Sequential Evolution of Vancomycin-Intermediate Resistance AltersVirulence in Staphylococcus aureus: Pharmacokinetic/PharmacodynamicTargets for Vancomycin Exposure

Justin R. Lenhard,a,b Tanya Brown,a,b* Michael J. Rybak,c Calvin J. Meaney,a,b Nicholas B. Norgard,a Zackery P. Bulman,a,b

Daniel A. Brazeau,e Steven R. Gill,d Brian T. Tsujia,b

Laboratory for Antimicrobial Pharmacodynamics, School of Pharmacy and Pharmaceutical Sciences,a and New York State Center of Excellence in Life Sciences andBioinformatics,b University at Buffalo, Buffalo, New York, USA; Anti-Infective Research Laboratory, Eugene Applebaum College of Pharmacy and Health Sciences, Detroit,Michigan, USAc; Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USAd; Department of PharmaceuticalSciences, University of New England, Portland, Maine, USAe

Staphylococcus aureus possesses exceptional virulence and a remarkable ability to adapt in the face of antibiotic therapy. Weexamined the in vitro evolution of S. aureus in response to escalating vancomycin exposure by evaluating bacterial killing andthe progression of resistance. A hollow-fiber infection model was utilized to simulate human doses of vancomycin increasingfrom 0.5 to 4 g every 12 h (q12h) versus a high inoculum (108 CFU/ml) of methicillin-resistant S. aureus (MRSA) USA300 andUSA400. Host-pathogen interactions using Galleria mellonella and accessory gene regulator (agr) expression were studied inserially obtained isolates. In both USA300 and USA400 MRSA isolates, vancomycin exposure up to 2 g q12h resulted in persis-tence and regrowth, whereas 4 g administered q12h achieved sustained killing against both strains. As vancomycin exposure in-creased from 0.5 to 2 g q12h, the bacterial population shifted toward vancomycin-intermediate resistance, and collateral in-creases in the MICs of daptomycin and televancin were observed over 10 days. Guideline-recommended exposure of a ratio ofthe area under the concentration-time curve for the free, unbound fraction of the drug to the MIC (fAUC/MIC ratio) of 200 dis-played a 0.344-log bacterial reduction in area, whereas fAUC/MICs of 371 and 554 were needed to achieve 1.00- and 2.00-log re-ductions in area, respectively. The stepwise increase in resistance paralleled a decrease in G. mellonella mortality (P � 0.021) anda gradual decline of RNAIII expression over 10 days. Currently recommended doses of vancomycin resulted in amplification ofresistance and collateral damage to other antibiotics. Decreases in agr expression and virulence during therapy may be an adap-tive mechanism of S. aureus persistence.

Staphylococcus aureus is a primary human pathogen capable ofexceptional virulence and an array of life-threatening infec-

tions ranging from necrotizing pneumonia to endocarditis (1). S.aureus also has a remarkable ability to adapt in the face of antimi-crobial therapy via a plethora of resistance mechanisms (2–6).Although there is a large body of information on the mechanismsof antibiotic resistance in S. aureus, the interplay between viru-lence and antibiotic resistance is not completely understood. Theresults of genome-wide sequencing analyses have been con-founded by the discovery that multiple genetic pathways may leadto similar levels of antibiotic resistance (7–9). Also, while the se-quential development of resistance mutations during the courseof an infection has been analyzed, the temporal link between re-sistance and virulence is poorly defined (10).

Despite the successful use of vancomycin to combat commu-nity-associated (CA) methicillin-resistant S. aureus (MRSA) forseveral decades, the spread of vancomycin-intermediate S. aureus(VISA) has brought the utility of vancomycin into question (4,11). The genetic basis for VISA is unknown, although reducedvancomycin susceptibility has been associated with dysfunction ofthe accessory gene regulator (agr), the master regulator of patho-genicity in S. aureus (12–14). Isolates of S. aureus that are defectivein agr have significantly reduced virulence profiles and low-levelvancomycin resistance conferred through a number of mecha-nisms, including biofilm formation, stationary-phase growth, andalterations in autolysis (15–17). At present, it is not known howmodulating vancomycin dosing regimens will alter the time scale

of agr expression or S. aureus virulence. Here, we attempted toresolve the link between vancomycin resistance and virulence bysimulating human dosing in an in vitro hollow-fiber infectionmodel (HFIM) to study the stepwise evolution of vancomycinresistance and also determine the impact that these mutationshave on virulence.

MATERIALS AND METHODSBacterial isolates. To represent the predominant clones of MRSA in theUnited States, Europe, and Canada, we selected the two pulsed-field gelelectrophoresis-type community-acquired strains USA300 (FPR 3757)and USA400 (MW2), of which the complete genome sequences have beenestablished (18–20). Both isolates were obtained from the Network ofAntimicrobial Resistance in Staphylococcus aureus (NARSA).

Received 3 November 2015 Returned for modification 29 November 2015Accepted 13 December 2015

Accepted manuscript posted online 28 December 2015

Citation Lenhard JR, Brown T, Rybak MJ, Meaney CJ, Norgard NB, Bulman ZP,Brazeau DA, Gill SR, Tsuji BT. 2016. Sequential evolution of vancomycin-intermediate resistance alters virulence in Staphylococcus aureus:pharmacokinetic/pharmacodynamic targets for vancomycin exposure.Antimicrob Agents Chemother 60:1584 –1591. doi:10.1128/AAC.02657-15.

Address correspondence to Brian T. Tsuji, btsuji@buffalo.edu.

* Present address: Tanya Brown, Florida International University, Miami, Florida,USA.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Antibiotics and medium. Vancomycin, ciprofloxacin, levofloxacin,gentamicin, nafcillin, and rifampin analytical-grade powders were com-mercially purchased (Sigma Chemical Company, St. Louis, MO). Dapto-mycin was obtained from Cubist (Lexington, MA), telavancin was ob-tained commercially from the University at Buffalo Pharmacy, andlinezolid was obtained from Pfizer (Groton, CT). Brain heart infusion(BHI) broth and BHI agar (Difco Laboratories, Detroit, MI) were used forall in vitro hollow experiments.

