disc diffusion, chequerboard titration and the time- killing method) were used to detect synergy of selected antibiotics (aminoglycosides, fluoroquinolones and third-generation cephalosporins) against clinical isolates of Pseudomonas spp. The results obtained by the three methods were subjected to a comparative evaluation.
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Materials and methods |
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The minimum inhibitory concentrations (MICs) of the antibiotics were determined in Mueller- Hinton broth with the microbroth dilution method. 12 The range of antibiotic dilutions was 0.031- 512 mg/L.
The synergic effects of the antibiotic combinations against the selected isolates were examined
by three methods: (i) disc diffusion test two discs, each containing one or other of the
two tested antibiotics, were placed at a distance of about 20 mm from each other on top of a Pseudomonas sp. isolate-covered agar plate. Synergy was considered to occur when there
was a well-observed change (2 mm) in the zone of inhibition. The synergy was classified
as
weak when a change of <2 mm was observed in the zone of inhibition.
12
,16 (ii) chequerboard titration
subcultures of the bacteria were made from fresh blood agar and incubated overnight at
37°C in Mueller- Hinton broth. Two-fold dilutions of the antibiotics were made and 50
µL aliquots were placed into the wells of a sterile plastic microtitre plate. The criterion of
synergy was an FIC index not higher than 0.5.
12 The ranges of antibiotic dilutions used in the
chequerboard titration were: 0.5- 512 mg/L for aminoglycosides, 0.031- 64 mg/L for
quinolones and 0.5- 256 mg/L for cephalosporins. (iii) Time- killing method
subcultures of the test bacteria were made from fresh blood agar culture in Mueller- Hinton broth
and were incubated overnight. The same inoculum was used in fresh Mueller- Hinton broth
containing no antibiotic, one or other of the antibiotics in ½ x MIC concentrations, or
the same amounts of antibiotics in combination, and the cultures were incubated for 24 h at
37°C. The cfu/mL values of the cultures were determined at the beginning of the incubation
and after 2, 4, 6 and 24 h of incubation. Synergy was defined as a
100-fold increase in
killing
at 24 h (as measured by colony counts) with the combination in comparison with the more active
single drug.
12
,17
The following antimicrobial agents were used: amikacin (Bristol Myers Squibb, Sermoneta, Italy), netilmicin (Schering-Plough Labo N.V., Heist-op-den-Berg, Belgium), ciprofloxacin (Bayer, Leverkusen, Switzerland), ofloxacin (Hoechst AG, Frankfurt am Main, Germany), pefloxacin (Rhône- Poulenc/EGIS Gyógyszergyár, Budapest, Hungary), cefoperazone (Pfizer Biogál Kft., Debrecen, Hungary), ceftazidime (Glaxo S.P.A., Verona, Italy) and ceftriaxone (Hoffmann- La Roche Ltd, Basel, Switzerland). The stock solutions were prepared in sterile distilled water.
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Results |
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Discussion |
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The application of antimicrobial combination therapy in such patients attempts to take advantage of synergic antibiotic interactions to enhance the efficacy in the treatment of the infections and to decrease the risk of development of resistance during therapy. Combinations of aminoglycosides and antipseudomonal penicillins have been well investigated. 17 ,19 ,20 Weiss & Lapointe 19 reported that a tobramycin- ceftazidime combination exerted a synergic effect against 39% of the tested P. aeruginosa isolates from patients with cystic fibrosis. Jenkins & Lewis, 8 however, found no significant in-vitro synergy between fluoroquinolones and other antimicrobial agents when they applied the chequerboard titration method against Pseudomonas(Burkholderia) cepacia isolates. Moody et al. (1987), 21 using the same method, demonstrated synergy for various Pseudomonas spp.when ciprofloxacin was administered in combination with different ß-lactam antibiotics. Enciso 22 found that ceftazidime in combination with ciprofloxacin exhibited synergy against 40% of P. aeruginosa isolates, whereas ceftazidime plus amikacin did so against 53% of the isolates. Gould & Milne 11 tested the efficacies of gentamicin and ciprofloxacin in combination with piperacillin- tazobactam against different Gram- positive and Gram-negative species, including Pseudomonas spp. isolates, and described a synergic effect. Neu 9 ,10 ,23 observed synergy when quinolones and antipseudomonal penicillins and ceftazidime were applied in combination. His results led him to suggest the use of these combinations in therapy in special cases. 9 ,10 ,23
The aim of the present study was to evaluate the effects of different aminoglycoside,
fluoroquinolone and ß-lactam combinations against selected Pseudomonas spp.
exhibiting different resistance patterns to these drugs. An evaluation of the synergic effects of
different antibiotic combinations is highly dependent on the method used.
24 Different methods were applied during this study and the
results were compared. Although 7- 12 of 18 isolates displayed expressed or weak synergy by the
disc diffusion screening test when amikacin or netilmicin and third- generation cephalosporins
were applied, only 2- 7 isolates did so when combinations of fluoroquinolones were tested. With
the chequerboard titration method and calculation of the FIC index, a synergic
effect (FIC index 0.5) was observed less frequently. In our experience, the changes in the
zone of inhibition can reveal synergy where chequerboard titration and the
time- killing method do not. Different combinations of the aminoglycosides- fluoroquinolones
and third-generation cephalosporins exhibited different effects, depending on the Pseudomonas spp. isolates and the antibiotic combination. Discrepancies were observed
between the FIC indices and the killing activities resulting from time- killing experiments. The
combination of amikacin and ceftriaxone exerted synergic killing activity after 6 and 24 h for ten
isolates where chequerboard titration did not indicate a synergic effect. In
contrast, chequerboard titration indicated synergy for six isolates where the
time- killing method did not. Differences were likewise observed between the disc diffusion and
time- killing methods (data not shown). Haller
24 tested the effects of combinations of ciprofloxacin and
aminoglycosides against Escherichia coli, Serratia marcescens and Pseudomonas spp. isolates, and his results appear to demonstrate methodology-based
differences in the in-vitro activities of these combinations. His study revealed less than 5%
synergy for ciprofloxacin plus azlocillin and less than 1% synergy for the ciprofloxacin plus
aminoglycoside combination against P. aeruginosa isolates. On the other hand, Chin et al.
25 detected synergy and/or an additive beneficial effect
against 78% of P. aeruginosa when ciprofloxacin was combined with azlocillin.
In the present study, the fluoroquinolone ciprofloxacin in combination with third-generation cephalosporins had the highest activity. However, all combinations used exerted a synergic effect against some of the 18 Pseudomonas spp. isolates belonging to the different species and selected on the basis of their different resistance patterns.
In conclusion, these in-vitro comparative studies demonstrate that, besides the well-known synergic effects of aminoglycoside and ß-lactam antibiotics, quinolones (primarily ciprofloxacin) in combination with ceftazidime may exert enhanced activity against clinical isolates of Pseudomonas spp. However, our data suggest that measurement of the time- killing of the bacteria is the most reliable means of assessing the existence of a synergic effect.
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Acknowledgments |
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Notes |
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References |
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Received 9 July 1998; returned 26 October 1998; revised 4 December 1998; accepted 29 December 1998