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Interactions of colistin and rifampin on multidrug-resistant Acinetobacter baumannii
Diagnostic Microbiology and Infectious Disease 40 (2001) 117–120
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Interactions of colistin and rifampin on multidrug-resistant Acinetobacter baumannii Evangelos J. Giamarellos-Bourboulis, Evangelia Xirouchaki, Helen Giamarellou* 4th Department of Internal Medicine, University of Athens, Medical School, Greece Received 14 March 2001; accepted 29 May 2001
Abstract The increased incidence of nosocomial infections by multidrug-resistant Acinetobacter spp creates demand on the application of some combinations of older antimicrobials on that species. The in vitro activities of colistin and of rifampin and of their interaction were tested on 39 nosocomial isolates of Acinetobacter baumannii. All isolates were resistant to ampicillin/sulbactam, to 3rd and 4th generation cephalosporins, to amikacin and to ciprofloxacin. MICs were determined by a microdilution technique and interactive studies between 1⫻ or 4⫻ MIC of colistin and rifampin were performed by the time-kill assay. Rifampin was applied at a concentration of 2g/mL which is equal to its mean serum level. All isolates were inhibited by colistin and only 15.2% by rifampin. Synergy between 1⫻ MIC of colistin and rifampin was detected in 15.4% of isolates at 6 h of growth and in 51.3% of isolates at 24 h of growth. Synergy between 4⫻ MIC of colistin and rifampin was detected in 15.4% of isolates at 6 h of growth and in 66.7% of isolates at 24 h of growth. It is concluded that colistin is highly active on multidrug-resistant Acinetobacter spp and its activity on A.baumannii is increased in the presence of rifampin, so that their administration might be proposed for nosocomial infections by these isolates. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Colistin; Rifampin; Acinetobacter
1. Introduction
2. Material and methods
Acinetobacter baumannii has been recognized as a cause of outbreaks of nosocomial infections in the Intensive Care Unit with considerable case fatality. There are limited therapeutic alternatives against that species particularly after the evolution of resistance for antimicrobial agents like ampicillin/sulbactam, imipenem and the fluoroquinolones over the last decade (Garcia-Arata et al., 1996; Ruiz et al., 1999). Colistin has been shown to remain active on that species and despite its poor pharmacokinetic profile and its adverse effects it has been proposed as an alternative for infections by multidrug-resistant Gram-negative pathogens (Evans et al., 1999; Levin et al., 1999). Although it has been proposed that colistin combined with rifampin may be in vitro active on A.baumannii (Hogg et al., 1998), this is the first study on the in vitro interaction of colistin and of rifampin on a large number of multidrug-resistant isolates of Acinetobacter baumannii.
2.1. Selection of isolates
* Corresponding author. Tel.: ⫹1-301-80-39-542; fax.: ⫹1-301-80-39543 E-mail address: [email protected].
A total of 39 A. baumannii isolates were included in the study derived from different patients with nosocomial infections. They were isolated in five different hospitals of the territory of Athens over the five-year period between January 1995 and January 2000 and they were identified by the API 20E test (bioMe´rieux, Paris, France). They were isolated from the following specimens: pus 17; urine 10; blood six; bronchoalveolar lavage four; and cerebrospinal fluid two. Isolates were selected to be resistant to ampicillin/ sulbactam, cefotaxime, ceftriaxone, ceftazidime, cefepime, amikacin and ciprofloxacin according to their susceptibilities by the Kirby-Bauer method. 2.2. Susceptibility testing Colistin sulfomethane sodium (Sigma Co, St. Louis, USA), rifampin (Marion Merrell Dow, Ontario, Canada) and meropenem (Zeneca, Macclesfiled, UK) were provided as amorphous powders. Colictin and meropenem were wa-
0732-8893/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 1 ) 0 0 2 5 8 - 9
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ter-soluble and rifampin was dissolved in methanol 99% (Merck, Darmstadt, Germany) in order to become watersoluble. Minimal Inhibitory Concentrations (MICs) of colistin, rifampin and meropenem were determined by a microdilution technique at a final volume of 0.1 mL using dilutions of 0.03 to 128g/mL of each tested antimicrobial. MICs of ampicillin/sulbactam, cefotaxime, ceftriaxone, ceftazidime, cefepime, imipenem, amikacin and ciprofloxacin were determined on ready-made plates (Sensititre Ltd, West Sussex, UK). A 5 ⫻ 105 cfu/mL log-phase inoculum of each tested isolate was applied. MIC was defined as the lowest concentration of an antimicrobial limiting visible bacterial growth after 18 h of incubation at 35°C. The following concentrations were considered as the susceptibility breakpoints of each tested antimicrobial: colistin 4g/mL (Catchpole et al., 1997); rifampin 2g/mL (Hogg et al., 1998); meropenem 4g/mL (MacGowan et al., 1995); ampicillin/sulbactam 8/4g/mL; cefotaxime, ceftriaxone, ceftazidime, and cefepime 8g/mL; imipenem 4g/mL; amikacin 16g/mL (Giamarellos-Bourboulis et al., 2000); and ciprofloxacin 2g/mL (Heinemann et al., 2000).