Determining alterations in MIC. Antimicrobial agents commonlyused to treat MRSA were tested with CLSI broth microdilution methodsto evaluate the potential collateral damage of vancomycin treatment toother antibiotics. Isolates were initially evaluated for MICs at baselineprior to vancomycin exposure. Selective postexposure mutants obtainedfrom the HFIM were also evaluated for MICs. CLSI interpretive criteriawere used to categorize the isolates as susceptible, intermediate, or resis-tant. S. aureus ATCC 29213 bacteria were utilized as quality control (QC)organisms. All QC results were within published limits.

HFIM. As previously described, an HFIM was used to evaluate howclinically relevant vancomycin regimens alter the bacterial burden ofMRSA and influence the amplification of antibiotic resistance over 240 h(21). The HFIM used a C3008 cellulosic cartridge (FiberCell Systems,Frederick, MD). Bacteria were trapped in the extracapillary space of thecartridge, and the large surface area from the hollow fibers was used tofacilitate nutrient exchange and antibiotic exposure. Over 10 days, vanco-mycin dosing regimens were administered to achieve the same values forareas under the concentration-time curve (AUCs) as those expected inhumans. The clinical scenario of a high bacterial burden infection such as abilobar pneumonia or endocarditis was simulated using a 108-CFU/ml inoc-ulum achieved from overnight MRSA cultures. Samples were taken from the10-day HFIM experiment at 0, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240h and subsequently incubated and quantified to assess bacterial growth.

To simulate human antibiotic dosing, four different vancomycin reg-imens were administered in the HFIM assuming a free (ƒ) fraction of 50%and a 6-h half-life. The following dosing schemes were chosen to target the

pharmacokinetic parameters of maximal concentration (Cmax), mini-mum concentration (Cmin), and an area-under-the-curve/MIC (AUC/MIC) ratio expected in human plasma: (i) 500 mg every 12 h (q12h)(ƒCmax of 10 mg/liter, ƒCmin of 2.5 mg/liter, and ƒAUC/MIC of 112.5); (ii)1,000 mg q12h (ƒCmax of 20 mg/liter, ƒCmin of 5 mg/liter, and ƒAUC/MICof 225); (iii) 2,000 mg q12h (ƒCmax of 40 mg/liter, ƒCmin of 10 mg/liter,and ƒAUC/MIC of 450); and (iv) 4,000 mg q12h (ƒCmax of 80 mg/liter,ƒCmin of 20 mg/liter, and ƒAUC/MIC of 900).

Samples from the HFIM were also obtained serially for confirmationof vancomycin pharmacokinetics using a standard agar diffusion bioassayprocedure. Mueller-Hinton agar (MHA) with Micrococcus luteus ATCC9341 as an indicator organism was utilized to validate vancomycin con-centrations as previously described (43). All observed pharmacokineticparameters were within 12% of targeted values.

Population analysis profiles. Quantitative cultures from the HFIMwere determined in “real time” in mini-population analysis profiles(PAPs) that utilized BHI agar containing 0, 2, 4, and 6 mg/liter of vanco-mycin, which enabled the daily detection of the total bacterial population,as well as vancomycin-resistant subpopulations in all four dosing regi-mens. Full PAPs were also completed using 0, 0.5, 1, 2, 3, 4, 6, 8, and 16mg/liter of vancomycin to quantify daily evolution of the resistant sub-populations in the 2-g q12h regimen, as well as the 240-h time point of allfour dosing regimens. PAP cultures were incubated for 48 h, and thecolonies were then counted and plotted against either vancomycin con-centration or time.

RNA extraction and quantitative real-time PCR. Cells were collectedat 240 h from the HFIM for MRSA USA300 before and after exposure tothe four vancomycin regimens. RNAIII expression was then assessed withreal-time PCR as described previously (22). Briefly, the collected sampleswere centrifuged at 14,000 rpm for 5 min at room temperature. The su-pernatant was aspirated, and the pellet was immediately frozen at �80°Cuntil RNA isolation. The total RNA was isolated from the pellet (SV totalRNA isolation system; Promega, Madison, WI) according to the manu-facturer’s protocol for Gram-positive bacteria. From the total RNA pool,

FIG 1 Humanized dosing regimens of vancomycin (Vanco) against MRSA USA300 quantifying the total population (blue) and the sequential emergence ofresistance secondary to drug exposure (red, gray, and pink) over a 10-day period.

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mRNA was purified (MICROBExpress; Ambion, Austin, TX). Reversetranscription (RT) of the mRNA (315 ng) was carried out using randomhexamer primers and the AccuScript high-fidelity RT-PCR system (Strat-agene, La Jolla, CA).

G. mellonella virulence assays. Galleria mellonella was utilized to in-vestigate the pathogenicity of resistant mutants which evolved in theHFIM, as detailed previously (23, 24). In the 10-day HFIM utilizing avancomycin dose of 2 g q12h, USA300 mutants were collected dailyand stored at �80°C prior to the virulence assessment. Twenty ran-domly chosen caterpillars 200 to 300 mg in weight in the final instarlarval stage (Vanderhorst, Inc., St. Mary’s, OH) were used in eachgroup. A 10-�l Hamilton syringe was used to inject 10-�l aliquots ofthe inoculum into the hemocoel of each caterpillar via the last left proleg.Bacterial colony counts were used to confirm all inocula, and appropriatecontrol arms with caterpillars receiving no injection or an injection ofphosphate-buffered saline were included.

