JAC Advance Access originally published online on May 31, 2007
Journal of Antimicrobial Chemotherapy 2007 60(2):421-423; doi:10.1093/jac/dkm178
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In vitro effect of minocycline and colistin combinations on imipenem-resistant Acinetobacter baumannii clinical isolates
1 Laboratory Medicine Services, Changi General Hospital, 2 Simei Street 3, Singapore 529889; 2 Ngee Ann Polytechnic, 535 Clementi Road, Singapore 599489
* Corresponding author. Tel: +65-68504934; Fax: +65-64269507; E-mail: thean_yen_tan{at}cgh.com.sg
Received 7 March 2007; returned 23 April 2007; revised 6 April 2007; accepted 25 April 2007
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Abstract |
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Objectives: The study investigated the effect of colistin and minocycline when tested singly and in combination against Acinetobacter baumannii.
Methods: Thirteen unrelated imipenem-resistant A. baumannii clinical isolates were included in the study. MICs of colistin sulphate and minocycline were determined by broth macrodilution and Etest. Organisms were also tested against the two antibiotics singly and in combination using time–kill methods and an Etest-based method.
Results: Neither colistin nor minocycline when tested alone demonstrated bactericidal activity. However, the combination of colistin and minocycline demonstrated bactericidal activity against most of the isolates tested. At 24 h, the combination of antibiotics demonstrated synergy in 12 of the 13 isolates by time–kill methods. None of the isolates demonstrated synergy by Etest methods.
Conclusions: The combination of colistin and minocycline was found to be bactericidal and synergistic against A. baumannii by time–kill methods. There was no agreement between time–kill and Etest methods for synergy testing.
Keywords: synergy , tetracyclines , Etest
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Introduction |
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Acinetobacter baumannii is well documented as a multiresistant nosocomial pathogen. Carbapenems are usually recommended as the antibiotics of choice. However, the increasing incidence of carbapenem-resistant strains and a lack of viable treatment alternatives have prompted the use of unconventional antibiotics such as the polymyxins, minocycline and rifampicin for the treatment of infections with multidrug-resistant isolates.
The administration of combinations of antibiotics has been proposed for three main reasons: to broaden the spectrum of activity, to minimize the appearance of antibiotic resistance and to achieve antibiotic synergy. The latter may be important if an antibiotic with marginal activity is used against the infecting bacterium.
Colistin has been increasingly used for the treatment of infections caused by multidrug-resistant Acinetobacter spp., despite initial concerns regarding potential nephrotoxicity and neurotoxicity. There are limited in vitro data1 to support the susceptibility of Acinetobacter spp. to minocycline and anecdotal evidence to support clinical effectiveness.2 Newer glycylcycline derivatives such as tigecycline have also demonstrated in vitro activity against Acinetobacter spp.,1 but clinical experience is still limited.
This study examined the effect of combinations of colistin and minocycline against unrelated strains of imipenem-resistant A. baumannii, using time–kill methods and an Etest-based method.
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Materials and methods |
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Thirteen unique imipenem-resistant (MIC > 8 mg/L) A. baumannii clinical isolates were included in this study. Isolates were collected over a 2 year period and originated from the urinary tract, blood and respiratory tract. Genus identity was determined using conventional biochemical methods and ID-GN cards (Vitek 2, bioMérieux, France), and isolates were phenotypically confirmed as A. baumannii-calcoaceticus group using a published method.3 Susceptibilities to gentamicin, piperacillin/tazobactam, ciprofloxacin, ceftazidime, imipenem, ampicillin/sulbactam, co-trimoxazole and amikacin were determined by disc diffusion and Vitek 2 (bioMérieux). Multidrug resistance was defined as absence of susceptibility to the aminoglycosides, ciprofloxacin, ceftazidime and ß-lactam/ß-lactamase combinations. All study isolates were genotyped using a PCR-based method.4 Digital images of the DNA fingerprints were processed using Gene Profiler 4.05 (Scanalytics, BD Biosciences, USA) and similarity analysis, distance estimation and cluster analysis using UPGMA were performed using Treecon software.
MICs of minocycline and colistin were determined for each test isolate by broth macrodilution, using standards published by the CLSI (formerly NCCLS).5 Colistin sulphate and minocycline powders used for susceptibility testing and combination testing were obtained from Sigma-Aldrich (Singapore). MICs were also determined using minocycline and colistin Etest strips (AB Biodisk, Solna, Sweden) for each study isolate, using the methods recommended by the manufacturer. In general, there was good agreement between the MICs obtained by both methods.
