Zbl. Bakt. 283, 69-78 (1995) © Gustav Fischer Verlag Stutrgart : Jena . New York
Imipenem Inhibits in vitro Activity of Amphotericin B Directed against Aspergillus fumigatus PETER-MICHAEL RATH, JORG WERNER HOLSEWEDE, and RAINER ANSORG Institut fiir Medizinische Mikrobiologie, Universitiit-GH Essen, HufelandstraBe 55,
45147 Essen, Germany
Received March 15, 1995 . Accepted May 16, 1995
Summary A decrease of the susceptibility of Aspergillus fumigatus strains to amphotericin B was found when tested in combination with high concentrations (128-2048 mg/ll of the antibacterial agent imipenem by checkerboard titration and agar diffusion assay. Only bacteriologically active imipenem showed the antagonism. The mechanism of action is unknown. However, imipenern and amphotericin B did not react directly, as shown by checkerboard titration with bacterial strains as well as by HPLC analysis . It is concluded that imipenem medication may influence the efficacy of amphotericin B treatment in aspergillosis .
Introduction Imipenem (N-formimidoyl thienamycin monohydrate) is a highly potent antibiotic with a broad spectrum of activity against both grampositive and gramnegative bacteria as well as Mycobacterium spp., Actinomyces spp. and Nocardia spp. (7). It is a semisynthetic derivative of thienamycin produced by Streptomyces catteya (3). Imipenem covalently binds to penicillin-binding proteins and induces rapid cell death. For in vivo use it is formulated in combination with cilastatin to inhibit hydropeptidase-I which is located in the proximal tubule (2). Minimum inhibitory concentrations (MICs) of rapidly growing bacteria were determined to be in the range of 0.008-4 mg/I (8.9). Amphotericin B is a polyene macrolide antibiotic produced by Streptomyces nodosum. It is used in the treatment of life-threatening fungal infections. The substance acts by binding to ergosterol resulting in specific pores of the fungal membrane (1). Am photericin B is formulated in a deoxycholate micellar solution for in vivo use (11). MICs were determinated to be in the range of 0.06-1 mg/I for yeasts and Aspergillus fumigatus (4, 11). A combination of both agents might be indicated in neutropenic patients with fever of unknown origin and lacking response to imipenem alone (6) as well as in patients suffering from both severe bacterial and fungal infections. However, no data exist on
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P.-M. Rath,]. W. Hiilsewede, and R. Ansorg
the interaction of imipenem with amphotericin B. Therefore, the susceptibility of A. fumigatus to amphotericin B was tested in combination with imipenem.
Materials and Methods
Strains and growth conditions. An Aspergillus fumigatus strain (strain 6/93) isolated from tracheal secretion of a patient with suspected invasive aspergillosis was used for most of the experiments. Additionally, nine A. fumigatus strains isolated from patients were used in checkerboard titration. Identificat ion of the strains was based on their typical macroscopic and microscopic morphology and the ability to grow at 48°C on Sabouraud dextrose agar. Conidia were harvested from cultures grown for 4-6 days at 37°C on Czapeck-Dox agar (Difeo, Detroit, USA), using swabs, and were suspended in PBS (pH 7.0), NaCl 0.9%, or broth. Three bacterial reference strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Streptococcus faecalis NCTC 771) were used to investigate the effect of amphotericin B on imipenem activity. Microbroth assayfor checkerboard titration. Serial twofold dilutions of imipenem (MSD Sharpe & Dohme, Munich, Germany) or imipenem/cilastatin (Zienam for infusion, MSD) alone and in combinat ion with amphotericin B (amphotericin B/deoxycholate for infusion, Squibb-von Heyden, Munich, Germany) were tested against A. fumigatus (strain 6/93) by using micro-well plates (final volume 200 ul/well, approximately 10 4 conidia/well). The test medium was yeast-nitrogen broth (Difco yeast-nitrogen base supplemented with glucose and asparagine as described (4)). The ranges of finally tested concentrations were 82048 mg/l for imipenem and imipenem/cilastatin, respectively,and 0.06-32 mg/l for amphotericin B. The antibiotics were diluted in PBS (pH 7.0, imipenem), NaCl 0.9% (imipenem/cilastatin) or distilled water (amphotericin B/deoxycholate) as recommended by the distributors. Nine additional A. fumigatus strains were tested with minor modifications of the procedure: Tested concentrations of imipenem were 4-256 mg/I and the