BSAC standardized disc susceptibility testing method (version 4)

J. M. Andrews* for the BSAC Working Party on Susceptibility Testing

Department of Microbiology, City Hospital NHS Trust, Birmingham B18 7QH, UK


* Tel: +44-121-507-5693; Fax: +44-121-507-5521; Email: jenny.andrews{at}swbh.nhs.uk

Received 1 March 2005; accepted 6 March 2005

Keywords: breakpoints , disc testing , MICs


    Preface
 Top
 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 
In 2002 the BSAC agreed to participate with several other European national groups, namely CA-SFM (Comité de l'Antibiogramme de la Société Française de Microbiologie, France), the CRG (Commissie Richtlijnen Gevoeligheidsbepalingen, The Netherlands), DIN (Deutsches Institut für Normung, Germany), NWGA (Norwegian Working Group on Antimicrobials, Norway) and the SRGA (Swedish Reference Group of Antibiotics, Sweden), in a project to harmonize antimicrobial breakpoints, including previously established breakpoints. This work is being undertaken by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) with the support and collaboration of the national committees, and is funded by the European Union, the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) and the national committees, including the BSAC. The Clinical and Laboratory Standards Institute (CLSI; formerly the NCCLS) is also in the process of reviewing some breakpoints and has currently agreed to work with EUCAST on breakpoints for cephalosporins in Enterobacteriaceae. The review process includes application of more recent techniques, such as pharmacodynamic analysis, and current data, where available, on susceptibility distributions, resistance mechanisms and clinical outcome related to in vitro tests. There is also extensive discussion within EUCAST and the national committees, including the BSAC Working Party on antimicrobial susceptibility testing, and wide consultation on proposals.1,2 In the interests of international standardization of susceptibility testing, and the need to update older breakpoints in the light of current knowledge, these developments are welcomed by the BSAC.

The implication of such harmonization is that over time some MIC breakpoints will change slightly and these changes will be reflected, where necessary, in corresponding changes to zone diameter breakpoints in the BSAC disc diffusion method. The first of these will be included in this update to the method (version 4) and will be highlighted in the interpretative tables.

It is appreciated that changes in the method require additional work for laboratories in changing templates and laboratory information systems, but the benefits of international standardization are considerable, and review of some older breakpoints is undoubtedly warranted.

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 (http://www.bsac.org.uk), where the latest updates will be made available.

New or altered text compared with version 33 is indicated in bold.


    Introduction
 Top
 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 
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.4 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,5 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.6

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).7


    1 Preparation of plates
 Top
 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 

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) that can be obtained from Mast Group (Merseyside, UK) as in Table 1.
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.


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Table 1. A summary of media for testing depending on organism type

 

    2 Selection of control organisms
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 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 
2.1 The control strains listed in Table 2 should be included, as appropriate, with every batch of susceptibility tests.


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Table 2. Recommended control strains for inclusion, as appropriate, with every batch of susceptibility tests

 

    3 Preparation of inoculum
 Top
 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 
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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Figure 1. Example of the acceptable inoculum density range for a Gram-negative rod.

 
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. Thoroughly mix the McFarland standard to ensure that it is evenly suspended. Using matched cuvettes with a 1 cm light path and water as a blank standard, measure the absorbance in a spectrophotometer at a wavelength of 625 nm. The acceptable range for the turbidity standard is 0.08–0.13. 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), but commercial standards should be checked to ensure that absorbance is within the acceptable range—see above.

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 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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Table 3. Dilution ratios for suspensions using sterile distilled water

 
3.3 Photometric standardization of turbidity of suspensions

A photometric method of preparing inocula was described by Moosdeen et al.9 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. (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.)


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Table 4. Preparation of inoculum

 

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Table 5. Conditions of incubation

 

    4 Inoculation of agar plates
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 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 
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
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 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 

5.1 Disc contents are given in Tables 6GoGoGoGoGoGoGoGoGoGoGoGo19.


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Table 6. MIC and zone breakpoints for Enterobacteriaceae and Acinetobacter spp.

 

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Table 7. MIC and zone breakpoints for Pseudomonas spp. and S. maltophilia

 

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Table 8. MIC and zone breakpoints for staphylococci

 

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Table 9. MIC and zone breakpoints for S. pneumoniae

 

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Table 10. MIC and zone breakpoints for enterococci

 

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Table 11. MIC and zone breakpoints for {alpha}-haemolytic streptococci

 

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Table 12. MIC and zone breakpoints for ß-haemolytic streptococci

 

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Table 13. MIC and zone breakpoints for M. catarrhalis

 

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Table 14. MIC and zone breakpoints for N. gonorrhoeae

 

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Table 15. MIC and zone breakpoints for N. meningitidis

 

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Table 16. MIC and zone breakpoints for H. influenzae

 

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Table 17. MIC and zone breakpoints for P. multocida

 

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Table 18. MIC and zone breakpoints for urinary tract infections (Gram-negative rods)ad

 

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Table 19. MIC and zone breakpoints for urinary tract infections (Gram-positive cocci)ac

 
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 protect 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 discs on the expiry date shown on the side of the container.


