Journal of Antimicrobial Chemotherapy (1999) 44, 689-691
© 1999 The British Society for Antimicrobial Chemotherapy
Brief report |
In-vitro effects of a combination of antipseudomonal antibiotics against multi-drug resistant Pseudomonas aeruginosa
a Department of Pharmacy b Clinical Laboratory, Yamaguchi University Hospital, 1144 Kogushi, Ube 755, Japan
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Abstract |
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We evaluated the in-vitro effects of various combinations of five types of widely used antipseudomonal antibiotics (piperacillin, meropenem, ceftazidime, aztreonam and amikacin) against six Pseudomonas aeruginosa strains that were resistant to each of these antibiotics. Among two-drug combinations, the combinations of two ß-lactam antibiotics inhibited growth of one to three P. aeruginosa strains, while those of one ß-lactam antibiotic and amikacin inhibited growth of two to four strains. Among three-drug combinations, the combinations of three ß-lactam antibiotics inhibited growth of four to five strains, and those of two ß-lactam antibiotics and amikacin inhibited growth of five strains. These results suggest the potential usefulness of a combination of two ß-lactam antibiotics and amikacin or that of three ß-lactam antibiotics in treating multi-drug resistant P. aeruginosa infections.
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Introduction |
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In the treatment of Pseudomonas aeruginosa infections, ß-lactam antibiotics such as ceftazidime, aminoglycoside antibiotics such as amikacin, or a combination of two drugs from different chemical classes is widely used.1,2 However, P. aeruginosa strains that are resistant to ß-lactam antibiotics, aminoglycoside antibiotics or both have appeared,3 ,4 ,5 ,6 ,7 presenting major clinical problems. In this study, we examined the in-vitro effects of various combinations of piperacillin, meropenem, ceftazidime, aztreonam and amikacin against multi-drug resistant P. aeruginosa strains that were resistant to each of these antibiotics. Intravenous ciprofloxacin is not available in Japan, and for this reason was not included in this study.
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Materials and methods |
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Bacterial strains
We requested five hospitals in Yamaguchi Prefecture, Japan to supply non-replicate strains of P. aeruginosa, isolated from patients with infection between December 1994 and May 1998, that were possibly resistant to piperacillin, meropenem, ceftazidime, aztreonam andamikacin. A total of 36 strains were provided and re-examined for identification (Gram's stain, morphology, oxidation–fermentation test, cytochrome oxidase test and ‘API’ system) and antimicrobial sensitivity. Of the 36 strains, six multi-drug resistant P. aeruginosa strains showing the following antibiotic resistance pattern were used in this experiment: piperacillin, MIC> 64 mg/L; meropenem, MIC> 8 mg/L; ceftazidime, MIC> 16 mg/L; aztreonam, MIC> 16 mg/L; amikacin, MIC> 8 mg/L. The sources of the six strains were bronchial secretion (four strains), urine (one) and blood (one).
Susceptibility tests
MICs were determined after 18 h incubation at 37°C by dilution on Sensitivity Disc Agar-N (Nissui Pharm., Tokyo, Japan). The following antibiotics were tested: piperacillin (Toyama Chemicals, Tokyo, Japan), meropenem (Sumitomo Pharmaceuticals, Tokyo), ceftazidime (Glaxo Japan Co., Tokyo), aztreonam (Eisai Co., Tokyo) and amikacin (Banyu Pharmaceuticals, Tokyo). These antibiotics were provided in the form of a freeze-dried amorphous powder. The inocula (104 cfu/spot) were plated using a multipoint inoculator (Sakuma Co., Tokyo). The MIC was defined as the lowest antibiotic concentration that inhibited visible growth. P. aeruginosa IFO 3919 was used as a reference strain.
The effects of the combination of two or three drugs among the above five types of antibiotics were evaluated. The antibiotic concentration was set as follows: piperacillin, 64 mg/L; meropenem, 8 mg/L; ceftazidime, 16 mg/L; aztreonam, 16 mg/L; amikacin, 4 mg/L. The presence or absence of growth on Sensitivity Disc Agar-N was determined by a method similar to that used for the measurement of MIC.
