Skip Navigation


JAC Advance Access originally published online on August 10, 2006
Journal of Antimicrobial Chemotherapy 2006 58(4):864-867; doi:10.1093/jac/dkl330
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/4/864    most recent
dkl330v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Tan, T. Y.
Right arrow Articles by Ng, L. S. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tan, T. Y.
Right arrow Articles by Ng, L. S. Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2006. 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

Comparison of three standardized disc susceptibility testing methods for colistin

Thean Yen Tan* and Lily Siew Yong Ng

Laboratory Medicine Services, Changi General Hospital 2 Simei Street 3, Singapore 529889

*Corresponding author. Tel: +65-68504934; Fax: +65-64269507; E-mail: thean_yen_tan{at}cgh.com.sg

Received 14 May 2006; returned 15 June 2006; revised 18 July 2006; accepted 18 July 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Objectives: With increasing antibiotic resistance in Gram-negative bacteria, the use of the polymyxins has increased in recent years. Antibiotic disc susceptibility testing remains the most widely used method in clinical laboratories, but there is very little data on the accuracy of disc testing methods for colistin. In this study, the accuracy of three standardized methods of disc susceptibility testing for colistin was compared with agar dilution.

Methods: A total of 228 clinical isolates of Acinetobacter spp., Pseudomonas aeruginosa and Enterobacteriaceae were included in the study. Isolates were tested by agar dilution for susceptibility to colistin, and results were compared with those obtained by three disc susceptibility testing methods (product insert based on CLSI methodology, British BSAC and French SFM).

Results: Colistin displayed good activity against Acinetobacter spp., Klebsiella spp. and Escherichia coli (MIC90 2 mg/L) but was less active against P. aeruginosa (MIC90 4 mg/L) and Enterobacter spp. (MIC90 ≥ 128 mg/L). Totally, 81%, 79% and 89% of colistin-resistant isolates were falsely reported as susceptible when tested by the product insert, BSAC and SFM testing methods, respectively. There were no false-resistant results.

Conclusions: Disc susceptibility testing methods are unreliable at detecting colistin resistance. Dilution methods should be the method of choice for susceptibility testing of colistin.

Keywords: disc diffusion , susceptibility , resistance , antimicrobial activity , polymyxin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
Colistin is a polymyxin with bactericidal activity against most strains of Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter spp. Therapeutic use of parenteral colistin was minimal due to concerns about the high incidence of side effects, notably nephrotoxicity. The recent increase in multidrug-resistant strains of Gram-negative bacilli, particularly P. aeruginosa and Acinetobacter spp., has prompted renewed interest in the use of colistin.

Disc susceptibility testing methods remain the most commonly used techniques in clinical microbiology laboratories. Interpretative criteria for disc susceptibility testing of colistin are not available from the CLSI and zone size interpretations are made based on product literature. Gales et al.1 recommended the use of amended zone diameters for better correlation with reference methods. In contrast, European guidelines for colistin disc susceptibility testing are published by the British Society for Antimicrobial Chemotherapy (BSAC), the Société Française de Microbiologie (SFM) and the German Deutsches Institut für Normung (DIN).

The present study set out to evaluate the accuracy of disc susceptibility methods for colistin using a method based on the product insert2 and published literature,1 and information available from BSAC3 and SFM.4


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Transparency declarations
 References
 
Bacterial strains

A total of 228 non-duplicate isolates of Acinetobacter spp., P. aeruginosa and Enterobacteriaceae were randomly collected from clinical specimens over an 18 month period from July 2004, with an emphasis on multiresistant strains. Bacterial identification was performed using standard laboratory methods and commercial identification kits.