Table 1 In vitro susceptibilities of 39 multidrug-resistant isolates of Acinetobacter baumannii to 11 antimicrobial agents Antimicrobial (susceptibility breakpoint)
MIC (g/mL) Range
50%
90%
Ampicillin/ sulbactam (8/4) Cefotaxime (8) Ceftriaxone (8) Ceftazidime (8) Cefepime (8) Imipenem (4) Meropenem (4) Ciprofloxacin (2) Amikacin (16) Rifampin (2) Colistin (4)
16/4–⬎64/4
32/4
⬎64/4
0.0
32–⬎64 32–⬎64 32–⬎64 32–⬎256 0.5–16 1–16 4–128 32–⬎64 2–64 ⱕ0.03–2
⬎64 ⬎64 ⬎64 ⬎256 4 4 64 ⬎64 4 0.12
⬎64 ⬎64 ⬎64 ⬎256 16 8 ⬎128 ⬎64 64 1
0.0 0.0 0.0 0.0 89.7 74.4 0.0 0.0 15.4 100.0
% susceptible
changes achieved by single antimicrobials and their interactions were performed by paired “t test.” Any value of P below 0.05 was considered as significant.
2.3. Interactive time-kill studies
3. Results
Colistin and/or rifampin were added into tubes with Mueller-Hinton broth (Oxoid Ltd, London, UK) of a 10 mL final volume. Colistin was applied at concentrations equal to 1⫻ MIC and 4⫻ MIC and rifampin at a concentration of 2g/mL which is equal to its mean serum level after application of its conventional dose (Sambatakou et al., 1998). A 5 ⫻ 105 cfu/mL log-phase inoculum of each tested isolate was added to tubes with Mueller-Hinton broth (Oxoid Ltd, London, UK) containing single antimicrobials or their combinations. In tubes with rifampin the final concentration of methanol 99% was 0.1v/v. One growth control was applied per tested isolate. Tubes were left to incubate at 37°C in a water bath and at 2, 4, 6 and 24 h of growth viable cell counts were determined as follows: a 0.1 mL aliquot was sampled from each tube and diluted five serial times 1:10 in sterile NaCl 0.9% whereas a 0.1 ml aliquot of each dilution was plated onto McConkey agar (Becton Dickinson, Cockeysville, Md). These serial dilutions permitted to avoid any antimicrobial carry-over effect and offered a lowest detection limit of 30 cfu/mL. All experiments were performed in duplicate. The total number of time-kill studies was 195. At each time interval the log10 change of the viable cell count compared to the starting inoculum was determined. Synergy was defined as any more or equal to a 2log10 decrease of viable cells compared to the most active single agent (Hindler, 1992).
