Disc susceptibility testing of staphylococci-implications for the BSAC standardized method

J Antimicrob Chemother 1999; 44: 717–718

John D. Perry*, Lynne A. Butterworth, Gavin Riley and F. Kate Gould

Microbiology Department, Freeman Hospital, Newcastle upon Tyne NE7 7DN, UK

Sir,

Successful treatment of infections caused by staphylococci can be particularly challenging, especially if the causative organism is multiply resistant.1 Alternatives to ‘standard’ therapy have to be sought,2 and in most laboratories the initial screening test for an appropriate agent is by the disc diffusion method.

The proposal for a standardized method for disc susceptibility testing by the Working Party of the British Society for Antimicrobial Therapy3 is welcome and should eventually lead to an improvement in the quality of laboratory results. A major limitation of the scheme at present is the lack of data for many organism/antibiotic combinations and consequently many of the specified zone sizes are ‘tentative’ and have not been validated in the field. We critically examined the tentative zone diameter breakpoints for three antimicrobial agents useful in the treatment of difficult infections; fusidic acid, ciprofloxacin and chloramphenicol, against staphylococci.

Chloramphenicol and fusidic acid were purchased from the Sigma Chemical Company, Poole, UK. Ciprofloxacin was kindly provided by Bayer plc. Each of the three drugs were prepared as soluble dilutions in IsoSensitest agar (Oxoid, Basingstoke, UK) using a standard method (BSAC Working Party 1991). The range for each antibiotic was 32–0.125 mg/L. Eighty distinct staphylococci, deemed to be of clinical significance, were isolated from specimens received in our laboratory. These comprised 52 Staphylococcus aureus (including 14 MRSA), 12 Staphylococcus epidermidis, nine Staphylococcus haemolyticus and six other miscellaneous coagulase-negative staphylococci. S. aureus strains were confirmed using a standard tube coagulase test, and coagulase-negative strains were identified using API staph (bioMérieux, la Balme-les-Grottes, France). Each strain was cultivated overnight on Columbia blood agar and suspended in IsoSensitest broth (Oxoid) to an inoculum density equivalent to McFarland 1.0 using a densitometer (Densimat, bioMérieux). This suspension was diluted 1/30 and 1 µL was inoculated using a multi-point inoculator on to the various dilutions of antibiotic to produce an inoculum of approximately 104 cfu/spot. All plates, including antibiotic-free controls, were incubated for 18 h at 37°C before reading MIC results.

On the same day each strain was tested in strict accordance with the BSAC standardized disc sensitivity testing method with the following discs (Oxoid): fusidic acid (10 µg), chloramphenicol (10 µg and 30 µg) and ciprofloxacin (1 µg and 5 µg). After incubation for 18 h zone sizes were measured and recorded. Any strains that did not yield semi-confluent growth were repeated using an adjusted inoculum. A control strain of S. aureus (NCTC 6571) was run simultaneously with MIC and disc tests and produced expected MIC results. The relationships between zone size and MIC for 80 staphylococci for the three antimicrobials are given in the Table.


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Table. Relationship between zone size (mm) and MIC for 80 staphylococci with three antimicrobials
 
For testing fusidic acid the BSAC quote a breakpoint of <=1 mg/L and a corresponding tentative zone diameter of >=30 mm to define susceptibility. Using this zone diameter, 95% of the strains were correctly classified as susceptible or resistant with respect to a breakpoint of 1 mg/L. Two strains were incorrectly classified as sensitive by disc testing and two were incorrectly classified as resistant. Despite these anomalies, this zone diameter proved to be optimal for the accurate classification of sensitive and resistant staphylococci and adjustment of the zone diameter breakpoint led to higher rates of error.

For ciprofloxacin the Working Party have quoted a breakpoint of <=1 mg/L and a corresponding tentative zone diameter of >=18 mm to define susceptibility. Using this zone diameter 16 strains (20%) which were sensitive to ciprofloxacin (MIC <= 1 mg/L) were mis-classified as resistant. No false-sensitive results were achieved. Our data show that a lower zone diameter cut-off as low as >=10 mm for a ciprofloxacin 1 µg disc would mean that only two strains (2.5%) were mis-classified as resistant with no false-sensitive results. Using a ciprofloxacin 5 µg disc and applying a zone diameter of >=20 mm resulted in three strains of S. haemolyticus (3.75%) being wrongly classified including one falsely sensitive strain (MIC 2 mg/L, zone diameter 23 mm). These lower zone diameter breakpoints would appear to offer a significant improvement in the accuracy of ciprofloxacin testing. We would advocate the use of ciprofloxacin 5 µg discs for testing staphylococci, and indeed other Gram-positive organisms4 as it offers a greater differentiation between sensitive and resistant strains.

For chloramphenicol the Working Party advocate a breakpoint of <=2 mg/L and a corresponding tentative zone diameter of >=20 mm to define susceptibility. Using this breakpoint MIC only three strains of staphylococci (3.75%) could be classified as sensitive. However, 69 strains (86%) produced a zone size >=20 mm and were erroneously classified as sensitive according to the MIC breakpoint. The NCCLS5 recommend a breakpoint of >=8 mg/L for staphylococci to define susceptibility as did a BSAC Working Party in 19916 based on a chloramphenicol dosage of 500 mg po. If this breakpoint were applied, the staphylococci in this study fell into a well defined bi-modal distribution. The four resistant strains had MICs of >32 mg/L and produced zone sizes of <10 mm with both 30 µg and 10 µg discs. All sensitive strains produced zone sizes of >=18 mm with both strengths of disc. We believe the breakpoint quoted by the Working Party is unnecessarily low and effectively classified all strains of S. aureus in this study as resistant to chloramphenicol. Based on these results we would advocate a tentative zone size breakpoint of 18 mm using a chloramphenicol 30 µg disc.

Whilst we accept that in the treatment of complicated staphylococcal infections MIC testing of antibiotics for the causative organisms must remain the ‘gold-standard’, we believe that modification of the zone diameter breakpoints of chloramphenicol and ciprofloxacin for staphylococci will allow for a significant improvement in accuracy when using the BSAC standardized method. It is likely that further work will need to be performed as a matter of urgency on various other organism/antibiotic combinations which are currently subject to tentative breakpoints in order to further develop the accuracy of the BSAC standardized method.

Notes

* Corresponding author: Tel: +44-191-2231226; Fax: +44-191-2231224; E-mail: homeresearch{at}compuserve.com Back

References

1 . Mulligan, M. E., Murray-Leisure, K. A., Ribner, B. S., Standiford, H. C., John, J. F., Korvick, J. A. et al. (1993). Methicillin-resistant Straphylococcus aureus: a consensus review of the microbiology, pathogenesis, and epidemiology with implications for prevention and management.American Journal of Infection 94, 313–28.

2 . Rahman, M. (1998). Alternatives to vancomycin in treating methicillin-resistant Staphylococcus aureus infections. Journal of Antimicrobial Chemotherapy, 41,325 –8.[Free Full Text]

3 . British Society for Antimicrobial Chemotherapy Newsletter, (1998).

4 . Perry, J. D., Ford, M. & Gould, F. K. (1994). Susceptibility of enterococci to ciprofloxacin. Journal of Antimicrobial Chemotherapy 34, 297–8.[ISI][Medline]

5 . National Committee for Clinical Laboratory Standards. (1998). Performance Standards for Antimicrobial Susceptibility Testing—Eighth Informational Supplement. NCCLS, Villanova, PA.

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





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