JAC Advance Access originally published online on January 23, 2006
Journal of Antimicrobial Chemotherapy 2006 57(3):573-576; doi:10.1093/jac/dki477
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In vitro antibacterial activities of tigecycline in combination with other antimicrobial agents determined by chequerboard and time-kill kinetic analysis
Infectious Disease Discovery Research, Wyeth Research, Pearl River, NY 10965, USA
* Corresponding author. Tel: +1-845-602-4612; Fax: +1-845-602-5671; E-mail: jonesh3{at}wyeth.com
Received 7 October 2005; returned 16 November 2005; revised 30 November 2005; accepted 12 December 2005
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Abstract |
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Objectives: This study was undertaken to determine the interaction of tigecycline with 13 select antimicrobial agents against a wide variety of Gram-negative and Gram-positive bacterial isolates.
Methods: Antibiotic interactions were assayed using the chequerboard MIC format and selected synergistic combinations were confirmed using time-kill kinetic analysis.
Results: Microdilution chequerboard analysis of tigecycline in combination with amikacin, ampicillin/sulbactam, azithromycin, ciprofloxacin, colistin, imipenem, levofloxacin, piperacillin, piperacillin/tazobactam, polymyxin B, rifampicin, minocycline and vancomycin resulted in an interpretation of either no interaction or synergy. Time-kill kinetic analysis resulted in an interpretation of no interaction for all but one of the drug combinations that resulted in an interpretation of synergy by the chequerboard analysis. Antagonism was not observed for any combination when assayed by either method.
Conclusions: The lack of antagonism seen with tigecycline combinations in both chequerboard and time-kill kinetic studies is an encouraging outcome, suggesting that tigecycline may prove to be effective in combination therapy as well as in monotherapy.
Keywords: antibiotics , synergy , antagonism , susceptibility
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Introduction |
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Tigecycline, the 9-glycylamido derivative of minocycline, is the first member of the glycylcycline class of antibiotics to enter the clinic. Tigecycline acts by preventing translation through a reversible binding interaction that blocks the association of charged tRNA with the ribosome. Tigecycline has a distinct advantage over tetracycline and minocycline in that it is not subject to either the efflux or ribosomal protection mechanisms of tetracycline resistance.1,2 Preclinical studies have demonstrated the potent in vitro activity of tigecycline against a broad spectrum of Gram-positive, Gram-negative, anaerobic and atypical pathogens, including those organisms expressing tetracycline resistance determinants.1,2 Moreover, tigecycline is active against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp. (VRE), penicillin-resistant Streptococcus pneumoniae (PRSP) and extended spectrum ß-lactamase (ESBL) producing Klebsiella pneumoniae and Escherichia coli.1 Despite the potent broad spectrum of activity of tigecycline, supported by both preclinical and clinical studies, it is important to characterize tigecycline in combination with other antibiotics in order to identify synergistic and/or antagonistic combinations providing guidance for empirical use as well as for treatment of poly-microbial infections where combination therapy is warranted.3
Due to the emergence of multidrug-resistant pathogens, treatment with combination therapy, using two or more antibacterials, has become commonplace.3 Two of the most widely used in vitro methodologies to assess drug–drug interactions are the chequerboard MIC technique, yielding the fractional inhibitory concentration index (FICI), and time-kill kinetics.3,4 The chequerboard MIC method is prone to error5 and, by necessity, results from the chequerboard MIC are often confirmed with the more dynamic interaction provided by the time-kill kinetic study format.6–8
This study was undertaken to determine the interaction of tigecycline with other antimicrobial agents against a variety of bacterial isolates collected during clinical trials in the United States and Canada between 1990 and 2000.
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Materials and methods |
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Bacterial strains
Representative isolates of clinically relevant species, collected during clinical trials, from various medical centres in the United States and Canada between 1990 and 2000, were used in this study. The Gram-negative organisms used were chosen from the collection at random and do not represent any specific resistance mechanism. The Gram-positive organisms were also chosen from the clinical collection; however, these were chosen to represent important resistance mechanisms (MRSA, PRSP and VRE).
