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Antimicrobial Agents and Chemotherapy, February 2009, p. 814-817, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01132-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Activity of Levofloxacin Alone and in Combination with a DnaK Inhibitor against Gram-Negative Rods, Including Levofloxacin-Resistant Strains

Kim Credito,¹ Gengrong Lin,¹ Laura Koeth,² Michael A. Sturgess,³ and Peter C. Appelbaum^1*

Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania 17033,¹ Laboratory Specialists, Inc., Cleveland, Ohio 44106,² Chaperone Technologies, Inc., East Stroudsburg, Pennsylvania 18301³

Received 22 August 2008/ Returned for modification 22 October 2008/ Accepted 5 November 2008

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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 µg/ml).

Top ABSTRACT INTRODUCTION REFERENCES

 
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).

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FIG. 1. CHP-105 is a pyrrhocoricin-derived DnaK inhibitor.

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 drug-resistant 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.

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 2 µg/ml) and 22 were quinolone resistant (including three strains with intermediate levofloxacin MICs of 4 µg/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 x 10⁵ CFU/ml to 5 x 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 2 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 2 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 number 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 µg/ml and 4 to 256 µg/ml for E. coli strains and from 0.06 to 64 µg/ml and 4 to 128 µg/ml for Klebsiella pneumoniae strains; these MICS were 0.06 and 16 µg/ml and 4 and 8 µg/ml for Enterobacter cloacae strains, 0.125 and 32 µg/ml and 8 and 512 µg/ml for Citrobacter freundii strains, and 2 and 16 µg/ml and 128 and 256 µg/ml for Pseudomonas aeruginosa strains.

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TABLE 1. MIC and time-kill synergy test results for Klebsiella pneumoniae strains

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TABLE 2. MIC and time-kill synergy test results for Escherichia coli strains

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TABLE 3. MIC and time-kill synergy test results for other gram-negative rods

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 of both compounds. Subinhibitory concentrations of CHP-105 and levofloxacin in synergistic combinations ranged from 4 to 128 µg/ml and 0.008 to 16 µg/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 µg/ml and 0.016 to 32 µg/ml, respectively. One quinolone-susceptible Citrobacter freundii strain showed synergy at 12 and 24 h at CHP-105 and levofloxacin concentrations of 128 µg/ml and 0.03 µg/ml, respectively, and one quinolone-resistant Pseudomonas aeruginosa strain demonstrated synergy at 12 and 24 h at CHP-105 and levofloxacin concentrations of 64 µg/ml and 8 µg/ml, respectively; synergy in both strains was found at subinhibitory drug concentrations. All other drug-drug interactions 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.

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FIG. 2. Activity of CHP-105-levofloxacin against Klebsiella pneumoniae E368.

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 quinolone-resistant 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 quinolone-susceptible 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.

 
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.

 
* 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: pappelbaum{at}psu.edu

Published ahead of print on 17 November 2008.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, February 2009, p. 814-817, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01132-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Credito, K. Articles by Appelbaum, P. C. Search for Related Content PubMed PubMed Citation Articles by Credito, K. Articles by Appelbaum, P. C.