Characterization of High-Level Daptomycin Resistance in ViridansGroup Streptococci Developed upon In Vitro Exposure to Daptomycin

Ronda L. Akins,a,b Bradley D. Katz,c Catherine Monahan,c Dylan Alexanderc

Methodist Charlton Medical Center, Dallas, Texas, USAa; Louisiana State University Health Sciences Center–Shreveport, School of Medicine, Shreveport, Louisiana, USAb;Cubist Pharmaceuticals, Lexington, Massachusetts, USAc

Viridans group streptococci (VGS) are part of the normal flora that may cause bacteremia, often leading to endocarditis. Weevaluated daptomycin against four clinical strains of VGS (MICs � 1 or 2 �g/ml) using an in vitro-simulated endocardial vegeta-tion model, a simulated bacteremia model, and kill curves. Daptomycin exposure was simulated at 6 mg/kg of body weight and 8mg/kg every 24 h for endocardial and bacteremia models. Total drug concentrations were used for analyses containing protein(albumin and pooled human serum), and free (unbound) drug concentrations (93% protein bound) were used for analyses notcontaining protein. Daptomycin MICs in the presence of protein were significantly higher than those in the absence of protein.Despite MICs below or at the susceptible breakpoint, all daptomycin regimens demonstrated limited kill in both pharmacody-namic models. A reduction of approximately 1 to 2 log10 CFU was seen for all isolates and dosages except daptomycin at 6 mg/kg,which achieved a reduction of 2.7 log10 CFU/g against one strain (Streptococcus gordonii 1649) in the endocardial model. Activitywas similar in both pharmacodynamic models in the presence or absence of protein. Similar activity was noted in the kill curvesover all multiples of the MIC. Regrowth by 24 h was seen even at 8� MIC. Postexposure daptomycin MICs for both pharmacody-namic models increased to >256 �g/ml for all isolates by 24 and 72 h. Despite susceptibility to daptomycin by standard MICmethods, these VGS developed high-level daptomycin resistance (HLDR) after a short duration following drug exposure not at-tributed to modification or inactivation of daptomycin. Further evaluation is warranted to determine the mechanism of resis-tance and clinical implications.

Viridans group streptococci (VGS) include a number of speciesand are commensal Gram-positive bacteria often responsible

for human disease, particularly infective endocarditis and bac-teremia in neutropenic patients (1–5). Historically, these organ-isms have been relatively susceptible to most antibiotics. How-ever, the frequency of multidrug resistance has been increasing(6–17). Much of the resistance and virulence associated with theseorganisms can be exchanged among multiple streptococcal spe-cies via horizontal transfer of genetic material (13, 18, 19). Anti-biotic resistance has been reported primarily for penicillin, mac-rolides, and fluoroquinolones (4, 6, 7, 9, 12). The resistancerelated to the mef and erm genes, which confer resistance to mac-rolides, lincosamides, and streptogramin B, has been well charac-terized in VGS (3, 4, 7, 12). More recently, reports of daptomycinand linezolid resistance have also been published; these reportsinclude both in vitro studies and in vivo studies with 2 clinical casesfor each agent (20–25). Vancomycin resistance in VGS has notpreviously been identified (10, 11, 25); however, recently a Strep-tococcus anginosus isolate was found to contain the vanG element,conferring resistance to vancomycin in this strain (55).

Daptomycin (DAP) is a cyclic lipopeptide antibiotic with ac-tivity against multidrug-resistant Gram-positive organisms, in-cluding Staphylococcus aureus resistant to vancomycin (VAN) andlinezolid (LZD)- and vancomycin-resistant enterococci. To date,DAP has not exhibited any cross-resistance with other antibioticclasses nor has any plasmid-mediated resistance occurred (20). AsGram-positive bacterial resistance increases, empirical usage ofDAP continues to rise, particularly in patients at high risk of en-docarditis or S. aureus infection. With VGS as a common cause ofendocarditis and limited data with DAP against these strains, weevaluated the in vitro activity of DAP at various dosages (6 and 8mg/kg of body weight) or multiples of the MIC against four

clinical isolates of VGS with elevated daptomycin MICs (1 or 2�g/ml).

(Portions of this study were presented at the 21st EuropeanCongress of Clinical Microbiology and Infectious Diseases[ECCMID], May 2011, Milan, Italy.)

MATERIALS AND METHODSBacterial strains. Four clinical strains of VGS, all in the mitis subgroup(Streptococcus mitis strain 1643, Streptococcus oralis strains 1647 and 1648,and Streptococcus gordonii strain 1649) were evaluated. These strains, pro-vided by Cubist Pharmaceuticals (Lexington, MA), obtained from SentrySurveillance archived cultures (JMI Laboratories, North Liberty, IA) col-lected between 1999 and 2003, were isolated from hospitalized patientswith infective endocarditis. The isolates were selected based on a MIC toDAP at the high end of the wild-type distribution (up to 2 �g/ml). Isolateswere verified by Molecular Epidemiology Inc. (MEI) (Lake Forest Park,WA) using 16S rRNA analysis supported by multiple phenotypic tests,including but not limited to Gram stain, catalase/oxidase reaction, mul-tiple sugar fermentation, and optochin/bacitracin susceptibility. The ref-erence strains for pre- and postexposure isolates are listed in Table 1.Preexposure isolates refer to all testing or results prior to exposure to

Received 3 September 2014 Returned for modification 10 December 2014Accepted 21 January 2015

Accepted manuscript posted online 26 January 2015

Citation Akins RL, Katz BD, Monahan C, Alexander D. 2015. Characterization ofhigh-level daptomycin resistance in viridans group streptococci developed uponin vitro exposure to daptomycin. Antimicrob Agents Chemother 59:2102–2112.doi:10.1128/AAC.04219-14.

Address correspondence to Ronda L. Akins, rondaakins@mhd.com.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.04219-14

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daptomycin, and postexposure isolates refer to all testing or results sub-sequent to any treatment-based exposure to daptomycin.

Antibiotics. DAP (lot no. 850753A, CDCX01, MCB2009) analyticalpowder was provided by Cubist Pharmaceuticals (Lexington, MA). LZDwas purchased commercially from the manufacturer. VAN, rifampin(RIF), and gentamicin (GEN) were purchased from Sigma-Aldrich (St.Louis, MO).

