JAC Advance Access originally published online on February 8, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1099-1102; doi:10.1093/jac/dkn037
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Original research |
Teicoplanin pharmacodynamics in reference to the accessory gene regulator (agr) in Staphylococcus aureus using an in vitro pharmacodynamic model
1 Anti-Infective Research Laboratory, Department of Pharmacy Practice, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI 48201, USA; 2 Detroit Receiving Hospital and University Health Center, Detroit, MI, USA; 3 University of Wisconsin School of Pharmacy, Madison, WI 53705, USA; 4 School of Medicine, Wayne State University, Detroit, MI, USA; 5 John D. Dingell Department of Veteran’s Affairs Medical Center, Detroit, MI, USA; 6 Department of Medicine, Division of Infectious Diseases, New York Medical College, Munger Pavilion 245, Vahalla, NY 10595, USA
* Corresponding author. Tel: +1-313-993-4673; Fax: +1-313-577-8915. E-mail: m.rybak{at}wayne.edu
Received 3 August 2007; returned 8 October 2007; revised 7 January 2008; accepted 13 January 2008
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Abstract |
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Objectives: The accessory gene regulator (agr) has been identified as playing a role in the expression of reduced glycopeptide susceptibility in Staphylococcus aureus. Previous studies indicate that strains with agr dysfunctional group II polymorphism have a higher propensity for reduced vancomycin activity. We investigated this relationship in agr groups I–IV using another glycopeptide, teicoplanin, in an in vitro pharmacodynamic model.
Methods: Teicoplanin doses ranging from 1.88 to 30 mg/kg daily (fAUC/MIC 26.1–380.7) were simulated and evaluated for activity and the development of reduced susceptibility over 72 h.
Results: A dose–response relationship in activity was noted as doses escalated up to 30 mg/kg daily, but regrowth was identified with all doses. Teicoplanin doses of 3.75 and 1.88 mg/kg daily resulted in isolates with intermediate teicoplanin susceptibility (MIC = 16 mg/L) in agr groups II, IV (MIC = 16 mg/L) and III (MIC = 24 mg/L), regardless of function of the agr operon. Resistance to teicoplanin (
32 mg/L) occurred in agr group I functional and dysfunctional isolates. Minimal changes in MIC were noted with 7.5 mg/kg daily doses in agr groups II–IV. However, this dose resulted in variable susceptibility (4–24 mg/L) in agr group I+/– isolates. Higher doses of 15 and 30 mg/kg daily did not produce changes in MIC in any isolate tested.
Conclusions: Agr function did not determine teicoplanin resistance proclivity and is consistent with the previously described higher mutation rate in S. aureus to teicoplanin. Further investigation of agr group and function is warranted for all glycopeptides and compounds with a similar mechanism of action.
Keywords: quorum sensing , glycopeptides , resistance
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Introduction |
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The emergence of methicillin-resistant Staphylococcus aureus (MRSA) with heterogeneous (hGISA) and intermediate susceptibility to glycopeptides (GISA) presents new therapeutic challenges.1 A relationship has now been identified linking reduced glycopeptide susceptibility and the accessory gene regulator (agr), a quorum-sensing global regulator of virulence factors and adhesion molecules in S. aureus.2 We previously reported that although reduced susceptibility to the glycopeptide vancomycin can occur with all agr groups regardless of function, a higher propensity exists for hGISA and GISA strains to emerge from dysfunctional S. aureus, in particular agr group II.3 Only vancomycin has been investigated in this regard, and it is unknown whether other glycopeptides also display this effect.
Another glycopeptide, teicoplanin, displays a similar mechanism of action and spectrum of activity to that of vancomycin, but because of its extended half-life, it can be given as a once-daily regimen. Although previous studies have indicated that teicoplanin more easily selects for first-step mutants in S. aureus compared with vancomycin,4–6 the relationship between exposure and agr group and function in developing reduced susceptibility is unknown. The propensity towards reduced susceptibility to teicoplanin was evaluated in agr wild-type and agr dysfunctional pairs of isogenic S. aureus isolates in agr groups I–IV in an in vitro pharmacodynamic model.
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Materials and methods |
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S. aureus RN6390b, RN6607, RN3984 and RN8540, corresponding to agr functional groups I, II, III and IV, were obtained from the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA). The respective agr dysfunctional groups I, II and IV isolates RN6911, RN9120 and RN9121 were also obtained from NARSA. RN3984-M is the agr III dysfunctional derivative of RN3984. Analytical grade teicoplanin was obtained in powder form from the manufacturer.