RESULTSMRSA USA300 and USA400 HFIMs. To study the temporal pro-file of MRSA USA300’s response to increasing antibiotic exposure,an HFIM was utilized to simulate human vancomycin dosing overa 10-day period (Fig. 1). Despite high doses of up to 2 g q12h,mimicking exposure profiles in administered humans, USA300demonstrated persistence and tolerance, maintaining bacterialcounts of �1010 CFU/ml with growth similar to the control by the240-h endpoint. The lower-dose vancomycin regimen of 500 mgq12h was nearly identical to that of the control throughout the 240h. The 1-g q12h scheme, which is the regimen administered to themajority of patients with S. aureus bloodstream infections, dem-onstrated initial stasis at �5 � 107 CFU/ml for approximately 24h, followed by gradual regrowth that plateaued at �3.5 � 1010

CFU/ml by 120 h. Similarly, the 2-g q12h regimen demonstrated

stepwise increases in bacterial counts beginning with an initialstasis phase from 0 to 48 h at �108 CFU/ml, followed by a re-growth phase from 48 h to 96 h at �109 CFU/ml, followed in turnby a final regrowth phase at 144 h to �1010 CFU/ml that contin-ued to 240 h. Unlike the lower-dose regimens, the 4-g q12hscheme achieved a �3-log reduction in bacterial counts by 72 hand, after 240 h of continuous killing, resulted in a final popula-tion of �3 � 102 CFU/ml.

An additional HFIM analysis was conducted on USA400 toconfirm that the levels of activity of vancomycin are comparableamong common CA-MRSA strains (Fig. 2). Similar to USA300,vancomycin doses up to 2 g q12h were unable to prevent USA400from regrowing by 48 h. While the 500-mg and 1-g q12h regimensmirrored the growth control by 96 h, the 2-g q12h scheme main-tained counts below 1010 CFU/ml for the duration of the experi-ment. The 4-g q12h regimen was once again the only simulatedvancomycin therapy capable of achieving bactericidal activity,with a �3-log reduction conferred by 120 h and a final populationcount of �104 CFU/ml at 240 h. Comodeling the reduction in thelog ratio area of both USA300 and USA400 as a function of van-comycin exposure confirmed that the performances of vancomy-cin were similar for the two strains (Fig. 3). In both USA300 andUSA400, ratios of the AUC for the free, unbound fraction of thedrug to the MIC (fAUC24/MIC ratios) of approximately 200, 371,554, and 757 achieved a reduction in the total population’s logratio area of 0.344, 1.00, 2.00, and 3.00, respectively (R2 � 0.990),as described by a Hill-type function utilized previously (25).

Sequential emergence of antibiotic resistance. Throughoutthe 10-day HFIM experiments, subpopulations capable of grow-ing on 2, 4, and 6 mg/liter of vancomycin were tracked for both

FIG 2 Humanized dosing regimens of vancomycin against MRSA USA400 quantifying the total population (blue) and the sequential emergence of resistancesecondary to drug exposure (red, gray, and pink) over a 10-day period.

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USA300 and USA400 (Fig. 1 and 2). In both investigationalstrains, lower vancomycin doses of 500 mg and 1 g q12h did notsubstantially amplify vancomycin resistance, with �1% of thepopulation ever growing on 2 mg/liter of vancomycin. However,when the dose of vancomycin was increased to 2 g q12h, �10% ofUSA300’s population and �50% of USA400’s total populationwere capable of growing on 2 mg/liter of vancomycin by 240 h.Increasing the vancomycin dose even further to 4 g q12h resultedin drastic killing of the total bacterial populations, with resis-tant subpopulations growing on 2 mg/liter of vancomycincomprising �1% of the population for the duration of theexperiments.

A more comprehensive PAP scheme utilizing vancomycinconcentrations up to 16 mg/liter was also performed for the entire2-g q12h regimen and the 240-h terminal time points of all theUSA300 HFIMs (Fig. 4). After several days of vancomycin expo-

sure, subpopulations of USA300 began to grow on 6 mg/liter ofvancomycin beginning at 120 h. Resistance to vancomycin inten-sified as the 2-g q12h regimen continued, eventually resulting inisolates capable of growing on 8 mg/liter of vancomycin by 240 h.Comparing the dose responses among different regimens, doses of500 mg up to 2 g q12h produced similar resistance profiles by 240h, characterized by bacterial growth on 8 mg/liter of vancomycin.In contrast, the 4-g q12h regimen prevented the growth of anycolonies in the presence of �4 mg/liter of vancomycin at 240 h.

In order to track the emergence of resistance to other agentsduring vancomycin therapy, the MICs of common antibioticswere determined for USA300 isolates collected every 24 h duringthe 2-g q12h HFIM experiment and are presented in Table 1.Secondary to vancomycin exposure, significant alterations in thesusceptibility to the lipopeptide daptomycin and lipoglycopeptidetelavancin were observed. For daptomycin, sequential increaseswere noted from 0.125 to 0.25 mg/liter on day 4, to 0.5 mg/liter onday 7, and 1.0 mg/liter on day 10. For telavancin, sequential in-creases from 1.0 to 2.0 on day 2, and to 4.0 mg/liter on day 5occurred. There was also a consistent trend of decreased rifampinsusceptibility from 0.015 to 0.03 mg/liter, albeit only a 1-fold dif-ference

G. mellonella survival. To determine what impact vancomy-cin resistance has on S. aureus virulence, G. mellonella moths wereinoculated with USA300 isolates collected daily throughout the2-g q12h regimen (Fig. 5A) and also isolates obtained at the 240-hterminal time point of each USA300 HFIM experiment (Fig. 5B).In the 2-g q12h regimen, isolates collected after �96 h of vanco-mycin exposure displayed attenuated virulence that paralleled theagr dysfunctional control, with significantly higher G. mellonellasurvival rates after 6 days compared to the results seen with thebaseline isolate (G. mellonella survival of �80% versus 45% onday 6, P � 0.021, log-rank test). Conversely, isolates obtainedprior to 96 h of vancomycin exposure demonstrated killing thatmirrored the profile of the baseline isolate (G. mellonella survivalof �60% on day 6). When all four vancomycin regimens werecompared to one another, doses of 500 mg to 2 g q12h resulted insignificantly better G. mellonella survival by day 6 relative to theresults seen with the baseline isolate (G. mellonella survival of

FIG 3 Bacterial reductions (represented as log ratio areas) of MRSA USA300and USA400 are plotted as a function of vancomycin exposure. The fAUC/MICs needed to confer a reduction in the log ratio areas of 0.344, 1, 2, and 3(black, blue, green, and purple, respectively) are listed above the correspond-ing vancomycin exposures. The data were described by a Hill-type function asdescribed previously (25).

