Synergy of Daptomycin with Oxacillin and Other {beta}-Lactams against Methicillin-Resistant Staphylococcus aureus

We previously observed marked synergy between daptomycin andboth rifampin and ampicillin against vancomycin-resistant enterococci(VRE). Because the synergy between daptomycin and ampicillinwas observed for 100% of VRE strains with high-level ampicillinresistance (ampicillin MIC of $≥$ 128 µg/ml), we looked forsynergy between daptomycin and other ß-lactams against18 strains of methicillin-resistant Staphylococcus aureus (MRSA)by employing a time-kill method using Mueller-Hinton broth supplementedto 50 mg of Ca²⁺/liter. All strains were resistant to oxacillin(16 of 18 strains were resistant at drug concentrations of $≥$ 256µg/ml), and all strains were susceptible to daptomycin(the MIC at which 90% of the tested isolates were inhibitedwas 1 µg/ml). Daptomycin was tested at concentrationsof 2, 1, 0.5, 0.25, 0.125, and 0.0625 µg/ml alone or incombination with oxacillin at a fixed concentration of 32 µg/ml.Synergy was found for all 18 strains with daptomycin at one-halfthe MIC in combination with 32 µg of oxacillin/ml, andsynergy was found for 11 of 18 strains (61%) with daptomycinat one-fourth the MIC or less in combination with oxacillin.At 24 h, the daptomycin-oxacillin combination with daptomycinat one-half the MIC showed bactericidal activity against all18 strains, and the combination with one-fourth the daptomycinMIC showed bactericidal activity against 9 of 18 strains. Wealso used a novel screening method to look for synergy betweendaptomycin and other ß-lactams. In this approach,daptomycin was incorporated into Ca²⁺-supplemented Mueller-Hintonagar at subinhibitory concentrations, and synergy was screenedby comparing test antibiotic Kirby-Bauer disks on agar withand without daptomycin. By this method, daptomycin with ampicillin-sulbactam,ticarcillin-clavulanate, or piperacillin-tazobactam showed synergycomparable to or greater than daptomycin with oxacillin. Forseven of the eight strains tested, time-kill studies confirmedsynergy between daptomycin and ampicillin-sulbactam with ampicillinin the range of 2 to 8 µg/ml. The combination of daptomycinand ß-lactams may be useful for the treatment of MRSAinfection, but further studies are needed to elucidate the mechanismsand to determine the in vivo efficacy of the combination.

INTRODUCTION

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Introduction

Daptomycin is a novel lipopeptide antibiotic with bactericidalactivity against a wide range of clinically important gram-positivebacteria, including vancomycin-resistant enterococci (VRE) andmethicillin-resistant Staphylococcus aureus (MRSA). In a recentstudy, we found synergy between daptomycin and both rifampinand ampicillin against VRE (12a). In that work, a screeningtechnique was used to test daptomycin with 18 other antibiotics,and promising combinations were further tested by time-killstudies. In brief, daptomycin was added to Ca²⁺-supplementedMueller-Hinton broth at subinhibitory concentrations, and theEtest-determined MIC of individual antibiotics on agar containingdaptomycin was compared with the MIC on agar without daptomycin.When MRSA strains were studied with this method, combinationsof daptomycin and ß-lactams were found to warrantfurther study. In this report, we present comparative resultsfor several daptomycin-ß-lactam combinations by thescreening method and confirmative time-kill studies using combinationsof daptomycin and oxacillin or ampicillin-sulbactam for 18 strainsof MRSA.

MATERIALS AND METHODS

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Materials and Methods

Eighteen randomly selected strains of MRSA were obtained fromthe clinical microbiology laboratory at Shands Hospital at theUniversity of Florida, Gainesville, between January and March2002. All isolates were tested for relatedness by pulsed-fieldgel electrophoresis at the Mayo Medical Laboratories, Rochester,Minn. Five isolates were different from each other and all otherstrains. The remaining 13 isolates were divided into six uniquegroups, five pairs and one group of three identical strains,making a total of 11 unique strains. All were identified byMicroScan as MRSA strains, and oxacillin resistance was confirmedby growth on agar containing 6 µg of oxacillin/ml (BectonDickinson, Cockeysville, Md.). By Etest, the oxacillin MICsfor all strains were $≥$ 96 µg/ml (for 16 of the 18 strains,they were $≥$ 256 µg/ml). The daptomycin MICs for all 18 strainswere $≤$ 1 µg/ml by both broth and agar dilution (the MICat which 50% of the isolates were inhibited was 0.5 µg/mland the MIC at which 90% of the isolates were inhibited was1.0 µg/ml in both broth and agar). The ampicillin-sulbactam(2:1) MICs for eight unique strains of MRSA in Mueller-Hintonbroth were determined by using macrodilution. The MICs of theampicillin component of ampicillin-sulbactam ranged from 8 to32 µg/ml. The minimum bactericidal concentrations weregenerally equal to the MICs and were never more than twice theMIC. S. aureus ATCC 25923 and S. aureus ATCC 29213 were includedfor quality control testing of daptomycin, and the MICs forthese strains were always within the NCCLS acceptable rangeof 0.25 to 1.0 µg/ml.

