Reproducibility of control organism zone diameters for batches of IsoSensitest agar manufactured from 1996 to 2000 using the BSAC disc susceptibility test method

Janine Landrygana, Peter A. Jamesa,*, Dawn Brooksb and Elizabeth M. Kubiaka

a Department of Microbiology, Royal Gwent Hospital, Gwent Healthcare NHS Trust, Newport NP20 2UB; b Oxoid Limited, Basingstoke, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The BSAC Working Party on Susceptibility Testing has recently suggested that the performance of IsoSensitest agar has changed since 1991. Twenty batches of IsoSensitest agar that had been manufactured between 1996 and 2000 were tested using the BSAC standardized disc susceptibility testing method. Antibiotic discs containing amoxicillin 10 µg, ceftazidime 30 µg, gentamicin 10 µg, ciprofloxacin 1 µg and colistin sulphate 25 µg were tested on each batch of media 12 times against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 where appropriate. There was a small reduction in zone sizes for most antibiotics on batches of media that were near their expiry date, but otherwise zone sizes were remarkably consistent. We could find no evidence to suggest that a change in the performance of IsoSensitest agar for the disc diffusion method had occurred since 1996.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
With the introduction of the BSAC standardized susceptibility testing method1 into many laboratories in the UK, attention is focused on the performance of the new method and factors that may affect reproducibility. Most of the methodological variations have been addressed by virtue of standardized methodology, and attention is now moving to other variables such as the culture media and antibiotic discs. Recently,2 the BSAC Working Party on Susceptibility Testing has suggested that the performance of IsoSensitest agar, which was used to derive the original MIC distribution data and breakpoints,3 may have changed since 1991. Concern that the MIC of gentamicin for Pseudomonas aeruginosa NCTC 10662, when tested on IsoSensitest agar, has increased has led the Working Party to tentatively raise the MIC breakpoint from 1 to 2 mg/L, and to change the corresponding zone diameter breakpoint from 22 to 20 mm to avoid gentamicin being unnecessarily withheld from therapeutic use.2 Experience has shown that IsoSensitest-like agar media from different manufacturers can vary considerably.4 We were able to obtain limited samples of batches of IsoSensitest dehydrated culture media that had been stored by Oxoid Ltd since 1996, and we were therefore able to assess variation in zone sizes of control organisms to a range of antibiotics. The antibiotics used were selected as representatives from different antibiotic classes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Control organisms Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and P. aeruginosa ATCC 27853 were tested where appropriate using amoxicillin 10 µg, ceftazidime 30 µg, gentamicin 10 µg, ciprofloxacin 1 µg and colistin sulphate 25 µg discs (Oxoid, Basingstoke, UK), in strict accordance with the recommendations of the BSAC.1 Antibiotic discs used were from the same lot number for each antibiotic tested. IsoSensitest agar (Oxoid) from 20 different batches of dehydrated powder, stored by Oxoid at intervals between 1996 and 2000, was prepared according to the manufacturer's instructions. Agar was poured into 90 mm plastic Petri dishes using an MP 1000 automatic plate pourer (New Brunswick, Hatfield, UK), which was recalibrated before each batch to ensure a 25 mL plate fill. Plates were dried, stored at 4°C and used 2 days later, after sterility checks on sample plates from each batch. Inocula were prepared in sterile distilled water by dilution from a turbidity equivalent to 0.5 McFarland standard, to achieve semi-confluent growth as directed by the BSAC methodology. Six identical antibiotic discs (four in the case of ceftazidime against E. coli) were applied to each plate using a disc dispenser (Oxoid), such that each antibiotic was tested 12 times against appropriate control organisms for each of the 20 batches of IsoSensitest agar and additionally on a batch of commercially prepared (25 mL fill) IsoSensitest agar plates.

Plates were incubated in air at 36°C for 18 h, and read using an automated plate reader5 (aura Image System; Oxoid). Manual adjustments were made where zone edges were not accurately detected. MICs were determined using Etests (AB Biodisk, Solna, Sweden) on commercially prepared IsoSensitest agar on three separate occasions for each antibiotic.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One batch of agar failed to grow any organisms and was excluded. The mean value (± 2 s.d.) of 12 determinations for each antibiotic tested against E. coli ATCC 25922, S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 for the remaining batches of IsoSensitest agar are shown in the FigureGo. Negative trends in mean zone sizes for some antibiotics were noted for the older batches of culture media (labelled 1–4 on the abscissas) in the FigureGo, for example E. coli with gentamicin (Figure, cGo). Calculated control zone limits (±2 s.d.) from our data were within the BSAC published limits1 (TableGo), with the following exceptions: amoxicillin and colistin for E. coli (where our calculated lower control zone limit exceeded BSAC lower limits by 1.5 and 0.2 mm, respectively), ciprofloxacin for P. aeruginosa (upper and lower control zone limits exceeded BSAC limits by 1.2 and 0.3 mm, respectively), ceftazidime for P. aeruginosa (upper control zone 0.6 mm greater than BSAC limit) and gentamicin for S. aureus (upper control limit 5.8 mm greater than BSAC limit). Overall reproducibility was generally good, with ±2 s.d. of all readings within 4 mm for E. coli with amoxicillin, ceftazidime, gentamicin and colistin, S. aureus with ciprofloxacin, and P. aeruginosa with ceftazidime and gentamicin. However, for S. aureus with amoxicillin, ceftazidime and gentamicin and for P. aeruginosa with ciprofloxacin, ±2 s.d. exceeded 6 mm.