The in vitro susceptibilities of the tested isolates to 11 antimicrobial agents is shown in Table 1. All isolates were resistant to ampicillin/sulbactam, cefotaxime, ceftriaxone, ceftazidime, cefepime, amikacin and ciprofloxacin and susceptible to colistin. Imipenem inhibited 89.7% of isolates, meropenem 74.4%, and rifampin 15.4%. Synergy was found between 1⫻ MIC of colistin and rifampin in six isolates (15.4%) at 6 h of growth and in 20 isolates (51.3%) at 24 h of growth. Respective results for synergy between 4⫻ MIC of colistin and rifampin was six isolates (15.4%) and 26 isolates (66.7%). No synergy was found at 2 and 4 h of growth. Interactive results between colistin and rifampin are shown in Table 2. The time-kill curves of colistin and of rifampin and of their interaction on two isolates are shown in Fig. 1.
2.4. Statistical analysis Log10 changes of viable cell counts were expressed as their mean value ( ⫾ SD) values. Comparisons between
4. Discussion A.baumannii is nowadays recognized as an important nosocomial pathogen especially in the Intensive Care Unit giving rise to infections particularly difficult to manage. Few drugs may be active on that species; as a consequence the need for an active combination of antimicrobials is obvious (Wolff et al., 1999). A large number of nosocomial A.baumanni isolates were included in the present study, all resistant to ampicillin/sulbactam, to 3rd and 4th generation cephalosporins, to amikacin and to ciprofloxacin. The reported series is considerably larger than that of other authors. The in vitro interaction of colistin and rifampin was tested on these isolates.
E.J. Giamarellos-Bourboulis et al. / Diagnostic Microbiology and Infectious Disease 40 (2001) 117–120
119
Table 2 In vitro interactions between 1⫻MIC or 4⫻MIC of colistin and 2 g/mL of rifampin on 39 nosocomial multiple drug-resistant Acinetobacter baumannii isolates Time (hours)
Colistin 1⫻MIC
2 4 6 24
⫺3.72 ⫾ 1.54 ⫺3.40 ⫾ 1.72 ⫺2.34 ⫾ 2.35 ⫹1.13 ⫾ 1.46
Mean (⫾SD) log10 change of viable cell counts
Synergy [No (%) of isolates]
Colistin 4⫻MIC
Rifampin
Colistin 1⫻ MIC ⫹ Rifampin
Colistin 4⫻ MIC ⫹ Rifampin
Colistin 1⫻ MIC ⫹ Rifampin
Colistin 4⫻ MIC ⫹ Rifampin
⫺4.32 ⫾ 0.72 ⫺4.49 ⫾ 0.52 ⫺4.44 ⫾ 0.50 ⫺2.55 ⫾ 2.42
⫹0.14 ⫾ 1.01 ⫹0.90 ⫾ 1.06 ⫹0.64 ⫾ 1.14 ⫹1.05 ⫾ 1.17
⫺4.38 ⫾ 0.59a ⫺4.31 ⫾ 0.75b,e ⫺4.45 ⫾ 0.55c,e ⫺2.96 ⫾ 2.30c,f
⫺4.44 ⫾ 1.12a ⫺4.26 ⫾ 1.32a,e ⫺4.27 ⫾ 1.27a,e ⫺3.84 ⫾ 1.77d,e
0 0 6 (15.4) 20 (51.3)
0 0 6 (15.3) 26 (66.7)
a
P NS compared to the activity of the same concentration of colistin in the absence of rifampin P: 0.004 compared to the activity of 1⫻MIC of colistin in the absence of rifampin c P ⬍ 0.0001 compared to the activity of 1⫻MIC of colistin in the absence of rifampin d P: 0.0015 compared to the activity of 4⫻MIC of colistin in the absence of rifampin e P NS compared to the activity of the combination of colistin and rifampin at the previous time of growth f P: 0.002 compared to the activity of the combination of 1⫻MIC of colistin and rifampin at the previous time of growth b
The presented results (Table 1) revealed that colistin possessed a considerable intrinsic activity on the majority of isolates with values of MIC50 and MIC90 lower than those reported elsewhere (Catchpole et al., 1997). On the contrary MIC50s and MIC90s of rifampin and meropenem were higher than those reported by other authors (MacGowan et al., 1995; Ruiz et al., 1999). This is probably reflecting the multidrug-resistance of the applied isolates compared to isolates applied in other studies. The in vitro activity of colistin was highly increased in
the presence of rifampin. Synergy between colistin and rifampin was dependent from the applied concentration of colistin. It was however expressed with concentrations of rifampin equal to those achieved in serum and it involved 66.7% of A.baumannii isolates (Table 2). Single colistin was active on A.baumannii over the first six hours of growth after which regrowth was observed, a phenomenon restrained in the presence of rifampin. (Table 2, Fig. 1). Synergy between colistin and rifampin has also been reported elsewhere but it involved only 13 isolates and it was
Fig. 1. Time-kill curves of Acinetobacter baumannii isolates no34 and 29 exposed to 1⫻ MIC and 4⫻ MIC of colistin, to 2g/mL of rifampin and to their interaction.
