JAC Advance Access originally published online on March 17, 2004
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Journal of Antimicrobial Chemotherapy (2004) 53, 713-728
© 2004 The British Society for Antimicrobial Chemotherapy
BSAC standardized disc susceptibility testing method (version 3)
Department of Microbiology, City Hospital NHS Trust, Birmingham B18 7QH, UK
Received 2 September 2003; accepted 16 December 2003
Keywords: breakpoints, disc testing, MICs
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Preface |
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 Top Preface IntroductionAppendix 1. Amendments to...Appendix 2. Review of...Appendix 3. Advice on...Appendix 4. Susceptibility...References |
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Since the Journal of Antimicrobial Chemotherapy Supplement containing the BSAC standardized disc susceptibility testing method was published in 2001, there have been various changes to the recommendations and these have been posted on the BSAC website (http://www.bsac.org.uk). Two major organizational changes have been made to the interpretative tables. First is the separation of the acceptable ranges for the control strains from the interpretative criteria. The Working Party felt that it was appropriate to do this, as the control ranges are a tool for controlling the performance of the test and are not used for interpreting susceptibility. Second, in line with the European consensus, the presentation of MIC breakpoints (mg/L) has been amended as follows:
MIC 
(as previously) MIC breakpoint concentration = organism-susceptible
MIC > (previously 
) MIC breakpoint concentration = organism-resistant.
In practice, this does not make any changes to breakpoint systems based on two-fold dilutions, but it avoids the theoretical ‘gap’ inherent in the previous system. However, the appearance of the tables will change, e.g. R 16, S 8 will change to R > 8, S 8.
The interpretative criteria have been expanded to include some new agents. Some criteria have been modified as new mechanisms of resistance have been recognized, and some have been amended to improve the reliability of reporting, and these changes have been posted on the website (current recommendations and archived versions are also available on the website). New or altered text is indicated in bold.
It is the intention of the BSAC and the Journal of Antimicrobial Chemotherapy to publish this document annually. However, as with all methods, it will require constant review and updating. We therefore advise that all interested parties frequently consult the BSAC website where the latest updates will be made available.
For nearly a decade, microbiologists have used the MIC breakpoints published in the BSAC Guide to Sensitivity Testing to interpret susceptibility. Historically, and unlike the rest of Europe, the UK and Ireland have used a comparative method of disc testing to interpret susceptibility rather than one based on a correlation between MIC and zone of inhibition. Although innovative when introduced in the 1970s, Stokes’ comparative method has evolved ad hoc and it has become increasingly apparent that there is a need for a standardized method of disc testing that is correlated with BSAC MIC breakpoints. The method described here, like all other standardized methods of disc testing, cannot be adapted by the user, and interpretative criteria are only applicable if the method is adhered to fully. A major advantage of this approach to susceptibility testing is that data from several sources can be combined for surveillance of resistance, a task that has been made much easier by the introduction of this method and coincides with the availability of automated zone-measuring devices. It is hoped that the method described here will provide the core document for standard operating procedures; however, changes will necessarily occur over time as the method is developed and refined.
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Introduction |
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TopPrefaceIntroductionAppendix 1. Amendments to...Appendix 2. Review of...Appendix 3. Advice on...Appendix 4. Susceptibility...References |
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The BSAC Guide to Sensitivity Testing was first published in 1991 and one of its most important sections was that dealing with breakpoints for clinically relevant bacteria.1 These breakpoints have been used extensively to interpret MIC results and for single concentration ‘breakpoint’ tests. However, a criticism of the guidelines was that they did not provide a standardized method of disc diffusion testing with zone limits that correlated with these MIC breakpoints. The limitations of the widely used Stokes’ comparative method were also a cause for concern.
The task of developing such a method of disc testing is immense and the Working Party and the Council of the BSAC needed evidence that there was sufficient interest to warrant the investment required not only in the short term, but also for continuing support and development. This necessary confirmation was obtained from a questionnaire survey,2 which indicated that 90.6% of UK laboratories would be prepared to switch to an upgraded disc test, and the development and ‘field testing’ of the standardized method was therefore undertaken.3
Fortuitously, the introduction of the standardized method has coincided with the availability of automated zone-measuring devices, which aid measuring and interpretation considerably. With laboratories using the same method, there is a real opportunity to combine zone diameter data, so that levels of resistance in the UK and Ireland can be surveyed, and subtle changes in susceptibility detected.
