Combined effects of meropenem and aminoglycosides on Pseudomonas aeruginosa in vitro

Atsushi Nakamuraa,*, Masayoshi Hosodaa, Takashi Katoa, Yasuo Yamadaa, Makoto Itoha, Katsunori Kanazawab and Hiroshi Noudab

a First Department of Internal Medicine, Nagoya City University, Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan; b Sumitomo Pharmaceuticals Research Center, 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-0022, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
To investigate combinations of antibiotics against Pseudomonas aeruginosa, the in vitro effects of combinations of meropenem with each of three aminoglycosides, arbekacin, amikacin and netilmicin, were evaluated using an agar dilution chequerboard technique. The combinations of meropenem and aminoglycosides were effective against almost all P. aeruginosa strains tested, which included meropenem-resistant strains. Increased synergic effects were observed in combinations that included arbekacin or amikacin. None of the combinations had an antagonistic effect. Most of the synergic and additive effects were achieved at clinically relevant concentrations.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Meropenem, a frequently used carbapenem, is stable against most ß-lactamases and has strong antimicrobial activities with a broad spectrum covering most Gram-positive and Gram-negative bacteria.1 Carbapenem-resistant Pseudomonas aeruginosa can cause life-threatening infections,2 however, and other carbapenem-resistant bacteria could emerge as a result of frequent use of carbapenems. For these reasons, we investigated the effects of meropenem combined with each of the three aminoglycosides, arbekacin, amikacin and netilmicin, on P. aeruginosa in vitro.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains and antibiotics

The 50 clinical strains of P. aeruginosa used in this study were isolated from patients at Nagoya City University Hospital. Thirty-four were from sputum, seven from urine, four from decubitus ulcers, two from pharyngeal smears and three from other specimens.

Meropenem was obtained from Sumitomo Pharmaceuticals, Osaka, Japan; arbekacin and amikacin from Meiji Seika Kaisha, Ltd, Osaka, Japan; and netilmicin from Schering-Plough Co., Osaka, Japan.

Determination of MICs

Mueller–Hinton broth or agar (Nissui, Tokyo, Japan) was used in all studies. The MICs of antibiotics alone or in combination were determined using a two-fold serial agar dilution method according to the ‘Standard Method’ recommended by the Japanese Society of Chemotherapy.3 In summary, serial two-fold dilutions of the antibiotics in Mueller–Hinton agar were inoculated with an overnight broth culture of the organism to give a final concentration of c. 106 cfu/mL. After incubating at 37°C for 20 h, the MIC was defined as the lowest concentration of antibiotic that completely inhibited visible growth. Serial two-fold concentrations ranging from 0.05 to 25 mg/L for meropenem and 0.1 to 100 mg/L for arbekacin, amikacin and netilmicin were prepared.

Determination of the combined effect of meropenem and other antibiotics

The effects of antibiotic combinations were assessed using an agar dilution chequerboard technique and fractional inhibitory concentration (FIC) indices.4 The FIC for each agent was calculated by dividing the MIC of the antibiotics when used in combination by that of the drug alone. The FIC index is the sum of the FICs of each of two antibiotics when examined in combination. A minimum FIC index of <=0.5 indicates synergy, while an FIC index >2 indicates antagonism. If the minimum FIC index was >0.5 and <=1, the effect of the combination was classified as additive. If the minimum FIC index was >1 and <=2, the effect of the combination was classified as indifferent.


    Results
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 Materials and methods
 Results
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Antipseudomonal activity of antibiotics

The MICs of each antibiotic alone for P. aeruginosa are shown in Table IGo. Meropenem had low MICs for P. aeruginosa (MIC50 0.39 mg/L; MIC90 3.13 mg/L) except for two resistant strains (MIC >= 12.5 mg/L). For most of the strains tested, arbekacin had lower MICs than did amikacin or netilmicin.


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Table I. MICs of antibiotics for 50 strains of P. aeruginosa
 
Combined effect of meropenem and aminoglycosides against P. aeruginosa

Most minimum FIC indices were distributed in the synergic or additive range (arbekacin, 0.16–1.06; amikacin, 0.16– 1.03; netilmicin, 0.38–1.13). The combinations of meropenem and aminoglycosides were effective against most of the strains tested; combinations that contained arbekacin or amikacin were more synergic than those containing netilmicin. None of the combinations had an antagonistic effect (Table IIGo). Combinations of meropenem and aminoglycosides also had synergic or additive effects against each strain of meropenem-resistant P. aeruginosa.


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Table II. Combined effects of meropenem and aminoglycosides against 50 strains of P. aeruginosa
 
MICs of meropenem combined with each of three aminoglycosides for P. aeruginosa with a minimum FIC index are shown in Table IIGo. The MIC50 and MIC90 of meropenem were reduced to <=1.56 mg/L in combination with the aminoglycosides tested. The MIC50 and MIC90 of aminoglycosides tested were also reduced to <=6.25 mg/L in combination with meropenem.