Isolates were tested against colistin, minocycline, a combination of both and an antibiotic-free growth control, following methods published by the CLSI.6 Colistin sulphate and/or minocycline was added to cation-adjusted Mueller–Hinton broth (Becton Dickinson, USA) to make a final testing volume of 10 mL. For isolates susceptible to both antibiotics, a final concentration of 1 x MIC was used for each antibiotic. Minocycline-resistant strains were tested at a minocycline concentration of 4 mg/L. For each test strain, the MIC used for testing by time–kill methods was that determined by broth macrodilution. At the start of each test, a log-phase inoculum of 5 x 105 of each test isolate was added to the testing tubes. Tubes were incubated at 35°C for a total of 24 h. Aliquots of 0.1 mL were removed at intervals of 0, 2, 4, 6 and 24 h from the time of initial incubation, serial 10-fold dilutions performed and aliquots plated on to nutrient agar. Colonies were counted after 18–24 h of incubation. The lower limit of detection by this method was 20 cfu/mL. At each time interval, the log10 value of the viable colony count was determined. Synergy was defined as 
2 log10 decrease in cfu/mL for the antibiotic combination when compared with its more active constituent,6 while a bactericidal effect was defined as 3 log10 decrease in cfu over the measured time period.
The effect of antibiotic combinations by Etest was tested using a previously published method.7 In brief, for each test isolate, Etest strips of colistin and minocycline were placed perpendicular to each other on inoculated Mueller–Hinton plates (Becton Dickinson). The intersection of the two Etest strips corresponded to the MIC of the organism to colistin and minocycline, as determined by previous Etest results. Plates were incubated for 16–18 h at 35°C, and the MIC of each drug in combination was read from the zones of inhibition. The following formulae were used to calculate the fractional inhibitory concentration (FIC) index: the FIC of antibiotic = MIC of antibiotic in combination/MIC of antibiotic alone and FIC index = FIC of colistin + FIC of minocycline. The results of the FIC index were interpreted as follows: synergy, FIC 
0.5; antagonism, FIC > 4; indifference, 0.5 < FIC 4.8
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Results |
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All isolates were resistant to imipenem, whereas five isolates were defined as multidrug-resistant. All study isolates were susceptible to colistin (MIC range of 0.5–2 mg/L, modal MIC 0.5 mg/L), whereas nine (70%) isolates were susceptible to minocycline (MIC range of 0.06–16 mg/L, modal MIC 2 mg/L). PCR typing results showed that all study isolates had similarity indices of < 90% and were considered unrelated.
At 1 x MIC, neither colistin nor minocycline when tested alone demonstrated bactericidal activity, although the average fall in cfu/mL was greater for colistin at 4 and 6 h (Table 1). There was also evidence of bacterial regrowth at 24 h for both antibiotics, when tested singly.
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The use of colistin and minocycline in combination showed a rapidly bactericidal effect, with four (31%), seven (54%) and nine (69%) isolates showing a
3 log10 reduction in cfu at 2, 4 and 24 h, respectively. Once achieved, this reduction in cfu was maintained for all test isolates throughout the testing period, with minimal evidence of bacterial regrowth at 24 h. At the tested antibiotic concentrations, synergy was detected in one (8%) isolate at 2 h, two isolates (15%) at 4 and 6 h and 12 (92%) isolates at 24 h. One isolate did not fulfil the criteria for synergy, because at 24 h the colony count in the combined antibiotic test fell below the detectable threshold. Synergy was also detected in three out of four minocycline-resistant isolates at 24 h. Time–kill curves for a representative isolate are demonstrated in Figure 1.
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Results from the Etest synergy studies showed that the FIC index for study isolates ranged between 1.1 and 1.5. On the basis of the FIC index, antibiotic synergy between colistin and minocycline was not present in any of the test isolates.
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Discussion |
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The results from this study are the first to demonstrate synergistic activity between minocycline and colistin for A. baumannii as tested by the time–kill method. The study also adds to current knowledge about the kinetics of colistin and minocycline against A. baumannii. Similar to other studies,9 the phenomenon of delayed bacterial regrowth was noted for colistin and minocycline when tested alone. The clinical relevance of this delayed regrowth remains uncertain, although Li et al.10 caution that the presence of heteroresistant subpopulations in colistin-susceptible A. baumannii may pose future therapeutic issues.
