JAC Advance Access originally published online on April 13, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1190-1193; doi:10.1093/jac/dkm091
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Correlation of vancomycin and daptomycin susceptibility in Staphylococcus aureus in reference to accessory gene regulator (agr) polymorphism and function
1 Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy and Health Sciences 2 Detroit Receiving Hospital and University Health Center 3 School of Medicine, Wayne State University, Detroit, MI 48201, USA 4 School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, NY 14260, USA 5 John D. Dingell VA Medical Center, Detroit, MI 48201, USA 6 Department of Medicine, Division of Infectious Diseases, New York Medical College, Munger Pavilion 245, Valhalla, NY 10595, USA
* Corresponding author. Tel: +1-313-993-4673; Fax: +1-313-577-8915; E-mail: m.rybak{at}wayne.edu
Received 20 November 2006; returned 31 January 2007; revised 5 March 2007; accepted 6 March 2007
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Abstract |
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Objectives: Recently, an association between the accessory gene regulator (agr) in Staphylococcus aureus and the development of vancomycin resistance secondary to suboptimal exposure has been demonstrated. We investigated the relationship of vancomycin with and without gentamicin or rifampicin, and daptomycin in the development of resistance in agr groups I and II.
Methods: S. aureus belonging to agr groups I and II was exposed to varying concentrations of vancomycin and daptomycin simulating an fAUC/MIC of 14–460 and 30–239, respectively, in an in vitro pharmacodynamic model.
Results: Vancomycin regimens resulting in fAUC/MIC of 16.1–107.0 resulted in resistance in agr I and agr II knockout strains, whereas regimens resulting in fAUC/MIC of 16.1 resulted in emergence of resistance in agr I- and agr II-positive strains. Overall, agr-null strains demonstrated a higher likelihood of resistance and a greater change in vancomycin susceptibility. The addition of gentamicin and rifampicin to vancomycin at these same exposures prevented the emergence of resistance. At extremely low daptomycin exposures of fAUC/MIC of 22–66, an increase in MIC of 2–3-fold up to a maximum of 0.75 mg/L was observed. However, this was independent of agr group and/or function and still within the susceptible range of daptomycin.
Conclusions: The combination of vancomycin with either rifampicin or gentamicin prevented the emergence of vancomycin resistance in agr I and II S. aureus isolates. Changes in daptomycin susceptibility were independent of agr group and function. The association between agr and vancomycin resistance in S. aureus requires further investigation.
Keywords: S. aureus , pharmacodynamics , vancomycin resistance
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Introduction |
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The accessory gene regulator (agr) of Staphylococcus aureus is a global regulator that modulates the expression of numerous virulence factors in a growth phase-dependent manner.1 Previous studies have demonstrated a relationship between loss of agr function in an agr group II strain and attenuated bactericidal activity. Although agr group II isolates predominate in the hospital, we have documented the incorporation of agr group I strains into this setting.2 We have noted that all agr groups develop intermediate resistance to vancomycin with subtherapeutic exposures, with a higher propensity for dysfunctional isolates to display reduced susceptibility.3 This led us to evaluate the relationship of vancomycin with and without gentamicin or rifampicin and the development of intermediate-level vancomycin heteroresistance. The effect of varying daptomycin exposures on the emergence of daptomycin resistance with these agr pairs was also evaluated.
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Materials and methods |
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RN6607 and RN9120, corresponding to agr II-positive and -null, respectively, were obtained from the Network on Antimicrobial Resistance in S. aureus. Methicillin-resistant Staphylococcus aureus (MRSA) 3436 and 3402, corresponding to agr I-positive and -null clinical isolates, respectively, were obtained from Detroit Receiving Hospital and University Health Center. Powdered vancomycin, gentamicin and rifampicin of analytical grade were commercially purchased (Sigma, St Louis, MO, USA). Daptomycin analytical powder was provided by the manufacturer (Cubist Pharmaceuticals, Lexington, MA, USA).