Media. Multiple medium broths were studied to determine the mostappropriate media to conduct time-kill analysis with these strains basedon the strict medium requirements for VGS growth and calcium contentfor DAP activity. Four types of broth (Todd-Hewitt broth with and with-out 5% yeast extract, Columbia broth, brain heart infusion broth, andMueller-Hinton broth) were tested for their free (unbound) calcium con-centration with and without 50 mg/liter calcium supplementation (Lab-oratory Specialists, Inc., Westlake, Ohio) via a calcium probe. The con-centrations of free calcium for each broth type are listed in Table 2 withMICs determined for DAP against all strains and VAN for a representativestrain to determine the impact of broth type on antibiotic susceptibility.There was a significant increase in DAP MICs noted in all broth types

(except calcium-supplemented Mueller-Hinton broth) even with suffi-cient calcium supplementation up to 50 mg/liter. Based on this analysisand DAP dependency on calcium for activity, all DAP models utilizedcalcium-supplemented Mueller-Hinton broth (CA-SMHB) with 50 mg/liter calcium and 12.5 mg/liter magnesium (26–29). All other experimentsutilized Mueller-Hinton broth (Difco, Detroit, Michigan) supplementedwith 25 mg/liter calcium and 12.5 mg/liter magnesium (SMHB). Colonycounts were determined using tryptic soy agar with 5% sheep blood (TSA-SB) (Becton, Dickinson and Company, Sparks, MD) plates.

Susceptibility testing. MICs and minimum bactericidal concentra-tions (MBCs) of the antibiotics were determined by broth microdilutionin SMHB (28). DAP MICs and MBCs were determined in supplementedbroth (CA-SMHB) as described above. DAP MICs and MBCs were alsodetermined in the presence of albumin (3.5 to 4 g/dl) (human albumin25%; CSL Behring LLC, Kankakee, IL) and pooled human serum (PHS).Five-microliter samples from clear wells in the MIC experiments wereplated onto TSA-SB plates for determination of MBCs, and all sampleswere incubated at 35°C for 24 h. Postexposure isolate MICs were alsodetermined by Etest or broth microdilution methodology.

SEVs. Organism stocks were prepared by inoculating 5-ml test tubesof SMHB with colonies harvested from fresh overnight growth on TSA-SB. The test tubes were then incubated for 24 h on a rotator at 35°C. Thetest tubes were then centrifuged for 15 min at 3,500 � g, and the super-natant was removed. The remaining pellet of organism was collected andresuspended to achieve a concentration of 1 � 1010 CFU/ml. Simulatedendocardial vegetations (SEVs) were prepared by mixing 0.1 ml of organ-ism suspension (final inoculum, 1 � 109 CFU/0.5 g), 0.4 ml of humancryoprecipitate from volunteer donors (Gulf Coast Regional Blood Cen-ter, Houston, TX), and 0.025 ml of platelet suspension (platelets mixedwith normal saline, 250,000 to 500,000 platelets per clot in 1.5-ml sili-conized Eppendorf tubes; platelets obtained and pooled from volunteerdonors, LifeShare Blood Center, Shreveport, LA). Bovine thrombin(5,000 units/ml; lot R114A1048, Jones Pharma Inc., St. Louis, MO) (0.05ml) was added to each tube, after insertion of a sterile monofilament lineinto the mixture. The resultant simulated vegetation was then removedfrom the Eppendorf tubes with a sterile 21-gauge needle.

Simulated endocardial vegetation model (SEVM). An in vitro infec-tion model consisting of a 250-ml one-compartment glass apparatus withsample ports incorporated, in which the simulated fibrin clots were sus-pended and sealed with a rubber stopper, was utilized. The apparatus wasprefilled with SMHB or CA-SMHB, and antibiotics were administered asboluses over a 72-h period into the central compartment via an injectionport. The model apparatus was placed in a 35°C water bath throughoutthe procedure, and a magnetic stir bar was placed in the media for thor-ough mixing of the drug in the model. Fresh medium was continuously

TABLE 1 Summary of pre- and postexposure MIC testing results under standard testing conditions

Referencestrain Genetic IDa

Pre/postexposurestatusb

MIC (�g/ml)

DAPb,c DAPd,e RIFb,c VANd GENc,e LZDf

1643 S. mitis Pre 1 NA 0.5 0.5 4 15064 S. mitis Post �32 �256 �1 0.5 NA 11647 S. oralis Pre 2 NA 0.5 0.5 4 15065 S. oralis Post �32 �256 �1 0.5 NA 11648 S. oralis Pre 1 NA 256 0.5 4 15066 S. oralis Post �32 �256 �4 0.5 NA 11649 S. gordonii Pre 2 NA 0.125 0.5 1 15067 S. gordonii Post �32 �256 �4 0.5 NA 1a Genetically identified species.b Postexposure strains were collected after 72 h of daptomycin treatment at 8 mg/kg/day.c MICs were determined by the investigator using broth microdilution (preexposure strains) or by Laboratory Specialists, Inc. (Westlake, OH) (postexposure strains).d MICs were determined by the investigator using Etest methodology (postexposure strains).e NA, not available (MICs were not determined for this organism and antibiotic).f All MICs were determined by Laboratory Specialists, Inc., using broth microdilution.

TABLE 2 Free calcium concentration with MIC comparisons of VGS indifferent media

Mediuma

Free Ca�

concn

MIC (�g/ml) for the following drug/referencestrainb:

DAP/1643

DAP/1647

DAP/1648

DAP/1649

VAN/1648

CA-SMHB 56.608 1 2 1 2 0.5THB 5.370 64 �64 64 64 2STHB 16.565 4 �64 64 8 1THB�Y 29.079 32 �64 �64 64 1STHB�Y 17.133 4 �64 16 8 2CB 7.055 64 �64 �64 �64 0.5SCB 16.565 8 64 16 16 1BHI 4.669 �64 �64 �64 �64 1SBHI 35.252 4 �64 32 16 1a The different media tested were as follows: CA-SMHB, calcium-supplementedMueller-Hinton broth with 50 mg/liter Ca and 12.5 mg/liter Mg; THB, Todd-Hewittbroth; STHB, Todd-Hewitt broth with 50 mg/liter Ca and 12.5 mg/liter Mg; THB�Y,Todd-Hewitt broth plus 5% yeast extract; STHB�Y, Todd-Hewitt broth with 50 mg/liter Ca and 12.5 mg/liter Mg plus 5% yeast extract; CB, Columbia broth; SCB,Columbia broth with 50 mg/liter Ca and 12.5 mg/liter Mg; BHI, brain heart infusionbroth; SBHI, brain heart infusion broth with 50 mg/liter Ca and 12.5 mg/liter Mg.b The drugs were daptomycin (DAP) and vancomycin (VAN). The organisms testedwere S. mitis strain 1643, S. oralis strains 1647 and 1648, and S. gordonii strain 1649.

