Journal of Antimicrobial Chemotherapy (2006) 57, 573576

doi:10.1093/jac/dki477
Advance Access publication 23 January 2006

In vitro antibacterial activities of tigecycline in combination

with other antimicrobial agents determined by chequerboard and
time-kill kinetic analysis
Peter J. Petersen, Ponpen Labthavikul, C. Hal Jones* and Patricia A. Bradford
Infectious Disease Discovery Research, Wyeth Research, Pearl River, NY 10965, USA

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 timekill 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

Introduction
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 methicillinresistant Staphylococcus aureus (MRSA), vancomycin-resistant
Enterococcus spp. (VRE), penicillin-resistant Streptococcus
pneumoniae (PRSP) and extended spectrum b-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 drugdrug 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.68
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.

.............................................................................................................................................................................................................................................................................................................................................................................................................................

*Corresponding author. Tel: +1-845-602-4612; Fax: +1-845-602-5671; E-mail: jonesh3@wyeth.com

.............................................................................................................................................................................................................................................................................................................................................................................................................................

573

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Received 7 October 2005; returned 16 November 2005; revised 30 November 2005; accepted 12 December 2005

Petersen et al.

Materials and methods

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

Chequerboard MIC
Antibiotic interactions were determined using the chequerboard MIC
assay as previously described.5 MuellerHinton 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 mL final volume and
incubated for 1822 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 >0.54;
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.

Results and discussion

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

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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).

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.
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.
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 1 and 0.5 the MIC and in combination at 0.5 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

Tigecycline synergy
Table 1. Results of chequerboard testing of tigecycline and a second
antibacterial agent against Gram-negative bacteria

Organism
Acinetobacter
baumannii

Second agent

No. of strains
showing synergy/total
no. of strainsa

amikacin
ampicillin/sulbactam
azithromycin
ciprofloxacin
colistin
piperacillin/tazobactam
polymyxin B
rifampicin

4/9
1/9
0/9
0/9
1/9
2/9
2/9
1/9
3/11
0/11
3/11

Enterobacter
aerogenes

amikacin
ampicillin/sulbactam
azithromycin
ciprofloxacin
colistin
imipenem
levofloxacin
piperacillin
piperacillin/tazobactam
polymyxin B
rifampicin

1/1
0/1
0/1
0/1
1/1
1/5
4/5
3/5
1/1
0/1
1/1

amikacin
ampicillin/sulbactam
azithromycin
ciprofloxacin
colistin
imipenem
levofloxacin
piperacillin
piperacillin/tazobactam
polymyxin B
rifampicin

2/2
2/2
1/2
0/2
0/2
1/5
1/5
3/5
1/2
0/2
2/2

imipenem
levofloxacin
piperacillin

1/11
0/11
2/11

amikacin
ampicillin/sulbactam
azithromycin
ciprofloxacin
colistin
imipenem
levofloxacin
piperacillin
piperacillin/tazobactam
polymyxin B
rifampicin

3/3
2/3
0/3
0/3
0/3
3/10
3/10
1/10
3/3
0/3
2/3

imipenem
levofloxacin
minocycline
piperacillin

2/4
0/4
3/4
2/4

Enterobacter
cloacae

Escherichia coli

Klebsiella
pneumoniae

Proteus mirabilis

Organism

Second agent

No. of strains
showing synergy/total
no. of strainsa

Proteus vulgaris

imipenem
levofloxacin
minocycline
piperacillin

0/4
1/4
3/4
3/4

Providencia
rettgeri

imipenem
levofloxacin
minocycline
piperacillin

1/3
0/3
2/3
1/3

Pseudomonas
aeruginosa

amikacin
ampicillin/sulbactam
azithromycin
ciprofloxacin
colistin
imipenem
levofloxacin
piperacillin
piperacillin/tazobactam
polymyxin B
rifampicin

0/3
2/3
0/3
0/3
0/3
1/11
0/11
3/11
2/3
0/3
0/3

Stenotrophomonas
maltophilia

imipenem
levofloxacin
piperacillin

0/10
0/10
0/10

None of the strains tested showed antagonism.

Acinetobacter spp. includes: A. baumannii (13), A. anitratus (4), A. lwoffi (3).

Table 2. Results of chequerboard testing of tigecycline and a second

antibacterial agent against Gram-positive bacteria

Organism

Second agent

No. of strains
showing
synergy/total
no. of strainsa

Staphylococcus
aureus (MRSA)

vancomycin
rifampicin

1/10
2/10

Enterococcus
faecium (VRE)

vancomycin
rifampicin

3/5
4/5

Enterococcus
faecium

vancomycin
rifampicin

1/5
4/5

Enterococcus
faecalis (VRE)

vancomycin

0/3

rifampicin
vancomycin

2/3
0/8

rifampicin
vancomycin
rifampicin

5/8
7/10
10/10

Enterococcus
faecalis
Streptococcus
pneumoniae (PRSP)
a

None of the strains tested showed antagonism.

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Acinetobacter spp.b imipenem

levofloxacin
piperacillin

Table 1. (continued)

Petersen et al.
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Chemother 2002; 46: 2595601.
3. Rybak MJ McGrath BJ. Combination antimicrobial therapy
for bacterial infections. Guidelines for the clinician. Drugs 1996; 52:
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4. 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: 191418.
5. Rand KH, Houck HJ, Brown P et al. Reproducibility of the microdilution checkerboard method for antibiotic synergy. Antimicrob Agents
Chemother 1993; 37: 613615.
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Transparency declarations
None to declare.

References
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: S31532.

9. National Committee for Clinical Laboratory Standards. Methods

for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
AerobicallySixth Edition: Approved Standard M7-A6. NCCLS,
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Williams and Wilkins, 1996; 330-96.

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a screening test.38 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.