Susceptibility testing. Daptomycin (lot 710403A) was obtained from Cubist Pharmaceuticals,Lexington, Mass. Oxacillin was obtained from Sigma Scientific,St. Louis, Mo. Etest strips were obtained from AB Biodisk, Solna,Sweden. Ampicillin lot 44H0176 was obtained from Sigma Scientific,and sulbactam lot 052301-008-06 was generously provided by Pfizer,Inc., Groton, Conn. All testing was carried out with Mueller-Hintonagar or Mueller-Hinton broth (Becton Dickinson), both supplementedto 50 mg of Ca²⁺/liter as previously suggested (8).

Antimicrobial agents. For synergy screening, the MICs of the following antibioticswere measured by Etest with and without daptomycin in the agar:oxacillin, piperacillin, ceftriaxone, cefepime, imipenem, gentamicin,amikacin, azithromycin, tetracycline, chloramphenicol, clindamycin,linezolid, quinupristin-dalfopristin, rifampin, trimethoprim-sulfamethoxazole,vancomycin, amoxicillin-clavulanate, and levofloxacin (AB Biodisk).Cloxacillin, ampicillin-sulbactam, piperacillin-tazobactam,and ticarcillin-clavulanate were tested with Kirby-Bauer disks(Becton Dickinson). Synergy screening was performed as describedbelow.

Synergy screening. Daptomycin was incorporated into Mueller-Hinton agar supplementedto 50 mg of Ca²⁺/liter at one-eighth the MIC, one-fourth theMIC, one-half the MIC, the MIC, and two times the MIC in agarfor each strain to be tested. Etest strips (or Kirby-Bauer disks)were placed on agar containing no daptomycin or containing daptomycinat one-eighth or one-fourth the MIC. For the plates with eitherno daptomycin or daptomycin at one-eighth or one-fourth theMIC, six different Etest strips were placed on each 150-mm-diameterplate after inoculation with a suspension equivalent to a McFarlandstandard of 0.5 prepared by the direct colony suspension method(12). The plates containing one-half, one times, and two timesthe daptomycin MIC did not have Etest strips placed on themand were included so that an experimentally accurate daptomycinMIC would be obtained within each test run. Etest-determinedMICs were read after 20 to 24 h of incubation at 36°C inair. The Etest-determined MIC results for the plates with one-eighthand one-fourth the daptomycin MIC were compared with those forthe plates without daptomycin. The decrease in the MIC determinedby the Etest at one-fourth or one-eighth the daptomycin MICin agar was used to calculate the fractional inhibitory concentration(FIC) for the combinations. Synergy was defined as an FIC indexof $≤$ 0.5 as conventionally used in checkerboard synergy studies(5). Kirby-Bauer disks were used to test some antibiotics: cloxacillin,ampicillin-sulbactam, piperacillin-tazobactam, and ticarcillin-clavulanate.Here the data were analyzed to compare the relative increasesin zone diameter between the different ß-lactams onagar with and without daptomycin. A 30-µg daptomycin diskwas included in these experiments to represent additivity, sincedaptomycin in the agar could not be synergistic with the daptomycinin a disk. The paired-differences t test was used to determinethe statistical significance of the increase in zone size aroundKirby-Bauer disks as the daptomycin concentration was increased(between no daptomycin and one-fourth the MIC or one-fourthand one-half the MIC).