Agar dilution method

MICs of colistin were obtained by the agar dilution method, performed according to CLSI methods.5 Colistin sulphate powder (Sigma-Aldrich, Singapore) was dissolved in sterile water and added to molten Mueller–Hinton II agar (Becton-Dickinson, USA) to provide 2-fold concentrations ranging from 0.25 to 128 mg/L. Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were used as quality control, and test values obtained were in line with published standards.6

Disc diffusion method

Three methods of disc diffusion susceptibility testing using the Kirby Bauer inoculation method were performed. In brief, bacterial suspensions were first adjusted to a turbidity equivalent to that of a 0.5 McFarland standard using a calibrated turbidimeter, followed by dilutions of the test suspensions (where appropriate) prior to inoculation of the suspension onto recommended media. Details of the differing parameters used for each method are listed in Table 1. Quality control performed using the previously listed ATCC strains was within recommended limits for each batch of testing. Following incubation, zone diameters were measured and recorded. Categorical interpretations were made with reference to the appropriate standards (see Table 1).


View this table:
[in this window]
[in a new window]

  		Table 1. Parameters used for disc diffusion testing

 
Analysis of results

Zone diameters and MIC values were entered in Excel (Microsoft, USA) and WHONET (WHO). Calculation of MIC distributions, categorical interpretations and categorical errors were performed in both Excel and WHONET.

The categorical interpretation for each isolate obtained by the three disc testing methods was compared with the categorical interpretation obtained by agar dilution, using the resistance breakpoints specified by each method. A very major error was defined when isolates were catagorized as susceptible by disc diffusion but resistant by agar dilution and a major error was defined as organisms that were resistant by disc diffusion but susceptible by agar dilution. A categorical interpretation of intermediate by disc diffusion when compared with a resistant or susceptible category by agar dilution was defined as a minor error.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Transparency declarations
 References
 
Antimicrobial spectrum

The activity of colistin against the 228 bacterial strains, as tested by agar dilution, is shown in Table 2. Generally, colistin exhibited excellent activity against Acinetobacter spp. (MIC90 2 mg/L), E. coli (MIC90 2 mg/L) and Klebsiella spp. (MIC90 2 mg/L). In contrast, colistin was less consistently active against P. aeruginosa (MIC90 4 mg/L) and Enterobacter spp. (MIC90 ≥ 128 mg/L). When an interpretative breakpoint for resistance of ≥4 mg/L was used, resistance rates varied from 0% in E. coli and Acinetobacter baumannii to 27% in P. aeruginosa.


View this table:
[in this window]
[in a new window]

  		Table 2. MIC distributions for tested isolates

 
Categorical errors by disc diffusion

Method based on product literature.. Isolates with MIC ≥4 mg/L were considered resistant (n = 27). When disc zone diameters were interpreted according to the manufacturer’s guidelines2 (resistant ≤ 8 mm; susceptible ≥ 11 mm), there were 22 (10%) very major errors. Disc testing failed to detect colistin resistance in P. aeruginosa (n = 15), Klebsiella spp. (n = 3) and Enterobacter spp. (n = 4); 81% of colistin-resistant organisms were reported as falsely susceptible by this testing method.

When disc zone diameters were interpreted according to the criteria by Gales et al.1 (resistant ≤ 10 mm; susceptible ≥ 14 mm), the number of very major errors was reduced to 12 (44%). However, an unacceptably large number of isolates (161 isolates, 71%) fell into the intermediate category.

BSAC method.. Isolates with MIC >4 mg/L were considered resistant (n = 14). Zone diameters were interpreted according to the published 2005 standards,3 with different breakpoints used for P. aeruginosa (resistant ≤ 13 mm) and the Enterobacteriaceae and Acinetobacter spp. (resistant ≤ 14 mm). Strains of Enterobacteriaceae that produced clear zones of inhibition with small colonies around the colistin discs were interpreted as resistant. There were 11 (5%) very major errors. Disc testing failed to detect colistin resistance in P. aeruginosa (n = 5) and Enterobacter spp. (n = 6); 79% of colistin-resistant organisms were reported as falsely susceptible by this testing method.