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supplied and removed from the compartment along with the drug via aperistaltic pump (Masterflex; Cole-Parmer Instrument Company, Chi-cago, IL) set to simulate the half-lives of the antibiotics. The pH wasmonitored throughout all experiments with DAP due to the possible ef-fects on its activity (26). SEVs were removed at various time points over a72-h period. Samples were homogenized, diluted, plated onto TSA-SB,and incubated at 35°C for 24 h. Colonies were counted after 24 h ofincubation, and the number of CFU/g was calculated. DAP was adminis-tered at dosages of 6 mg/kg or 8 mg/kg every 24 h for an estimated humantotal peak concentration/trough concentration of 80/10 and 133/17 �g/ml, respectively, with an average half-life of 8 h and 93% protein binding.Total drug concentrations were simulated, due to the significant effects ofprotein binding on DAP and since each SEV contains an average totalprotein concentration of 6.8 to 7.4 g/dl and 3.0 to 3.5 g/dl of albumin (26,30). LZD was given in 600-mg doses every 12 h with an estimated peakconcentration/trough concentration of 18.3/3.6 �g/ml and an averagehalf-life of 5 h and 31% protein binding (31). VAN was administered tosimulate 1 g every 12 h for peak concentration/trough concentration of 30to 40/10 to 15 �g/ml with a half-life of 6 h and 55% protein binding.

All infection model experiments were performed in triplicate to en-sure reproducibility. In addition, model experiments in the absence ofantibiotics were performed over 72 h to ensure adequate growth of theorganisms in the model.

SBM. An in vitro pharmacodynamic infection model consisting of aone-compartment glass chamber with multiple ports for the removal ofmedium, delivery of antibiotics, and collection of bacterial and antimi-crobial samples was utilized to determine the in vitro activity of the anti-biotic regimens without any protein components. The apparatus was pre-filled with SMHB or CA-SMHB, and antibiotics were administered asboluses over a 72-h period. The simulated bacteremia model (SBM) wasplaced in a 35°C water bath for the duration of the experiment, and amagnetic stir bar was placed in the medium for continuous mixing of themedium. A peristaltic pump (Masterflex; Cole-Parmer Instrument Com-pany, Chicago, IL) was used to replace antibiotic-containing mediumwith fresh medium at a rate to clear the various antibiotics in patients withnormal renal function by utilizing the drugs’ half-lives. The pH was mon-itored throughout all experiments with DAP due to the possible effects onits activity (26). Bacterial colonies after overnight growth on TSA-SB wereadded to SMHB to obtain a 0.5 McFarland suspension. An aliquot of thissuspension was added to the model to produce an initial inoculum ofapproximately 1 � 106 CFU/ml. Colony counts were measured at 0 (base-line), 8, 24, 32, 48, and 72 h after introduction of antibiotic. Bacterialdensity in CFU/ml was calculated after 24 h of incubation at 35°C. Free(unbound) drug concentrations were simulated in the SBM, since there isno protein within the model based on the dosages, half-lives, and proteinbinding indicated above in the SEVM. Free drug concentrations used wereas follows. The DAP fCmax/fCmin values (maximum concentration of thefree, unbound fraction of a drug/minimum concentration of the free,unbound fraction of a drug) were 5.6/0.7 and 9.3/1.2 �g/ml for a 6-mg/kgand 8-mg/kg dose given every 24 h, respectively. The LZD fCmax/fCmin was12.6/2.5 for a 600-mg dose every 12 h, and the VAN fCmax/fCmin was15.8/5.6 for a 1-g dose every 12 h. All infection model experiments wereperformed in triplicate to ensure reproducibility. In addition, model ex-periments with no antibiotics were performed over 72 h to ensure ade-quate growth of the organisms in these models.

FIC synergy determination. Approximately 1 � 107 CFU/ml of eachorganism was pipetted into microtiter assay plates containing concentra-tions of each antibiotic combination (DAP plus GEN and DAP plus RIF)ranging from 1/32 MIC to 4� MIC. Microtiter plates were incubated at35°C for 24 h. After 6, 12, and 24 h, the plates were read for detection ofinhibition of bacterial growth. The experiments were performed in trip-licate. The fractional inhibitory concentration (FIC) indices were calcu-lated by the following formulas: FIC of drug A � MIC of drug A incombination/MIC of drug A alone, FIC of drug B � MIC of drug B incombination/MIC of drug B alone, and FIC index � FIC of drug A � FIC

of drug B. Synergy was defined as an FIC index of �0.5, an additive effector indifference was defined as an FIC index of �0.5 to �4, and antago-nism was defined as an FIC index of �4 (32). Synergy screening wasconducted on each antibiotic pair.

Pharmacokinetic analysis. Samples were obtained from broth (0.5 mlfrom each infection model), through the injection port, at 0.5, 1, 2, 4, 8,24, 32, 48, and 72 h for determination of the antibiotic concentrationsbased on a pharmacodynamic model (SEVM or SBM). Samples were alsotaken from the homogenized SEVs obtained at 0, 8, 24, 32, 48, and 72 h fordetermination of the antibiotic concentration within the vegetation. Sam-ples were stored at �70°C until analysis. DAP concentrations were deter-mined by microbioassay utilizing Micrococcus luteus ATCC 9341 as de-scribed below (33). The vegetation drug concentrations were alsodetermined via microbioassay using the same indicator organism. Blank0.25-in. disks were spotted with 20 �l of the standards or samples. Eachstandard was tested in triplicate by placing the disk on Antibiotic AssayMedium 1 agar plates, which was preswabbed with a 0.5 McFarland sus-pension of the test organism. The plates were incubated for 18 to 24 h at35°C, at which time the zone sizes were measured. The standard DAPantibiotic concentrations used were 150, 100, and 10 �g/ml for total drugconcentration dosing and 10, 2.5, and 1.25 �g/ml for free (unbound) drugconcentration dosing, with the lower limit of detection of 1.25 �g/ml dueto the limitation of the blank disk size. The intraday and interday coeffi-cients of variation percentage (CV%) for the DAP microbioassay were�7.4 and �9.8 for total drug concentration standards and �9.1 and�10.4 for free drug concentration standards, respectively. VAN concen-trations were determined by fluorescence polarization immunoassay(TDx assay; Abbott Diagnostics, Irving, TX) with a lower limit of detec-tion of 2.0 �g/ml and an intraday CV% of �5.4. LZD concentrations weredetermined by high-performance liquid chromatography (HPLC) as pre-viously described (34) with a lower level of detection of 0.5 �g/ml and anintraday and interday CV% of �5.7 and �11.8. The antibiotic peak andtrough concentrations, half-lives, and area under the curve were deter-mined by the trapezoidal method using PK Analyst (version 1.10;MicroMath Scientific Software, Salt Lake City, UT).