    Discussion
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 Materials and methods
 Results
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 References
 
Combinations of ß-lactams and aminoglycosides have synergic effects against P. aeruginosa. The underlying mechanism is thought to be the destruction of cell wall peptidoglycan polymers by ß-lactams and enhancement of the entry of the aminoglycosides into the bacterial cell.5,6 We investigated the effect of combining meropenem with three aminoglycosides, namely arbekacin, amikacin and netilmicin, which are frequently used in Japan. The combinations of meropenem and aminoglycosides were effective against almost all P. aeruginosa strains tested, which included meropenem-resistant strains. Ferrara et al.7 studied the interaction of meropenem with other antibiotics against P. aeruginosa, determined using the chequerboard technique, and found synergic effects of meropenem in combination with amikacin or tobramycin, but antagonistic effects in combination with cefotaxime, ceftazidime, azlocillin or aztreonam. In contrast, another study reported no synergy against P. aeruginosa when meropenem was combined with aminoglycosides (netilmicin, tobramycin or gentamicin).8 In these studies, MICs of antibiotics were determined using a microdilution technique, so the discrepancies between these and our data could be due to the different methods used. Although arbekacin has been used to treat infections with methicillin-resistant Staphylococcus aureus (MRSA) in Japan, it is a broad-spectrum aminoglycoside antibiotic. It binds to both the 50S and 30S ribosomal subunits, inhibits protein synthesis at bacterial ribosomes, causes misreading of codons5 and has bactericidal activities against some gentamicin- or amikacin-resistant bacteria.9 The synergy between meropenem and arbekacin could be related to these bactericidal activities.

Oie et al.10 reported the usefulness of a combination of two ß-lactams and amikacin or that of three ß-lactams in treating multi-drug resistant P. aeruginosa. In the present study, a combination of meropenem and aminoglycosides was also effective against two strains of meropenem-resistant P. aeruginosa. The combined effect of meropenem and arbekacin or amikacin was also examined for two more strains of meropenem-resistant P. aeruginosa; these two strains were excluded, however, because the MIC of netilmicin alone could not be determined for these strains. Synergy between meropenem and arbekacin was observed in both strains, as was either synergy or additivity between meropenem and amikacin (data not shown).

The MIC90 of meropenem in all combinations with aminoglycosides was <=1.56 mg/L (Table IIGo), while the MIC90 of aminoglycosides in combination with meropenem was <=6.25 mg/L. Such concentrations are readily achieved in human plasma after a standard dose of each antibiotic.

In conclusion, combinations of meropenem and aminoglycosides were effective against almost all P. aeruginosa strains tested, including meropenem-resistant strains, but further evaluations of bactericidal activity, in vivo activity and clinical efficacy are needed.


    Notes
 
* Corresponding author. Tel: +81-52-853-8211; Fax: +81-52-852-0952; E-mail: anakamur{at}med.nagoya-cu.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Fish, D. N. (1997). Meropenem, a new carbapenem antibiotic. Pharmacotherapy 17, 644–69.[ISI][Medline]

2 . Deguchi, K., Yokota, N., Koguchi, M., Suzuki, Y., Suzuki, K., Fukayama, S. et al. (1992). Antimicrobial activities of major oral antimicrobial agents against clinically isolated microbial strains from inpatients. Japanese Journal of Antibiotics 45, 965–79.[Medline]

3 . Goto, S., Jo, K., Kawakita, T., Kosakai, N., Mitsuhashi, S., Nishino, T. et al. (1981). Determination method of minimum inhibitory concentrations. Japanese Journal of Chemotherapy 29, 76–9.

4 . Totsuka, K., Shiseki, M., Kikuchi, K. & Matsui, Y. (1999). Combined effects of vancomycin and imipenem against methicillin- resistant Staphylococcus aureus (MRSA) in vitro and in vivo. Journal of Antimicrobial Chemotherapy 44, 455–60.[Abstract/Free Full Text]

5 . Watanabe, T., Obayashi, K., Matsui, K. & Kubota, T. (1997). Comparative studies of the bactericidal morphological and post-antibiotic effects of arbekacin and vancomycin against methicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 39, 471–6.[Abstract]

6 . Wise, R., Ashby, J. P. & Andrews, J. M. (1989). The antibacterial activity of meropenem in combination with gentamicin or vancomicin. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 233–8.[ISI][Medline]

7 . Ferrara, A., Grassi, G., Grassi, F. A., Piccioni, P. D. & Gialdroni Grassi, G. (1989). Bactericidal activity of meropenem and interactions with other antibiotics. Journal of Antimicrobial Chemotherapy 24, Suppl. A, 239–50.[ISI][Medline]

8 . Kropec, A., Lemmen, S., Wursthorn, M. & Daschner, F. D. (1994). Combination effect of meropenem with aminoglycosides and teicoplanin on Pseudomonas aeruginosa and enterococci. Infection 22, 306–8.[ISI][Medline]

9 . Fukuchi, K., Takeda, K., Takagi, Y. & Gomi, K. (1992). Synergism of imipenem in combination with arbekacin against methicillinresistant Staphylococcus aureus and Pseudomonas aeruginosa. Japanese Journal of Chemotherapy 40, 780–8.

10 . Oie, S., Sawa, A., Kamiya, A. & Mizuno, H. (1999). In-vitro effects of a combination antipseudomonal antibiotics against multi-drug resistant Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 44, 689–91.[Abstract/Free Full Text]

Received 14 February 2000; returned 21 May 2000; revised 12 July 2000; accepted 19 August 2000