SFM method.. Isolates with MIC >2 mg/L were considered resistant (n = 27). Zone diameters were interpreted according to the published 2005 criteria (resistant ≤ 14 mm).4 There were 24 (11%) very major errors. Disc testing failed to detect colistin resistance in P. aeruginosa, (n = 14), Klebsiella spp. (n = 3) and Enterobacter spp. (n = 7); 89% of colistin-resistant organisms were reported as falsely susceptible by this testing method.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Transparency declarations
 References
 
Analysis of the performance of each of the disc susceptibility testing methods evaluated in this study was complicated by the differing resistance breakpoints adopted for colistin. The SFM specifies that isolates with MIC >2 mg/L are considered resistant, while the BSAC adopts a resistance breakpoint of >4 mg/L. In 2005, the CLSI published resistance breakpoints of ≥4 mg/L for P. aeruginosa and non-Enterobacteriaceae,6 but in the 2006 edition subsequently withdrew these breakpoints for organisms other than Acinetobacter spp. We also note that the reference MIC method used in the present study was performed according to CLSI methods, while the BSAC specify the use of Iso-Sensitest medium for MIC testing.

The three disc methods differed in various parameters that are known to affect susceptibility testing, which include inoculum size, colistin concentration in the disc and the type of medium used. However, resistance to colistin is poorly detected by disc diffusion susceptibility testing regardless of the method used. Disc susceptibility testing by all three methods produced an unacceptably high rate of very major errors (5–11%).

Some inherent properties of the polymyxins make agar-based disc susceptibility testing difficult. The polymyxins diffuse poorly in agar, resulting in small zones of inhibition. This results in poor categorical differentiation of susceptible and resistant isolates. Utilizing higher concentrations of colistin in the discs does not appear to improve the accuracy of test results. Early studies performed on Gram-negative bacteria using colistin sulphate7 documented poor correlation between zone sizes and MIC values. More recent work by Gales et al.1 demonstrated a 3.5–6% false-susceptibility rate. In vitro activity of the polymyxins may be affected by cation levels in agar.5,7 Variations in Iso-Sensitest medium performance have also been demonstrated to affect colistin susceptibility testing for P. aeruginosa.8 This may not apply to Mueller–Hinton agar, as testing of QC results from three separate lots of medium showed no substantial variability.9

The optimum method for in vitro susceptibility testing of the polymyxins remains unsettled. Although the colistimethate sodium preparation is used for intravenous formulations of colistin,10 the sulphate preparation is used for susceptibility testing. The sulphomethyl derivatives for colistin are generally 2–8 times less active than the sulphate counterparts.10 Dilution methods remain the ‘gold standards’ for susceptibility testing, but some data suggest that agar-based testing methods generate higher MIC values than equivalent broth-based methods,11 which may be due to the binding of the polymyxins to the agar. Although the Etest (AB Biodisk, Sweden) is a commonly used MIC-equivalent testing method in clinical microbiology laboratories, limited data on the performance of polymyxin susceptibility testing by this method are available. Arroyo et al.12 reported a 1.7% very major error rate for A. baumannii, with good concordance of MIC values between 0.25 and 1 mg/L. However, no similar data are available for P. aeruginosa and the Enterobacteriaceae.

The present study shows that disc diffusion remains an inherently unreliable susceptibility testing method for the polymyxins, particularly in P. aeruginosa. Of significant concern is that current disc diffusion testing methods fail to reliably detect resistance to the polymyxins. Based on the available data, we recommend that dilution-based methods be used for testing whenever parenteral use of the polymyxins is considered in clinical practice.


   Transparency declarations
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
References
 
None to declare.


   Acknowledgements
 
This study was funded by the National Medical Research Council, Singapore.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Transparency declarations
 References
 
1 Gales AC, Reis AO, Jones RN. (2001) Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines. J Clin Microbiol 39:183–90.[Abstract/Free Full Text]

2 Package Insert. (2003) BD BBL sensi-Disc Antimicrobial Susceptibility Test Discs. (Dickinson and Company, Maryland, USA: Becton).

3 British Society for Antimicrobial Chemotherapy. BSAC Disc Diffusion Method for Antimicrobial Susceptibility Testing, Version 4. http://www.bsac.org.uk/_db/_documents/version_4_january_2005_final_NH_april_2.pdf (28 April 2006, date last accessed).