Time-kill study. Daptomycin MIC measurements and time-kill studies were carriedout with Mueller-Hinton broth supplemented to 50 mg of Ca²⁺/literby using an inoculum of 9 x 10⁵ to 2.7 x 10⁶ cells/ml preparedby the direct colony suspension method (12). For time-kill synergystudies, the following antibiotic concentrations were prepared:daptomycin at 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 µg/mlwith or without oxacillin at 32 µg/ml. A no-antibioticgrowth control was also included. Since the oxacillin MICs forthe strains used were so high, the choice of 32 µg/mlwas somewhat arbitrary (1). Time-kill studies were carried outwith 2-ml volumes of Ca²⁺-supplemented Mueller-Hinton broth,and 0.1-ml volumes of 1:100, 1:1,000, and 1:10,000 dilutionswere subcultured at 0, 4, and 24 h. Synergy was defined as $≥$ 100-fold-greaterkilling at 24 h by the combination than by the more active ofthe individual antibiotics alone at the appropriate concentration(5) and also required $≥$ 100-fold killing of the input bacterialCFU. Bactericidal activity (killing) was defined as a $≥$ 10³-folddecrease in the number of CFU from input CFU at 4 or 24 h.

RESULTS

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Results

At subinhibitory concentrations of daptomycin, Etest screeningresults suggested that further studies of daptomycin combinedwith ß-lactams should be pursued. By this method,the FIC index of the combination of oxacillin and daptomycinfor 6 of the 18 strains of MRSA (5 of which were clonally unique)was $≤$ 0.5. No other antibiotic combinations had FIC indices of $≤$ 0.5 for more than two strains. Increases in zone size for oxacillin,cloxacillin, ampicillin-sulbactam, piperacillin-tazobactam,and ticarcillin-clavulanate were compared by using Kirby-Bauerdisks. A daptomycin disk was included as a control for additivity.Figure 1 shows that when the daptomycin concentration in theagar is raised from one-fourth to one-half the MIC, there areincreases in average zone sizes of 7.3 mm for oxacillin, 10.3mm for ampicillin-sulbactam, 7.8 mm for piperacillin-tazobactam,and 10.9 mm for ticarcillin-clavulanate. In contrast, a daptomycindisk showed only a 4.8-mm increase in zone size after the daptomycinconcentration in the agar was increased from one-fourth to one-halfthe MIC. Since the increased size of the zone around the daptomycindisk on daptomycin-containing agar can only be additive, thegreater increases in zone size around the ß-lactamdisks suggested possible synergy. These differences were highlystatistically significant for ampicillin-sulbactam (P < 0.0001,paired-differences t test) and ticarcillin-clavulanate (P <0.0001), significant for piperacillin-tazobactam (P = 0.05),and not significant for oxacillin (P = 0.15) and cloxacillin(P = 0.21). While analyzing the results of disk synergy testing,we noted that "clonally identical" strains did not always behaveidentically, particularly for oxacillin, cloxacillin, and piperacillin-tazobactam.We typically observed discrepancies in one to three antibioticcombinations between pairs of "identical" strains. In essence,the clonally related strains showed as much variability in thesynergy screening tests as did unrelated strains. However, isolatesfrom all 11 unique clones displayed highly significant increasesin zone size with daptomycin in the agar for all ß-lactamstested.

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FIG. 1. Increase in zone sizes surrounding disks with indicated ß-lactams as daptomycin concentration in agar is increased from zero to one-fourth the MIC and one-half the MIC. Mean zone sizes + standard deviations are given in millimeters. Seventeen strains were used for the ß-lactam zone assays, and eight strains were used for daptomycin zone assays. The increases in zone sizes for all antibiotics are significantly greater with daptomycin in the agar at one-fourth the MIC than with no daptomycin (all P values were $≤$ 0.006 by the paired-differences t test) and with daptomycin in the agar at one-half the MIC than with no daptomycin (all P values were $≤$ 0.001 by the paired-differences t test). Based on the paired-differences t test, the increases in zone sizes for ampicillin-sulbactam (P < 0.0001) and ticarcillin-clavulanate (P < 0.0001) are significantly greater than the increases in zone sizes around the daptomycin disks when the daptomycin in the agar is increased to one-half from one-fourth the MIC; these results are followed in significance by those for piperacillin-tazobactam (P = 0.05), while the increases are not significant for oxacillin (P = 0.15) and cloxacillin (P = 0.21) (see Materials and Methods for details). Clox, cloxacillin; Amp/Sul, ampicillin-sulbactam; Pip/Tazo, piperacillin-tazobactam; Tic/Clav, ticarcillin-clavulanate.

