ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2009, p. 814–817 0066-4804/09/$08.00⫹0 doi:10.1128/AAC.01132-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 2
Activity of Levofloxacin Alone and in Combination with a DnaK Inhibitor against Gram-Negative Rods, Including Levofloxacin-Resistant Strains䌤 Kim Credito,1 Gengrong Lin,1 Laura Koeth,2 Michael A. Sturgess,3 and Peter C. Appelbaum1* Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania 170331; Laboratory Specialists, Inc., Cleveland, Ohio 441062; and Chaperone Technologies, Inc., East Stroudsburg, Pennsylvania 183013 Received 22 August 2008/Returned for modification 22 October 2008/Accepted 5 November 2008
Synergy time-kill testing of levofloxacin alone and in combination with CHP-105, a representative DnaK inhibitor, against 50 gram-negative rods demonstrated that 34 of the 50 strains tested showed significant synergy between levofloxacin and CHP-105 after 12 h and 24 h. Fourteen of these 34 organisms were quinolone resistant (levofloxacin MICs of >4 g/ml). Organisms tested included 22 Escherichia coli, 22 Klebsiella pneumoniae, 2 Citrobacter freundii, 2 Enterobacter cloacae, and 2 Pseudomonas aeruginosa strains. Of these, 28 were quinolone susceptible (taken as having levofloxacin MICs of ⱕ2 g/ml) and 22 were quinolone resistant (including three strains with intermediate levofloxacin MICs of 4 g/ml). Some of the organisms were provided by Ronald Jones (JMI Laboratories, Liberty City, IA) and Kenneth Thompson (Creighton University School of Medicine, Omaha, NE). Strains were frozen at ⫺70°C in double-strength skim milk (Difco Laboratories, Detroit, MI) before testing. Clinical and Laboratory Standards Institute-approved broth macrodilution in one-quarter-strength cation-adjusted Mueller Hinton broth (BBL Microbiology Systems, Cockeysville, MD) was used to test for MICs of all drugs alone against each of the 50 organisms (7). Prior studies (9) have demonstrated the reduced potency of cationic antimicrobial peptides in the presence of high-salt-containing media, such as full-strength Mueller-Hinton broth. This effect is thought to be mediated through divalent ion complexation of the active peptides and a subsequently reduced interaction of peptides with the bacterial membrane. For time-kill, all compounds were tested alone at concentrations up to three times above and three times below the MIC. The inoculum amounts ranged from 5 ⫻ 105 CFU/ml to 5 ⫻ 106 CFU/ml. The concentrations selected for synergy testing were one to two dilutions below the MIC of each drug tested alone. Suspensions were incubated in a shaking water bath at 35°C, and viability counts for time-kill and synergy testing were performed at 0, 3, 6, 12, and 24 h. For the purposes of this study, synergy was defined as a decrease of ⱖ2 log10 in CFU/ml between the combination and its more-active constituent after 3 h, 6 h, 12 h, and 24 h, with the number of surviving organisms in the presence of the combination being ⱖ2 log10 CFU/ml below the starting inoculum. At least one of the drugs was present in a concentration which did not significantly affect the growth curve of the organism when used alone. When the effect of a second drug was similar to that of the single more-effective compound, this interaction was termed “indifferent”; antagonism was taken as the combination yielding higher colony counts than those seen with the more-active single drug alone. The minimum countable num-
The number of multiresistant gram-negative rod strains is increasing all over the world, and there are no new drugs in development to improve therapeutic choices (4, 11, 12). In the absence of new drugs, combination therapy may currently be the only option to treat these resistant strains. Previous studies by our and other groups have demonstrated that time-kill is more discriminatory than checkerboard for determining synergy in vitro (1–3, 6, 8, 17, 19). CHP-105 (Fig. 1) is an example of a novel series of pyrrhocoricin-derived peptide inhibitors of the bacterial chaperone DnaK. The insect-derived parent peptide has been shown to bind to the multihelical lid and inhibit the refolding activity of Escherichia coli-derived DnaK (13). Conversely, pyrrhocoricin does not interact with the corresponding lid sequence derived from Staphylococcus aureus. These peptides rapidly penetrate both gram-negative and gram-positive bacteria but only show growth inhibition against gram-negative species. However, pyrrhocoricin has limited utility due to its low proteolytic stability and its cellular-membrane-disrupting tendencies at high doses (15). Initial efforts to overcome these issues resulted in the identification of the dimeric analog CHP-105. This peptide has been extensively studied in an effort to explore the generic utility of DnaK inhibitors as antibacterial agents. More-recent synthesis efforts have focused upon more drug-like next-generation small-molecule and low-molecular-weight DnaK inhibitors exhibiting far greater potential utility as antimicrobial agents (5, 14, 16). We have recently reported that peptide inhibitors of DnaK, such as CHP-105 and pyrrhocoricin, when combined with levofloxacin acted in a synergistic manner when tested against a small panel of gram-negative organisms (18). To further establish the utility of such combinations in the treatment of drugresistant infections, we have expanded upon these preliminary results by using time-kill synergy analysis to examine the activity of levofloxacin, with and without CHP-105, against 50 quinolone-susceptible and -resistant gram-negative rods.
