Variation in the content of ciprofloxacin susceptibility testing discs: its effect on the BSAC disc method

J. M. Darville and A. M. Lovering*

Bristol Centre for Antimicrobial Research & Evaluation, Department of Medical Microbiology, Southmead Hospital, Bristol BS10 5NB, UK

Keywords: ciprofloxacin, susceptibility testing

Sir,

Over the past few years, medical microbiology laboratories both in the UK and elsewhere have adopted the British Society for Antimicrobial Chemotherapy (BSAC) method for antimicrobial disc susceptibility testing.1 In common with a number of other disc susceptibility testing methods,2,3 the BSAC method is based on defined zone diameters for differentiation between susceptible and resistant strains, unlike earlier methods where the relative zone diameters for test and control strains were used.4

Despite the widespread adoption of the BSAC method, there are still a number of gaps to be filled in the recommendations,5 one of which is the appropriate disc content of ciprofloxacin to use for the testing of Pseudomonas spp. Currently, a disc content of either 1 or 5 µg can be used, with zone diameters for use with both given in the method.1 However, recently we have become aware of variability in the zone diameters obtained when using the lower content ciprofloxacin discs and difficulty in achieving acceptable zone diameters for reference Pseudomonas aeruginosa strains when using certain batches of discs.

At present, the appropriate manufacturing standards for the preparation of antimicrobial susceptibility testing discs permit a range of content from 90% to 120% of the stated disc content.6 This allows manufacturers to load discs above the stated content to allow for loss on storage and prolong shelf lives. In this study, we have measured the ciprofloxacin content of 1 µg discs from three commercial suppliers and looked at the potential impact of variability in disc content on the susceptibility testing of P. aeruginosa using the BSAC method.

Ten ciprofloxacin discs (1 µg content) from each of three commercial suppliers (Mast Diagnostics, Bootle, UK; Oxoid Ltd, Basingstoke, UK; Becton Dickinson, Oxford, UK) were placed, individually, into plastic micro-centrifuge tubes and 1 mL volumes of phosphate-buffered saline (PBS; pH 7.2) added. After overnight extraction at 4°C, ciprofloxacin extracted from the discs was assayed by a validated HPLC method and the actual ciprofloxacin content of the discs calculated.7 Experiments showed that elution in deionized water gave only 50–67% recovery relative to PBS, and that blank disc material absorbed ciprofloxacin from solution in water, but not PBS (data not shown). The mean zone diameters found for the three batches of discs are shown in Table 1. These ranged from a disc content of 0.71 µg (below the manufacturing tolerance limits) to 1.14 µg, depending on manufacturer. The discs with the low ciprofloxacin content were supplied from a batch that produced smaller zone diameters than expected but that, nevertheless, was in general supply at the time of the study.


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Table 1.  Mean disc content of ciprofloxacin 1 µg discs from three manufacturers and the mean zone diameters obtained with P. aeruginosa ATCC 27853 and E. coli ATCC 25922
 
To assess the potential impact of variability in disc content, six replicates of each batch of discs, along with ‘in-house’ discs made from the addition of 1 µg of ciprofloxacin (Bayer Pharmaceutical, Newbury, UK) to Whatman AA discs, were tested by the BSAC method1 against P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922. For the P. aeruginosa strain, mean zone diameters ranged from 24.7 to 28.3 mm (Table 1), depending on disc content, and although the mean zone diameters were within the acceptable range of 24–30 mm, for the discs with a mean content of 0.71 µg, several of the replicates were on the limits of this range. For the E. coli strain, the mean zone diameters for the different batches of discs were a lot closer than seen with the P. aeruginosa strain and ranged from 33.3 to 34.5 mm (Table 1). These were all within the acceptable range of 31–37 mm and no individual zone diameter was outside of this range.

To investigate the relationship between disc content and the diameter of zones of inhibition, discs containing 0.25–6 µg of ciprofloxacin were prepared, as described earlier. These were tested using the BSAC method against P. aeruginosa ATCC 27853 and the results are shown in Figure 1. In keeping with standard curves from bioassays, the relationship between disc content and zone diameter shows a distinct flattening at increasing disc content or concentration. A disc content of 1 µg, compared with 5 µg, is on the steep part of the zone size to content curve, and small differences in disc content are likely to have a greater influence on zone diameter than those at higher disc content. In contrast, the zone diameter to disc content curve for E. coli ATCC 25922 is much flatter at the 1 µg disc content, and slight variation in disc content is unlikely to have such an impact on zone diameter as with P. aeruginosa ATCC 27853 (Figure 1), as observed with the mean zone diameters (Table 1).



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Figure 1. Mean zone diameter (n = 6) and S.D. bars for the susceptibility testing of P. aeruginosa ATCC 27853 (diamonds) and E. coli ATCC 25922 (squares) by the BSAC method, using different ciprofloxacin content discs. Dashed lines indicate the manufacturing tolerance limits for 1 and 5 µg discs.

 
Although the supplier of the discs with the low ciprofloxacin content has informed us that their discs have been reformulated to prevent loss of ciprofloxacin during the manufacturing process, we have been able to establish that the content of ciprofloxacin 1 µg discs does vary between manufacturers. However, as a result of the shape of the zone diameter to content response curves, for the testing of E. coli our results would suggest that such variation is unlikely to have a significant effect. In contrast, for testing of P. aeruginosa, variation in disc content may lead to significant variability in zone diameters when using a 1 µg disc but probably not when using a 5 µg disc. Although determination of the optimum conditions for susceptibility testing is a complex process that involves a number of factors, we are not aware whether the impact of variability in disc content has been considered before. Certainly, our results would suggest that the impact of variability in disc content is likely to be lower if a 5 µg rather than a 1 µg disc is used to test Pseudomonas spp., which may be helpful for laboratories when assessing which of the two disc contents permitted in the method they should use.

Footnotes

* Corresponding author. Tel: +44-117-9595653; Fax: +44-117-9593217; E-mail: Lovering_a{at}southmead.swest.nhs.uk Back

References

1 . Andrews, J. M. (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 43–57.[Abstract/Free Full Text]

2 . National Committee for Clinical Laboratory Standards. (1999). Performance Standards for Antimicrobial Susceptibility Testing: Approved Standard M100-S9. NCCLS, Villanova, PA, USA.

3 . Ringertz, S., Olsson-Liljequist, B., Kahlmeter, G. & Kronvall, G. (1997). Antimicrobial susceptibility testing in Sweden. II. Species-related zone diameter breakpoints to avoid interpretive errors and guard against unrecognized evolution of resistance. Scandinavian Journal of Infectious Diseases Supplementum 105, 8–12.[Medline]

4 . Pearson, C. H. & Whitehead, J. E. (1974). Antibiotic sensitivity testing: a modification of the Stokes method using a rotary plater. Journal of Clinical Pathology 27, 430–1.[ISI][Medline]

5 . Andrews, J. M. & Wise, R. (2002). Reply to: Ciprofloxacin susceptibility testing of enterococcal urinary isolates in accordance with BSAC guidelines. Journal of Antimicrobial Chemotherapy 50, 433.[Free Full Text]

6 . WHO Expert Committee on Biological Standardisation. (1977). Technical Report Serial No. 610. WHO, Geneva, Switzerland.

7 . White, L. O., Bowyer, H. M., Lovering, A. M. & Reeves, D. S. (1985). Assay of ciprofloxacin in body fluids by HPLC with fluorimetric detection. In Second European Congress of Clinical Microbiology, Brighton, UK, 1985. Abstract 22/16. European Society of Clinical Microbiology and Infectious Diseases, Basel, Switzerland.





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