The method, like all standardized disc tests, cannot be adapted by the user, with the exception that various methods of inoculum preparation can be used to achieve semi-confluent growth.
For microorganisms not included in this section, work is either ongoing (e.g. anaerobes) or reported elsewhere (e.g. mycobacteria).4
1 Preparation of plates
1.1 Prepare Iso-Sensitest agar (ISA; Oxoid, Basingstoke, UK), or media shown to have the same performance as ISA, according to the manufacturer’s instructions. Media for fastidious organisms are supplemented with 5% defibrinated horse blood or 5% defibrinated horse blood + 20 mg/L ß-nicotinamide adenine dinucleotide (NAD; can be obtained from Mast Group, Merseyside, UK) as in Table 1.
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1.2 Pour sufficient molten agar into sterile Petri dishes to give a depth of 4 ± 0.5 mm (25 mL in a 90 mm Petri dish).
1.3 Dry the surface of the agar to remove excess moisture. The length of time needed to dry the surface of the agar depends on whether a fan-assisted drying cabinet (approximately 5–10 min) or a ‘still air’ incubator is used. Important: do not over-dry plates.
1.4 Ideally, the plates should be stored in vented plastic boxes at 8–10°C before use. Storage can extend for up to 1 week. Alternatively the plates may be stored at 4–8°C in sealed plastic bags. Acceptable methods of drying plates and durations of storage should be validated by individual laboratories as part of their quality assurance programme. In particular, tests should confirm that excess moisture is not produced in a sealed environment and that plates are not over-dried in an unsealed environment.
2 Selection of control organisms
2.1 The control strains listed in Table 2 should be included, as appropriate, with every batch of susceptibility tests.
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3 Preparation of inoculum
An inoculum giving semi-confluent growth of colonies after overnight incubation should be used. A denser inoculum will result in reduced zones of inhibition and a decreased inoculum will have the opposite effect. Use of an inoculum that yields semi-confluent growth has the advantage that an incorrect inoculum can readily be seen. Figure 1 shows the acceptable range of densities.
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The following method reliably gives semi-confluent growth. Other methods, such as photometric measurement of turbidity of suspensions, may be adequate but must be shown to be equivalent to the following.
3.1 Comparison with 0.5 McFarland Standard
3.1.1 Preparation of the McFarland Standard
Add 0.5 mL of 0.048 M BaCl2 (1.17% w/v BaCl2·2H2O) to 99.5 mL of 0.18 M H2SO4 (1% w/v) with constant stirring. Distribute the standard into screw-cap tubes of the same size and volume as those used to prepare the test inoculum. Seal the tubes tightly to prevent loss by evaporation. Store protected from light at room temperature. Vigorously agitate the turbidity standard on a vortex mixer before use. Standards may be stored for up to 6 months, after which time they should be discarded. Alternatively, prepared standards can be purchased (e.g. from bioMérieux, Basingstoke, UK).
3.1.2 Inoculum preparation by the growth method (for non-fastidious organisms, e.g. Enterobacteriaceae, Pseudomonas spp. and staphylococci)
Touch at least four morphologically similar colonies with a sterile loop. Transfer the growth into Iso-Sensitest broth or an equivalent that has been shown to have no adverse effect on the test. Incubate the broth with shaking at 35–37°C, until the visible turbidity is equal to or greater than that of the 0.5 McFarland Standard.
3.1.3 Inoculum preparation by the direct colony suspension method (the method of choice for fastidious organisms, e.g. Haemophilus spp., Neisseria gonorrhoeae and Streptococcus pneumoniae)
Colonies are taken directly from the plate into Iso-Sensitest broth (or equivalent) or distilled water. The suspension should match or exceed the density of the 0.5 McFarland Standard. Note that with some organisms, production of an even suspension of the required turbidity is difficult, and growth in broth is a more satisfactory option.
3.1.4 Adjustment of the organism suspension to the density of the 0.5 McFarland Standard
Adjust the density of the organism suspension prepared, as in 3.1.2 or 3.1.3, to equal that of the 0.5 McFarland Standard by adding sterile distilled water. To aid comparison, compare the test and standard against a white background with a contrasting black line. Note that the suspension should be used within 15 min.
3.2 Dilution of suspension adjusted to the turbidity of a 0.5 McFarland Standard
See Table 3 for details. These suspensions should be used within 15 min of preparation.