* Corresponding author. Mailing address: Department of Pathology, Hershey Medical Center, P.O. Box 850, Hershey, PA 17033. Phone: (717) 531-5113. Fax: (717) 531-7953. E-mail:
[email protected]. 䌤 Published ahead of print on 17 November 2008. 814
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FIG. 1. CHP-105 is a pyrrhocoricin-derived DnaK inhibitor.
ber of CFU/ml was approximately 30 to 300, and drug carryover was addressed by dilution, as we have previously described (1, 2, 6, 8, 10, 17, 19). Results are presented in Tables 1 to 3. As can be seen, levofloxacin and CHP-105 MICs, respectively, ranged from 0.03 to 32 g/ml and 4 to 256 g/ml for E. coli strains and from 0.06 to 64 g/ml and 4 to 128 g/ml for Klebsiella pneumoniae strains; these MICS were 0.06 and 16 g/ml and 4 and 8 g/ml for Enterobacter cloacae strains, 0.125 and 32
g/ml and 8 and 512 g/ml for Citrobacter freundii strains, and 2 and 16 g/ml and 128 and 256 g/ml for Pseudomonas aeruginosa strains. Levofloxacin and CHP-105 showed synergy against 34 of 50 organisms (14 were quinolone resistant) after 12 and 24 h when tested at subinhibitory concentrations. Ten E. coli strains (6 were quinolone resistant) showed synergy at 12 h, and 15 strains (7 were quinolone resistant) showed synergy at 24 h, all at subinhibitory concentrations
TABLE 1. MIC and time-kill synergy test results for Klebsiella pneumoniae strains
TABLE 2. MIC and time-kill synergy test results for Escherichia coli strains
MIC (g/ml) Strain
E353 E354 E356 E362 E363 E364 E366 E367 E368 E369 E370 E371 E372 E411 E418 E422 E426 E427 E428 E431 E432 E433
CHP-105 ⫹ levofloxacin time-kill synergy test resulta at indicated time point (h)
CHP-105
Levofloxacin
12
24
4 8 8 4 4 8 16 16 8 8 4 32 4 8 8 32 4 8 8 4 128 8
0.25 16 0.5 0.125 0.06 2 0.125 0.25 8 64 32 32 0.25 2 1 16 4 2 1 4 1 16
Indifferent Indifferent Indifferent Synergy (2/0.03) Synergy (2/0.016) Synergy (2/1) Synergy (4/0.03) Synergy (4/0.06) Synergy (2/4) Indifferent Synergy (1/16) Synergy (16/8) Synergy (2/0.06) Indifferent Indifferent Synergy (8/8) Indifferent Synergy (2/0.5) Indifferent Indifferent Indifferent Synergy (4/4)
Synergy (1/0.125) Indifferent Indifferent Indifferent Synergy (2/0.03) Synergy (2/0.5) Synergy (4/0.06) Synergy (4/0.06) Synergy (2/2)b Synergy (4/32) Synergy (2/16) Synergy (8/8) Synergy (2/0.06) Synergy (2/1) Synergy (4/0.5) Synergy (16/4) Indifferent Synergy (4/0.5) Indifferent Indifferent Synergy (64/0.5) Synergy (4/4)
a The respective drug concentrations (g/ml) at which synergy was found are listed in parentheses. b Strain became levofloxacin susceptible with the combination.
MIC (g/ml) Strain
E341 E342 E346 E351 E393 E394 E395 E396 E408 E409 E410 E412 E413 E424 E425 E430 E434 E435 E436 E437 E438 E439
CHP-105 ⫹ levofloxacin time-kill synergy test resulta at indicated time point (h)
CHP-105
Levofloxacin
12
24
16 256 64 32 16 32 32 16 16 16 4 8 32 64 32 16 8 8 8 4 8 8
0.06 0.5 0.03 16 16 32 16 16 32 8 4 8 8 0.25 2 0.125 0.125 0.125 0.125 0.03 0.06 0.5
Synergy (4/0.03) Indifferent Indifferent Synergy (8/4) Indifferent Synergy (8/8) Synergy (16/4) Synergy (8/4) Synergy (4/16) Synergy (8/2)b Indifferent Indifferent Indifferent Synergy (16/0.125) Indifferent Indifferent Indifferent Indifferent Indifferent Indifferent Synergy (4/0.03) Synergy (4/0.25)
Synergy (4/0.03) Synergy (64/0.125) Synergy (16/0.016) Synergy (8/4) Indifferent Synergy (8/8) Synergy (16/4) Synergy (8/4) Synergy (8/8) Synergy (4/4) Indifferent Indifferent Synergy (8/4) Synergy (16/0.125) Synergy (16/1) Indifferent Indifferent Indifferent Synergy (4/0.06) Indifferent Synergy (4/0.03) Synergy (4/0.25)
a The respective drug concentrations (g/ml) at which synergy was found are listed in parentheses. b Strain became levofloxacin susceptible with the combination.
