Investigation of the synergic effects of aminoglycoside- fluoroquinolone and third-generation cephalosporin combinations against clinical isolates of Pseudomonas spp.

I. Mayer and E. Nagy*

Department of Clinical Microbiology, Albert Szent-Györgyi Medical University, H-6720 Szeged, PO Box 482, Hungary


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial synergy resulting from antibiotic combination therapy is often important in the treatment of serious bacterial infections. Previous studies have demonstrated synergy between an aminoglycoside and ß-lactam antibiotics in the treatment of Pseudomonas aeruginosa infections. The present paper investigates the synergic effects of aminoglycosides (amikacin and netilmicin) and fluoroquinolones (ciprofloxacin, ofloxacin and pefloxacin) in combination with third-generation cephalosporins (cefoperazone, ceftriaxone and ceftazidime) against 18 clinical isolates of Pseudomonas spp. The effects of these drugs were examined by three methods (disc diffusion, ‘chequerboard’ titration and the time- killing method), to evaluate the activities of the antibiotics alone and in combination against selected isolates. Fractional inhibitory concentration indices were calculated for all isolates with all combinations. Use of the disc diffusion method revealed that amikacin and netilmicin in combination with the three cephalosporins exhibited synergy against 7- 12 isolates, whereas the combinations of quinolones and ceftazidime displayed synergic effects only in the case of 3- 5 isolates. On ‘chequerboard’ titration, amikacin and ceftriaxone exerted synergy against seven of the isolates. The other combinations showed synergy against fewer isolates, but every combination demonstrated synergic effect against some of the isolates. The tested combinations had different effects against various Pseudomonas spp. With the time- killing method, the ½ xMIC of amikacin or ciprofloxacin in combination with the ½ xMIC of third-generation cephalosporins proved to be most effective. No antagonism was found with these combinations. Discrepancies in the detection of synergy were observed for the different methods.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa and other Pseudomonas spp. continue to be a significant cause of morbidity and mortality, especially in intensive care units, and they are frequent isolates causing nosocomial epidemics. 1 ,2 ,3 ,4 ,5 ,6 The multiresistance of these isolates plays an important role in the colonization or infection of chronically hospitalized patients. 2 ,6 ,7 ,8 For effective treatment of nosocomial infections caused by these multiresistant isolates, clinicians often have to resort to the most potent fluoroquinolones or combinations of different antibiotics. 6 ,7 ,9 ,10 The most widely documented synergy is to be found when ß-lactams are combined with aminoglycosides. These combinations are synergic against most species, including P. aeruginosa. Although these combinations are clinically well proven to provide effective treatment, there are limitations, such as an increasing resistance of Pseudomonas spp. to ß-lactam antibiotics and toxicity associated with aminoglycoside therapy. 11 For this reason, there is a continuous search for alternative combinations. One such group of combinations is fluorinated quinolones with ß-lactams, where synergy has been demonstrated against clinical isolates of P. aeruginosa. 11 Various in-vitro methods are available for measurement of the synergic effects of combinations of antibiotics, but the results may exhibit discrepancies when different tests are used. 12 ,13 ,14 ,15

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The 18 Pseudomonassp. isolates used in this study were isolated from severe infections in different patients from different clinics. Routine isolation and identification methods were applied. 13 These isolates belonged in different Pseudomonas spp. and their antibiotic sensitivities to aminoglycosides, fluoroquinolones and third-generation cephalosporins were also different.

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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eighteen isolates belonging to various Pseudomonas spp. with different resistance patterns were used for this study. The MICs of the tested antibiotics were determined (Table I). According to the resistance breakpoints, on the basis of the NCCLS performance standards, 18 2- 12 isolates were resistant to the tested individual antibiotics. Ceftazidime, amikacin and ciprofloxacin proved to be the most active agents in the different groups of antibiotics against these isolates. The P. aeruginosa isolates were more sensitive to aminoglycosides, ceftazidime and fluoroquinolones than the isolates of the other species.