Results of the time-kill studies for daptomycin in combinationwith oxacillin are shown for 18 strains of MRSA in Fig. 2. Asdescribed in Materials and Methods, the combination of daptomycinat one-half the MIC and oxacillin at 32 µg/ml was foundto be synergistic for 18 of 18 strains at 24 h. The combinationof daptomycin at one-fourth the MIC and oxacillin at 32 µg/mlwas found to be synergistic for 11 of 18 (61%) strains at 24h. Even for the combination with one-eighth the daptomycin MIC,synergy was found for 6 of 18 (33%) strains in comparison tothe results with daptomycin or oxacillin alone (data not shown).Killing (i.e., a $≥$ 10³-fold decrease from the input CFU at 24h) was observed for 18 of 18 strains for the daptomycin-oxacillincombination with one-half the daptomycin MIC and for 9 of 18strains with one-fourth the daptomycin MIC. These data are bestappreciated by comparing the results shown in Fig. 2B and Cfor one-half the daptomycin MIC and those shown in Fig. 2D and Efor one-fourth the daptomycin MIC. In the time-kill studies,the behaviors of related clones were identical. The six strainsthat exhibited regrowth at 24 h after inhibition of growth at4 h (Fig. 2E) came from four unique clones.

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FIG. 2. Time-kill study results for 18 strains of MRSA (see Materials and Methods for details). (A) Oxacillin at 32 µg/ml alone. At 24 h, all tubes were grossly turbid, a condition represented by a titer of 10^8.7 CFU/ml. (B) Daptomycin alone at one-half its MIC. (C) Daptomycin at one-half its MIC plus oxacillin at 32 µg/ml. At 24 h, there was no growth on subculture plates, a condition represented by the lowest level of detection, $≤$ 10³ CFU/ml. (D) Daptomycin alone at one-fourth its MIC. (E) Daptomycin at one-fourth its MIC plus oxacillin at 32 µg/ml. The growth control for these experiments is not shown but was grossly turbid at 24 h for all strains.

Figure 3 shows the distribution of bactericidal concentrations(expressed as a multiple of the daptomycin MIC) with and withoutthe addition of oxacillin, measured at 4 and 24 h. In essence,the median concentration showing $≥$ 10³-fold killing of the inputCFU at 4 h was two times the daptomycin MIC when daptomycinwas used alone but one times the daptomycin MIC when daptomycinwas combined with oxacillin. At 24 h, the median concentrationto achieve $≥$ 10³-fold killing for daptomycin alone was one timesthe MIC but was one-fourth the MIC for daptomycin in the presenceof oxacillin.

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FIG. 3. Distribution of bactericidal concentrations (expressed as a multiple of the daptomycin MIC) at 4 (A) and 24 (B) h with ( ${square}$ ) and without ( ${blacksquare}$ ) the addition of oxacillin (see Materials and Methods for details). The distributions of bactericidal activity with and without daptomycin are statistically significantly different at 4 h (P = 0.005) and at 24 h (P < 0.0001) by ${chi}$ ² analysis.

For the combination of ampicillin-sulbactam and daptomycin,synergy was observed by time-kill studies at one-half the daptomycinMIC for seven of eight clonally unique strains of MRSA and atone-fourth the daptomycin MIC for six of eight strains. Synergywas observed for ampicillin concentrations of 2 to 8 µg/mlfor the seven strains for which synergy was observed at one-halfthe daptomycin MIC and in the same concentration range for thesix strains for which synergy was found at one-fourth the daptomycinMIC.

DISCUSSION

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Discussion

In view of the very high level of resistance of these strainsof MRSA to oxacillin (MICs are $≥$ 256 µg/ml for 16 of 18strains), the synergy between daptomycin at one-half the MICand oxacillin at 32 µg/ml for 18 of 18 strains is surprising.Even at one-fourth and one-eighth the daptomycin MIC, synergywith oxacillin at 32 µg/ml was observed for 61 and 33%of MRSA strains, respectively. In addition, $≥$ 10³-fold killingat 24 h of the input CFU was also observed for all strains withthe combination of daptomycin at one-half its MIC and oxacillinand for 50% of strains with the combination of daptomycin atone-fourth its MIC and oxacillin. Based on the results (Fig.1) with subinhibitory concentrations of daptomycin in agar andeither disks or Etests to screen for antibiotic synergy, allß-lactams appear to show a marked increase in activitywhen combined with daptomycin. In a previous study of the activityof daptomycin and ampicillin against VRE, synergy was foundagainst 19 of 19 strains in time-kill studies (12a). While $≥$ 10³-foldkilling was observed for only 2 of 19 strains of VRE, 10 of19 strains did show 100-fold killing of the input CFU. Daptomycin-ß-lactamsynergy appears to be consistent across the gram-positive speciestested.

In contrast to the results for VRE, no synergy between daptomycinand rifampin against MRSA strains was observed in the screeningstudy, although all MRSA strains were highly susceptible torifampin. No synergy between daptomycin and rifampin was foundagainst three laboratory-derived rifampin-resistant MRSA strains(data not shown).