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performed by the checkerboard technique and not by the time-kill method of the present study (Hogg et al., 1998). The presented in vitro results might be promising for a new existing alternative in the therapy of infections by multidrug-resistant A.baumannii. However it should always be kept in mind that the in vivo situation might differ from the in vitro condition. A lot of consideration is suggested for the interpretation of interactive results occurring after 24 h of growth since the time-kill study offers an in vivo simulation to the reality whenever bacterial killing is achieved over the first hours of growth (Hindler, 1992). Few in vivo results exist where rifampin is found to increase the activity of imipenem and of ampicillin/sulbactam against A.baumannii in a mouse pneumonia model (Wolff et al., 1999). The present study revealed that colistin is an antimicrobial agent possessing a considerable in vitro activity on multidrug-resistant A.baumannii which is increased in the presence of rifampin. Colistin has already been reported as an adequate alternative in sporadic cases of nosocomial infections by multidrug-resistant A.baumannii (FernandezVilladrich et al., 1999; Levin et al., 1999). Since the panel of its adverse effects limits its application only for infections by multidrug-resistant isolates, the presented results may propose its administration with rifampin for such infections.
References Catchpole, C.R., Andrews, J.M., & Wise, R. (1997). A reassessment of the in vitro activity of colistin sulphomethane sodium. J Antimicrob Chemother, 39, 255–260. Evans, M. E., Feola, D. J., & Rapp, R. P. (1999). Polymixin B sulfate and colistin: old antibiotics for emerging multiresistant gram-negative bacteria. Ann Pharmacother, 33, 960 –967. Fernandez-Villadrich, P., Corbella, X., Corral, L., Tubau, F., & Mateu, A. (1999). Successful treatment of ventriculitis due to carbapenem-resistant Acinetobacter baumannii with intraventricular colistin sulfomethane sodium. Clin Infect Dis, 28, 9216 –917.
Garcia-Arata, M. I., Alarc´on, T., & Lo´pez-Brea, M. (1996). Emergence of resistant isolates of Acinetobacter calcoaceticus –A.baumannii complex in a Spanish hospital over a five-year period. Eur J Clin Microbiol Infect Dis, 15, 512–515. Giamarellos-Bourboulis, E. J., Grecka, P., Tsitsika, A., Tympanidou, C., & Giamarellou, H. (2000). In vitro activity of FK 037 (cefoselis), a novel 4th generation cephalosporin, compared to cefepime and cefpirome on nosocomial staphylococci and gram-negative isolates. Diagn Microbiol Infect Dis, 36, 185–191. Heinemann, B., Wisplinghoff, H., Edmond, M., & Seifert, H. (2000). Comparative activities of ciprofloxacin, clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against epidemiologically defined Acinetobacter baumannii strains. Antimicrob Agents Chemother, 44, 2211–2213. Hindler, J. (1992). Tests to assess bactericidal activity. In H.D. Eisenberg (Ed.), Clinical Microbiology Procedures Hanbook. (pp. 5.16.14 – 5.16.24). Washington, DC: American Society for Microbiology. Hogg, G. M., Barr, J. G., & Webb, C. H. (1998). In vitro activity of the combination of colistin and rifampin against multidrug-resistant strains of Acinetobacter baumannii. J Antimicrob Chemother, 41, 494 – 495. Levin, A. S., Barone, A. A., Penc¸o, J., Santos, M. V., Marinho, I. S., Arruda, A. G., Manrique, E. I., & Costa, S. F. (1999). Intravenous colistin as therapy for nosocomial infections caused by multidrugresistant Pseudomonas aeruginosa and Acinetobacter baumannii. Clin Infect Dis, 28, 1008 –1011. MacGowan, A. P., Bowker, K. A., Holt, H. A., Reeves, D. S. & Hedges, A. (1995). The comparative inhibitory and bactericidal activities