    6 Incubation
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 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 

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, and higher stacks are acceptable if it can be shown that they do not affect zone diameter.


    7 Measuring zones and interpretation
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 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 

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 (see 7.3). 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. When using cefoxitin for the detection of methicillin/oxacillin/cefoxitin resistance in Staphylococcus aureus, measure the obvious zone, taking care to examine zones carefully in good light to detect minute colonies that may be present within the zone of inhibition.
7.2 Confirm that the zone of inhibition for the control strain falls within the acceptable ranges in Tables 20GoGo23 before interpreting the test.
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.



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Figure 2. Example of a template for interpreting susceptibility.

 

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Table 20. Acceptable zone ranges for Iso-Sensitest agar, plates incubated at 35–37°C in air for 18–20 h

 

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Table 21. ISA supplemented with 5% defibrinated horse blood, with or without the addition of NAD, plates incubated at 35–37°C in air for 18–20 h

 

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Table 22. Detection of methicillin/oxacillin/cefoxitin resistance in staphylococci

 

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Table 23. Iso-Sensitest agar supplemented with 5% defibrinated horse blood, with or without the addition of NAD, plates incubated at 35–37°C in 4–6% CO2 for 18–20 h

 

    8 Direct susceptibility testing
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 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 
8.1 Direct susceptibility testing of urines

The Working Party does not advocate direct susceptibility testing, as the control of 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.

  1. 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.
  2. 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 diptheroids
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.

Appendix 1. Amendments to the MIC and zone diameter breakpoints for ampicillin, amoxicillin and co-amoxiclav for interpreting the susceptibility of Enterobacteriaceae
In February 2003, after reviewing recent MIC and zone diameter data, the susceptible MIC breakpoints for ampicillin, amoxicillin and co-amoxiclav were raised to ≤16 mg/L for isolates from systemic infections. This change was made to avoid interpreting organisms at the top end of the normal susceptible population as resistant.

It has come to the attention of the BSAC Working Party on Susceptibility Testing that Enterobacteriaceae that are chromosomal AmpC enzyme producers have been misclassified as susceptible to these agents using the modified criteria. The breakpoints have therefore been amended again to include an intermediate category. Thus, the susceptible MIC breakpoint for ampicillin, amoxicillin and co-amoxiclav is ≤8 mg/L with an intermediate category of 16 mg/L and resistant >16 mg/L. The zone diameter breakpoints have been amended accordingly, thus the zone diameter breakpoint are as follows: ≥15 mm = susceptible, 12–14 mm = intermediate and ≤11 mm = resistant.

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.

Review of the zone diameter breakpoints for piperacillin/ tazobactam for interpreting the susceptibility of Enterobacteriaceae
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.

Advice on testing the susceptibility to co-trimoxazole is provided; however, the following are the UK Committee on the Safety of Medicines (CSM) recommendations
‘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.’

Appendix 4. Susceptibility testing H. influenzae to ß-lactam antibiotics
During the last year, several users of the method have reported problems when testing susceptibility of clinical isolates of H. 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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    References
 Top
 Preface
 Introduction
 1 Preparation of plates
 2 Selection of control...
 3 Preparation of inoculum
 4 Inoculation of agar...
 5 Antimicrobial discs
 6 Incubation
 7 Measuring zones and...
 8 Direct susceptibility testing
 References
 
1. Kahlmeter G, Brown DFJ, Goldstein FW et al. European harmonisation of MIC breakpoints for antimicrobial susceptibility testing of bacteria. J Antimicrob Chemother 2003; 52: 145–8.[Free Full Text]

2. Kahlmeter G, Brown DFJ. Harmonisation of antimicrobial breakpoints in Europe—can it be achieved? Clin Microbiol Newsl 2004; 24: 187–92.

3. Andrews JM. BSAC standardized disc susceptibility testing method (version 3). J Antimicrob Chemother 2004; 53: 713–28.[Free Full Text]

4. Report of the Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. A guide to sensitivity testing. J Antimicrob Chemother 1991; 27 Suppl D: 1–50.[ISI][Medline]

5. Andrews JM, Brown DFJ, Wise R. A survey of antimicrobial susceptibility testing in the United Kingdom. J Antimicrob Chemother 1996; 37: 187–8.[ISI][Medline]

6. Andrews JM. The development of the BSAC standardized method of disc diffusion testing. J Antimicrob Chemother 2001; 48 Suppl 1: 29–42.[Abstract/Free Full Text]

7. Inderlied CB, Nash KA. Antimycobacterial agents: in vitro susceptibility testing, spectra of activity, mechanisms of action and resistance, and assays for activity in biologic fluids. In: Lorian V, ed. Antibiotics in Laboratory Medicine. Baltimore, MD, USA: Williams and Wilkins, 1996; 127–75.

8. Brown DFJ. Detection of methicillin/oxacillin resistance in staphylococci. J Antimicrob Chemother 2001; 48 Suppl 1: 65–70.[Abstract/Free Full Text]

9. Moosdeen F, Williams JD, Secker A. Standardization of inoculum size for disc susceptibility testing: a preliminary report of a spectrophotometric method. J Antimicrob Chemother 1988; 21: 439–43.[Abstract]