final volume in the assay was 100 ul/well, The test media were either yeast-nitrogen broth or Mueller-Hinton broth (Unipath, Basingstoke, England) . Testing of strain 6/93 gave similar results in both test procedures. The bacterial strains were tested in Mueller-Hinton broth . MICs were determined after approximately 20 h incubation at 37°C for the bacterial strains and after 24, 48, 72, and 96 h for the A. [umigatus strains. Agar diffusion assay. Yeast-morphology agar (Difco) supplemented with 2mg/1 amphotericin B (YMA-A) was used for the agar diffusion assay. Conidia of the ten A. fumigatus strains were flooded onto the agar-plates. Wells of 6 mm in diameter were punched in the agar and filled with 50 ~I of imipenem and imipenem/cilastatin solution, respectively,in three concentrations (2048, 1024,512 mg/I). PBS and NaCI 0.9% served as controls. After incubation for 24 to 72 h, the zone of A. fumigatus growth was measured. A subculture on YMAA without imipenem was performed to exclude development or selection of resistance. In order to investigate the spontaneous decrease of imipenem activity during incubation at 37°C, the substance was diluted in PBS (8192 mg/l), After 6, 24, 48, 72, 100, and 125 h of incubation, respectively, 50 ~I of the solution were filled into wells on YMA-A. A second aliquot was filled into wells on Mueller-Hinton agar for testing with E. coli. After an incubation period of 24 hat 37°C, the zones of growth (A. fumigatus strain 6/93) and zones of inhibition (E. coli) were measured in mm. The action of five other antibiotics (7500-8000mg/l) on A. fumigatus (strain 6/93) was tested by using the YMA-A assay: Piperacillin (Lederle Arzneimittel, Wolfratshausen, Germany), ceftriaxone (Hoffmann-LaRoche, Grenzach-Wyhlen, Germany), gentamicin (Merck, Darmstadt, Germany), ciprofloxacin (Bayer, Leverkusen, Germany), and vancom ycin (Lilly, Giessen, Germany) .
Amphotericin B inhibition by Imipenem
71
Effect of imipenem on growth of A. fumigatus . A. fumigatus (strain 6/93) was cultured in yeast-nitrogen broth containing 5000 mg/l imipenernlcilastatin. Cultures without imipenem served as controls . After 60 h incubat ion at 37°C, mycelia were harvested by filtration and washed with PBS. The dry weights of five parallel cultures were determined after lyophilization. Interaction ofimipenem with A. fumigatus conidia. Conidia of A. fumigatus (strain 6/93) were preincubated in imipenem solution (8192mg/1 in PBS) prior to determination of the MIC of amphotericin B, to investigate a possible coating effect of imipenem. After 1.5 h incubation at 37°C in imipenem, the conidia were either centrifuged (5 min, 8000 g) or washed with PBS. Thereafter, conidia were resuspended in PBS solution and inoculated in a microbroth assay to determine the MIC of amphotericin B. Control vials without imipenem were run in parallel. HPLC. Samples were prepared by dissolving imipenem in PBS (pH 7.0) until a final concentration of 8192 mg/I was reached. This stock solution was diluted 1 : 2 with either PBS or with a solution of 3.12 mg/l amphotericin B/deoxycholate and incubated at 37°C for 4 h. Prior to HPLC analysis, the samples were diluted 20 times with PBS for analysis of imipenem or 3 times with methanol for analysis of amphotericin B. The HPLL system (Pharmacia, Broma, Sweden) consisted of a gradient pump 2249M, a variable wave-length detector VWM 2141, a column oven 2155, an auto sampler 2157, and a helium degassing equipment . The detector response was monitored by a 486 computer (Pyramid 403 i plus). The results of a run were analysed by using the program PEAKMASTER (Pharmacia). Detection of imipenem was done at 289 nm with +0.5000 absorbance units set at full scale. Separation was evaluated by injection of 20 ~l of the sample on a C-18 reverse phase column (E. Merck, Highbar RT, Lichrospher 100, 5 mm, 250 mrnl4mm) at 30°C. The main column was protected with a 30 mrnl4mm pre-column filled with Lichrospher 100 RP-18, 5 m (E. Merck). The mobile phase was an isocratic gradient of phosphate buffer (0.25 M K2HP0 4 , adjusted to pH 7.0 with phosphor ic acid)/methanol 93 : 3 (vol : vol), flow rate 0.800 ml/min. Detection of amphotericin B was done at 405 nm with +0.1000 absorbance units set at full scale, using an isocratic gradient of phosphate buffer (0.01 M K2HP0 4 , 0.005 M EDTA-Na2 adjusted to pH 4.0 with phosphoric acid)/methanol 25 : 75 (vol : vol) , Injection volume was 100 ~1. Quantification was done by measurement of the peak area.