The use of two antibiotics in combination showed bactericidal effect on test organisms, and the effect was maintained throughout the 24 h testing period. Synergy was detected predominantly at 24 h, and this was mainly derived from the suppression of organism regrowth in the presence of antibiotic combinations. However, it should be noted that only a single antibiotic concentration was employed in the study, and concentrations of antibiotics within the test solutions were not monitored.
We did not find any concordance between the Etest method and conventional time–kill studies at 24 h. A previous study has also shown that the synergistic antibiotic combinations for A. baumannii demonstrated by time–kill or chequerboard methods are not reproducible by the Etest method.11 These data suggest that the results of Etest synergy testing methods for A. baumannii may not show reliable agreement with time–kill or chequerboard testing methods.
The clinical application of synergy testing to the treatment of infections with multidrug-resistant A. baumannii remains uncertain. Time–kill and chequerboard methods are too labour-intensive for routine use and will not provide results in a clinically relevant time frame. Furthermore, the relevance of laboratory synergy testing methods to therapeutic outcome remains unresolved. For example, some animal models report the in vitro effectiveness of antibiotic combinations,12 but published clinical experience provides a cautionary note.13
The optimal susceptibility testing method for the polymyxins is also not clearly defined. Thus, although colistin sulphate is used for in vitro susceptibility testing, the colistimethate sodium form is used for intravenous therapy.
In conclusion, the results of this study demonstrate synergistic effects of colistin and minocycline combinations against unrelated strains of A. baumannii when tested by time–kill methods, but not when an Etest method was used. There was no agreement between the results of Etest and time–kill methodology.
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T. Y. T. has received funding from Wyeth Pharmaceuticals for unrelated research studies. L. S. Y. N., E. T. and G. H. have none to declare.
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Acknowledgements |
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This study was partially supported by an educational grant from Ngee Ann Polytechnic, Singapore.
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References |
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1 Scheetz MH, Qi C, Warren JR, et al. In vitro activities of various antimicrobials alone and in combination with tigecycline against carbapenem-intermediate or -resistant Acinetobacter baumannii. Antimicrob Agents Chemother (2007) 51:1621–6.
2 Wood GC, Hanes SD, Boucher BA, et al. Tetracyclines for treating multidrug-resistant Acinetobacter baumannii ventilator-associated pneumonia. Intensive Care Med (2003) 29:2072–6.[CrossRef][ISI][Medline]
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Gerner-Smidt P, Tjernberg I, Ursing J. Reliability of phenotypic tests for identification of Acinetobacter species. J Clin Microbiol (1991) 29:277–82.
4 Grundmann HJ, Towner KJ, Dijkshoorn L, et al. Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp. J Clin Microbiol (1997) 35:3071–7.[Abstract]
5 National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6 (2003) Wayne, PA, USA: NCCLS.
6 National Committee for Clinical Laboratory Standards. Methods for Determining Bactericidal Activity of Antimicrobial Agents: Approved Guideline M26-A (1999) Wayne, PA, USA: NCCLS.
7 White RL, Burgess DS, Manduru M, et al. Comparison of three different in vitro methods of detecting synergy: time–kill, checkerboard, and Etest. Antimicrob Agents Chemother (1996) 40:1914–8.[Abstract]
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Odds FC. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother (2003) 52:1.
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Owen RJ, Li J, Nation RL, et al. In vitro pharmacodynamics of colistin against Acinetobacter baumannii clinical isolates. J Antimicrob Chemother (2007) 59:473–7.
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Li J, Rayner CR, Nation RL, et al. Heteroresistance to colistin in multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother (2006) 50:2946–50.
11 Bonapace CR, White RL, Friedrich LV, et al. Evaluation of antibiotic synergy against Acinetobacter baumannii: a comparison with Etest, time–kill, and checkerboard methods. Diagn Microbiol Infect Dis (2000) 38:43–50.[CrossRef][ISI][Medline]
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Montero A, Ariza J, Corbella X, et al. Antibiotic combinations for serious infections caused by carbapenem-resistant Acinetobacter baumannii in a mouse pneumonia model. J Antimicrob Chemother (2004) 54:1085–91.
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Saballs M, Pujol M, Tubau F, et al. Rifampicin/imipenem combination in the treatment of carbapenem-resistant Acinetobacter baumannii infections. J Antimicrob Chemother (2006) 58:697–700.
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