FIG 4 (A) Stepwise evolution of resistance, as shown in population analysis profiles of vancomycin quantified every 24 h over a 10-day period in response tosimulated human exposure to vancomycin at 2 g q12h. (B) Comparative population analysis profiles among different vancomycin dosing regimens of 500, 1,000,2,000, and 4,000 mg q12h at the 240-h study endpoint.

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�80% versus 45% on day 6, P � 0.029), whereas the 4-g q12hregimen resulted in an isolate with comparable virulence to thebaseline isolate (P � 0.231).

RNAIII expression and comodeling with total counts, viru-lence, and resistance plots. Real-time PCR of RNAIII was per-formed on all USA300 isolates recovered from 0 to 240 h duringthe 2-g q12h regimen to delineate the temporal pattern of agrexpression during antibiotic therapy and its relationship to resis-tance and virulence in S. aureus (Fig. 6A). The quantity of RNAIIIinitially surged to �10� baseline between 0 and 24 h, following asteep decline to almost undetectable amounts of RNAIII at 96 h,after which agr expression hovered around baseline until 240 h.When agr expression was comodeled with bacterial counts and G.mellonella mortality, a trend was observed in which the initialsurge in agr expression coincided with higher levels of G. mello-nella mortality (�50%), whereas the low levels of agr expressionbeginning at 96 h corresponded to attenuated mortality ratesof �20% (Fig. 6B). The relationship between bacterial counts andagr expression was not as clearly defined in the 2 g of vancomycinq12h regimen. However, when agr expression and bacterial countswere plotted as a function of vancomycin exposure, an inverse

relationship was observed in which an fAUC24/MIC of 900 mg·h/liter resulted in a bacterial count of �3 � 102 CFU/ml and agrexpression �10� baseline at 240 h (Fig. 6C).

DISCUSSION

Although vancomycin was the drug of choice for MRSA infectionsfor several decades, increased treatment failure rates coincidingwith the spread of VISA have cast doubt on the reliable use ofvancomycin (26–30). As S. aureus MICs continue to creep upwardfor vancomycin, understanding how drug exposure influencesboth resistance and virulence is critical to the judicious use ofanti-MRSA agents. Here, we investigated clinically relevantvancomycin regimens in a 10-day HFIM to translate how van-comycin dosing alters MRSA population dynamics and glyco-peptide resistance. In both of the investigational CA-MRSAstrains, administration of 500 mg or 1 g of vancomycin q12h wasunable to achieve bacteriostatic activity. When the dose of vanco-mycin was increased to 2 g q12h, a regimen yielding twice the drugexposure of the most common vancomycin scheme, bacterialcounts still rose steadily in the face of intensified antibiotic dosing.

TABLE 1 Bacterial isolates of MRSA USA300 collected during the 10-day HFIM investigating a 2-g q12h vancomycin regimena

StrainTime (h) of VAN 2-gq12h exposure

MIC (mg/liter)

VAN LZD DAP RIF LEV CIP GEN NAF

LAD201 0 (baseline) 1 2 0.5 0.015 8 32 1 32LAD202 24 1 2 0.5 0.015 8 32 2 32LAD203 48 1 2 0.5 0.015 8 32 2 32LAD204 72 2 2 0.5 0.015 16 32 2 32LAD205 96 2 2 0.25 0.015 8 32 2 32LAD206 120 4 2 0.5 0.03 8 32 2 32LAD207 144 4 2 1 0.03 16 32 0.5 32LAD208 168 4 2 1 0.03 16 16 2 32LAD209 192 4 2 2 0.03 8 16 2 16LAD210 216 4 2 2 0.03 8 16 1 32LAD211 240 4 2 2 0.03 8 16 2 32a MICs to vancomycin and other antibiotic of interest are listed for each isolate. VAN, vancomycin; LZD, linezolid; DAP, daptomycin; RIF, rifampin, LEV, levofloxacin; CIP,ciprofloxacin; GEN, gentamicin; NAF, nafcillin.

FIG 5 (A) G. mellonella virulence assay in response to simulated human vancomycin exposure. Worms were inoculated with MRSA USA300 isolates collectedthroughout the 2-g q12h vancomycin regimen in the HFIM, and the subsequent survival of the worms was recorded. G. mellonella inoculated with isolatescollected after �96 h of vancomycin treatment displayed significantly better survival than worms inoculated with the baseline isolate (P � 0.021, log-rank test).(B) The assay was repeated for the terminal MRSA USA300 isolates collected at the end of the 10-day HFIM experiments for each vancomycin regimen.Vancomycin doses of �2 g q12h resulted in significantly better survival of the G. mellonella relative to the baseline isolate (P � 0.029), whereas the isolate collectedafter 240 h of exposure to the 4-g q12h regimen was comparably virulent to the baseline isolate (P � 0.231).

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Only the 4-g q12h regimen achieved bactericidal activity, withsustained killing over 10 days for both investigational strains.