Pharmacodynamic analysis. Three vegetations were removed fromeach model (for a total of nine vegetations) at 0, 8, 24, 32, 48, and 72 h.Vegetations were homogenized and diluted in cold saline, and 20 �l of thehomogenate was plated in triplicate onto TSA-SB plates. The plates wereincubated at 35°C for 24 h, at which time colony counts were performed.The total reduction in log10 CFU/g over 72 h was determined by plottingtime-kill curves based on the number of remaining organisms over the72-h time period. The time to achieve a 99.9% bacterial load reductionwas determined by linear regression (if r2 � 0.9) or by visual inspection.The Cmax-to-MIC ratio (Cmax/MIC), time above the MIC for 24 h (T �MIC24), and the area under the concentration-time curve from 0 to 24 h(AUC0 –24)/MIC ratio were determined for the different dosing regimens.

Resistance. The 100-�l samples from each time point were plated onTSA-SB plates containing 4� and 8� the MIC of the respective antibioticto assess the development of resistance. The plates were then examined forgrowth after 48 h of incubation at 35°C. Development of resistance wasevaluated for each model at the 24-, 48-, and 72-h time points. Any growthof organisms observed on the antibiotic-containing plates after 48 h ofincubation was considered to show resistance. If resistance developed,further MIC and MBC testing was performed to determine the level ofresistance. Pre- and postexposure strains were also subtyped and analyzedfor clonal relatedness by restriction fragment length polymorphism(RFLP) using either XbaI or ApaI restriction endonuclease followed bypulsed-field gel electrophoresis (PFGE) at MEI.

Statistical analysis. Changes in the CFU/g for SEVM or CFU/ml forSBM at 72 h were compared by two-way analysis of variance with Tukey’spost hoc test. A P value of �0.05 was considered significant. All statisticalanalyses were performed using SPSS statistical software (release 21.0;SPSS, Inc., Chicago, IL).

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Time-kill curve experiments. Upon completion of SEVMs and SBMs,time-kill curve experiments (KCs) were utilized with DAP only to allowfor further evaluation of the extent of resistance development with expo-sure to multiple drug concentrations (up to 8� MIC) and to examinewhether differences existed between studies containing protein (albuminand pooled human serum) and studies not containing protein. KCs wereperformed by mixing colonies from an overnight growth of organism onTSA-SB plates into an appropriate volume of CA-SMHB to obtain a 0.5McFarland suspension. This suspension was then diluted to achieve astarting inoculum of 1 � 106 CFU/ml. The following conditions werecompared: broth alone, broth plus albumin (3.5 to 4 g/dl), and broth pluspooled human serum (PHS) (50:50). The DAP concentrations run in theKCs were therapeutic (6 mg/kg/day) and multiples of 0.5�, 1�, 2�, 4�,and 8� MIC. Total DAP concentrations (26, 30) were used in all protein-containing experiments, and free drug concentrations were used in exper-iments containing broth alone. Each regimen was tested in triplicate.Samples (100 �l) were taken at 0, 4, 8, and 24 h, serially diluted with coldnormal saline, and plated in triplicate on drug-free TSA-SB plates anddrug-containing (2�, 4�, and 8� DAP MICs) TSA with 5% lysed horseblood plates for determination of the numbers of CFU/ml. Colony countson drug-free and drug-containing plates were compared over the experi-mental period. For situations in which use of the first dilution was neces-sary for bacterial enumeration, samples were filtered through a 0.45-�m-pore-size polysulfone filter and washed with cold normal saline, and thefilter was applied aseptically to a TSA-SB plate to minimize the potentialeffects of antibiotic carryover.

Resistance characterization. Numerous studies were performed onpostexposure resistant isolates to characterize the mechanism of resistance.

(i) Determination of heterogeneous resistance in preexposurestrains. Preexposure strains were screened for heterogeneous DAP resis-tance via a modified macro-Etest and by direct plating of high inocula ondaptomycin-containing agar media.

A modified DAP macro-Etest was performed on the preexposurestrains according to methods previously described for the detectionof heterogeneous vancomycin-intermediate Staphylococcus aureus(hVISA) (35–37). The modified method substituted Mueller-Hinton agarsupplemented with 5% sheep blood (MHAB) to support the growth ofVGS in place of brain heart infusion agar. Briefly, 200 �l of a 2 McFarlandinoculum was placed on MHAB, streaked in three directions with a sterileswab, and allowed to dry for at least 5 to 10 min. A DAP Etest was thenadded to the plate and incubated for 24 to 48 h at 35°C and 5% CO2. Atboth 24 and 48 h, the plates were examined for colonies within the ellipse(zone of inhibition). The presence of colonies within the ellipse indicateda positive screen, while no colonies indicated a negative screen (Fig. 1).

Heterogeneity screens for other antibiotics used in the in vitro pharmaco-dynamic models and synergy assays (i.e., LZD, VAN, and RIF) were per-formed for the S. oralis 1647 and 1648 strains using the same methodology.

(ii) Heterogeneous resistance. Heterogeneous DAP resistance wasalso examined by plating a high inoculum on DAP-containing MHAB andincubating the culture overnight at 35°C and 5% CO2. Briefly, an over-night culture of each organism was suspended in 1� phosphate-bufferedsaline (PBS) and serially diluted. A volume of 100 �l of the undiluted cellsuspension and 100 �l of the first 10-fold dilution was spread on each ofthree plates at each DAP concentration, 0, 4, 8, 16, and 64 �g/ml of dap-tomycin, respectively. The plates were incubated at 35°C and 5% CO2 andexamined at 24 and 48 h. Growth was subcultured on drug-free MHABfrom each plate (15 colonies per DAP concentration). MIC values werethen determined by standard Etest methodology in accordance with themanufacturers’ recommendations.

