JAC Advance Access originally published online on April 26, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):186-189; doi:10.1093/jac/dkl151
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Comparison of four methods for detection of teicoplanin resistance in methicillin-resistant Staphylococcus aureus
1 Department of Clinical Microbiology, University College London Hospitals, Windeyer Institute of Medical Sciences 46 Cleveland Street, London W1T 4JF, UK 2 Antibiotic Reference and Monitoring Reference Laboratory, Health Protection Agency, Centre for Infections Colindale, UK
*Corresponding author. Tel: +44-207-380-9516; Fax: +44-207-636-6482; E-mail: peter.wilson{at}uclh.nhs.uk
Received 11 February 2006; returned 21 March 2006; revised 28 March 2006; accepted 30 March 2006
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Abstract |
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Objectives: To determine which method of determining the MIC of teicoplanin produces a result closely related to outcome in the critically ill patient.
Methods: Four methods of teicoplanin susceptibility testing—disc diffusion, Etest, VITEK (Legacy and VITEK 2) and agar incorporation—were compared for 47 methicillin-resistant Staphylococcus aureus (MRSA) isolates from invasive intensive care unit (ICU) infections and 83 isolates from ICU patients colonized with the organism. Clinical outcome was recorded prospectively for all the patients. Another 13 reference laboratory strains of MRSA with reduced susceptibility to teicoplanin were tested.
Results: Both VITEK systems failed to demonstrate resistance in the three isolates identified as resistant by Etest or agar incorporation, and disc testing detected only one resistant isolate. A higher MIC, as found by Etest or agar incorporation, was associated with lower survival (n = 130, 95% CI –0.082 to –0.006, P = 0.023, Etest; n = 130, 95% CI –0.156 to –0.020, P = 0.011, agar). The findings for the 13 reference strains were similar, with a 
4-fold reduction in MIC between agar incorporation or Etest and VITEK2 for six isolates.
Conclusions: Neither disc diffusion nor the VITEK systems are reliable for detection of teicoplanin resistance in MRSA. Etest and agar incorporation remain the methods of choice.
Keywords: S. aureus , Etest , agar incorporation
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Introduction |
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Despite experience over 15 years, the correct dosage of teicoplanin remains controversial, as does the need for routine monitoring of serum levels. Harding et al.1 suggested that only 20% of patients were cured if the trough serum concentrations of teicoplanin were below 5 mg/L, compared with 90% if they exceeded 25 mg/L. Nevertheless, although it results in trough levels of only 5 to 10 mg/L, the standard regimen (6 mg/kg/day) was not associated with inferior cure rates in comparison with linezolid in a double-blind trial.2,3 In any event, cost prohibits many centres from undertaking routine assay of levels or from greatly increasing dosage, although there is little or no concern over toxicity.
In these circumstances, it is important to use a susceptibility test method that can reliably detect resistance and predict patient outcome. Unfortunately, detection of teicoplanin resistance by conventional disc diffusion methods is difficult because of limited diffusion of its large molecule in agar;4 moreover, resistance demonstrated by one method often is not confirmed by another.5 These problems have become more pressing with the appearance of glycopeptide-intermediate resistant Staphylococcus aureus (GISA), especially since more of these have reduced susceptibility or frank resistance to teicoplanin than to vancomycin itself. We examined isolates of methicillin-resistant S. aureus (MRSA) collected from critically ill patients over a 1 year period, testing their susceptibility to teicoplanin by four methods and comparing with outcome.
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Materials and methods |
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Isolates of MRSA were collected, with ethical approval, over 1 year in the intensive care unit (ICU) at this hospital as part of a separate study, which had the approval of the UCLH Ethics Committee.6 In addition to clinical samples, all patients were screened on admission and at weekly intervals during ICU stay to detect colonization with MRSA. Isolates from patients with MRSA infection (i.e. those who required treatment) were separated from those representing colonization (who required no treatment). Data concerning treatment and survival were collected prospectively for all patients. A separate collection of 13 S. aureus isolates from the Antibiotic Reference and Monitoring Reference Laboratory, Health Protection Agency Centre for Infection, was also tested. These organisms were clinical isolates for which the MIC of teicoplanin was determined by agar incorporation to be >4 mg/L. There were no clinical outcome data available for the source patients of these organisms.
Etest method
The high-inoculum Etest method is recommended by the manufacturer to detect intermediate glycopeptide susceptibility (AB Biodisk, Solna, Sweden). A bacterial suspension equivalent to a 2 McFarland standard (200 µL) was spread on to the surface of brain heart infusion agar (Oxoid, Basingstoke, UK). One Etest teicoplanin strip was applied per plate and incubated for 48 h in air at 35–37°C. Results were read at the point of complete inhibition of growth. The standard method was also used, with inocula based on a 0.5 McFarland standard suspension applied to Mueller–Hinton agar (Oxoid) and incubated for 48 h.