Antimicrobial agents
This study was designed to evaluate tigecycline in combination with a wide variety of antimicrobial agents in support of the use of tigecycline in combination therapy for a compassionate use clinical protocol. The antimicrobial agents used in the study were: tigecycline, piperacillin, tazobactam (Wyeth Research, Pearl River, NY, USA), ampicillin, minocycline, amikacin, ciprofloxacin, vancomycin, rifampicin, polymyxin B, colistin (Sigma-Aldrich Co., St Louis, MO, USA), azithromycin, sulbactam, imipenem (USP, Rockville, MD, USA) and levofloxacin (R. W. Johnson, Princeton, NJ, USA).
Chequerboard MIC
Antibiotic interactions were determined using the chequerboard MIC assay as previously described.5 Mueller–Hinton II broth (MHB) was used for the Enterobacteriaceae, staphylococci and enterococci and was supplemented with 5% lysed horse blood for streptococci. Seven doubling dilutions of tigecycline and 11 doubling dilutions of the test antimicrobial agent were tested. After drug dilution, microbroth dilution plates were inoculated with each organism to yield the appropriate density (105 cfu/mL) in a 100 µL final volume and incubated for 18–22 h at 35°C in ambient air.
The FICI was calculated for each combination using the following formula: FICA + FICB = FICI, where FICA = MIC of drug A in combination/MIC of drug A alone, and FICB = MIC of drug B in combination/MIC of drug B alone. The FICI was interpreted as follows: synergy = FICI 
0.5; no interaction = FICI >0.5–4; antagonism = FICI > 4.
Time-kill assays
Flasks containing MHB and drug were inoculated with test organism to a density of 
106 cfu/mL in a final volume of 100 mL and incubated in a shaking water bath at 35°C in ambient air. Aliquots were removed at time 0 and 3, 6 and 24 h post-inoculation and serially diluted in 0.85% sodium chloride solution for determination of viable counts. Diluted samples, 0.05 mL, were plated in duplicate on trypticase soy agar plates using a spiral plater (Don Whitley Scientific Ltd). Total bacterial cfu/mL (log10cfu/mL) were determined after 18 h of incubation at 35°C.
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Results and discussion |
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The in vitro interactive effects of the antibiotics were determined by the broth microdilution chequerboard method as previously described.5 The range of drug concentrations used in the chequerboard analysis was such that the dilution range encompassed the MIC of each drug used in the analysis.
The combination of tigecycline and another antibiotic demonstrated either synergy (24%) or no interaction (76%) against the panel of Gram-negative bacteria; antagonism was not observed for any combination with tigecycline, against any of the strains tested (Table 1). A higher percentage of synergistic combinations with tigecycline were observed with amikacin (56%), ampicillin/sulbactam (33%), piperacillin/tazobactam (50%) and rifampicin (33%). Interestingly, 73% of the Proteus spp. showed synergy when tigecycline was tested in combination with minocycline. No other clear trend could be established for synergy occurring with any other bacterial species and drug combinations.
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With the Gram-positive isolates, rifampicin displayed a synergistic effect with tigecycline for 66% of the isolates tested (Table 2). The majority of these strains showing synergy were Enterococcus spp. including vancomycin-resistant (VRE) strains and penicillin-resistant Streptococcus pneumoniae (PRSP). Conversely, the combination of vancomycin and tigecycline resulted in a larger percentage of no interaction (71%) than synergistic effects (29%) against the Gram-positive isolates. Antagonism was not observed in this analysis.
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In order to confirm a result of synergy (FICI
0.5) by the chequerboard MIC method, time-kill kinetic studies were performed with tigecycline combinations against selected bacterial species.9 The antibiotics were tested at concentrations based on the MIC determined from microbroth chequerboard testing: alone at 1x and 0.5x the MIC and in combination at 0.5x the MIC. The concentrations of the antibiotics used in the time-kill assays were, at a minimum, one dilution higher than the synergistic combination shown by chequerboard MIC analysis.
As determined by Eliopoulos and Moellering10 an interpretation of ‘synergy’ required a 
2 log10 decrease in cfu/mL by the drug combination when compared with its most active constituent after 24 h and a 2 log10 decrease in the cfu/mL below the starting inoculum. Likewise, the drug combination was considered to be ‘antagonistic’ if there was a 2 log10 increase in cfu/mL and ‘no interaction’ was the interpretation of a <2 log10 change in cfu/mL.