Results The A. fumigatus strains showed an MIC of 0.25-0.5 mg/I amphotericin B in yeast nitrogen broth. MICs did not vary within 96 h of incubation. When testing the com bination of amphotericin Band imipenem in checkerboard titration, high concentrations of imipenem (1024-2048 mg/l) resulted in a 16-fold to 32-fold increase of MICs of amphotericin B within a 24 h incubation period. After prolonged incubation (72 h) this effect was also found at lower concentrations of imipenem (128-256 mg/l) , with an increase of the MIC of amphotericin B from 0.25-0.5 mg/l to 2-4 mg/l. Table 1 shows a representative example. Similar results were found when imipenernlcilastatin and Mueller-Hinton broth were used, respectively (data not shown). However, the MICs of amphotericin B (without imipenem) increased in Mueller-Hinton broth during prolonged incubation (> 24 h), while the effect of imipenem resulted in an additional increase of the MIC. Three bacterial strains (E. coli, S. aureus, S. faecalis) showed for imipenem MICs of 0.125, 0.016, and 0.5 mg/I, respectively. Amphotericin B did not alter the susceptibility to imipenem of these strains in checkerboard titration, not even when high concentrations of the antifungal drug (3.12-400 mg/I) were used.
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Table 1. Effect of imipenem on the MIC of amphotericin B for A. fumiga tus (strain 6/93) in checkerboard titra tion Imipenem concentration
MIC of amphotericin B (rng/l)"
(mg/l]
24h"""
48 h
72h
96h
8 16 32 64 128 256 512 1028 2048
0.25/0.5 0.25/0.2 5 0.25/0.25 0.25/0.25 0.25/0 .25 0.25/0.5 0.5/1 2/2 4/4 8/8
0.25/0.5 0.25/0 .25 0.25/0.25 0.25/0 .25 0.5/0.5 1/2 2/4 4/4 8/8 16/16
0.25/0.5 0.25/0 .25 0.25/0.25 0.25/0.5 1/2 2/4 4/4 8/8 8/8 16/16
0.25/0 .5 0.25/0.25 0.25/0.25 0.5/0.5 1/2 4/4 4/8 8/8 16/16 16/16
" Results of two parallel tests
** Incubation period
Fig. 1. Growth of A. fumi gatus on yeast morphology agar containing 2 mg/I amphotericin B (YMA-A). The wells were filled with 50 III of imipenem solutions of 2048 mg/l (1), 1024mg/1 (2), and 512mg/1 (3). Well No.4 contained PBS.
Amphotericin B inhibition by Imipenem
73
The antagonistic effect of imipenem on the amphotericin B sensitivity of A. [umigatus was also seen in a diffusion assay, using yeast morphology agar, supplemented with 2 mg/l amphotericin B (YMA-A). Circular growth of A. fumigatus around the wells containing imipenem (512-2048 mg/l) was found as shown in Figure 1. Subculturing on YMA-A without imipenem revealed growth inhibition, indicating that no selection of resistent fungal cells had occurred. The other antibiotics (piperacillin, ceftriaxone, gentamicin, ciprofloxacin, and vancomycin) did not show an effect in this assay. As shown in Table 2, the antagonistic effect of imipenem was only found when bacteriologically active substance was used . Prolonged incubation of imipenem at 37°C before testing in Mueller-Hinton agar resulted in a time-dependent decrease of activity directed against E. coli. Zones of growth of A. fumigatus on YMA-A decreased as well. After 125 h preincubation, no effect of imipenem was found, neither on E. coli nor on A. fumigatus. Imipenem/cilastatin itself did not support the growth of A. fumigatus . Five cultures supplemented with imipenem/cilastatin (5000 mg/l) yielded a mean dry weight of 160.76mg (range 150.5-166.0mg) compared to 156.34mg without the substance (range 154.2-171.6 mg). In order to investigate the mechanism of action, conidia of A. fumigatus were preincubated in imipenem solution (8192 mg/l) for 1.5 h at 37°C. After centrifugation, conidia were resuspended in PBS and inoculated into yeast-nitrogen broth to determine the MIC of amphotericin B. After 24 hat 37°C, the MIC of amphotericin B was 2 mg/l compared to 0.25 mg/l of the untreated conidia. When the preincubated conidia were washed with PBS, no difference in susceptibility was found to exist between preincubated and untreated cultures. HPLC analysis showed that concentrations of imipenem did not alter over an incubation period of 4 h, when combined with amphotericin B. No significant shift in retention time (RT = 6.77 ± 1 min) or peak shape has been observed, so that there is no indication of a direct interaction of imipenem with amphotericin B (Fig. 2-4) . Correspondingly, the same results were obtained when the concentrations of amphotericin B were measured (RT = 7.20 ± 1 min) .
