chequerboard and time-kill kinetic analysis

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Journal of Antimicrobial Chemotherapy (2006) 57, 573–576

doi:10.1093/jac/dki477

Advance Access publication 23 January 2006

In vitro antibacterial activities of tigecycline in combinationwith 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

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

Objectives : This study was undertaken to determine the interaction of tigecycline with 13 selectantimicrobial 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 syner-

gistic 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 eitherno interaction or synergy. Time-kill kinetic analysis resulted in an interpretation of no interaction for all butone 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 incombination 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 minocyclinein 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 patho-

gens, including those organisms expressing tetracycline resistance

determinants.1,2 Moreover,tigecyclineis active against methicillin-

resistant 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 wherecombination therapy is warranted.3

Due to the emergence of multidrug-resistant pathogens,

treatment with combination therapy, using two or more anti-

bacterials, 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 inhi-

bitory 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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*Corresponding author. Tel: +1-845-602-4612; Fax: +1-845-602-5671; E-mail: [email protected].............................................................................................................................................................................................................................................................................................................................................................................................................................

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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

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, micro-

broth dilution plates were inoculated with each organism to yield

the appropriate density (105 cfu/mL) in a 100 mL final volume andincubated for 18–22 h at 35C 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 incuba-

ted in a shaking water bath at 35C 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 35C.

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 chequer-

board analysis was such that the dilution range encompassed

the MIC of each drug used in the analysis.

The combination of tigecycline and another antibiotic demon-

strated 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/sulbac-

tam (33%), piperacillin/tazobactam (50%) and rifampicin (33%).

Interestingly, 73% of the Proteus spp. showed synergy when tige-

cyclinewas 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). Con-

versely, 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 thechequerboard MIC method, time-kill kinetic studies were per-

formed 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-killassays 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 drugcombination when compared with its most active constituent

after 24 h and a ‡2 log10 decrease in the cfu/mL below the startinginoculum. Likewise, the drug combination was considered to be

‘antagonistic’ if there was a ‡2 log10 increase in cfu/mL and ‘nointeraction’ was the interpretation of a


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Table 1. Results of chequerboard testing of tigecycline and a second

antibacterial agent against Gram-negative bacteria

Organism Second agent

No. of strains

showing synergy/total

no. of strainsa

Acinetobacter

baumannii

amikacin 4/9

ampicillin/sulbactam 1/9azithromycin 0/9

ciprofloxacin 0/9

colistin 1/9

piperacillin/tazobactam 2/9

polymyxin B 2/9

rifampicin 1/9

Acinetobacter spp.b imipenem 3/11

levofloxacin 0/11

piperacillin 3/11

Enterobacter

aerogenes

amikacin 1/1

ampicillin/sulbactam 0/1

azithromycin 0/1

ciprofloxacin 0/1

colistin 1/1

imipenem 1/5

levofloxacin 4/5

piperacillin 3/5


polymyxin B 0/1

rifampicin 1/1

Enterobacter

cloacae

amikacin 2/2


azithromycin 1/2

ciprofloxacin 0/2

colistin 0/2

imipenem 1/5

levofloxacin 1/5

piperacillin 3/5


polymyxin B 0/2

rifampicin 2/2

Escherichia coli imipenem 1/11

levofloxacin 0/11

piperacillin 2/11

Klebsiella

pneumoniae

amikacin 3/3


azithromycin 0/3

ciprofloxacin 0/3

colistin 0/3

imipenem 3/10

levofloxacin 3/10piperacillin 1/10


polymyxin B 0/3

rifampicin 2/3

Proteus mirabilis imipenem 2/4

levofloxacin 0/4

minocycline 3/4

piperacillin 2/4

Table 1. (continued )


No. of strains

showing synergy/total

no. of strainsa

Proteus vulgaris imipenem 0/4

levofloxacin 1/4

minocycline 3/4

piperacillin 3/4

Providencia

rettgeri

imipenem 1/3

levofloxacin 0/3

minocycline 2/3

piperacillin 1/3

Pseudomonas

aeruginosa

amikacin 0/3


azithromycin 0/3

ciprofloxacin 0/3

colistin 0/3

imipenem 1/11

levofloxacin 0/11

piperacillin 3/11

piperacillin/tazobactam 2/3polymyxin B 0/3

rifampicin 0/3

Stenotrophomonas

maltophilia

imipenem 0/10

levofloxacin 0/10

piperacillin 0/10

aNone of the strains tested showed antagonism.b 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


No. of strains

showing

synergy/total

no. of strainsa

Staphylococcus

aureus (MRSA)

vancomycin 1/10

rifampicin 2/10

Enterococcus

faecium (VRE)

vancomycin 3/5

rifampicin 4/5

Enterococcus

faecium

vancomycin 1/5

rifampicin 4/5

Enterococcus

faecalis (VRE)

vancomycin 0/3

rifampicin 2/3

Enterococcus

faecalis

vancomycin 0/8

rifampicin 5/8

Streptococcus

pneumoniae (PRSP)

vancomycin 7/10

rifampicin 10/10

aNone of the strains tested showed antagonism.

Tigecycline synergy

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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 concen-

trations do not result in synergy in the more constrained experi-

mental 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 com-

binations in both studies is an encouraging outcome suggesting

that tigecycline may prove to be effective in combination therapy

as well as in monotherapy.

Transparency declarationsNone to declare.

References

1. Bradford PA, Weaver-Sands DT Petersen PJ. In vitro activity oftigecycline 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 therapyfor bacterial infections. Guidelines for the clinician. Drugs 1996; 52:

390–405.

4. WhiteR, Burgess D, Manduru M etal . Comparisonof threedifferent

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 micro-dilution 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.

7. Jacqueline C, Navas D, Batard E et al . In vitro and in vivo synergistic activities of linezolid combined with subinhibitory concentra-

tions 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.

Petersen et al.

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