Mueller–Hinton broth (Difco, Detroit, MI, USA) supplemented with 25 mg/L calcium and 12.5 mg/L magnesium (CAMHB) was used for in vitro pharmacodynamic models and susceptibility testing involving vancomycin with and without gentamicin and rifampicin. Owing to the dependency of daptomycin on calcium for its activity, all simulations with daptomycin were performed in the presence of CAMHB containing 50 mg/L calcium. Colony counts were determined on tryptic soy agar (TSA; Difco). MICs were determined by broth microdilution according to CLSI (formerly NCCLS) standards and confirmed by the Etest.4 The function of the agr operon was assessed as described previously.1
A previously described in vitro pharmacodynamic model (250 mL) was utilized and simulations were conducted over 72 h in duplicate to ensure reproducibility.3,5 A two-compartment hollow fibre model (FiberCell Systems Inc., Frederick, MD, USA) was used for resistance breakpoint verification experiments.6 The following regimens were evaluated: vancomycin 62.5–2000 mg every 12 h (fAUC/MIC24 14–450; t1/2 6 h); daptomycin 0.75–6 mg/kg every 24 h (fAUC/MIC24 30–239; t1/2 8 h); gentamicin 5 mg/kg every 24 h (Cmax 15 mg/L; t1/2 3 h) and rifampicin 300 mg every 8 h (Cmax 4–5 mg/L; t1/2 3 h). Protein binding levels of 55% and 92% were utilized for vancomycin- and daptomycin-free concentration exposures, respectively.5,7
Antibiotic concentrations were determined in duplicate in each model between 0 and 72 h. Vancomycin and gentamicin concentrations were determined using fluorescence polarization immunoassay (Abbott Diagnostics TDx). Concentrations of daptomycin and rifampicin were determined using a microbioassay with Micrococcus luteus ATCC 9341.5 For those regimens outside the bioassay range, pharmacokinetics were extrapolated from known regimens. The AUC was determined using the linear trapezoidal method. Differences between regimens in log10 cfu/mL at 72 h were determined using analysis of variance with Tukey's test for multiple comparisons. For all experiments, a P value of 
0.05 was considered statistically significant.
The emergence of resistance was screened for at multiple time points throughout the simulations. Samples were plated on brain heart infusion agar (for vancomycin) or on Mueller–Hinton agar supplemented with 50 mg/L calcium (for daptomycin) containing 3 x and 6 x MIC. Susceptibility was confirmed by the Etest to detect subsequent changes in MIC.
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Results and discussion |
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Pre-exposure MICs of vancomycin for agr I-positive and -null and agr II-positive and -null isolates were 0.75/1 and 1/1 mg/L, respectively. All isolates were susceptible to daptomycin (MIC 0.25 mg/L). Observed pharmacokinetic parameters (±SD) for vancomycin and daptomycin are shown in Table 1.
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The effect of varying vancomycin concentrations on killing and the emergence of resistance in agr-null group II are displayed in Figure 1. A dose–response relationship was noted throughout the 72 h testing period against all strains tested. An fAUC/MIC of 16.1 resulted in the development of resistance, with detection of 4–8-fold changes in the MIC with all strains. The agr-null isolates displayed higher MIC changes at this dose when compared with agr-positive isolates, regardless of agr type with MICs as high as 8 mg/L (8-fold) (Table 1). Only regimens with an fAUC/MIC greater than 107.0 were able to suppress resistance emergence. The agr-positive isolates displayed up to a 4-fold increase in MIC with an fAUC/MIC of 16.1 and 31.2. Verification of resistance was confirmed in the hollow fibre model by reproducing the lowest concentration that resulted in resistance from the PK/PD model. Resistance breakpoints were conducted with simulation of an fAUC/MIC of 16.1 for vancomycin, resulting in an MIC of 8 mg/L.
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The combination of gentamicin and vancomycin displayed in Figure 1 resulted in bacterial killing at or near detection limits against agr I- and II-null and -positive strains. Vancomycin plus gentamicin produced no resistance in any regimen or strain. The combination of rifampicin and vancomycin resulted in improved activity and less regrowth when compared with vancomycin alone only with the agr I-null strain, displaying a 2-fold increase in MIC (3 mg/L) at 72 h. No MIC changes were detected in the agr I- and II-positive and agr II-null strains with the addition of rifampicin.
The pharmacodynamic effects of daptomycin exposures of 0.75–6 mg/kg against RN9120 are displayed in Figure 1. Subtherapeutic regimens of 0.75, 1.5 and 3 mg/kg every 24 h (fAUC/MIC 22, 66 and 132) demonstrated early bactericidal kill followed by considerable regrowth in all isolates tested. Minimal regrowth was noted with 6 mg/kg every 24 h (fAUC/MIC 265). Increased MIC values were observed at suboptimal doses irrespective of agr type or function. Regimens with an fAUC/MIC of 22 and 66 resulted in an MIC value of 0.75 and 0.5 mg/L (3- and 2-fold increase), respectively, in the agr II-null strain. Similar results were noted with these regimens in the other isolates. All isolates recovered were susceptible to daptomycin (MIC 1 mg/L). In the hollow fibre model, the fAUC/MIC of 22 had no effect on inoculum reduction, resulting in no changes in the MIC.