All in vitro model experiments and susceptibility testing utilized Mueller–Hinton broth (Difco, Detroit, MI, USA) supplemented with 25 mg/L calcium and 12.5 mg/L magnesium. Colony counts were determined on Tryptic Soy Agar (Difco). Susceptibility testing was determined by broth microdilution and Etest methodology in accordance with the CLSI guidelines.7 Functional status of the agr operon was assayed qualitatively by 
-haemolysin activity as described previously.2
The in vitro model is a previously described apparatus consisting of a one-compartment (250 mL) chamber for administration and removal of organisms and antimicrobial agents.3 All therapeutic regimens were simulated over 72 h using this model in duplicate to ensure reproducibility. Regimen simulations were based on the following in vitro concentrations: Teicoplanin 30 mg/kg (targeted fCmax 16, fCmin 9.2; fAUC 304), 15 mg/kg (fCmax 8, fCmin 4.6; fAUC 152), 7.5 mg/kg (fCmax 4, fCmin 2.3; fAUC 76), 3.75 mg/kg (fCmax 2, fCmin 1.1; fAUC 37), 1.88 mg/kg (fCmax 1, fCmin 0.6; fAUC 19.3) given every 24 h (t
30 h). Free concentrations used take into account the in vivo binding of teicoplanin to proteins (
90%).
Teicoplanin concentrations were determined in duplicate between 0 and 72 h using Mueller–Hinton II agar supplemented with sodium and calcium and adjusted to an acidic pH of 5.1 ± 0.1.8 Bacillus subtilis ATCC 6633 was used as the indicator organism. The lower limit of detection of this assay is 0.19 mg/L (CV < 10%). Pharmacokinetic parameters were determined by PK Analyst software (version 1.10; MicroMath Scientific Software, Salt Lake City, UT, USA) including 24 h area-under-the-curve (AUC0–24) by linear trapezoidal methodology. Tukey’s post hoc test for multiple comparisons was used to determine differences in regimens based on log10 cfu/mL at 72 h. Comparisons with a P value of 
0.05 were considered statistically significant.
Reduced susceptibility to teicoplanin was screened at multiple time points during the antibiotic therapy simulations by plating on Brain-Heart Infusion agar containing 3x or 6x the MIC followed by 48 h of incubation. Etest methodology was used to verify changes in MIC in colonies from the screening plates.
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Results |
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All isolates prior to teicoplanin exposure were susceptible with an MIC of 2 mg/L. The mean pharmacokinetic parameters achieved with each dosing regimen are displayed in Table 1. Figure 1 displays the antistaphylococcal activity of teicoplanin over 72 h in agr II functional and dysfunction isolates, and is similar to the other agr groups and function evaluated. Teicoplanin demonstrated a dose–response relationship with doses of 30 and 15 mg/kg daily achieving the highest activity. However, regrowth was noted even with the higher dosage regimens after 24 h. Regimens ranging from 1.88 to 7.5 mg/kg daily resulted in reduced activity and higher bacterial numbers by 72 h. This activity was displayed in all regimens regardless of agr group or function.
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The susceptibility results for each isolate tested after 72 h teicoplanin exposure are displayed in Table 1. Decreased teicoplanin susceptibility was noted as early as 24 h post exposure ranging from minor (2-fold increase) to considerable (3.5-fold increase) alterations in the MIC. Teicoplanin doses of 3.75 and 1.88 mg/kg daily resulted in isolates with intermediate teicoplanin susceptibility in agr groups II, IV (MIC = 16 mg/L) and III (MIC = 24 mg/L), regardless of function of the agr operon. Isolates ranging from intermediate susceptibility to resistant (MIC
32 mg/L) to teicoplanin occurred in agr I functional and dysfunctional isolates, representing a 16-fold increase in MIC, although bacterial counts and regrowth were similar to those of other isolates evaluated. The MIC elevations were stable to passage over 3 days and corresponded to a decrease in vancomycin susceptibility, albeit to a much lesser extent (5- to 8-fold lower) than teicoplanin. Doses of 7.5 mg/kg daily resulted in MIC changes in agr groups II, III and IV functional and dysfunctional isolates (3 mg/L) and varying susceptibility in agr I isolates (4–24 mg/L). Although doses of 15 and 30 mg/kg daily displayed initial kill followed by regrowth by 72 h in all strains, no changes in MIC value were found by the end of therapy. |
Discussion |
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Glycopeptides such as vancomycin have long been utilized as the mainstay of treatment for MRSA infections. Their utility in treating these infections has recently become challenged due to the emergence of strains with reduced susceptibility. The agr locus in S. aureus has been demonstrated to play an important role in these strains. Moise-Broder et al.9 noted a significantly larger percentage of agr group II isolates in patients failing vancomycin therapy. The loss of function of the agr operon has also been reported to be linked to reduced vancomycin susceptibility by Sakoulas et al.,2 who demonstrated that a down-regulated or defective agr locus corresponded with GISA and hGISA and potential for enhanced vancomycin tolerance. A recent study employing whole genomic sequencing has identified, among a series of mutations, a loss-of-function mutation in the agr locus of MRSA treated with vancomycin and developing intermediate vancomycin resistance.1 We recently expanded upon this finding using a series of in vitro models with agr dysfunctional strains requiring 4-fold higher vancomycin fAUC/MIC (112–169) concentrations to prevent the emergence of intermediate resistance compared with agr functional strains.3