of meropenem and imipenem against Acinetobacter spp. and enterobacteriaceae resistant to second generation cephalosporins. J Antimicrob Chemother, 35, 333–337. Ruiz, J., Nu´n˜ez, M. L., Pe´rez, J., Simarro, E., Martı´nez-Campos, L., & Go´mez, J. (1999). Evolution of resistance among clinical isolates of Acinetobacter over a 6-year period. Eur J Clin Microbiol Infect Dis, 18, 292–295. Sambatakou, H., Giamarellos-Bourboulis, E. J., Grecka, P., Chryssouli, Z., & Giamarellou, H. (1998). In vitro activity and killing effect of quinupristin/dalfopristin (RP59500) on nosocomial Staphylococcus aureus and interactions with rifampicin and ciprofloxacin against methicillinresistant isolates. J Antimicrob Chemother, 41, 349 –355. Wolff, M., Joly-Guillou, M. L., Farinotti, R., & Carbon, C. (1999). In vivo efficacies of combinations of -lactams, -lactamase inhibitors, and rifampin against Acinetobacter baumannii in a mouse pneumonia model. Antimicrob Agents Chemother, 43, 1406 –1411.
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Interactions of colistin and rifampin on multidrug-resistant Acinetobacter baumannii Evangelos J. Giamarellos-Bourboulis, Evangelia Xirouchaki, Helen Giamarellou* 4th Department of Internal Medicine, University of Athens, Medical School, Greece Received 14 March 2001; accepted 29 May 2001
Abstract The increased incidence of nosocomial infections by multidrug-resistant Acinetobacter spp creates demand on the application of some combinations of older antimicrobials on that species. The in vitro activities of colistin and of rifampin and of their interaction were tested on 39 nosocomial isolates of Acinetobacter baumannii. All isolates were resistant to ampicillin/sulbactam, to 3rd and 4th generation cephalosporins, to amikacin and to ciprofloxacin. MICs were determined by a microdilution technique and interactive studies between 1⫻ or 4⫻ MIC of colistin and rifampin were performed by the time-kill assay. Rifampin was applied at a concentration of 2g/mL which is equal to its mean serum level. All isolates were inhibited by colistin and only 15.2% by rifampin. Synergy between 1⫻ MIC of colistin and rifampin was detected in 15.4% of isolates at 6 h of growth and in 51.3% of isolates at 24 h of growth. Synergy between 4⫻ MIC of colistin and rifampin was detected in 15.4% of isolates at 6 h of growth and in 66.7% of isolates at 24 h of growth. It is concluded that colistin is highly active on multidrug-resistant Acinetobacter spp and its activity on A.baumannii is increased in the presence of rifampin, so that their administration might be proposed for nosocomial infections by these isolates. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Colistin; Rifampin; Acinetobacter
1. Introduction
2. Material and methods
Acinetobacter baumannii has been recognized as a cause of outbreaks of nosocomial infections in the Intensive Care Unit with considerable case fatality. There are limited therapeutic alternatives against that species particularly after the evolution of resistance for antimicrobial agents like ampicillin/sulbactam, imipenem and the fluoroquinolones over the last decade (Garcia-Arata et al., 1996; Ruiz et al., 1999). Colistin has been shown to remain active on that species and despite its poor pharmacokinetic profile and its adverse effects it has been proposed as an alternative for infections by multidrug-resistant Gram-negative pathogens (Evans et al., 1999; Levin et al., 1999). Although it has been proposed that colistin combined with rifampin may be in vitro active on A.baumannii (Hogg et al., 1998), this is the first study on the in vitro interaction of colistin and of rifampin on a large number of multidrug-resistant isolates of Acinetobacter baumannii.
