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JAC Advance Access published online on August 18, 2007
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm297
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Determination of disc breakpoints and evaluation of Etests for tigecycline susceptibility testing by the BSAC method

R. Hope*, T. Parsons, S. Mushtaq, D. James and D. M. Livermore

Antibiotic Resistance and Monitoring Reference Laboratory, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK

* Corresponding author. Tel: +44-20-8327-6493; Fax: +44-20-8327-6264; E-mail: russell.hope{at}hpa.org.uk

Received 25 April 2007; returned 7 June 2007; revised 12 July 2007; accepted 13 July 2007


   Abstract
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Background: Tigecycline has been recently licensed in Europe for intra-abdominal and complicated skin and soft tissue infections. We determined zone breakpoints for use with 15 µg tigecycline discs and evaluated Etests for the routine determination of tigecycline susceptibility by BSAC methods.

Methods: Disc zones for 2236 isolates and MICs by Etest for 531 isolates were compared with MICs obtained by the BSAC agar dilution method.

Results: Based on error minimization, we propose zone breakpoints for 15 µg tigecycline discs as follows: {alpha}/ß-haemolytic streptococci, S ≥ 25 mm, R ≤ 19 mm; Acinetobacter spp. and Enterobacteriaceae, S ≥ 24 mm, R ≤ 19 mm; Enterococcus spp., S ≥ 21 mm, R ≤ 20 mm; Haemophilus spp., S ≥ 28 mm, R ≤ 27; Streptococcus pneumoniae, S ≥ 24 mm, R ≤ 23 mm; and staphylococci, S ≥ 26 mm, R ≤ 25 mm. These criteria gave overall false resistance rates of ≤0.8% and false susceptibility rates of ≤0.7%. Tigecycline Etests, used on Iso-Sensitest agar, gave MICs within one doubling dilution of those by agar dilution in 97% of cases. Categorization agreement was good for isolates with borderline susceptibility or resistance—a group where Etests are likely to be used in order to verify disc-based results. MICs for highly susceptible {alpha}-haemolytic streptococci were underestimated by Etest, but this seems unlikely to be significant.

Conclusions: Disc breakpoints corresponding to BSAC MIC breakpoints were defined for 15 µg tigecycline discs and have been adopted by the BSAC. Tigecycline Etest gave results in good agreement with agar dilution.

Key Words: diagnostic tests , disc diffusion , MIC determination


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Tigecycline was registered for use in the European Union (EU) in April 2006, licensed by the European Medicines Agency (EMEA) for parenteral use in complicated skin and soft tissue infections and complicated intra-abdominal infections caused by Enterobacteriaceae (excluding Proteeae, which are resistant to tigecycline), enterococci, staphylococci and streptococci. Organisms against which it is active include Escherichia coli and Klebsiella spp. with blaCTX-M plasmids, as well as methicillin-resistant staphylococci and other multiresistant Gram-positive bacteria. Acquired ribosomal protection [tet(M)]1 and efflux [tet(A–E)]2,3 mechanisms affect tetracyclines but not tigecycline.

Tigecycline MIC breakpoints have been proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST—www.escmid.org) and adopted by BSAC, but are of limited immediate use to the UK routine microbiology departments, which mostly use disc testing. A 15 µg tigecycline disc has been proposed for routine susceptibility testing worldwide, and we calibrated zone breakpoints for these when using the BSAC disc diffusion method. In addition, we compared MICs determined by Etest with those found by the BSAC agar dilution method.


   Materials and methods
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The 15 µg tigecycline discs were from Oxoid (Basingstoke, Hants, UK) and Etests were from AB Biodisk (Solna, Sweden). Tigecycline laboratory reference powder was provided by Wyeth (Taplow, UK). Isolates from the 2004 BSAC bacteraemia surveillance (http://www.bsacsurv.org) were used; these were supplemented with more-resistant isolates from among recent reference isolates. The test panel consisted of 161 {alpha}-haemolytic streptococci, 224 ß-haemolytic streptococci, 233 Streptococcus pneumoniae, 243 Staphylococcus aureus, 185 coagulase-negative staphylococci, 235 enterococci, 29 Citrobacter spp., 228 Enterobacter spp., 257 E. coli, 284 Klebsiella spp., 74 Serratia spp., 142 Acinetobacter spp. and 102 Haemophilus spp. Controls were obtained from the National Collection of Type Cultures (NCTC; Health Protection Agency, London, UK) or the American Type Culture Collection (ATCC; Middlesex, UK) as follows: E. coli NCTC 10418 and ATCC 25922, Enterococcus faecalis ATCC 29212, S. aureus ATCC 29213, S. pneumoniae ATCC 49619 and Haemophilus influenzae NCTC 11931 and ATCC 49247.