Data analysis
The effects against the six P. aeruginosa strains were compared (between the combinations of two drugs and those of three drugs, and between the combinations of three drugs not including amikacin and three drugs including amikacin) by the chi-square test, using the number of growth-inhibited strains as a variable.
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Results |
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A total number of approximately 7000 P. aeruginosa strains were isolated from patients over a 3 year period at the five hospitals that were requested to provide multi-drug resistant P. aeruginosa isolates. The isolation incidence of multi-drug resistant P. aeruginosa strains that were resistant to all five of the drugs (piperacillin, meropenem, ceftazidime, aztreonam and amikacin) was approximately 0.08% (6/7000).
The Table shows the MICs of the five types of drugs used for the six multi-drug resistant P. aeruginosa strains, and the presence or absence of growth inhibition by the combination of two or three drugs among the five drugs. P. aeruginosa Nos 1 and 3 were obtained from patients treated in the same hospital, but four other isolates were obtained, respectively, from patients treated in four different hospitals. The combinations of two ß-lactam antibiotics inhibited growth of one to three strains. The combinations of one ß-lactam antibiotic and amikacin inhibited growth of two to four strains. Among the combinations of three ß-lactam antibiotics, piperacillin + meropenem + ceftazidime and piperacillin + meropenem + aztreonam inhibited growth of four strains, while piperacillin + ceftazidime + aztreonam and meropenem + ceftazidime + aztreonam inhibited growth of five strains. The combinations of two ß-lactam drugs and amikacin also inhibited growth of five strains.
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Statistical comparison of the combination effects of antibiotics against the strains used showed a significant difference in the number of growth-inhibited strains between the combinations of two drugs and those of three drugs (P < 0.01). However, no difference was observed between the combinations of three drugs not including amikacin and those of three drugs including amikacin.
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Discussion |
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The isolation incidence of multi-drug resistant P. aeruginosa strains may still be low in Japan. However, since multi-drug resistant P. aeruginosa strains that are resistant to all widely used antipseudomonal antibiotics have appeared, the establishment of treatment methods for P. aeruginosa infection is of major importance. The studies of antibiotic combinations should be carried out now rather than when it is too late. Breakpoints used for all antibiotics tested except amikacin were according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria.8 However, in Japan, the administration dose of amikacin is 200–400 mg/day (in one to two divided doses) in adults, which is lower than that used in Western countries. Therefore, our own criteria were used for amikacin.
In this study, the combinations of two ß-lactam antibiotics and amikacin were moderately effective against multi-drug resistant P. aeruginosa. Roussel-Delvallez et al.,9 who performed a 24 h time–kill study, also showed potentiation effects of a three-drug combination of imipenem + (ticarcillin + clavulanic acid) + amikacin on multi-drug resistant P. aeruginosa. Therefore, the combination of two ß-lactam antibiotics and amikacin is worth considering for multi-drug resistant P. aeruginosa infection. In some patients receiving nephrotoxic drugs such as amphotericin B, cisplatin or cyclosporin, since administration of amikacin is inappropriate, piperacillin + ceftazidime + aztreonam or meropenem + ceftazidime + aztreonam may be effective.
In this study, the three-drug combinations of two ß-lactam antibiotics and amikacin was ineffective against one of the six multi-drug resistant P. aeruginosa isolates. Even when the amikacin concentration was increased from 4 to 32 mg/L, these three-drug combinations were ineffective against this strain (data not shown). A future question may be what combinations of antibiotics should be used for such multi-drug resistant P. aeruginosa strains?
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Acknowledgments |
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The authors are grateful to Clinical Laboratories, Shimonoseki Municipal Hospital, Shimonoseki Kosei Hospital, Saiseikai Yamaguchi General Hospital and Yamato General Hospital for the kind donation of P. aeruginosa strains.
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Notes |
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* Corresponding author. Tel:+81-836-22-2665; Fax: +81-836-22-2705.
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References |
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Received 18 February 1999; returned 29 April 1999; revised 19 May 1999; accepted 27 June 1999
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