2.1. Selection of isolates
* Corresponding author. Tel.: ⫹1-301-80-39-542; fax.: ⫹1-301-80-39543 E-mail address: [email protected].
A total of 39 A. baumannii isolates were included in the study derived from different patients with nosocomial infections. They were isolated in five different hospitals of the territory of Athens over the five-year period between January 1995 and January 2000 and they were identified by the API 20E test (bioMe´rieux, Paris, France). They were isolated from the following specimens: pus 17; urine 10; blood six; bronchoalveolar lavage four; and cerebrospinal fluid two. Isolates were selected to be resistant to ampicillin/ sulbactam, cefotaxime, ceftriaxone, ceftazidime, cefepime, amikacin and ciprofloxacin according to their susceptibilities by the Kirby-Bauer method. 2.2. Susceptibility testing Colistin sulfomethane sodium (Sigma Co, St. Louis, USA), rifampin (Marion Merrell Dow, Ontario, Canada) and meropenem (Zeneca, Macclesfiled, UK) were provided as amorphous powders. Colictin and meropenem were wa-
0732-8893/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 1 ) 0 0 2 5 8 - 9
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E.J. Giamarellos-Bourboulis et al. / Diagnostic Microbiology and Infectious Disease 40 (2001) 117–120
ter-soluble and rifampin was dissolved in methanol 99% (Merck, Darmstadt, Germany) in order to become watersoluble. Minimal Inhibitory Concentrations (MICs) of colistin, rifampin and meropenem were determined by a microdilution technique at a final volume of 0.1 mL using dilutions of 0.03 to 128g/mL of each tested antimicrobial. MICs of ampicillin/sulbactam, cefotaxime, ceftriaxone, ceftazidime, cefepime, imipenem, amikacin and ciprofloxacin were determined on ready-made plates (Sensititre Ltd, West Sussex, UK). A 5 ⫻ 105 cfu/mL log-phase inoculum of each tested isolate was applied. MIC was defined as the lowest concentration of an antimicrobial limiting visible bacterial growth after 18 h of incubation at 35°C. The following concentrations were considered as the susceptibility breakpoints of each tested antimicrobial: colistin 4g/mL (Catchpole et al., 1997); rifampin 2g/mL (Hogg et al., 1998); meropenem 4g/mL (MacGowan et al., 1995); ampicillin/sulbactam 8/4g/mL; cefotaxime, ceftriaxone, ceftazidime, and cefepime 8g/mL; imipenem 4g/mL; amikacin 16g/mL (Giamarellos-Bourboulis et al., 2000); and ciprofloxacin 2g/mL (Heinemann et al., 2000).
Table 1 In vitro susceptibilities of 39 multidrug-resistant isolates of Acinetobacter baumannii to 11 antimicrobial agents Antimicrobial (susceptibility breakpoint)
MIC (g/mL) Range
50%
90%
Ampicillin/ sulbactam (8/4) Cefotaxime (8) Ceftriaxone (8) Ceftazidime (8) Cefepime (8) Imipenem (4) Meropenem (4) Ciprofloxacin (2) Amikacin (16) Rifampin (2) Colistin (4)
16/4–⬎64/4
32/4
⬎64/4
0.0
32–⬎64 32–⬎64 32–⬎64 32–⬎256 0.5–16 1–16 4–128 32–⬎64 2–64 ⱕ0.03–2
⬎64 ⬎64 ⬎64 ⬎256 4 4 64 ⬎64 4 0.12
⬎64 ⬎64 ⬎64 ⬎256 16 8 ⬎128 ⬎64 64 1
0.0 0.0 0.0 0.0 89.7 74.4 0.0 0.0 15.4 100.0
% susceptible
changes achieved by single antimicrobials and their interactions were performed by paired “t test.” Any value of P below 0.05 was considered as significant.