Unlike the AUC/MIC target of 400 (fAUC/MIC � 200) advo-cated by current vancomycin guidelines, higher vancomycin ex-posures were needed to kill the CA-MRSA strains investigated inthe present study (31). As current guidelines recommend vanco-mycin use for MRSA infections in which the causative organism’svancomycin MIC is �1 mg/liter, the isolates investigated in thepresent study are an accurate representation of MRSA strainscommonly treated with vancomycin (MIC � 1 mg/liter forUSA300 and USA400). However, the 2-g q12h vancomycin regi-men produced an fAUC/MIC �2� the suggested AUC/MIC of400 (fAUC/MIC of 200), and negligible killing was achieved inboth investigational strains. An alarmingly high fAUC/MIC of 554was necessary to achieve a 2-log bacterial reduction in area. Van-comycin regimens commonly used in the clinic may therefore beunable to fully overcome S. aureus resistance mechanisms at highinocula, and alternative regimens may need to be considered evenwhen an organism’s vancomycin MIC is deceptively below 2 mg/liter.

Not only were the 500 mg to 2 g of vancomycin q12h regimensincapable of killing S. aureus, but the suboptimal vancomycinexposure also amplified antibiotic resistance. Similar to other in-vestigations concerning antibiotic resistance, an inverted “U”phenomenon was observed in which rising vancomycin concen-trations resulted in higher levels of antibiotic resistance until ex-treme concentrations were capable of killing the entire population(32). Vancomycin exposure was also found to augment resistanceto other antimicrobials, including daptomycin, telavancin, andrifampin. Taken together, these results emphasize the importanceof utilizing optimal vancomycin dosing practices against MRSAthat is capable of being killed by vancomycin. If a high enoughvancomycin exposure cannot be achieved to confer bactericidalactivity, resistance to glycopeptides and other antibiotic classeswill increase in a manner that is proportionate to the amount ofvancomycin exposure.

Although the amplification of antibiotic resistance is a con-cern, agr expression and G. mellonella mortality demonstratedthat the ability of S. aureus to resist glycopeptide treatment ap-pears to come at a cost to its virulence. Lower vancomycin dosing

schemes of 500 mg to 2 g q12h resulted in reduced agr activity anda higher survival rate of the G. mellonella, whereas the bactericidal4-g q12h regimen suppressed vancomycin resistance and corre-spondingly did not significantly alter the survival rate of G. mello-nella. The emergence of vancomycin resistance appears to be aheterogenous process, but two observations commonly madeupon rising vancomycin MICs are thicker cell walls and dysfunc-tional agr profiles (4, 16, 33, 34). It has been proposed that alter-ations in the cell wall of S. aureus may interfere with the cell’sability to bind the autoinducing peptide of the agr system used forquorum sensing and activation of S. aureus toxins and other vir-ulence factors (35). It is likely that suboptimal vancomycin dosingin the clinic will drive MRSA from a more antibiotic-susceptibleand virulent state toward a more resistant but less virulent popu-lation.

Another consequence of vancomycin exposure is the potentialconversion of S. aureus from a virulent phenotype into a persistentstate better adapted to survive antimicrobial therapy and the hostimmune response. In the present study, the survival of G. mello-nella improved as the expression of agr decreased, suggesting thatthe reduction in virulence factor release reduced the pathogenicityof S. aureus. A prior investigation that compared S. aureus isolatescollected from a patient before and after 6 weeks of vancomycintreatment found that vancomycin exposure resulted in down-regulated agr, slower autolysis, increased cell wall thickness, and areduction in the secretion of alpha-toxin and phenol-solublemodulins (36). It has been established that alpha-toxin releaseactivates the NLRP3-inflammasome and induces interleukin-1and interleukin-6 secretion, while the VISA phenotype has beenshown to provoke less tumor necrosis factor alpha and interleukinrelease than its vancomycin-susceptible counterparts (9, 37, 38).An investigation of the selective pressure of vancomycin on MRSAalso found that vancomycin exposure amplifies the relative abun-dance of the small colony variant phenotype, which is a slow-growing phenotype implicated in chronic and recurrent S. aureusinfections, as well as intracellular persistence (25, 39). The conse-quence of suboptimal vancomycin exposure is therefore not re-stricted to proliferating antibiotic resistance but is also seen in theshifting of the population dynamics of S. aureus toward a persis-

FIG 6 (A) RNAIII profiling to measure activity of agr, the primary quorum-sensing response regulator of virulence in S. aureus, quantified every 24 h in responseto MRSA USA300 exposed to vancomycin at 2 g q12h for 10 days. (B) Comodeling of the agr expression in panel A (pink) with MRSA USA300’s total population(black), vancomycin-resistant subpopulations growing on 4.0 mg/liter of vancomycin (red), and G. mellonella mortality (blue). (C) The same analysis is repeatedusing the 240-h terminal time point for vancomycin doses of 500 mg to 4 g q12h.

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tent state that is capable of enduring host countermeasures andexogenous antimicrobials.

The present study has several meaningful limitations to con-sider before the results can be fully translated into the clinicalsetting. Similar to other in vitro investigations, not only does theHFIM fail to account for host defenses against S. aureus, but theuse of Mueller-Hinton broth provides a nutrient-rich environ-ment that may improve bacterial survival relative to in vivoexperiments. Most importantly, the poor performance of stan-dard vancomycin regimens is likely ascribable to the high in-oculum investigated in the present study. A previous in vitroinvestigation not only demonstrated a large inoculum effect forvancomycin against heteroresistant-VISA but also found thatsimulated doses of vancomycin up to 5 g q12h were unable toachieve sustained killing (40). A murine thigh infection model waslater used to characterize the magnitude of inoculum effects forMRSA and heteroresistant-VISA exposed to several antimicrobi-als (41); the authors concluded that the inoculum effect was muchmore severe for vancomycin than for other antistaphylococcalagents, such as daptomycin and linezolid. It is therefore prudentto view the results of the current investigation as representative ofa worst-case clinical scenario, since vancomycin may achievemuch better activity in more favorable conditions.