Susceptibility tests were performed for all isolates using the BSAC disc4 and agar dilution methods.5 A proportion of isolates, selected to represent a wide distribution of MICs, was tested with Etests on Iso-Sensitest agar, using inocula corresponding to a 0.5 McFarland standard. Results were read as the point where 80% of growth was inhibited, as recommended for bacteriostatic agents.


   Results and discussion
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Disc zone breakpoint calibration

Tables 1–9 show the zones of 15 µg tigecycline discs in relation to agar dilution MICs for the various bacterial groups. Using these data, zone breakpoints were estimated, corresponding to published EUCAST/BSAC MIC breakpoints and were optimized by error-rate-bounded analysis.6


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  		Table 1. MIC correlation plot for tigecycline 15 µg discs versus {alpha}-haemolytic streptococci (modal zones for each MIC are shown in bold)

 


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  		Table 2. MIC correlation plot for tigecycline 15 µg discs versus ß-haemolytic streptococci (modal zones for each MIC are shown in bold)

 


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  		Table 3. MIC correlation plot for tigecycline 15 µg discs versus coagulase-negative staphylococci (modal zones for each MIC are shown in bold)

 


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  		Table 4. MIC correlation plot for tigecycline 15 µg discs versus S. aureus (modal zones for each MIC are shown in bold)

 


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  		Table 5. MIC correlation plot for tigecycline 15 µg discs versus enterococci (modal zones for each MIC are shown in bold)

 


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  		Table 6. MIC correlation plot for tigecycline 15 µg discs versus Enterobacteriaceae (not including Proteeae) (modal zones for each MIC are shown in bold)

 


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  		Table 7. MIC correlation plot for tigecycline 15 µg discs versus Acinetobacter spp. (modal zones for each MIC are shown in bold)

 


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  		Table 8. MIC correlation plot for tigecycline 15 µg discs versus Haemophilus spp. (modal zones for each MIC are shown in bold)

 


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  		Table 9. MIC correlation plot for tigecycline 15 µg discs versus S. pneumoniae (modal zones for each MIC are shown in bold)

 
For the Gram-positive species, we lacked tigecycline-resistant organisms, based on MIC criteria (virtually none is published) and so could only define zone breakpoints as the lower zone limits for the normal distribution of the susceptible and intermediate populations. These breakpoints were: {alpha}/ß-haemolytic streptococci, S ≥ 25 mm, R ≤ 19 mm (Tables 1 and 2); coagulase-negative staphylococci and S. aureus, S ≥ 26 mm, R ≤ 25 mm (Tables 3 and 4); and Enterococcus spp., S ≥ 21 mm, R ≤ 20 mm (Table 5).

Assigning breakpoints for the Enterobacteriaceae excluding Proteeae (Table 6) was challenging because MICs of 2 mg/L, which are intermediate by EUCAST/BSAC criteria, fall within the upper end of the normal distribution for Klebsiella spp. thus making differentiation between susceptible and intermediate isolates difficult. Error minimization analysis indicated that zone breakpoints of S ≥ 24 mm, R ≤ 19 mm produced the best agreement with MIC categorization, with only 49/872 (5.6%) isolates miscategorized and with 48 of these 49 being minor errors only. No tigecycline-resistant (MIC >2 mg/L) Enterobacteriaceae isolates were misidentified as susceptible at these zone breakpoints and only one susceptible isolate was miscategorized as resistant. These zone breakpoints thus corresponded to a false resistance rate of ≤0.8% and a false susceptibility rate of ≤0.7%, whereas the BSAC considers <5% false resistant and <1% false susceptible to be acceptable.7 This performance, it should be noted, was with a collection loaded with resistant and borderline organisms; rather better performance might be expected from a random collection, dominated by fully susceptible isolates.