The relationship between agr group and function and resistance to teicoplanin has not been fully elucidated. Previous reports have noted that compared with vancomycin, teicoplanin more easily selects for first-step mutants in S. aureus4,6 and thus may have an increased propensity to develop hGISA. Clinical resistance to teicoplanin was first described in 1990 in the selection of a spontaneous mutant in the treatment of endocarditis.6 Further analysis of this resistant strain revealed an increase in the expression of both PBP2 and a novel 35 kDa protein in the membrane.5 The pharmacodynamic parameters of teicoplanin appear to play a significant role in perpetuating this occurrence. The high degree of protein binding, which approaches 90%, once-daily dosing and lack of aggressive clinical serum monitoring all may contribute to the potential for suboptimal teicoplanin concentrations. In our study, we were able to demonstrate a similar characteristic with teicoplanin. Intermediate susceptibility was displayed in doses as high as 7.5 mg/kg daily and fully resistant strains with 3.75 and 1.88 mg/kg daily. The finding that glycopeptide-resistant strains result from the in vitro model was not noted in previous studies with varying exposure of vancomycin.3 This higher affinity for the development of reduced susceptibility is important in the clinical detection of hGISA since teicoplanin, along with vancomycin, is used as a marker for hGISA detection.
The molecular changes involved in reduced susceptibility to teicoplanin have been evaluated by Renzoni et al. Utilizing an agr group I isolate, the investigators discovered that the derived teicoplanin-resistant strain exhibited marked reduction in RNA II and RNA III, suggesting a dysfunctional agr locus.10 Although the results of our study indicate that agr function did not determine teicoplanin resistance proclivity and thus differ from our previous results with vancomycin, this is likely attributable to the higher mutation rate associated with teicoplanin.4–6 Furthermore, it appeared that the highest MIC increases occurred with agr group I isolates. This is in contrast to our previous report with vancomycin, in which agr group II displayed the highest rate and largest MIC shifts. However, these findings are consistent with the previously described variation of teicoplanin susceptibility within S. aureus.9
The current limited treatment options for MRSA infections are alarmingly sparse.10 With the advent of new glycopeptide-like compounds in investigational use, understanding of the role agr has in the development of resistance is crucial. Investigations in this area may assist in optimizing antimicrobial selection and dosing practices, perhaps guiding patient-specific therapy based on the molecular characteristics of the infecting organism.
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Funding |
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No specific funding was received for this study.
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None to declare.
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Acknowledgements |
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A portion of this work was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007 (Abstract # A-15).
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References |
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1 Mwangi MM, Wu SW, Zhou Y, et al. Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing. Proc Natl Acad Sci USA (2007) 104:9451–6.
2
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.
3
Rose WE, Rybak MJ, Tsuji BT, et al. Correlation of vancomycin and daptomycin susceptibility in Staphylococcus aureus in reference to accessory gene regulator (agr) polymorphism and function. J Antimicrob Chemother (2007) 59:1190–3.
4 Shlaes DM, Shlaes JH. Teicoplanin selects for Staphylococcus aureus that is resistant to vancomycin. Clin Infect Dis (1995) 20:1071–3.[Web of Science][Medline]
5
Shlaes DM, Shlaes JH, Vincent S, et al. Teicoplanin-resistant Staphylococcus aureus expresses a novel membrane protein and increases expression of penicillin-binding protein 2 complex. Antimicrob Agents Chemother (1993) 37:2432–7.
6 Kaatz GW, Seo SM, Dorman NJ, et al. Emergence of teicoplanin resistance during therapy of Staphylococcus aureus endocarditis. J Infect Dis (1990) 162:103–8.[Web of Science][Medline]
7 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing—Sixteenth Edition: Approved Standard M100-S16 (2006) Wayne, PA, USA: CLSI.
8 Erickson RC, Hildebrand AR, Hoffman PF, et al. A sensitive bioassay for teicoplanin in serum in the presence or absence of other antibiotics. Diagn Microbiol Infect Dis (1989) 12:235–41.[Web of Science][Medline]
9 Moise-Broder PA, Sakoulas G, Eliopoulos GM, et al. Accessory gene regulator group II polymorphism in methicillin-resistant Staphylococcus aureus is predictive of failure of vancomycin therapy. Clin Infect Dis (2004) 38:1700–5.[CrossRef][Web of Science][Medline]
10
Renzoni A, Francois P, Li D, et al. Modulation of fibronectin adhesins and other virulence factors in a teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother (2004) 48:2958–65.
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