4 Comité de l'Antibiogramme de la Société Française de Microbiologie. Recommandations du CASFM, Communiqué 2005 (Edition de Janvier 2005). http://www.sfm.asso.fr/doc/download.php?doc=DiU8C&fic=Communiqu%E9_2005.pdf (28 April 2006, date last accessed).

5 National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition: Approved Standard M7-A6. NCCLS, Wayne, Pennsylvania, USA, 2003.

6 Clinical Laboratory Standards Institute. (2005) Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth Informational Supplement M100-S15. , CLSI, Wayne, Pennsylvania, USA.

7 Barry AL and Effinger LJ. (1974) Evaluation of two standardized disk methods for testing antimicrobial susceptibility of Pseudomonas aeruginosa and of the Enterobacteriaceae. Antimicrob Agents Chemother 6:452–9.[Abstract/Free Full Text]

8 Andrews J, Walker R, King A. (2002) Evaluation of media available for testing the susceptibility of Pseudomonas aeruginosa by BSAC methodology. J Antimicrob Chemother 50:479–86.[Abstract/Free Full Text]

9 Jones RN, Anderegg TR, Swenson JM. (2005) Quality control guidelines for testing gram-negative control strains with polymyxin B and colistin (polymyxin E) by standardized methods. J Clin Microbiol 43:925–7.[Abstract/Free Full Text]

10 Greenwood D. (2003) Miscellaneous antibacterial agents. In Finch RG, Greenwood D, Ragnar Norrby S (Eds.), et al. Antibiotic and Chemotherapy: anti-infective agents and their use in therapy (Churchill Livingstone, Edinburgh) pp. 407–13.

11 Gales AC, Jones RN, Sader HS. (2006) Global assessment of the antimicrobial activity of polymyxin B against 54731 clinical isolates of Gram-negative bacilli: report from the SENTRY antimicrobial surveillance programme (2001–2004). Clin Microbiol Infect 12:315–21.[CrossRef][Web of Science][Medline]

12 Arroyo LA, Garcia-Curiel A, Pachon-Ibanez ME, et al. (2005) Reliability of the E-test method for detection of colistin resistance in clinical isolates of Acinetobacter baumannii. J Clin Microbiol 43:903–5.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Clin. Microbiol. Rev.Home page
D. Landman, C. Georgescu, D. A. Martin, and J. Quale
Polymyxins Revisited
Clin. Microbiol. Rev., July 1, 2008; 21(3): 449 - 465.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Microbiol. Rev.Home page
A. Y. Peleg, H. Seifert, and D. L. Paterson
Acinetobacter baumannii: Emergence of a Successful Pathogen
Clin. Microbiol. Rev., July 1, 2008; 21(3): 538 - 582.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
F. Perez, A. M. Hujer, K. M. Hujer, B. K. Decker, P. N. Rather, and R. A. Bonomo
Global Challenge of Multidrug-Resistant Acinetobacter baumannii
Antimicrob. Agents Chemother., October 1, 2007; 51(10): 3471 - 3484.
[Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
J. R. Lo-Ten-Foe, A. M. G. A. de Smet, B. M. W. Diederen, J. A. J. W. Kluytmans, and P. H. J. van Keulen
Comparative Evaluation of the VITEK 2, Disk Diffusion, Etest, Broth Microdilution, and Agar Dilution Susceptibility Testing Methods for Colistin in Clinical Isolates, Including Heteroresistant Enterobacter cloacae and Acinetobacter baumannii Strains
Antimicrob. Agents Chemother., October 1, 2007; 51(10): 3726 - 3730.
[Abstract] [Full Text] [PDF]

Home page
 J Antimicrob ChemotherHome page
F. W. Goldstein, A. Ly, and M. D. Kitzis
Comparison of Etest with agar dilution for testing the susceptibility of Pseudomonas aeruginosa and other multidrug-resistant bacteria to colistin
J. Antimicrob. Chemother., May 1, 2007; 59(5): 1039 - 1040.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
58/4/864    most recent
dkl330v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (14)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Tan, T. Y.
Right arrow Articles by Ng, L. S. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tan, T. Y.
Right arrow Articles by Ng, L. S. Y.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?