The BSAC has advocated Enterobacteriaceae breakpoints for Acinetobacter spp. (http://www.bsac.org.uk), though EUCAST states that there is insufficient evidence and has not set breakpoints. We found that the present Enterobacteriaceae zone breakpoints of S ≥ 24 mm, R ≤ 19 mm were also adequate for Acinetobacter spp. (Table 7) based on the BSAC values. Clinical trials in pneumonia are ongoing and MIC breakpoints for H. influenzae and S. pneumoniae remain to be set, however we note that the values of S ≥ 28 mm, R ≤ 27 mm (Table 8) and S ≥ 24 mm, R ≤ 23 mm (Table 9), respectively, would correspond to 1 mg/L breakpoints for these species. Tigecycline MICs and zones obtained for the control strains are shown in Table 10.


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  		Table 10. Tigecycline MICs and zones for control strains

 
Tigecycline Etest evaluation

MICs by Etest and agar dilution were within experimental error (i.e. one doubling dilution) in 501/531 (94%) of cases and within two dilutions in all cases (Table 11). MICs by Etest nevertheless tended to be slightly lower than by agar dilution [lower in 32% (n = 171) of cases versus higher in 10.6% (n = 57)]. This effect was most evident for S. pneumoniae and other {alpha}-haemolytic streptococci, all of which were very susceptible, with MICs ≤0.5 mg/L. {alpha}-Haemolytic streptococci accounted for 21/26 cases where MICs by Etest were two tubes lower than by agar dilution, despite using a hand lens to seek microcolonies and the final termination of growth. Among Gram-negative isolates (n = 145) with agar dilution MICs in the range 1–4 mg/L (i.e. spanning the ‘top end’ of susceptible, through intermediate, to the ‘bottom end’ of resistant), categorization agreement between agar dilution and Etest MICs was 81%, with 19% minor errors, one major and no very major errors.


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  		Table 11. Comparison between MICs of tigecycline (TGC) determined by agar dilution and Etest

 
Conclusions

The 15 µg tigecycline discs differentiated adequately between susceptible, intermediate and resistant isolates for most species; though differentiation of the susceptible/intermediate border was difficult for Klebsiella spp. It may be prudent to do MIC determinations (e.g. by gradient diffusion test) on isolates giving zones around this border. Etests were shown to have good correlation with agar dilution MICs, including around this border.

We are currently conducting a survey of tigecycline activity in the UK with multiple sites providing disc tests using these breakpoints. The results from this study may allow us to further optimize the values proposed here.


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All funding for this work was provided by Wyeth.


   Transparency declarations
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All authors work in the field of antibiotic resistance and could be considered to have vested interests in investment in this area, whether by governments or charities or industry. D. M. L. is on a speakers’ bureau for Wyeth and has undertaken contract research for Oxoid and Bio-stat, both of which have major commercial interests in susceptibility testing.


   Acknowledgements
 
We thank Wyeth for sponsoring this work and the BSAC Bacteraemia Resistance Surveillance Programme for providing many of the isolates used. Aspects of this work were presented as posters at the Forty-sixth Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, and the Seventeenth European Congress of Clinical Microbiology and Infectious Diseases and Twenty-fifth International Congress of Chemotherapy, Munich, Germany. Many thanks to Dr Jenny Andrews for helpful comments on the manuscript.


   References
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1 . Petersen PJ, Jacobus NV, Weiss WJ, et al. In vitro and in vivo antibacterial activities of a novel glycylcycline, the 9-t-butylglycylamido derivative of minocycline (GAR-936). Antimicrob Agents Chemother (1999) 43:738–44.[Abstract/Free Full Text]

2 . Fluit AC, Florijn A, Verhoef J, et al. Presence of tetracycline resistance determinants and susceptibility to tigecycline and minocycline. Antimicrob Agents Chemother (2005) 49:1636–8.[Abstract/Free Full Text]

3 . Hirata T, Saito A, Nishino K, et al. Effects of efflux transporter genes on susceptibility of Escherichia coli to tigecycline (GAR-936). Antimicrob Agents Chemother (2004) 48:2179–84.[Abstract/Free Full Text]

4 . Andrews JM. BSAC standardized disc susceptibility testing method (version 6). J Antimicrob Chemother (2007) 60:20–41.[Free Full Text]

5 . Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother (2001) 48(Suppl 1):5–16.[Abstract]

6 . Metzler CM, DeHaan RM. Susceptibility tests of anaerobic bacteria: statistical and clinical considerations. J Infect Dis (1974) 130:588–94.[Web of Science][Medline]

7 . MacGowan AP, Wise R. Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. J Antimicrob Chemother (2001) 48(Suppl 1):17–28.[Abstract]


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