Table 2. Time-dependent decrease of imipenem activity Preincubation of imipenem solution
(h)
o
6 24 48 72 100 125
Zone of growth of A. fumigatus on YMA-A (mm)
Zone of inhibition of E. coli on MHA (mm)
21 20 20 16 12 < 6
51 50 47 35 26 18 < 6
< 6
YMA-A: Yeast morphology agar supplemented with 2 mg/l amphotericin B MHA : Mueller-Hinton agar
74 o
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= 0 at 37°C in
Amphoteric in B inhibition by Imipenem o
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Fig. 3. Chromatogram from the HPLC anal ysis of imipenem incubat ed for t = 0 hat 37°C in the presence of amphotericin B.
76
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Amphotericin B inhibition by Imipenem
77
Discussion The study has shown that imipenem causes an inhibition of the efficacy of amphotericin B on A. fumigatus. The susceptibility of A . fumigatus to amphotericin B decreased in a dose-dependent and time-dependent manner when tested in combination with imipenem and imipenem/cilastatin, respectively. In vivo, the therapeutic serum levels of amphotericin B ranged from 0.2 to 0.5 mg/I, with peak levels of approximately 1-3 mg/I (11). Intravenous administration of 1000 mg imipenem resulted in peak levels ranging from 60-70 mg/I, declining to 2 mg/l within 4 to 6 h (2). The effect of imipenem on the activity of amphotericin B in respect of A . fumigatus was found above these concentrations. However, this does not exclude that an in vivo effect may exist. Interestingly enough, Schmehl et al. (10) found that the viability of endothelial cells of canine saphenous veins after treatment with imipenem (12mg/l) combined with amphotericin B (5-10mg/1) was higher than the viability of cells incubated in amphotericin B alone. These findings indicate that imipenem is able to inhibit some of the toxic effects of amphotericin B. The basis of the protective effect might be similar to the antagonistic effect found in the present study. The mechanisms of action could not be clarified. The reactivity of imipenem seems to be specific as other antibiotics (piperacillin, ceftriaxone, gentamicin, ciprofloxacin, and vancomycin) did not show an effect. A direct action of imipenem on the growth of A. fumigatus was not found. Two results indicate that there has been no direct interaction between imipenem and amphotericin B: i) Amphotericin B did not alter the susceptibility of bacterial strains to imipenem, ii) HPLC analys is showed no interaction of the two substances. However, it might be possible that complexes were dissociated by the methanol extraction prior to HPLC analysis or by the mobile phases . A possible explanation of the antagonism could be, that imipenem blocks binding sites of amphotericin B on A . fumigatus . As outlined in a review by Brajtburg et al. (1) both the heptaene structure of amphotericin B and the carboxyl and amino groups are important for the antifungal activity of the agent. The authors discussed that heptaene antibiotics with free carboxyl and amino groups may interact with sterols similar to amphotericin B. Imipenem contains a carboxyl and an amino group (2). The effect of imipenem could be removed by washing conidia which had been preincubated in imipenem solution prior to testing, indicating that there was no strong competetive binding. A weak interaction, for example one that is ionic in nature, is not excluded. Furthermore, the chemical structure of spontaneously formed products of imipenem is not known (5). Therefore, such products may also interact with binding sites of amphotericin B on the fungal membrane. Even if the inhibitory concentrations of imipenem are beyond the peak serum levels, the effect may also exist in vivo. Combined administration of both drugs may include the risk of diminishing the amphotericin B efficacy in the treatment of aspergillosis. Acknowledgement. We thank Mr. D. Schmidt for skilful technical assistance.
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11. Walsh, T. J. and A. Pizzo: Treatment of systemic fungal infections: Recent progress and current problems. Europ. J. Clin , Microb iol. & Infect. Dis. 7 (1988) 460-475 Dr. med. Peter-Michael Rath, Institut fur Med. Mikrobiologie, Universitiit-GH Essen, Hufelandstr. 55,45147 Essen, Germany