Daptomycin is believed to act by Ca²⁺-dependent insertion ofits acyl tail into the gram-positive cell membrane, which isfollowed by the development of potassium efflux channels, depolarizationof the membrane, and cell death (15, 16). A recent study hasshown that membrane depolarization and potassium leakage correlatewell with decreased viability at concentrations above the daptomycinMIC (14). Sieradzki and Tomasz found that inhibitors of theearly stages of peptidoglycan synthesis (e.g., fosfomycin, ß-chloro-D-alanine,and D-cycloserine) markedly decreased the MIC of methicillinfor the COL strain of MRSA from $≥$ 800 µg/ml to as low as6 to 50 µg/ml depending on the specific second antibioticstudied (13). They proposed that inhibition of the early stagesof peptidoglycan synthesis resulted in the production of alteredpeptidoglycan precursors, which led to decreased effectivenessof penicillin binding protein 2' in cross-linking peptidoglycan.At its MIC, daptomycin is known to shut down macromolecularsynthesis, so subinhibitory concentrations may have significanteffects on peptidoglycan precursor levels. If insertion of daptomycininto the cell membrane at subinhibitory concentrations alteredpeptidoglycan precursors in a manner analogous to that of fosfomycin,ß-chloro-D-alanine, or D-cycloserine, penicillin bindingprotein 2' might be unable to perform its cross-linking functionin the presence of oxacillin. Another possibility is that subinhibitoryconcentrations of daptomycin somehow affect the levels or functionalityof one or more factors essential for methicillin resistance(fems) or auxillary (aux) factors required for the full expressionof mecA.

Alternatively, the activity of daptomycin might involve blockingthe induction of mecA by oxacillin itself. It is well knownthat mecA is normally only expressed in $~$ 1 in 10⁵ cells, butits expression rapidly increases when ß-lactams bindto the surface receptors for the derepression of mecA (10).If daptomycin were to block induction of mecA at subinhibitoryconcentrations, an apparent increase in killing might be observed.Finally, we cannot exclude the possibility that oxacillin somehowenhances the activity of daptomycin.

The consistency of both bactericidal activity and synergy makesthe combination of daptomycin and ß-lactams attractivefor the treatment of MRSA infection. It is reasonable to askwhether the antibiotic levels tested in this work are clinicallyachievable. For daptomycin at a dose of 4 mg/kg of body weight,the peak and trough concentrations are approximately 56 and7.5 µg/ml of plasma, respectively (J. Silverman [CubistPharmaceuticals], data on file), and daptomycin is $~$ 92% proteinbound, giving trough-free drug concentrations of $~$ 0.6 µg/ml.Thus, trough-free levels of daptomycin are in the same rangeas those studied here. Since oxacillin is >90% protein bound,it is unlikely that trough-free drug levels of 32 µg/ml(as used in this study) could be achieved in a clinical setting.However, depending on renal clearance, levels of ampicillinand piperacillin in blood of 75 to 80 µg/ml (with proportionalconcentrations of sulbactam and tazobactam) are achievable withhigh-dose continuous infusions (4, 6, 9), and levels of ampicillinas high as 103 to 130 µg/ml have been reported (11). Proteinbinding levels are reported to be 18% for both ampicillin andamoxicillin (7), 16% for piperacillin (2), 38% for sulbactam(3), and 20 to 23% for tazobactam (2). In this study, synergywas found against seven of eight MRSA strains by time-kill studieswith daptomycin and ampicillin-sulbactam with ampicillin concentrationsin the range of 2 to 8 µg/ml.

We conclude that the combination of daptomycin and ß-lactamsmay be useful in the treatment of MRSA infections but that furtherin vitro and in vivo studies should be carried out before clinicaluse can be recommended.

ACKNOWLEDGMENTS

We gratefully acknowledge the support of the Department of Pathology,Immunology and Laboratory Medicine, University of Florida, andthe staff of the Clinical Microbiology Laboratory, Shands Hospital,University of Florida. We thank Jared Silverman, Cubist Pharmaceuticals,for his detailed review of this manuscript and many helpfulsuggestions. We thank Claire Noegel for preparation of thismanuscript.

This study was supported in part by a grant from Cubist Pharmaceuticals.

FOOTNOTES

* Corresponding author. Mailing address: Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610. Phone: (352) 392-5621. Fax: (352) 392-4693. E-mail: Rand{at}pathology.ufl.edu.

REFERENCES

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