(iii) Resistance stability. The stability of resistance in postexposurestrains was assessed via serial passage. Strains from frozen glycerol stockswere cultured onto drug-free MHAB plates and incubated for 24 h at 35°Cand 5% CO2. The following day, the cells were resuspended in 1� PBS,and 100 �l of the cell preparation was inoculated into 10 ml of medium

(CA-SMHB) containing 0, 4, or 32 �g/ml of DAP. Cultures were incu-bated for 24 h at 35°C and 5% CO2 and were considered the first passage.

(iv) Cross-resistance. The development of cross-resistance to otherantibiotics was examined in S. oralis strain 1647 and its postexposurederivative (S. oralis 5065). MIC testing was performed by broth microdi-lution in accordance with CLSI guidelines for other cell wall active com-pounds, such as valinomycin, vancomycin, oxacillin, nisin, bacitracin,and gramicidin.

(v) Mechanism of resistance. The ability of the VGS strains to inacti-vate or modify DAP was investigated through a microbioassay and byHPLC. S. oralis 5065, the postexposure derivative of S. oralis 1647, wasgrown overnight on MHAB containing 10 �g/ml of DAP at 35°C and 5%CO2. The following day, growth was diluted in 1� PBS to an opticaldensity at 625 nm (OD625) of 0.1, and 30 �l of the cell preparation wasthen inoculated into 3-ml volumes of CA-SMHB containing DAP con-centrations of 0, 32, 64 and 128 �g/ml. Two hundred microliters of cul-ture supernatant for preexposure (0 h) and 6 and 24 h after daptomycinexposure was spot plated on a lawn of Streptococcus pneumoniae ATCC49619 grown to confluence on MHAB. Microbioassay plates were incu-bated at 35°C and 5% CO2 and examined 24 h later for zones of inhibitionindicating DAP activity. One hundred microliters of the same supernatantwas also subjected to HPLC analysis in an Agilent 1100 series ChemstationHPLC. HPLC traces were not subsequently examined to determine thepresence of breakdown product.

RESULTSSusceptibility testing. MICs were determined for all pre- andpostexposure isolates under standard testing conditions, using ei-ther broth microdilution or Etest (Table 1). The MICs/MBCs (�g/ml) of DAP tested in CA-SMHB were 1/16, 2/8, 1/8, and 2/16 forstrain 1643, 1647, 1648, and 1649, respectively, with tolerancenoted in strain 1648 with a ratio of 16. When the antibiotics weretested in the presence of albumin or PHS, the MICs increased to256 �g/ml for all organisms except for strain 1648, which had anMIC of 512 �g/ml. The MBCs were slightly lower in the presenceof albumin, with an MBC of 512 �g/ml for all strains, except forstrain 1643, for which it was 1,024 �g/ml. In the presence of PHS,

FIG 1 Modified DAP macro-Etest for S. oralis 1648 with a subsequent stan-dard Etest on the non-DAP-susceptible subpopulation. (a) Modified macro-Etest demonstrating non-DAP-susceptible subpopulations in S. oralis 1648 at48 h. (b) Standard Etest MIC of a purified colony (circled in panel a and labeledwith a dagger) from modified DAP macro-Etest of S. oralis 1648 at 48 h (DAPMIC � 256 �g/ml).

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the MBC was 2,048 �g/ml for all organisms except strain 1643(1,024 �g/ml). The MIC/MBC ratio was elevated for all organ-isms, ranging from 4 to 16 when the drugs were tested in theabsence of protein and 2 to 8 when the drugs were tested in thepresence of protein.

Pharmacokinetics. The pharmacokinetic parameters achievedfor DAP, VAN, and LZD are listed in Table 3.

Pharmacodynamics. The change in the initial and final bacte-rial inocula for the SEVM is shown in Table 4. A negative valueshows that bacteria were killed, and a positive value reflects bac-terial growth. The majority of DAP regimens achieved limited kill(0 to 2 log10 CFU/g reduction) with the greatest activity seen with6 mg/kg DAP against strain 1649 (2.7 log10 CFU/g reduction).However, two regimens and strains (6 mg/kg DAP against strain1643 and 8 mg/kg DAP against strain 1648) had an increase in thefinal colony count for 72 h compared to 0 h. VAN demonstratedmore activity and consistency in kill than DAP or LZD with �3log10 CFU/g reduction for two strains (1643 and 1648) and 1.6 and2.3 log10 CFU/g reduction for strains 1647 and 1649, respectively.LZD was static for all strains with a 1 to 2 log10 CFU/g reduction.A summary of activity in the SEVM is displayed in Fig. 2.

The SEVM model was selected as the initial and primary modelto assess DAP activity against the VGS, as this was the most rep-resentative infection type to simulate with these organisms. How-ever, when DAP activity data were not consistent with what hadbeen demonstrated against staphylococci and enterococci in thesame model, the SBM was then utilized to eliminate potentialinference from the protein components of the SEVM.

The SBM model utilized the same regimens except for usingthe free (unbound) concentration of the antibiotics. Bacterial re-duction over the experimental period is summarized in Table 4.DAP regimens achieved bacterial kill levels similar to those seen in theSEVM, with all strains having 1 to 2 log10 CFU/ml reduction at 72 h,except for 8 mg/kg DAP against strain 1643 (0.52 log10 CFU/ml in-crease). VAN achieved a �3 log10 CFU/ml reduction for all strainsexcept for strain 1643 (0.54 log10 CFU/ml reduction). LZD achievedless than a 2 log10 CFU/ml reduction for all strains.

Overall, VAN regimens (P � 0.05) were consistently betterthan LZD regimens (P � 0.05) compared to growth controlagainst all strains in both the SEVM and SBM. DAP displayedsignificant activity only against strain 1649 in both models andstrain 1648 in the SBM (P � 0.05).

Fractional inhibitory concentration. All RIF-DAP combina-tions showed indifference or additive activity with a FIC rangingfrom 0.75 to 2 for all strains. For GEN-DAP combinations, indif-ference or additive activity was observed for strains 1643 and 1647with a FIC ranging from 0.625 to 1.06. Strain 1648 demonstratedantagonism with a FIC of 8.0625, and strain 1649 displayed syn-ergy with a FIC of 0.3125.

Kill curves. Due to the continued regrowth noted in the DAPstudies despite removal of protein, additional time-kill studieswere conducted to determine whether increased drug concentra-tion could increase killing activity.

The KC results are displayed in Fig. 3. Of note, in some KCs, asthe drug concentration increases, more bacteria were initiallykilled, followed by significant regrowth. In contrast, lower drugconcentrations achieved some kill, but the curve was more staticover the experimental period. Even drug concentrations at 8 timesthe MIC showed significant regrowth by 24 h. Regardless of pro-tein content (albumin or PHS) or broth alone, the levels of bacte-ria killed were similar for all testing conditions.