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3.3 Photometric standardization of turbidity of suspensions
A photometric method of preparing inocula was described by Moosdeen et al.6 and from this the following simplified procedure has been developed.
3.3.1 Suspend colonies (touch 4–5 when possible) in 3 mL distilled water or broth in a 100 x 12 mm glass tube (note that tubes are not reused) to give turbidity that is just visible. Do not leave the organisms standing in water. It is essential to get an even suspension.
3.3.2 Zero the spectrophotometer with a sterile water or broth blank (as appropriate) at a wavelength of 500 nm. Measure the optical density of the bacterial suspension. [The spectrophotometer must have a cellholder for 100 x 12 mm test tubes. A much simpler photometer would also probably be acceptable. The 100 x 12 mm test tubes could also be replaced with another tube/cuvette system if required, but the dilutions would need to be recalibrated.]
3.3.3 From Table 4 select the volume to transfer (with the appropriate fixed volume micropipette) to 5 mL sterile distilled water.
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[As different spectrophotometers may differ slightly, it may be necessary to adjust the dilutions slightly to achieve semi-confluent growth with any individual set of laboratory conditions.]
4 Inoculation of agar plates
Dip a sterile cotton-wool swab into the suspension and remove the excess liquid by turning the swab against the side of the tube. Spread the inoculum evenly over the entire surface of the plate by swabbing in three directions. Allow the plate to dry before applying discs. Note that if inoculated plates are left at room temperature for any length of time before the discs are applied, the organism may begin to grow, and this will result in reduced zones of inhibition. Discs should therefore be applied to the surface of the agar within 15 min of inoculation.
4.1 Use of Rotary platers for susceptibility testing
Rotary platers can be used for inoculating susceptibility tests but care must be taken. The swab must be moved at an even pace to ensure that the inoculum is semi-confluent and that no gaps are present between the swab streaks.
5 Antimicrobial discs
5.1 Disc contents are given in Tables 6–17.
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5.2 Application of discs
Discs should be firmly applied to the surface of an agar plate that has been dried previously. The contact with the agar should be even. A 90 mm plate will accommodate six discs without unacceptable overlapping of zones.
5.3 Storage and handling of discs
Loss of potency from discs will result in reduced zones of inhibition. To avoid loss of potency as a result of improper handling the following procedures are essential.
5.3.1 Store discs in sealed containers with a desiccant and pro-tected from light (this is particularly important for some light-susceptible agents such as metronidazole, chloramphenicol and the quinolones).
5.3.2 Store stocks at –20°C except for drugs known to be unstable at this temperature. If this is not possible, store discs at <8°C.
5.3.3 Store working supplies of discs at <8°C.
5.3.4 To prevent condensation, allow discs to warm to room temperature before opening containers.
5.3.5 Store disc dispensers in sealed containers with an indicating desiccant.
5.3.6 Discard any discs on the expiry date shown on the side of the container.
6 Incubation
6.1 If the plates are left at room temperature after discs have been applied, larger zones of inhibition may be obtained compared with zones produced when plates are incubated immediately. Plates therefore should be incubated within 15 min of disc application.
6.2 Conditions of incubation
Conditions of incubation for different organisms are summarized in Table 5. In general, avoid stacking plates more than six high in the incubator, as this may affect results owing to uneven heating of plates. However, incubators differ in their efficiency, higher stacks are acceptable if it can be shown that they do not affect zone diameter.
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7 Measuring zones and interpretation
7.1 Measure the diameters of zones of inhibition to the nearest millimetre (zone edge should be taken as the point of inhibition as judged by the naked eye) with a ruler, callipers or an automated zone reader. A template (Figure 2) can also be used for interpreting susceptibility. Tiny colonies at the edge of the zone, films of growth as a result of the swarming of Proteus spp. and slight growth within sulphonamide or trimethoprim zones should be ignored. Colonies growing within the zone of inhibition should be subcultured and identified and the test repeated if necessary.
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7.2 Confirm that the zone of inhibition for the control strain falls within the acceptable ranges in Tables 18–21 before interpreting the test.
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7.3 A template can also be used for interpreting zone diameters (Figure 2). A program for preparing templates is available from the BSAC (http://www.bsac.org.uk). The test plate is placed over the template and the zones of inhibition are examined in relationship to the template zones. If the zone of inhibition of the test strain is within the area marked with an ‘R’, the organism is resistant. If the zone of inhibition is equal to or larger than the marked area, the organism is susceptible.