Many factors have been associated with vancomycin treatment failures. In one study, higher rates of morbidity were correlated with patients infected with hGISA strains. In addition, these patients were more likely to have a high bacterial load and a low initial vancomycin concentration (trough 10 mg/L).8 Factors correlating with reduced susceptibility to vancomycin have been verified in other in vivo and in vitro settings.9 The penetration of vancomycin into sequestered sites of infection such as pneumonia and endocarditis presents a difficult challenge to maintain therapeutic concentrations.7 Our findings suggest that vancomycin concentrations correlating with an fAUC/MIC of 107.0 (fCmin 2.4 mg/L) or lower did not suppress the emergence of resistance in an agr II-null strain. All strains, regardless of agr type and function, exhibited resistance with the fAUC/MIC 16.1 regimen.
The agr group and function has been noted to play an important role in the development of vancomycin heteroresistance. In vitro, hGISA has occurred in agr II-null populations exposed to subtherapeutic vancomycin concentrations, and loss of agr function may prove advantageous for organism survival.1 We have demonstrated the tendency of increased vancomycin resistance in all agr-null groups.3 This is important in hospital settings where we have reported that up to 48% of hospital-associated MRSA had defective agr function when compared with only 3.5% of community-associated MRSA strains.2
In this study, we were able to reduce the emergence of resistance at doses with an fAUC/MIC 
165.6 and to minimize or eliminate the emergence of vancomycin resistance with the addition of rifampicin or gentamicin. Although controversy exists regarding the risks and benefits of adding gentamicin or rifampicin to vancomycin to improve patient outcome,10 our results would suggest that these combinations may prevent the emergence of vancomycin resistance secondary to suboptimal exposures in serious infections such as pneumonia, bacteraemia or endocarditis. Suboptimal daptomycin exposures did not result in daptomycin resistance and increased MIC values did not correlate with agr group or function, which may be an advantage to the use of daptomycin against these strains.
The relationship between agr function and daptomycin was important to explore given recent reports suggesting that vancomycin MIC elevations found in GISA strains may correlate with reduced daptomycin susceptibility. It is important to note that our studies found no relationship with agr group or function and daptomycin susceptibility. Although addressing the most clinically relevant MRSA (USA100 agr II and USA300 agr I), this study was limited by not evaluating groups III and IV. The association between agr group, function, vancomycin resistance and potential therapeutic modalities to prevent resistance in S. aureus requires further investigation. Furthermore, the availability of several therapeutic agents to treat MRSA obligates further study to determine specific niches for these drugs with respect to variables such as vancomycin susceptibility, host factors and site of infection.
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Transparency declarations |
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M. J. R. and G. S. serve as consultants and have obtained grant funding from Cubist Pharmaceuticals. G. W. K. has obtained grant funding from Cubist Pharmaceuticals. The other authors have nothing to declare.
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References |
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1 Sakoulas G, Eliopoulos GM, Moellering RC Jr, et al. Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin. Antimicrob Agents Chemother (2002) 46:1492–502.
2 Tsuji BT, Rybak MJ, Cheung CM, et al. Community- and health care-associated methicillin-resistant Staphylococcus aureus: a comparison of molecular epidemiology and antimicrobial activities of various agents. Diagn Microbiol Infect Dis (2007) in press.
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Tsuji BT, Rybak MJ, Lau KL, et al. Evaluation of accessory gene regulator (agr) group and function in the proclivity towards vancomycin intermediate resistance in Staphylococcus aureus. Antimicrob Agents Chemother (2007) 51:1089–91.
4 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing Sixteenth Edition: Approved Standard M100-S16 (2006) Wayne, PA, USA: CLSI.
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Huang V, Rybak MJ. Evaluation of daptomycin activity against Staphylococcus aureus in an in vitro pharmacodynamic model under normal and simulated impaired renal function. J Antimicrob Chemother (2006) 57:116–21.
6 Rybak MJ, Allen GP, Hershberger E. In vitro antibiotic pharmacodynamic models. In: Antimicrobial Pharmacodynamics in Theory and Clinical Practice—Nightingale CH, Murakawa T, Ambrose PG, eds. (2002) New York, USA: Marcel Dekker, Inc. 48–51.
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Cruciani M, Gatti G, Lazzarini L, et al. Penetration of vancomycin into human lung tissue. J Antimicrob Chemother (1996) 38:865–9.
8 Charles PG, Ward PB, Johnson PD, et al. Clinical features associated with bacteremia due to heterogeneous vancomycin-intermediate Staphylococcus aureus. Clin Infect Dis (2004) 38:448–51.[CrossRef][ISI][Medline]
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Sakoulas G, Moise-Broder PA, Schentag J, et al. Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillin-resistant Staphylococcus aureus bacteremia. J Clin Microbiol (2004) 42:2398–402.
10 Levine DP, Fromm BS, Reddy BR. Slow response to vancomycin or vancomycin plus rifampin in methicillin-resistant Staphylococcus aureus endocarditis. Ann Intern Med (1991) 115:674–80.[CrossRef][ISI][Medline]
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