BSAC disc diffusion method
A bacterial suspension in saline, at a turbidity equivalent to that of a 0.5 McFarland standard, was prepared using a colorimeter. A 1:10 dilution was inoculated onto Iso-Sensitest agar (Oxoid) using a sterile cotton swab. Teicoplanin 30 µg discs (Oxoid) were applied, and the plates were incubated at 35–37°C for 18–20 h in air.
VITEK automated identification and susceptibility testing
Four morphologically similar colonies from an overnight agar plate culture were suspended in 3 mL of 0.45% saline and adjusted to a turbidity equivalent to that of a 0.5 McFarland standard using a colorimeter. A 1:10 dilution was loaded into VITEK (bioMérieux, Marcy l'Étoile, France) susceptibility cards. VITEK Legacy and VITEK 2 systems were tested. The results are reported relative to a BSAC breakpoint for teicoplanin of 4 mg/L; actual MIC values are generated only if they exceed 4 mg/L.
Agar incorporation method
Five well-isolated viable colonies from an overnight culture were inoculated into 5 mL of nutrient broth, which then was incubated at 37°C for 24 h or overnight. These broths were used to inoculate Sensitivity Test Agar plates containing teicoplanin at doubling concentrations from 0.5 to 64 mg/L using a pre-sterilized 96-pin multi-point inoculator delivering 0.3 µL per spot. The plates were incubated at 37°C in air for 18–24 h and MICs were defined as the lowest concentrations to inhibit visible growth.
Interpretation
For BSAC disc testing, a zone size 15 mm was taken as susceptible and <15 mm as resistant. For Etests used according to the manufacturer's directions, Clinical and Laboratory Standards Institute (CLSI) criteria are recommended to define teicoplanin susceptibility: an MIC of 8 mg/L is taken as susceptible, 16 mg/L as intermediate and 32 mg/L as resistant.7 For the VITEK systems, an MIC 
4 mg/L was taken as susceptible and >4 mg/L as intermediate or resistant. For agar incorporation, as recommended by the BSAC, an MIC 4 mg/L was taken as susceptible, an MIC = 8 mg/L as intermediate and MIC 16 mg/L as resistant. For purposes of comparison, all methods deem an MIC of 4 mg/L as susceptible, and this was taken as a general reference point.
Statistical analyses
Relationships between categorical data were tested by the 
2 test and analysis of variance (ANOVA) was applied to regression.
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Results |
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The test group comprised 47 patients who were treated for invasive MRSA infections, whilst the control group had 83 patients who were found to carry MRSA on routine screening with no clinical infection. The high-inoculum Etest identified five isolates with reduced teicoplanin susceptibility but only three of these were identified as requiring MICs > 4 mg/L by agar incorporation and two by low-inoculum Etest whereas three of the five were deemed susceptible (MIC
4 mg/L) by the low-inoculum test. VITEK (Legacy or 2) failed to demonstrate resistance or MIC > 4 mg/L in any isolate and disc testing detected only one resistant isolate (Table 1). MICs determined by agar incorporation and the low-inoculum Etest were similar and usually 4-fold lower than those determined by the high-inoculum Etest (Table 2).
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Death during hospital admission occurred in 21 of 47 (45%) test patients and 22 of 83 (27%) control patients. Using regression analysis there was no significant relationship between survival (1,0) and disc diffusion zone (n = 130, P = 0.47). However, a higher MIC measured by high-inoculum Etest or agar incorporation was associated with lower survival (n = 130, 95% CI –0.082 to –0.006, P = 0.023) (n = 130, 95% CI –0.156 to –0.020, P = 0.011). Using
2 tests (df = 4), the relationship between mortality and MIC was still significant as measured by high-inoculum Etest but not agar incorporation (2 11.4, df = 4, P < 0.025) (Table 2).
Among the 13 ‘teicoplanin-resistant’ isolates of MRSA from the ARMRL collection, the MIC by agar incorporation was 8 mg/L (intermediate) for eight isolates and 16 mg/L (resistant) for five (Table 1). Etest MICs, at standard inocula, were similar to those by agar incorporation, with only two being one dilution lower whereas BSAC disc susceptibility testing detected resistance in only one isolate. The VITEK 2 reported eight isolates as requiring MICs 4 mg/L and five with MICs of 8 mg/L. Among the isolates deemed susceptible by VITEK 2, MICs were 16-fold lower than by agar incorporation or Etest in three cases.