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Table I. MICs of selected antibiotics against 18 Pseudomonas isolates
 
Table II relates to the evaluation of the frequency of the synergic effects of the antibiotic combinations tested by the disc diffusion method. Amikacin and netilmicin in combination with cefoperazone, ceftriaxone and ceftazidime exhibited a synergic effect (expressed or weak) in the case of 7- 12 of the isolates, although ceftriaxone is not an everyday antipseudomonal antibiotic. When the combinations of ciprofloxacin, ofloxacin and pefloxacin with ceftazidime were tested by this method, synergy was observed for only 3- 5 isolates.


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Table II. Synergic effects of combinations of aminoglycosides and fluoroquinolones with third-generation cephalosporins against 18 selected Pseudomonas isolates by disc diffusion method
 
Somewhat different results were obtained when ‘chequerboard’ titration was used (Table III). The combination of amikacin with ceftriaxone exerted a synergic effect against seven isolates, whereas that of netilmicin with cefoperazone did so against six of the 18 Pseudomonas spp. isolates. The other combinations displayed synergic effects against fewer than six of the tested isolates. For some isolates and antibiotic combinations, only an additive effect was indicated by ‘chequerboard’ titration, whereas the disc diffusion method revealed weak synergy. Synergy was found in 61 cases by disc diffusion, but only in 37 cases with ‘chequerboard’ titration. The antibiotic combinations amikacin- ceftriaxone and netilmicin- cefoperazone exhibited the best synergic effect with both of these methods. No antagonism was detected with disc diffusion and ‘ chequerboard’ titration for any of the combinations used.


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Table III. Effects of combinations against isolates belonging in different categories, using ‘chequerboard’ titration
 
The FIC indices for the different antibiotic combinations are shown in Table IV for six Pseudomonas spp. isolates belonging to different species, which were selected according to their different sensitivities to the tested antimicrobials (Table I) and their different responses to the antibiotic combinations tested previously. Whereas seven and five combinations gave extremely low FIC indices for Pseudomonas testosteroni (new nomenclature Comomonas testosteroni) and Pseudomonas acidovorans (new nomenclature Comomonas acidovorans), respectively, only one combination proved synergic for each of three P. aeruginosa and Pseudomonas stutzeri isolates. The different quinolones in combination with ceftazidime displayed expressed synergic effects against P. testosteroni, whereas only the combination pefloxacin- ceftazidime had an FIC index of 0.5 for P. acidovorans. Those combinations which were not synergic for P. testosteroni and P. acidovorans, such as amikacin- ceftriaxone and netilmicin- ceftriaxone, revealed synergy against two P. aeruginosa isolates.


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Table IV. Fractional inhibitory concentration (FIC) indices of different antibiotic combinations for six Pseudomonas isolates, by ‘chequerboard’ titration
 
The Figure depicts the kinetics of killing by ciprofloxacin in combination with ceftazidime against P. testosteroni isolate 1 (a), P. aeruginosa isolate 3 (b) and P. stutzeri isolate 15 (c). A strong synergic effect was observed in the cases of P. testosteroni isolate 1 and P. aeruginosa isolate 3, whereas only an additive effect was seen against P. stutzeri isolate 15. In contrast, ‘chequerboard’ titration did not reveal synergism for P. aeruginosa isolate 3 and P. stutzeri isolate 15 (FIC index of 0.531 and 0.75, respectively), but synergy was found for P. testosteroni isolate 1 with this method.



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Figure. Time- killing effects of ciprofloxacin ({diamond}) and ceftazidime ({blacktriangleup}) alone and in combination ({circ}); control ({blacksquare}). (a) P. testosteroni isolate 1; (b) P. aeruginosa isolate 3; (c) P. stutzeri isolate 15.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite the recent introduction into clinical practice of highly potent antibiotics such as the newer aminoglycosides, fluoroquinolones and the third-generation cephalosporins or imipenem, Pseudomonas spp. infections (involving mostly nosocomial multiresistant isolates) pose a major therapeutic problem for clinicians world-wide.

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.


    Acknowledgments
 
This study was partly supported by the Hungarian National Scientific Research Fund (OTKA T-016222).


    Notes
 
* Corresponding author. Tel and Fax: +36-62-420981. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 9 July 1998; returned 26 October 1998; revised 4 December 1998; accepted 29 December 1998