Resistance. All DAP postexposure strains from the SEVM andSBM displayed growth on DAP-containing plates at 4�, 8�, and16� the preexposure strain MIC for all organisms. Subsequentsusceptibility determinations yielded postexposure MICs of �256�g/ml for all strains.

Resistance characterization. Determination of genetic related-ness in the pre- and postexposure strains by RFLP and PFGE yieldedidentical patterns with strains 1643 and 5064, closely related patternswith strains 1647 and 5065 and 1648 and 5066, and uniquely differentpatterns between strains 1649 and 5067 (Fig. 4 and 5).

The heterogeneity screen for DAP resistance by modified DAPmacro-Etest or plating on drug-containing media was positive forall of the preexposure strains by 48 h. The additional modifiedmacro-Etest assays performed were negative for S. oralis 1647 and1648 for both VAN and LZD at 48 h, while strain 1647 was positivefor RIF heterogeneity. Colonies that appeared within the ellipse ofthe modified DAP macro-Etest were tested for their DAP MICvalue by Etest, which resulted in a value 170 times the baselineMIC. Heterogeneity screening by plating on DAP-containing me-dia yielded results similar to those of the modified DAP macro-Etest (presence of colonies on drug-containing plates at 4 to 64�g/ml). Colonies that grew at 64 �g/ml from S. oralis 1647 and1648 were tested for DAP MIC by Etest and demonstrated thesame increase in MIC as seen with the modified DAP macro-Etest.

The resistant phenotype was maintained under selectivepressure when serially passaged on DAP-containing plates;however, resistance was lost in 7 to 13 serial passages in theabsence of DAP.

Cross-resistance, defined as a greater than three doubling di-lution increase in the MIC of the postexposure strain compared tothe preexposure strain, was not observed with any compoundtested other than DAP (i.e., oxacillin, VAN, etc.).

The supernatant of cultures containing the highly DAP-resis-tant S. oralis 5065 and various concentrations of DAP was able toeffectively inhibit the growth of S. pneumoniae in the DAP micro-bioassay at all test concentrations and time points without signif-icant changes in the zone of inhibition (reduction in zonediameter of 4.9 to 5.9 mm compared to 3.4 to 4.1 mm in theno-organism control). HPLC analysis demonstrated that 47.9 to62.1% of the original quantity of DAP remained after a 24-h pe-riod.

TABLE 3 Pharmacokinetic parameters for SEVM and SBM

Parameter andregimen

Mean Cmaxa

(mg/liter) (SD)Mean Cmin

b

(mg/liter) (SD)Mean half-life(h) (SD)

Total concns (SEVM)DAP (6 mg/kg) 83.2 (6.3) 11.3 (3.7) 8.6 (10.2)DAP (8 mg/kg) 138.4 (9.8) 18.6 (5.4) 7.5 (0.9)VAN 38.1 (2.1) 11.5 (1.3) 7.4 (2.3)LZD 19.6 (4.3) 3.2 (1.4) 4.6 (1.1)

Free concns (SBM)DAP (6 mg/kg) 6.03 (1.05) 0.96 (0.5)c 8.9 (1.5)DAP (8 mg/kg) 9.0 (1.5) 1.4 (0.6)c 7.7 (1.8)VAN 16.6 (1.9) 5.9 (1.5) 7.0 (1.4)LZD 13.3 (2.7) 2.03 (0.9) 5.2 (0.7)

a Cmax, maximum concentration.b Cmin, minimum concentration.c Extrapolated values.

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DISCUSSION

VGS are a clinically important group of Gram-positive patho-gens often associated with infective endocarditis, bloodstreaminfections, and systemic diseases, particularly in immunocom-promised patients. These commensal organisms are part of theupper respiratory and intestinal flora but predominantly residein the oral cavity. With alternation or damage to the mucosalbarrier, transient bacteremias may develop, potentially leading

to clinically significant bloodstream infections which havebeen reported to occur in up to 24% of patients (38). There-fore, VGS bacteremia may lead to more serious complications,as these organisms have been associated with the highest per-centage of native valve endocarditis (30 to 40%) compared to S.aureus (10 to 27%) (39).

Historically, penicillin has been the treatment of choice due tothe inherent susceptibility of these organisms to most �-lactams;

TABLE 4 Comparison of pharmacodynamic changes for in vitro models (SEVM and SBM)

Model and reference Regimen Initial inoculuma Final inoculuma

Change in log10 CFU/g(SEVM) or log10

CFU/ml (SBM)

SEVMS. mitis 1643 DAP (6 mg/kg) 8.22 0.11 8.72 0.24 �0.5

DAP (8 mg/kg) 8.0 0.34 7.93 0.60 �0.07VAN 8.2 0.16 4.81 0.76 �3.39LZD 7.6 0.16 6.13 0.12 �1.47None (growth control) 8.0 0.22 8.4 0.4 �0.4

S. oralis 1647 DAP (6 mg/kg) 8.51 0.13 8.12 0.21 �0.39DAP (8 mg/kg) 9.02 0.03 7.32 0.32 �1.7VAN 8.13 0.32 6.49 0.73 �1.64LZD 7.5 0.7 6.12 0.31 �1.38None (growth control) 8.43 0.13 8.2 0.4 �0.23

S. oralis 1648 DAP (6 mg/kg) 8.96 0.11 8.1 0.07 �0.86DAP (8 mg/kg) 8.41 0.13 8.64 0.14 �0.23VAN 8.6 0.12 5.54 0.34 �3.06LZD 7.97 0.13 5.85 0.84 �2.12None (growth control) 8.3 0.06 8.18 0.3 �0.12

S. gordonii 1649 DAP (6 mg/kg) 8.7 0.51 6.02 0.23 �2.68DAP (8 mg/kg) 8.8 0.06 7.65 0.24 �1.15VAN 8.78 0.08 6.51 0.15 �2.27LZD 8.56 0.05 6.34 0.44 �2.22None (growth control) 7.6 0.11 9.31 0.17 �1.71

SBMS. mitis 1643 DAP (6 mg/kg) 6.96 0.06 7.17 0.34 �0.21

DAP (8 mg/kg) 6.94 0.13 7.46 0.21 �0.52VAN 7.04 0.05 6.5 0.37 �0.54LZD 6.95 0.18 6.61 0.44 �0.34None (growth control) 6.58 0.28 8.21 0.13 �1.63