2.3. Interactive time-kill studies
3. Results
Colistin and/or rifampin were added into tubes with Mueller-Hinton broth (Oxoid Ltd, London, UK) of a 10 mL final volume. Colistin was applied at concentrations equal to 1⫻ MIC and 4⫻ MIC and rifampin at a concentration of 2g/mL which is equal to its mean serum level after application of its conventional dose (Sambatakou et al., 1998). A 5 ⫻ 105 cfu/mL log-phase inoculum of each tested isolate was added to tubes with Mueller-Hinton broth (Oxoid Ltd, London, UK) containing single antimicrobials or their combinations. In tubes with rifampin the final concentration of methanol 99% was 0.1v/v. One growth control was applied per tested isolate. Tubes were left to incubate at 37°C in a water bath and at 2, 4, 6 and 24 h of growth viable cell counts were determined as follows: a 0.1 mL aliquot was sampled from each tube and diluted five serial times 1:10 in sterile NaCl 0.9% whereas a 0.1 ml aliquot of each dilution was plated onto McConkey agar (Becton Dickinson, Cockeysville, Md). These serial dilutions permitted to avoid any antimicrobial carry-over effect and offered a lowest detection limit of 30 cfu/mL. All experiments were performed in duplicate. The total number of time-kill studies was 195. At each time interval the log10 change of the viable cell count compared to the starting inoculum was determined. Synergy was defined as any more or equal to a 2log10 decrease of viable cells compared to the most active single agent (Hindler, 1992).
The in vitro susceptibilities of the tested isolates to 11 antimicrobial agents is shown in Table 1. All isolates were resistant to ampicillin/sulbactam, cefotaxime, ceftriaxone, ceftazidime, cefepime, amikacin and ciprofloxacin and susceptible to colistin. Imipenem inhibited 89.7% of isolates, meropenem 74.4%, and rifampin 15.4%. Synergy was found between 1⫻ MIC of colistin and rifampin in six isolates (15.4%) at 6 h of growth and in 20 isolates (51.3%) at 24 h of growth. Respective results for synergy between 4⫻ MIC of colistin and rifampin was six isolates (15.4%) and 26 isolates (66.7%). No synergy was found at 2 and 4 h of growth. Interactive results between colistin and rifampin are shown in Table 2. The time-kill curves of colistin and of rifampin and of their interaction on two isolates are shown in Fig. 1.
2.4. Statistical analysis Log10 changes of viable cell counts were expressed as their mean value ( ⫾ SD) values. Comparisons between
4. Discussion A.baumannii is nowadays recognized as an important nosocomial pathogen especially in the Intensive Care Unit giving rise to infections particularly difficult to manage. Few drugs may be active on that species; as a consequence the need for an active combination of antimicrobials is obvious (Wolff et al., 1999). A large number of nosocomial A.baumanni isolates were included in the present study, all resistant to ampicillin/sulbactam, to 3rd and 4th generation cephalosporins, to amikacin and to ciprofloxacin. The reported series is considerably larger than that of other authors. The in vitro interaction of colistin and rifampin was tested on these isolates.