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. MIC and time-kill synergy test results for other gram-negative rods CHP-105 ⫹ levofloxacin time-kill synergy test resulta at indicated time point (h)
MIC (g/ml) Species, strain CHP-105
Enterobacter cloacae E373 E397
4.0 8.0
Levofloxacin
12
24
0.06 16.0
Indifferent Indifferent
Indifferent Indifferent
Citrobacter freundii E384 E400
512.0 8.0
0.125 32.0
Synergy (128.0/0.03) Indifferent
Synergy (128.0/0.03) Indifferent
Pseudomonas aeruginosa P330 P335
256.0 128.0
2.0 16.0
Indifferent Synergy (64.0/8.0)
Indifferent Synergy (64.0/8.0)
a
The respective drug concentrations (g/ml) at which synergy was found are listed in parentheses.
of both compounds. Subinhibitory concentrations of CHP105 and levofloxacin in synergistic combinations ranged from 4 to 128 g/ml and 0.008 to 16 g/ml, respectively. Against Klebsiella pneumoniae strains, 12 strains (5 were quinolone resistant) showed synergy at 12 h and 16 strains (6 were quinolone resistant) showed synergy at 24 h, all at subinhibitory concentrations of both drugs. Subinhibitory concentrations of CHP-105 and levofloxacin in synergistic combinations ranged from 1 to 64 g/ml and 0.016 to 32 g/ml, respectively. One quinolone-susceptible Citrobacter freundii strain showed synergy at 12 and 24 h at CHP-105 and levofloxacin concentrations of 128 g/ml and 0.03 g/ ml, respectively, and one quinolone-resistant Pseudomonas aeruginosa strain demonstrated synergy at 12 and 24 h at CHP-105 and levofloxacin concentrations of 64 g/ml and 8 g/ml, respectively; synergy in both strains was found at subinhibitory drug concentrations. All other drug-drug in-
teractions yielded indifferent results, and no antagonism between drugs was observed. The results of time-kill synergy tests for Klebsiella pneumoniae strain E368 are presented in Fig. 2. Our studies showed synergy in more than two-thirds of 50 gram-negative rods after 12 h and 24 h. An “indifferent” response was observed in the remaining one-third of strains tested, and in no case was there antagonism between the two drugs. The mechanism of levofloxacin resistance in quinoloneresistant strains tested was not determined, and we have no explanation as to why synergy was only observed after 12 h and 24 h and not at earlier time periods. Although these results need to be confirmed by testing a larger spectrum of quinolonesusceptible and -resistant gram-negative rods, the initial results are encouraging. More-potent DnaK inhibitors are currently in development and will be employed to further expand on the utility of this approach.
FIG. 2. Activity of CHP-105–levofloxacin against Klebsiella pneumoniae E368.
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This study was funded by Chaperone Technologies, Inc., East Stroudsburg, PA, through the Ben Franklin Technology Partners of Northeastern Pennsylvania. We thank Ronald Jones (JMI Laboratories, Liberty City, IA) and Kenneth Thompson (Creighton University School of Medicine, Omaha, NE) for providing some strains. REFERENCES 1. Bajaksouzian, S., M. A. Visalli, M. R. Jacobs, and P. C. Appelbaum. 1996. Antipneumococcal activities of cefpirome and cefotaxime, alone and in combination with vancomycin and teicoplanin, determined by checkerboard and time-kill methods. Antimicrob. Agents Chemother. 40:1973–1976. 2. Bajaksouzian, S., M. A. Visalli, M. R. Jacobs, and P. C. Appelbaum. 1997. Activities of levofloxacin, ofloxacin, and ciprofloxacin, alone and in combination with amikacin, against acinetobacters determined by checkerboard and time-kill studies. Antimicrob. Agents Chemother. 41:1073–1076. 3. Cappelletty, D. M., and M. R. Rybak. 1996. Comparison of methodologies for synergism testing of drug combinations against resistant strains of Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 40:677–683. 4. Carlet, J., A. Ben Ali, and A. Chalfine. 2004. Epidemiology and control of antibiotic resistance in the intensive care unit. Curr. Opin. Infect. Dis. 17: 309–316. 5. Chang, L., E. B. Bertelsen, S. Wisen, E. M. Larsen, E. R. Zuiderweg, and J. E. Gestwicki. 2008. High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK. Anal. Biochem. 372:167–176. 6. Clark, C. L., M. R. Jacobs, and P. C. Appelbaum. 1999. Activities of clinafloxacin, alone and in combination with other compounds, against 45 grampositive and -negative organisms for which clinafloxacin MICs are high. Antimicrob. Agents Chemother. 43:2295–2298. 7. Clinical and Laboratory Standards Institute. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7–A7, 7th ed. Clinical and Laboratory Standards Institute, Wayne, PA. 8. Credito, K., G. Lin, and P. C. Appelbaum. 2007. Activity of daptomycin alone and in combination with rifampin and gentamicin against Staphylococcus aureus by time-kill methodology. Antimicrob. Agents Chemother. 51: 1504–1507.
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