S. oralis 1647 DAP (6 mg/kg) 7.91 0.30 7.42 0.28 �0.49DAP (8 mg/kg) 7.17 0.17 5.98 0.23 �1.19VAN 7.31 0.90 3.11 1.1 �4.2LZD 6.90 0.23 4.95 0.30 �1.95None (growth control) 6.43 0.24 7.95 0.18 �1.52

S. oralis 1648 DAP (6 mg/kg) 7.12 0.10 6.33 0.18 �0.79DAP (8 mg/kg) 7.08 0.23 6.62 0.08 �0.46VAN 7.04 0.05 3.26 0.45 �3.78LZD 7.34 0.08 5.81 0.33 �1.53None (growth control) 7.32 0.03 7.81 0.16 �0.49

S. gordonii 1649 DAP (6 mg/kg) 7.41 0.18 5.64 0.27 �1.77DAP (8 mg/kg) 7.67 0.06 6.39 0.13 �1.28VAN 7.09 0.13 3.92 0.79 �3.17LZD 6.85 0.21 6.14 0.06 �0.71None (growth control) 7.23 0.15 8.07 0.21 �0.84

a The inoculum is given in log10 CFU/g for SEVM or log10 CFU/ml for SBM.

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however, resistance to penicillins, as well as other �-lactams, mac-rolides, and fluoroquinolones, is increasing (4, 6, 7, 9, 12). Peni-cillin and macrolide resistance rates as high as �50% have beenreported for VGS in some studies (4, 7, 40). Low levels of resis-tance to fluoroquinolones have been noted in most countries,with rates less than 3% (9, 41). However, this resistance rate in-creases significantly in cancer patients with previous exposure tofluoroquinolones, with resistance increasing to up to 64% for cip-rofloxacin against S. mitis (11). Resistance to various antibioticclasses is often strain dependent, with S. mitis, S. oralis, and S.sanguinis tending to be the least susceptible.

DAP is a potent antibiotic with activity against multidrug-re-sistant Gram-positive organisms. Although species within theVGS are not listed in the Cubicin (DAP) package insert, the CLSIhas recommended susceptibility interpretive criteria for Strepto-coccus viridans group at a broth microdilution MIC value of �1�g/ml (28). Using CLSI breakpoints as a guideline, two of the fourVGS strains (1647 and 1649) demonstrated baseline DAP MICvalues in excess of the susceptibility breakpoint. The DAP surveil-lance program has monitored DAP susceptibilities to variousGram-positive isolates since 2002, which has consistently demon-strated �99% susceptibility of all VGS strains (20, 41). TheMIC90s for these organisms are most commonly reported to be 0.5to 1 �g/ml with a MIC range of 0.06 to 2 �g/ml for the wild type.In vitro studies evaluating the activity of DAP against variousstreptococci, including VGS, have shown significant kill withoutdevelopment of resistance. There is only a single report for an invitro peritoneal dialysate model where DAP at 10 mg/liter against

Streptococcus sanguis (S. sanguinis) displayed more static activitywith only a 2-log10 CFU reduction compared to �3 log10 CFUreduction with cefazolin and VAN (42). In addition to reducedactivity of DAP, regrowth occurred in the model by 24 h with theauthors attributing this to the possibility of inadequate calciumsupplementation within the growth media despite noting that thecalcium concentration was greater than 50 mg/liter.

In our study, DAP regimens failed to achieve bactericidal ac-tivity (over a 24-h period) in the in vitro SEVM, even at high doses(8 mg/kg/day). VAN treatment more consistently reduced thebacterial burden and to a larger extent than LZD or DAP did,resulting in a 1.6- to �3-log10-unit reduction compared to a 1- to2-log10-unit reduction for LZD (Table 4 and Fig. 2). High-leveldaptomycin resistance (HLDR) was isolated postexposure, fromall strains, in all DAP treatment groups (except 6 mg/kg daptomy-cin against strain 1649). DAP MIC values of the postexposurestrains were greater than 16� the preexposure MIC (�256 �g/ml), demonstrating a rapid development of high-level resistance/selection by 24 h despite drug concentrations as high as 8 times theMIC. Similar activity was noted in the SBM utilizing free (un-bound) drug concentrations, thereby eliminating the potentialimpact of protein as a confounding factor for ineffectiveness. Pre-vious work has also demonstrated similar activity between SEVM,SBM, and KCs with DAP utilizing total drug concentrations inmodels containing protein versus free concentrations in modelsnot containing protein (30, 33).

The results of this study, using the same models, are uniquecompared to similar treatment regimens against methicillin-resis-

FIG 2 Comparison of antimicrobial activities of daptomycin at 6 mg/kg (open circle) or 8 mg/kg (closed inverted triangle), vancomycin (open triangle), linezolid(closed square), and growth control (closed circle) in the SEVM against 4 VGS strains.

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tant S. aureus, vancomycin-resistant Enterococcus faecium, andglycopeptide-intermediate S. aureus. In those studies, bactericidalactivity was achieved and maintained over a 24-h period, and noresistance was detected postexposure (30, 33).

The presence of DAP tolerance in S. aureus has been widelystudied and not detected in more than 900 isolates tested. All S.aureus isolates tested so far demonstrate an MBC/MIC ratio of 1or 2, indicating rapid bactericidal activity of DAP (43–45). TheDAP MBC/MIC ratios varied among the four VGS strains in thisstudy (from 4 to 16) and were higher than the values seen with S.aureus. If we apply a commonly used definition of antibiotic tol-erance (MBC/MIC ratio of �32 or MBC/MIC ratio of �16 withan MBC in the nonsusceptible/resistant range), strain 1643 wouldbe considered DAP tolerant (46). DAP tolerance in VGS has notbeen previously described. Despite three of the four organisms notdemonstrating tolerance, the elevated MBC/MIC ratio seems to beunique to these organisms. Tolerance, in addition to an elevatedMBC/MIC ratio, has previously been demonstrated in several

FIG 3 Activity of various concentrations of DAP in time-kill curve experiments evaluating the effect of protein (albumin and pooled human serum) on killingof bacteria. The testing condition and organism tested are indicated by a letter and number. Testing conditions were as follows: free (unbound) concentrationsof DAP tested alone in broth (A), total concentrations of DAP tested in broth and albumin (B), and total concentrations of DAP tested in broth and pooled humanserum (PHS) (C). Organisms tested were as follows: S. mitis 1643 (1), S. oralis 1647 (2), S. oralis 1648 (3), and S. gordonii 1649 (4). Regimens included growthcontrol (closed circle), therapeutic DAP (6 mg/kg/day) (open circle), 0.5� MIC (closed inverted triangle), 1� MIC (open triangle), 2� MIC (closed square), 4�MIC (open square), and 8� MIC (closed diamond). Free MICs to determine DAP testing concentrations were 1 �g/ml for strains 1643 and 1648 and 2 �g/mlfor strains 1647 and 1649, and total MICs were 256 �g/ml for strains 1643, 1647, and 1649 and 512 �g/ml for strain 1648.