The results of time-kill kinetic studies confirmed the chequerboard data in that none of the tigecycline combinations resulted in antagonism. However, synergy results by FICI were confirmed by time-kill kinetics for only one of the seventeen combinations examined, which was tigecycline, tested at 2 mg/L, combined with amikacin, tested at 8 mg/L against one strain of Acinetobacter baumannii (PT 9158). Indicative of the majority of strains tested was the finding that tigecycline, tested at 0.5 mg/L, in combination with azithromycin, tested at 16 mg/L, was synergistic by FICI against a strain of K. pneumoniae (PT 9266); however, when assayed by time-kill kinetics this combination failed to meet the criteria for synergy. In approximately half of the time-kill kinetics studies, the combinations demonstrated better killing at the 6 h time point than either drug alone; however, there were no changes in interpretations regarding synergy when measured at the earlier time point (data not shown).
Antimicrobial combinations are used frequently in the clinic to provide broad-spectrum coverage until the causative pathogens are isolated and identified.3 In the clinical setting, combination therapy is most often given empirically without the use of in vitro synergy data, as there is a lack of clinical data to correlate the results of in vitro synergy studies with patient outcome.3 Clearly, from a clinical viewpoint, antagonism is the least desirable outcome possible with an antimicrobial combination. Recent in vitro studies have demonstrated various antibiotic combinations that resulted in no interaction and or synergy.6,7 Although there is no consensus in the field as to the best methodology for measuring synergy, the most commonly used assay is the chequerboard MIC test; however, this is most often used only as a screening test.3–8 The chequerboard MIC test suffers due to lack of reproducibility and only measures bacteriostatic effects.5 Variability in the test as well as testing a bacteriostatic agent in combination with mostly bactericidal agents may be the cause for the overestimate of synergy experienced with the chequerboard test. Accordingly, synergy testing performed by time-kill kinetics was used to confirm the results of chequerboard MIC testing, as is standard protocol in many laboratories.7,8
The drug concentrations used in the time-kill kinetic studies, based on the MIC determined in the FICI analysis, were expected to have an effect in the growth assay. The fact that these concentrations do not result in synergy in the more constrained experimental format suggests again that the chequerboard analysis is overestimating synergy, possibly due to the reproducibility issue inherent in the test.5
Although synergy detected by in vitro chequerboard studies could not in the majority of cases be confirmed by time-kill kinetic analysis, the lack of antagonism seen with tigecycline combinations in both studies is an encouraging outcome suggesting that tigecycline may prove to be effective in combination therapy as well as in monotherapy.
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None to declare.
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References |
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1. Bradford PA, Weaver-Sands DT Petersen PJ. In vitro activity of tigecycline against isolates from patients enrolled in phase 3 clinical trials for complicated skin and skin structure infections and complicated intra-abdominal infections. Clin Infect Dis 2005; 41 Suppl 5: S315–32.
2.
Petersen PJ, Bradford PA, Weiss WJ et al. In vitro and in vivo activities of tigecycline (GAR-936), daptomycin, and comparative antimicrobial agents against glycopeptide-intermediate Staphylococcus aureus and other resistant Gram-positive pathogens. Antimicrob Agents Chemother 2002; 46: 2595–601.
3. Rybak MJ McGrath BJ. Combination antimicrobial therapy for bacterial infections. Guidelines for the clinician. Drugs 1996; 52: 390–405.[Web of Science][Medline]
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White R, Burgess D, Manduru M et al. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrob Agents Chemother 1996; 40: 1914–18.
5.
Rand KH, Houck HJ, Brown P et al. Reproducibility of the microdilution checkerboard method for antibiotic synergy. Antimicrob Agents Chemother 1993; 37: 613–615.
6. Alou L, Cafini F, Sevillano D et al. In vitro activity of mupirocin and amoxicillin-clavulanate alone and in combination against staphylococci including those resistant to methicillin. Int J Antimicrob Agents 2004; 23: 513–6.[CrossRef][Web of Science][Medline]
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Jacqueline C, Navas D, Batard E et al. In vitro and in vivo synergistic activities of linezolid combined with subinhibitory concentrations of imipenem against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2005; 49: 45–51.
8.
Cappelletty DM Rybak MJ. Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 1996; 40: 677–83.
9. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, PA, USA, 2003.
10. Eliopoulos GM Moellering RC Antimicrobial combinations. In: Lorian V, ed. Antibiotic in Laboratory Medicine, 4th edn. Baltimore, MD: Williams and Wilkins, 1996; 330–96.
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