E.J. Giamarellos-Bourboulis et al. / Diagnostic Microbiology and Infectious Disease 40 (2001) 117–120
119
Table 2 In vitro interactions between 1⫻MIC or 4⫻MIC of colistin and 2 g/mL of rifampin on 39 nosocomial multiple drug-resistant Acinetobacter baumannii isolates Time (hours)
Colistin 1⫻MIC
2 4 6 24
⫺3.72 ⫾ 1.54 ⫺3.40 ⫾ 1.72 ⫺2.34 ⫾ 2.35 ⫹1.13 ⫾ 1.46
Mean (⫾SD) log10 change of viable cell counts
Synergy [No (%) of isolates]
Colistin 4⫻MIC
Rifampin
Colistin 1⫻ MIC ⫹ Rifampin
Colistin 4⫻ MIC ⫹ Rifampin
Colistin 1⫻ MIC ⫹ Rifampin
Colistin 4⫻ MIC ⫹ Rifampin
⫺4.32 ⫾ 0.72 ⫺4.49 ⫾ 0.52 ⫺4.44 ⫾ 0.50 ⫺2.55 ⫾ 2.42
⫹0.14 ⫾ 1.01 ⫹0.90 ⫾ 1.06 ⫹0.64 ⫾ 1.14 ⫹1.05 ⫾ 1.17
⫺4.38 ⫾ 0.59a ⫺4.31 ⫾ 0.75b,e ⫺4.45 ⫾ 0.55c,e ⫺2.96 ⫾ 2.30c,f
⫺4.44 ⫾ 1.12a ⫺4.26 ⫾ 1.32a,e ⫺4.27 ⫾ 1.27a,e ⫺3.84 ⫾ 1.77d,e
0 0 6 (15.4) 20 (51.3)
0 0 6 (15.3) 26 (66.7)
a
P NS compared to the activity of the same concentration of colistin in the absence of rifampin P: 0.004 compared to the activity of 1⫻MIC of colistin in the absence of rifampin c P ⬍ 0.0001 compared to the activity of 1⫻MIC of colistin in the absence of rifampin d P: 0.0015 compared to the activity of 4⫻MIC of colistin in the absence of rifampin e P NS compared to the activity of the combination of colistin and rifampin at the previous time of growth f P: 0.002 compared to the activity of the combination of 1⫻MIC of colistin and rifampin at the previous time of growth b
The presented results (Table 1) revealed that colistin possessed a considerable intrinsic activity on the majority of isolates with values of MIC50 and MIC90 lower than those reported elsewhere (Catchpole et al., 1997). On the contrary MIC50s and MIC90s of rifampin and meropenem were higher than those reported by other authors (MacGowan et al., 1995; Ruiz et al., 1999). This is probably reflecting the multidrug-resistance of the applied isolates compared to isolates applied in other studies. The in vitro activity of colistin was highly increased in
the presence of rifampin. Synergy between colistin and rifampin was dependent from the applied concentration of colistin. It was however expressed with concentrations of rifampin equal to those achieved in serum and it involved 66.7% of A.baumannii isolates (Table 2). Single colistin was active on A.baumannii over the first six hours of growth after which regrowth was observed, a phenomenon restrained in the presence of rifampin. (Table 2, Fig. 1). Synergy between colistin and rifampin has also been reported elsewhere but it involved only 13 isolates and it was
Fig. 1. Time-kill curves of Acinetobacter baumannii isolates no34 and 29 exposed to 1⫻ MIC and 4⫻ MIC of colistin, to 2g/mL of rifampin and to their interaction.
120
E.J. Giamarellos-Bourboulis et al. / Diagnostic Microbiology and Infectious Disease 40 (2001) 117–120
performed by the checkerboard technique and not by the time-kill method of the present study (Hogg et al., 1998). The presented in vitro results might be promising for a new existing alternative in the therapy of infections by multidrug-resistant A.baumannii. However it should always be kept in mind that the in vivo situation might differ from the in vitro condition. A lot of consideration is suggested for the interpretation of interactive results occurring after 24 h of growth since the time-kill study offers an in vivo simulation to the reality whenever bacterial killing is achieved over the first hours of growth (Hindler, 1992). Few in vivo results exist where rifampin is found to increase the activity of imipenem and of ampicillin/sulbactam against A.baumannii in a mouse pneumonia model (Wolff et al., 1999). The present study revealed that colistin is an antimicrobial agent possessing a considerable in vitro activity on multidrug-resistant A.baumannii which is increased in the presence of rifampin. Colistin has already been reported as an adequate alternative in sporadic cases of nosocomial infections by multidrug-resistant A.baumannii (FernandezVilladrich et al., 1999; Levin et al., 1999). Since the panel of its adverse effects limits its application only for infections by multidrug-resistant isolates, the presented results may propose its administration with rifampin for such infections.
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