FIG 4 Genetic relatedness of pre- and postexposure isolates by PFGE by singledigestion for isolates 1643 and 5064 (S. mitis), isolates 1648 and 5066 andisolates 1647 and 5065 (S. oralis) and isolates 1649 and 5067 (S. gordonii).

FIG 5 Genetic relatedness of pre- and postexposure isolates 1648 and 5066 (S.oralis) by PFGE by single and double digestion.

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other drug classes, including penicillins, macrolides, lincos-amides, fluoroquinolones, and glycopeptides (47–50), although itwas not seen in the organisms used in this study. However, nomechanism of tolerance was determined in any study. Calciumconcentrations of �50 mg/liter were included in all media/exper-iments due to DAP’s calcium-dependent activity to ensure thiswas not a factor for the elevated MBC or MIC results. Addition-ally, the MBC/MIC ratios when drug was given in the presence ofprotein were also higher (from 2 to 8) than the ratio for S. aureus,although by definition, it was not DAP tolerant. All protein-con-taining experiments utilized total DAP concentrations, appropri-ately supplemented with calcium. Previous protein-containingexperiments utilizing total DAP concentrations have been com-parable to nonprotein experiments utilizing free (unbound) DAPconcentrations (30, 33). Regardless of protein content, the MBC/MIC ratios were similar across all tested media; consequently, theinclusion of protein was not believed to impact the elevated ratiosobtained.

The results of the KC experiments correspond well with thoseof the pharmacokinetic/pharmacodynamic models in that therewas some level of initial killing and then regrowth to baseline CFUlevels, an overall static effect at up to 8� the MIC value (Fig. 3).Isolates recovered at the 24-h time point had significantly elevatedDAP MIC values. These results make sense in light of the fact thatall of the preexposure strains were found to contain a DAP-resis-tant subpopulation as previously mentioned. This population ofcells (0.1 to 1%) is most likely able to thrive under selectivepressure and grow to baseline levels, while the majority of thesusceptible cells are killed within 8 h.

Garcia-de-la-Maria and colleagues provide supporting evi-dence that this in vitro phenomenon of HLDR occurs rapidly in astudy utilizing multiple pharmacodynamic models against nu-merous strains (23). Additionally, this study demonstrates thatdespite utilizing significant DAP concentrations, either free or to-tal, of up to 8� MIC, only static activity was achieved. The addi-tion of gentamicin resulted in enhanced activity and prevented thedevelopment of high-level resistance in several of the isolates stud-ied. However, the clinical implications for the development ofresistance seen in vitro are not currently known. Since the ap-proval of DAP over 10 years ago, there have been limited pub-lished cases of clinical failure and/or resistance in enterococci andstaphylococci but no reports regarding streptococci until recently(51, 52). The first case described was a clinical failure attributed tobreakthrough bacteremia with DAP-resistant Streptococcus angi-nosus from a patient with recurrent methicillin-resistant S. aureusosteomyelitis that had been previously unsuccessfully treated withVAN; the patient was then switched to DAP treatment at 6 mg/kg/day. After 15 days of DAP therapy, this patient presented in septicshock with positive blood cultures for S. anginosus with a DAPMIC of 4 �g/ml (51).

The recent reporting of this case is important. However, a base-line isolate was not saved; therefore, it cannot be confirmed thatthis was a case of resistance development or an infection attributedto an organism with a DAP MIC slightly above those seen in thewild-type population. The moderately elevated MIC pales in com-parison to the DAP MICs seen in this study and the study ofGarcia-de-la-Maria et al. (23), as well as the rapidity of resistancedevelopment that was demonstrated. Additional clinical data per-taining to the treatment of these types of organisms with DAP willbe essential in determining whether or not there is a clinical risk if

DAP is used off label to treat VGS. Another recent study evaluatedoutcomes of VGS treated with daptomycin (52). Out of 9,105patients, only 99 (1.1%) had VGS, for which DAP was utilized asthe first-line agent in 21 patients (21%). Only 33 patients receivedDAP monotherapy, and the assessment of clinical outcomes was73% success, 3% failure, and 24% nonevaluable. One patient, de-termined to be a clinical failure, was classified as such based onculture of a DAP-resistant isolate (DAP MIC of 2 �g/ml); a reasonfor failure was not specified for the other patients.

If these organisms can develop clinically and other therapeuticoptions are not available, combination therapy may be a potentialsolution to restrict the growth of the DAP-resistant subpopula-tion. However, the combinations examined in this study had dis-appointing results. The effects of the different antimicrobial com-binations were strain/species specific, and the combinations maynot be effective when applied to a larger collection of VGS. Theonly synergistic combination noted was GEN plus DAP againststrain 1649, which should be investigated further. Additionalcombination therapy, in particular DAP with �-lactam agents,should be considered and investigated, as current literature indi-cates that daptomycin in combination with a �-lactam againststaphylococci and enterococci exhibits enhanced activity and de-creased resistance selection (53, 54).

Conclusion. Certain VGS have the ability to rapidly developHLDR after drug exposure, but the clinical relevance is lacking.Indeed, after more than 10 years of clinical use, surveillance con-tinues to show very low resistance for VGS; however, a recentsingle clinical failure with a daptomycin-resistant strain of S. an-ginosus has been documented. Therefore, further studies are war-ranted to explore treatment options to prevent or minimize resis-tance development, particularly daptomycin in combination with�-lactams, given the evidence seen in staphylococci and entero-cocci. Additionally, studies should be conducted to elicit themechanism of DAP resistance and to determine the clinical signif-icance of this phenomenon.

ACKNOWLEDGMENTS

This work was supported by a research grant from Cubist Pharmaceuti-cals.

R.L.A. has received grant support from Cubist and Cerexa. B.D.K.,C.M., and D.A. are all employed by and own stock in Cubist Pharmaceu-ticals.

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