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Discussion |
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This study confirms the limitations of disc susceptibility testing for MRSA with teicoplanin, and demonstrates that the VITEK is unreliable for this purpose. Almost half of the isolates with reduced teicoplanin susceptibility were reported as susceptible by VITEK, in some cases with an MIC at 16-fold lower than by Etest or agar incorporation. The VITEK Legacy similarly failed to detect staphylococcal resistance to vancomycin in most Italian laboratories.8 By contrast, Etest (high inoculum) and agar incorporation both produced results predictive of poor clinical outcome.
The failure of disc testing is unsurprising; after a 6 h incubation using 30 µg discs, only 20–29% of the teicoplanin diffuses into the agar, compared with 72–74% of vancomycin.4 Moreover, as large molecules, both agents diffuse slowly. The Etest, in contrast, only requires limited antibiotic diffusion into agar to produce a stable and continuous antibiotic gradient beneath the strip and appears more reliable, especially in the high-inoculum test now widely used to seek GISA in diagnostic laboratories.9 This high-inoculum Etest method optimizes the chances of detecting resistance but does not give accurate MICs, whereas the standard Etest method (0.5 McFarland standard inoculum) can fail to detect low-level glycopeptide resistance.9 Thus, with a series of MRSA isolates from blood, the standard Etest found teicoplanin MICs 6 mg/L for 10/189 (5.2%) cases compared with 19/189 (10.1%) for the high-inoculum test.5 As a means of distinguishing GISA and hetero-GISA from MRSA, Etest has been shown to perform well with a sensitivity of 96% and specificity of 97% at a suspension density equivalent to that of a 2.0 McFarland standard.9
We conclude that heavy-inoculum Etest or agar incorporation should be used by clinical laboratories to detect teicoplanin resistance, which may be linked to poor outcome. Laboratories using disc or VITEK should consider alternative methods for teicoplanin. These data mirror those with vancomycin-intermediate S. aureus but are important because teicoplanin resistance is more likely to be encountered than intermediate vancomycin resistance among clinical S. aureus, being much more widely scattered, though still rare in absolute terms.10
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There are no conflicts of interest of any financial or other nature. The work was performed as part of an MSc project at Barts & the London School of Medicine. The original clinical trial published previously was supported by an educational unrestricted grant by Pfizer, UK.
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Acknowledgements |
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The original study was sponsored by an unrestricted educational grant from Pfizer.
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References |
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1 Harding I, MacGowan AP, White LO, et al. (2000) Teicoplanin therapy for Staphylococcus aureus septicaemia: relationship between pre-dose serum concentrations and outcome. J Antimicrob Chemother 45:835–41.
2
Cepeda JA, Whitehouse T, Cooper B, et al. (2004) Linezolid versus teicoplanin in the treatment of Gram-positive infections in the critically ill: a randomized, double-blind, multicentre study. J Antimicrob Chemother 53:345–55.
3
Whitehouse T, Cepeda JA, Shulman R, et al. (2005) Pharmacokinetic studies of linezolid and teicoplanin in the critically ill. J Antimicrob Chemother 55:333–40.
4 Cavenaghi LA, Biganzoli E, Danese A, et al. (1992) Diffusion of teicoplanin and vancomycin in agar. Diagn Microbiol Infect Dis 15:253–8.[Medline]
5 Bernard L, Vaudaux P, Rohner P, et al. (2004) Comparative analysis and validation of different assays for glycopeptide susceptibility among methicillin-resistant Staphylococcus aureus strains. J Microbiol Methods 57:231–9.[Medline]
6 Cepeda JA, Whitehouse T, Cooper B, et al. (2005) Isolation of patients in single rooms or cohorts to reduce spread of MRSA in intensive-care units: prospective two-centre study. Lancet 365:295–304.[Web of Science][Medline]
7 Schito GC, Auckenthaler R, Marchese A, et al. (1999) European survey of glycopeptide susceptibility in Staphylococcus spp. Clin Microbiol Infect 5:547–53.[Medline]
8 Jones ME, Gesu G, Ortisi G, et al. (2002) Proficiency of Italian clinical laboratories in detecting reduced glycopeptide susceptibility in Enterococcus and Staphylococcus spp. using routine laboratory methodologies. Clin Microbiol Infect 8:101–11.[Medline]
9
Walsh TR, Bolmstrom A, Qwarnstrom A, et al. (2001) Evaluation of current methods for detection of staphylococci with reduced susceptibility to glycopeptides. J Clin Microbiol 39:2439–44.
10 Johnson AP, Henwood C, Mushtaq S, et al. (2003) Susceptibility of Gram-positive bacteria from ICU patients in UK hospitals to antimicrobial agents. J Hosp Infect 54:179–87.[CrossRef][Web of Science][Medline]
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