In closing, clinically relevant vancomycin regimens simu-lated in an HFIM were largely ineffective against two commonCA-MRSA strains, with increasing vancomycin exposure con-ferring more substantial antibiotic resistance and severe lossesin virulence. The only investigational regimen capable of kill-ing USA300 and USA400 was the 4-g q12h regimen, which is adosing scheme neglected clinically due to the propensity of van-comycin for dose-related nephrotoxicity (42). In the model invitro system, simply obtaining the suggested AUC/MIC of 400 wasnot enough to overcome the resistance mechanisms of S. aureus.Owing to the inability of vancomycin to achieve bactericidal ac-tivity at clinical concentrations, clinicians are cautioned about se-lecting vancomycin for MRSA infections containing S. aureusstrains with questionable vancomycin susceptibility. Aggressivestewardship and the use of alternative antimicrobials may be nec-essary to stem the progressive upward creep of the MIC of vanco-mycin against S. aureus, since seemingly optimal vancomycin reg-imens may actually exacerbate vancomycin resistance. It will likelytake many years to fully define where the niche of vancomycinnow lies in the context of new anti-MRSA agents that offer clini-cians a more diverse armamentarium against S. aureus.

ACKNOWLEDGMENTS

We thank Alan Forrest for insight into pharmacokinetic/pharmacody-namic and statistical analyses.

B.T.T. was supported by National Institute of Allergy and InfectiousDiseases, National Institutes of Health (R01AI111990). The content issolely the responsibility of the authors and does not necessarily representthe official views of the National Institutes of Health.

FUNDING INFORMATIONHHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)provided funding to Brian T. Tsuji under grant number R01AI111990.

REFERENCES1. Lowy FD. 1998. Staphylococcus aureus infections. N Engl J Med 339:520 –

532. http://dx.doi.org/10.1056/NEJM199808203390806.2. Hanaki H, Kuwahara-Arai K, Boyle-Vavra S, Daum RS, Labischinski H,

Hiramatsu K. 1998. Activated cell wall synthesis is associated with vanco-mycin resistance in methicillin-resistant Staphylococcus aureus clinicalstrains Mu3 and Mu50. J Antimicrob Chemother 42:199 –209. http://dx.doi.org/10.1093/jac/42.2.199.

3. Hanaki H, Labischinski H, Inaba Y, Kondo N, Murakami H, HiramatsuK. 1998. Increase in glutamine-non-amidated muropeptides in the pep-tidoglycan of vancomycin-resistant Staphylococcus aureus strain Mu50. JAntimicrob Chemother 42:315–320. http://dx.doi.org/10.1093/jac/42.3.315.

4. Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC. 1997.Methicillin-resistant Staphylococcus aureus clinical strain with reducedvancomycin susceptibility. J Antimicrob Chemother 40:135–136. http://dx.doi.org/10.1093/jac/40.1.135.

5. Sieradzki K, Tomasz A. 2003. Alterations of cell wall structure and me-tabolism accompany reduced susceptibility to vancomycin in an isogenicseries of clinical isolates of Staphylococcus aureus. J Bacteriol 185:7103–7110. http://dx.doi.org/10.1128/JB.185.24.7103-7110.2003.

6. Tsuji BT, von Eiff C, Kelchlin PA, Forrest A, Smith PF. 2008.Attenuated vancomycin bactericidal activity against Staphylococcus au-reus hemB mutants expressing the small-colony-variant phenotype. Anti-microb Agents Chemother 52:1533–1537. http://dx.doi.org/10.1128/AAC.01254-07.

7. Alam MT, Petit RA, III, Crispell EK, Thornton TA, Conneely KN, JiangY, Satola SW, Read TD. 2014. Dissecting vancomycin-intermediate re-sistance in Staphylococcus aureus using genome-wide association. GenomeBiol Evol 6:1174 –1185. http://dx.doi.org/10.1093/gbe/evu092.

8. Vidaillac C, Gardete S, Tewhey R, Sakoulas G, Kaatz GW, Rose WE,Tomasz A, Rybak MJ. 2013. Alternative mutational pathways to inter-mediate resistance to vancomycin in methicillin-resistant Staphylococcusaureus. J Infect Dis 208:67–74. http://dx.doi.org/10.1093/infdis/jit127.

9. Howden BP, Smith DJ, Mansell A, Johnson PD, Ward PB, Stinear TP,Davies JK. 2008. Different bacterial gene expression patterns and attenu-ated host immune responses are associated with the evolution of low-levelvancomycin resistance during persistent methicillin-resistant Staphylo-coccus aureus bacteraemia. BMC Microbiol 8:39. http://dx.doi.org/10.1186/1471-2180-8-39.

10. Mwangi MM, Wu SW, Zhou Y, Sieradzki K, de Lencastre H, Richard-son P, Bruce D, Rubin E, Myers E, Siggia ED, Tomasz A. 2007. Trackingthe in vivo evolution of multidrug resistance in Staphylococcus aureus bywhole-genome sequencing. Proc Natl Acad Sci U S A 104:9451–9456. http://dx.doi.org/10.1073/pnas.0609839104.

11. Zhang S, Sun X, Chang W, Dai Y, Ma X. 2015. Systematic review andmeta-analysis of the epidemiology of vancomycin-intermediate and het-erogeneous vancomycin-intermediate Staphylococcus aureus isolates.PLoS One 10:e0136082. http://dx.doi.org/10.1371/journal.pone.0136082.

12. Novick RP. 2003. Autoinduction and signal transduction in the regula-tion of staphylococcal virulence. Mol Microbiol 48:1429 –1449. http://dx.doi.org/10.1046/j.1365-2958.2003.03526.x.

13. Lyon GJ, Wright JS, Muir TW, Novick RP. 2002. Key determinants ofreceptor activation in the agr autoinducing peptides of Staphylococcusaureus. Biochemistry 41:10095–10104. http://dx.doi.org/10.1021/bi026049u.