8 Direct susceptibility testing
8.1 Direct susceptibility testing of urines
The Working Party does not advocate direct susceptibility testing, as controlling the inoculum is impossible. However, we are aware that this is a common practice in many laboratories and therefore we are suggesting methods that will achieve the correct inoculum size for a reasonable proportion of infected urines. The following methods have been developed and recommended by laboratories that use the BSAC method and we suggest adopting whichever method best suits individual laboratory working practice. If the inoculum is not correct and growth is not semi-confluent, or the culture is mixed, the test must be repeated.
(i) Method 1: thoroughly mix the urine, place a 10 µL loop of urine in the centre of the susceptibility plate and spread with a dry swab.
(ii) Method 2: thoroughly mix the urine, then dip a sterile cotton-wool swab in the urine and remove excess. Make a cross in the centre of the susceptibility plate then spread with a sterile dry swab. If only small numbers of organisms are seen under the microscope, the initial cotton-wool swab may be used to inoculate and spread the susceptibility plate.
8.2 Direct susceptibility testing of positive blood cultures
The Working Party does not recommend direct susceptibility testing of positive blood cultures. However, we are aware that this is common practice in many laboratories and therefore suggest a method that gives the correct inoculum size for a reasonable proportion of positive blood cultures. The method varies according to the Gram reaction of the infecting organism.
8.2.1 Gram-negative bacilli
Using a venting needle, place one drop in 5 mL of sterile water and use this to inoculate Iso-Sensitest or equivalent agar.
8.2.2 Gram-positive organisms
It is not always possible to accurately assume the genera of Gram-positive organisms from the Gram’s stain. However, careful observation of the morphology, coupled with some clinical information should make an ‘educated guess’ correct most of the time.
8.2.2.1 Staphylococci and enterococci
Using a venting needle, place three drops in 5 mL of sterile water and use this to inoculate Iso-Sensitest or equivalent agar.
8.2.2.2 Pneumococci, ‘viridans’ streptococci and diphtheroids
Using a venting needle, place one drop in the centre of an Iso-Sensitest or equivalent agar supplemented with 5% horse-blood, and spread evenly over the entire surface of the plate. If the inoculum is not correct and growth is not semi-confluent, or the culture is mixed, the test must be repeated.
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Appendix 1. Amendments to the minimum inhibitory concentration and zone diameter breakpoints for ampicillin, amoxicillin and co-amoxiclav for interpreting the susceptibility of Enterobacteriaceae |
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TopPrefaceIntroductionAppendix 1. Amendments to...Appendix 2. Review of...Appendix 3. Advice on...Appendix 4. Susceptibility...References |
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After reviewing recently obtained MIC and zone diameter data, the following amendments have been made to the recommendations for interpreting susceptibility.
For isolates from systemic infections, the MIC breakpoint for ampicillin, amoxicillin and co-amoxiclav has been raised to 16 mg/L for susceptible strains to avoid interpreting organisms at the top end of the normal susceptible population as resistant. The corresponding zone diameter breakpoints have also been amended for these antibiotics so that a zone diameter 14 mm = susceptible with amoxicillin 10 µg, ampicillin 10 µg and co-amoxiclav 20/10 µg disc contents.
For coliforms isolated from urinary tract infections, the MIC breakpoint remains the same, but to improve reproducibility in disc diffusion tests the zone diameter breakpoints for ampicillin, amoxicillin and co-amoxiclav have been amended so that a zone diameter 12 mm = susceptible with amoxicillin 25 µg, ampicillin 25 µg and co-amoxiclav 20/10 µg disc contents.
Reminder: These interpretative standards apply only to species of Enterobacteriaceae that are naturally susceptible to ampicillin and not to species known to be AmpC enzyme producers.