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Figure. Mean ± 2 s.d. of 12 replicate disc susceptibility tests against E. coli ATCC 25922 (plots a–e), S. aureus ATCC 25923 (plots f–i) and P. aeruginosa ATCC 27853 (plots j–m), on 20 batches of IsoSensitest agar. Month and year of manufacture for each batch of agar as follows: 1, 3/1996; 2, 4/1996; 3, 9/1996; 4, 5/1997; 6, 9/1997; 7, 11/1997; 8, 3/1998; 9, 6/1998; 10, 9/1998; 11, 11/1998; 12, 2/1999; 13, 4/1999; 14, 5/1999; 15, 7/1999; 16, 1/2000; 17, 3/2000; 18, 6/2000; 19, 11/2000. For batch 20, commercially prepared pre-poured plates were used (manufactured 2/2001).

 

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Table. Comparison of BSAC control zone limits with calculated control zone limits from the present study with BSAC breakpoints
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The batches of IsoSensitest agar that we tested were samples from c. 50% of lot numbers manufactured by Oxoid Ltd between 1996 and 2001. We could find no evidence to suggest that the overall performance of IsoSensitest agar has changed since 1996, when utilizing the BSAC methodology. This is in contrast to the findings of Andrews,2 although we acknowledge that the time-span was not identical and we used different methodology and different control organisms.

Because the BSAC methodology is still relatively new and much of the data are still regarded as tentative, it is attracting considerable scrutiny and debate.6,7 It is important that any methodology is as robust as possible such that any minor change in any variable does not significantly alter the performance of the test. It follows that the selection of appropriate breakpoints has a direct influence on the performance of the methodology.

We appreciate that there is often a compromise when choosing appropriate breakpoints for some antibiotic– organism combinations. There is a potential conflict in not limiting the use of proven antibiotics for sick patients and reliably differentiating susceptible and resistant populations. The inability to reliably detect clinically relevant antibiotic resistance would be a failure for any method. However, we note that the BSAC test zone limit for gentamicin against P. aeruginosa (before amendment) relating to the original 1 mg/L breakpoint was 22 mm. The published zone limit for the control strain P. aeruginosa ATCC 27853, which has an MIC of 2 mg/L, is 22–28 mm. The close proximity of the lower zone limit for this strain and, more importantly, the lower end of zone distribution profiles of susceptible clinical isolates8 to the selected MIC breakpoint would suggest that the method is unlikely to be a reliable discriminator of truly susceptible and resistant strains. This is also true for E. coli ATCC 25922 with gentamicin, where the acceptable control zone lower limit is within 1 mm of the test zone breakpoint. It is of interest that most other standardized susceptibility methodologies have a higher breakpoint for gentamicin of 4 mg/L against P. aeruginosa and Enterobacteriaceae (for example the NCCLS9).

All the samples we used had been opened soon after manufacture for quality control purposes and may not have performed as well as media from unopened containers, particularly towards the end of their shelf lives (typically 5 years from manufacture). Other factors that may potentially affect the apparent activity of antibiotics (particularly aminoglycosides), are changes in local media production conditions, e.g. quality of water supply (including pH) and leaching of metal ions from vessels used in media preparation. Agar incorporation MIC results may also be affected by a change in the source of the standard antibiotic solution.

All this said, there are clearly many benefits of a standardized approach such as the BSAC methodology provided, and in our opinion the method is fully deserving of its current increasing use and acceptance.


    Notes
 
* Corresponding author. Tel: +44-1633-234502; E-mail: peter.james{at}gwent.wales.nhs.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Andrews, J. M. for the BSAC Working Party on Susceptibility Testing. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.[Abstract/Free Full Text]

2 . Andrews, J. M. (2000). Effect of medium compostion on the MIC breakpoint for gentamicin. Journal of Antimicrobial Chemotherapy 46, 851–2.[Free Full Text]

3 . Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. (1991). A guide to sensitivity testing. Journal of Antimicrobial Chemotherapy 27, Suppl. D, 1–50.[ISI][Medline]

4 . Andrews, J. M. (2000). Performance and evaluation of Iso-Sensitest-like media. Standardized Disc Sensitivity Method User Day Report. British Society for Antimicrobial Chemotherapy, November 2000.

5 . Andrews, J. M., Boswell, F. J. & Wise, R. (2000). Evaluation of the Oxoid Aura image system for measuring zones of inhibition with the disc diffusion technique. Journal of Antimicrobial Chemotherapy 46, 535–40.[Abstract/Free Full Text]

6 . Gould, I. M. (2000). For debate. Towards a common susceptibility testing method? Journal of Antimicrobial Chemotherapy 45, 757–62.[Free Full Text]

7 . Wise, R. & Phillips, I. (2000). Towards a common susceptibility testing method? Journal of Antimicrobial Chemotherapy 45, 919–31.[Free Full Text]

8 . Henwood, C. J., Livermore, D. M., James, D., Warner, M. & the Pseudomonas Study Group. (2001). Antimicrobial susceptibility of Pseudomonas aeruginosa: results of a UK survey and evaluation of the British Society for Antimicrobial Chemotherapy disc susceptibility test. Journal of Antimicrobial Chemotherapy 47, 789–99.[Abstract/Free Full Text]

9 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Sensitivity Testing: Eleventh Informational Supplement M2-A5, M7-A5. NCCLS, Wayne, PA.

Received 4 July 2001; returned 20 August 2001; revised 27 September 2001; accepted 30 October 2001





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