14. Ji G, Beavis R, Novick RP. 1997. Bacterial interference caused by auto-inducing peptide variants. Science 276:2027–2030. http://dx.doi.org/10.1126/science.276.5321.2027.

15. Moise-Broder PA, Sakoulas G, Eliopoulos GM, Schentag JJ, Forrest A,Moellering RC, Jr. 2004. Accessory gene regulator group II polymor-phism in methicillin-resistant Staphylococcus aureus is predictive of failureof vancomycin therapy. Clin Infect Dis 38:1700 –1705. http://dx.doi.org/10.1086/421092.

16. Tsuji BT, Rybak MJ, Lau KL, Sakoulas G. 2007. Evaluation of acces-sory gene regulator (agr) group and function in the proclivity to-wards vancomycin intermediate resistance in Staphylococcus aureus.Antimicrob Agents Chemother 51:1089 –1091. http://dx.doi.org/10.1128/AAC.00671-06.

17. Sakoulas G, Eliopoulos GM, Moellering RC, Jr, Novick RP, Venkatara-man L, Wennersten C, DeGirolami PC, Schwaber MJ, Gold HS. 2003.Staphylococcus aureus accessory gene regulator (agr) group II: is there arelationship to the development of intermediate-level glycopeptide resis-tance? J Infect Dis 187:929 –938. http://dx.doi.org/10.1086/368128.

18. Diep BA, Gill SR, Chang RF, Phan TH, Chen JH, Davidson MG, Lin F,Lin J, Carleton HA, Mongodin EF, Sensabaugh GF, Perdreau-Remington F. 2006. Complete genome sequence of USA300, an epidemic

Lenhard et al.

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clone of community-acquired methicillin-resistant Staphylococcus aureus.Lancet 367:731–739. http://dx.doi.org/10.1016/S0140-6736(06)68231-7.

19. Baba T, Takeuchi F, Kuroda M, Yuzawa H, Aoki K, Oguchi A, Nagai Y,Iwama N, Asano K, Naimi T, Kuroda H, Cui L, Yamamoto K, Hira-matsu K. 2002. Genome and virulence determinants of high virulencecommunity-acquired MRSA. Lancet 359:1819 –1827. http://dx.doi.org/10.1016/S0140-6736(02)08713-5.

20. Holden MT, Feil EJ, Lindsay JA, Peacock SJ, Day NP, Enright MC,Foster TJ, Moore CE, Hurst L, Atkin R, Barron A, Bason N, Bentley SD,Chillingworth C, Chillingworth T, Churcher C, Clark L, Corton C,Cronin A, Doggett J, Dowd L, Feltwell T, Hance Z, Harris B, Hauser H,Holroyd S, Jagels K, James KD, Lennard N, Line A, Mayes R, Moule S,Mungall K, Ormond D, Quail MA, Rabbinowitsch E, Rutherford K,Sanders M, Sharp S, Simmonds M, Stevens K, Whitehead S, Barrell BG,Spratt BG, Parkhill J. 2004. Complete genomes of two clinical Staphylo-coccus aureus strains: evidence for the rapid evolution of virulence anddrug resistance. Proc Natl Acad Sci U S A 101:9786 –9791. http://dx.doi.org/10.1073/pnas.0402521101.

21. Gumbo T, Louie A, Deziel MR, Parsons LM, Salfinger M, Drusano GL.2004. Selection of a moxifloxacin dose that suppresses drug resistance inMycobacterium tuberculosis, by use of an in vitro pharmacodynamic infec-tion model and mathematical modeling. J Infect Dis 190:1642–1651. http://dx.doi.org/10.1086/424849.

22. Tsuji BT, Brown T, Parasrampuria R, Brazeau DA, Forrest A, KelchlinPA, Holden PN, Peloquin CA, Hanna D, Bulitta JB. 2012. Front-loadedlinezolid regimens result in increased killing and suppression of the acces-sory gene regulator system of Staphylococcus aureus. Antimicrob AgentsChemother 56:3712–3719. http://dx.doi.org/10.1128/AAC.05453-11.

23. Peleg AY, Jara S, Monga D, Eliopoulos GM, Moellering RC, Jr, My-lonakis E. 2009. Galleria mellonella as a model system to study Acineto-bacter baumannii pathogenesis and therapeutics. Antimicrob Agents Che-mother 53:2605–2609. http://dx.doi.org/10.1128/AAC.01533-08.

24. Bulman ZP, Sutton MD, Ly NS, Bulitta JB, Holden PN, Nation RL, LiJ, Tsuji BT. 2015. Emergence of polymyxin B resistance influences patho-genicity in Pseudomonas aeruginosa mutators. Antimicrob Agents Che-mother 59:4343– 4346. http://dx.doi.org/10.1128/AAC.04629-14.

25. Lenhard JR, von Eiff C, Hong IS, Holden PN, Bear MD, Suen A,Bulman ZP, Tsuji BT. 2015. Evolution of Staphylococcus aureus undervancomycin selective pressure: the role of the small-colony variant phe-notype. Antimicrob Agents Chemother 59:1347–1351. http://dx.doi.org/10.1128/AAC.04508-14.

26. Casapao AM, Leonard SN, Davis SL, Lodise TP, Patel N, Goff DA,Laplante KL, Potoski BA, Rybak MJ. 2013. Clinical outcomes in patientswith heterogeneous vancomycin-intermediate Staphylococcus aureus(hVISA) bloodstream infection. Antimicrob Agents Chemother http://dx.doi.org/10.1128/aac.00380-13.

27. Casapao AM, Davis SL, McRoberts JP, Lagnf AM, Patel S, Kullar R,Levine DP, Rybak MJ. 2014. Evaluation of vancomycin population sus-ceptibility analysis profile as a predictor of outcomes for patients withinfective endocarditis due to methicillin-resistant Staphylococcus aureus.Antimicrob Agents Chemother 58:4636 – 4641. http://dx.doi.org/10.1128/AAC.02820-13.