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Appendix 2. Review of the zone diameter breakpoints for piperacillin/tazobactam for interpreting the susceptibility of Enterobacteriaceae |
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TopPrefaceIntroductionAppendix 1. Amendments to...Appendix 2. Review of...Appendix 3. Advice on...Appendix 4. Susceptibility...References |
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Data from a recent study in the UK and Ireland, where susceptibility to piperacillin/tazobactam was determined by the BSAC standardized disc diffusion method, have been reviewed by the BSAC Working Party on Susceptibility Testing. In this study, 5% of the isolates and all resistant organisms were sent to a central laboratory for testing. Scattergram plots of MIC versus the zone diameter (mm) were constructed. To reduce the number of isolates falsely interpreted as resistant to piperacillin/tazobactam, the zone diameter breakpoints have been changed from susceptible =
24 mm, resistant 23 mm to susceptible 22 mm, resistant <21 mm. |
Appendix 3. Advice on testing the susceptibility to co-trimoxazole is provided, however the following are the UK Committee on the Safety of Medicines (CSM) recommendations |
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TopPrefaceIntroductionAppendix 1. Amendments to...Appendix 2. Review of...Appendix 3. Advice on...Appendix 4. Susceptibility...References |
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‘Co-trimoxazole should be limited to the role of drug of choice in Pneumocystis carinii pneumonia, it is also indicated for toxoplasmosis and nocardiasis. It should now only be considered for use in acute exacerbations of chronic bronchitis and infections of the urinary tract when there is good bacteriological evidence of sensitivity to co-trimoxazole and good reason to prefer this combination to a single antibiotic; similarly, it should only be used in acute otitis media in children when there is good reason to prefer it. Review of the safety of co-trimoxazole using spontaneous adverse drug reaction data has indicated that the profile of reported adverse reactions with trimethoprim is similar to that with co-trimoxazole; blood and generalized skin disorders are the most serious reactions with both drugs and predominantly have been reported to occur in elderly patients. A recent large post-marketing study has demonstrated that such reactions are very rare with co-trimoxazole; the study did not distinguish between co-trimoxazole and trimethoprim with respect to serious hepatic, renal, blood or skin disorders.’
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Appendix 4. Susceptibility testing Haemophilus influenzae to ß-lactam antibiotics |
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TopPrefaceIntroductionAppendix 1. Amendments to...Appendix 2. Review of...Appendix 3. Advice on...Appendix 4. Susceptibility...References |
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During the last year, several users of the method have reported problems when testing susceptibility of clinical isolates of Haemophilus influenzae to ß-lactam antibiotics. Of particular concern was co-amoxiclav, with which many ß-lactamase-positive isolates had zone diameters 1–2 mm smaller than the zone diameter breakpoint of 20 mm and were therefore incorrectly interpreted as resistant.
The problem was further highlighted by results with NEQAS specimen 5853. This ß-lactamase-positive isolate has a co-amoxiclav MIC of 0.5 mg/L. Amongst the laboratories that incorrectly interpreted this organism as being resistant to co-amoxiclav, centres using the BSAC method were common, and they reported zone diameters of 18–19 mm for the organism with co-amoxiclav.
The Working Party therefore reviewed the zone diameter breakpoints for ampicillin, amoxicillin, co-amoxiclav and cefuroxime and recommendations for the interpretation of susceptibility of H. influenzae are as follows:
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Footnotes |
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* Tel: +44-121-507-5693; Fax: +44-121-551-7763; E-mail: jenny.andrews{at}swbh.nhs.uk
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References |
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TopPrefaceIntroductionAppendix 1. Amendments to...Appendix 2. Review of...Appendix 3. Advice on...Appendix 4. Susceptibility...References |
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1 . Report of the 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.
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Andrews, J. M., Brown, D. F. J. & Wise, R. (1996). A survey of antimicrobial susceptibility testing in the United Kingdom. Journal of Antimicrobial Chemotherapy 37, 187–8.
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Andrews, J. M. (2001). The development of the BSAC standardized method of disc diffusion testing. Journal of Antimicrobial Chemotherapy 48, Suppl. 1, 29–42.
4 . Inderlied, C. B. & Nash, K. A. (1996). Antimycobacterial agents: in vitro susceptibility testing, spectra of activity, mechanisms of action and resistance, and assays for activity in biologic fluids. In Antibiotics in Laboratory Medicine (Lorian, V., Ed), pp. 127–75. Williams and Wilkins, Baltimore, MD, USA.
5 . Brown, D. F. J. (2001). Detection of methicillin/oxacillin resistance in staphylococci. Journal of Antimicrobial Chemotherapy 48, Suppl. 1, 65–70.[Abstract]
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Moosdeen, F., Williams, J. D. & Secker, A. (1988). Standardization of inoculum size for disc susceptibility testing: a preliminary report of a spectrophotometric method. Journal of Antimicrobial Chemotherapy 21, 439–43.
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