28. De Vriese AS, Vandecasteele SJ. 2014. Vancomycin: the tale of the van-quisher and the pyrrhic victory. Peritoneal Dial Int 34:154 –161.

29. Fong RK, Low J, Koh TH, Kurup A. 2009. Clinical features and treat-ment outcomes of vancomycin-intermediate Staphylococcus aureus(VISA) and heteroresistant vancomycin-intermediate Staphylococcus au-reus (hVISA) in a tertiary care institution in Singapore. Eur J Clin Micro-biol Infect Dis 28:983–987. http://dx.doi.org/10.1007/s10096-009-0741-5.

30. Moise PA, Schentag JJ. 2000. Vancomycin treatment failures in Staphy-lococcus aureus lower respiratory tract infections. Int J Antimicrob Agents16(Suppl 1):S31–S34.

31. Rybak M, Lomaestro B, Rotschafer JC, Moellering R, Jr, Craig W,

Billeter M, Dalovisio JR, Levine DP. 2009. Therapeutic monitoring ofvancomycin in adult patients: a consensus review of the American Societyof Health-System Pharmacists, the Infectious Diseases Society of America,and the Society of Infectious Diseases Pharmacists. Am J Health SystPharm 66:82–98. http://dx.doi.org/10.2146/ajhp080434.

32. Tam VH, Louie A, Deziel MR, Liu W, Drusano GL. 2007. The relation-ship between quinolone exposures and resistance amplification is charac-terized by an inverted U: a new paradigm for optimizing pharmacody-namics to counterselect resistance. Antimicrob Agents Chemother 51:744 –747. http://dx.doi.org/10.1128/AAC.00334-06.

33. Tsuji BT, Rybak MJ, Cheung CM, Amjad M, Kaatz GW. 2007. Com-munity- and health care-associated methicillin-resistant Staphylococcusaureus: a comparison of molecular epidemiology and antimicrobial activ-ities of various agents. Diagn Microbiol Infect Dis 58:41– 47. http://dx.doi.org/10.1016/j.diagmicrobio.2006.10.021.

34. Viedma E, Sanz F, Orellana MA, San Juan R, Aguado JM, Otero JR,Chaves F. 2014. Relationship between agr dysfunction and reduced van-comycin susceptibility in methicillin-susceptible Staphylococcus aureuscausing bacteraemia. J Antimicrob Chemother 69:51–58. http://dx.doi.org/10.1093/jac/dkt337.

35. Rudkin JK, Edwards AM, Bowden MG, Brown EL, Pozzi C, Waters EM,Chan WC, Williams P, O’Gara JP, Massey RC. 2012. Methicillin resis-tance reduces the virulence of healthcare-associated methicillin-resistantStaphylococcus aureus by interfering with the agr quorum sensing system.J Infect Dis 205:798 – 806. http://dx.doi.org/10.1093/infdis/jir845.

36. Gardete S, Kim C, Hartmann BM, Mwangi M, Roux CM, Dunman PM,Chambers HF, Tomasz A. 2012. Genetic pathway in acquisition and lossof vancomycin resistance in a methicillin-resistant Staphylococcus aureus(MRSA) strain of clonal type USA300. PLoS Pathog 8:e1002505. http://dx.doi.org/10.1371/journal.ppat.1002505.

37. Craven RR, Gao X, Allen IC, Gris D, Bubeck Wardenburg J, McElvania-Tekippe E, Ting JP, Duncan JA. 2009. Staphylococcus aureus alpha-hemolysin activates the NLRP3-inflammasome in human and mousemonocytic cells. PLoS One 4:e7446. http://dx.doi.org/10.1371/journal.pone.0007446.

38. Onogawa T. 2002. Staphylococcal alpha-toxin synergistically enhancesinflammation caused by bacterial components. FEMS Immunol Med Mi-crobiol 33:15–21. http://dx.doi.org/10.1016/S0928-8244(01)00308-X.

39. Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, HerrmannM, Peters G. 2006. Small colony variants: a pathogenic form of bacteriathat facilitates persistent and recurrent infections. Nat Rev Microbiol4:295–305. http://dx.doi.org/10.1038/nrmicro1384.

40. Rose WE, Leonard SN, Rossi KL, Kaatz GW, Rybak MJ. 2009. Impact ofinoculum size and heterogeneous vancomycin-intermediate Staphylococ-cus aureus (hVISA) on vancomycin activity and emergence of VISA in anin vitro pharmacodynamic model. Antimicrob Agents Chemother 53:805– 807. http://dx.doi.org/10.1128/AAC.01009-08.

41. Lee DG, Murakami Y, Andes DR, Craig WA. 2013. Inoculum effects ofceftobiprole, daptomycin, linezolid, and vancomycin with Staphylococcusaureus and Streptococcus pneumoniae at inocula of 105 and 107 CFU in-jected into opposite thighs of neutropenic mice. Antimicrob Agents Che-mother 57:1434 –1441. http://dx.doi.org/10.1128/AAC.00362-12.

42. Lodise TP, Lomaestro B, Graves J, Drusano GL. 2008. Larger vancomy-cin doses (at least four grams per day) are associated with an increasedincidence of nephrotoxicity. Antimicrob Agents Chemother 52:1330 –1336. http://dx.doi.org/10.1128/AAC.01602-07.

43. Harigaya Y, Bulitta JB, Forrest A, Sakoulas G, Lesse AJ, Mylotte JM,Tsuji BT. 2009. Pharmacodynamics of vancomycin at simulated epitheliallining fluid concentrations against methicillin-resistant Staphylococcusaureus (MRSA): implications for dosing in MRSA pneumonia. Antimi-crob Agents Chemother 53:3894 –3901. http://dx.doi.org/10.1128/AAC.01585-08.

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