JAC Advance Access originally published online on July 8, 2009
Journal of Antimicrobial Chemotherapy 2009 64(3):454-489; doi:10.1093/jac/dkp244
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Original research |
BSAC standardized disc susceptibility testing method (version 8)
Department of Microbiology, Sandwell and West Birmingham NHS Trust, City Hospital, Birmingham, B18 7QH, UK
* Corresponding author. Tel: +44-121-507-5693; Fax: +44-121-507-5521; E-mail: jenny.andrews{at}swbh.nhs.uk
Received 5 June 2009; returned 11 June 2009; revised 12 June 2009; accepted 18 June 2009
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Abstract |
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Top
Abstract
IntroductionThere have been considerable changes to the format of the recommendations since the previous version (version 7). The majority of the footnotes to the tables have been removed and the notations added to the end column; it is hoped that this change will avoid confusion in interpretation. Antibiotics have been separated into groups, e.g. β-lactams, aminoglycosides, etc. Recommendations for urinary tract infections (UTIs) have been removed for most agents except for those that are administered solely for the treatment of uncomplicated UTIs or where there are limited recommendations for specific organisms, e.g. trimethoprim. For agents that previously had dual recommendations, systemic recommendations remain and the intermediate category can be used for interpretation for UTIs because intermediate susceptibility infers that the infection may respond as the agent is concentrated at the site of infection. This change will also avoid errors in interpretation when an organism is isolated from multiple sites, e.g. blood and urine. The changes that have been made to version 7 are as follows: MIC and zone diameter breakpoints (BPs) for trimethoprim, fosfomycin and nitrofurantoin for UTIs (Table 7); MIC and zone diameter breakpoints (BPs) for doripenem (Tables 7–9); colistin MIC BPs for Pseudomonas spp. (Table 9), co-trimoxazole MIC BPs for Stenotrophomonas maltophilia (Table 10); staphylococci MIC and zone diameter BPs for clarithromycin, clindamycin, erythromycin, quinupristin/dalfopristin, trimethoprim UTI, nitrofurantoin UTI and rifampicin (Table 11); Streptococcus pneumoniae MIC and zone diameter BPs for azithromycin, clarithromycin, erythromycin, co-trimoxazole, linezolid, rifampicin and telithromycin (Table 12); addition of streptomycin recommendations for enterococci (Table 13); enterococcal MIC and zone diameter BPs for quinupristin/dalfopristin, nitrofurantoin UTI and trimethoprim UTI (Table 13); β-haemolytic streptococci MIC and zone diameter BPs for azithromycin, clarithromycin, erythromycin and telithromycin (Table 15); clarithromycin and erythromycin MIC and zone diameter BPs for Moraxella catarrhalis (Table 16); azithromycin MIC BPs for Neisseria gonorrhoeae (Table 17); chloramphenicol and rifampicin MIC BPs for Neisseria meningitidis (Table 18); azithromycin MIC BPs for Haemophilus influenzae (Table 19); MIC BPs for metronidazole for Bacteroides fragilis, Bacteroides thetaiotaomicron and Clostridium perfringens (Tables 23–25, respectively); susceptibility testing of Listeria spp. (Appendix 3); the acceptable range for ATCC 25923 to a 10 µg tobramycin disc (Table 26).
Keywords: breakpoints , disc testing , MICs
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Introduction |
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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 were 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 document, work is either ongoing (e.g. anaerobes) or reported elsewhere (e.g. mycobacteria).4
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1. Preparation of plates |
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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. Supplement media for fastidious organisms with 5% defibrinated horse blood or 5% defibrinated horse blood and 20 mg/L β-nicotinamide adenine dinucleotide (NAD) as indicated in Table 1. Use Columbia agar with 2% NaCl for methicillin/oxacillin susceptibility testing of staphylococci.
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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 before use. The length of time needed to dry the surface of the agar depends on the drying conditions, e.g. whether a fan-assisted drying cabinet or ‘still air’ incubator is used, whether plates are dried before storage and storage conditions. It is important that plates are not over-dried.
1.4 Store the plates in vented plastic boxes at 8–10°C prior to use. Alternatively the plates may be stored at 4–8°C in sealed plastic bags. Plate drying, method of storage and storage time should be determined by individual laboratories as part of their quality assurance programme. In particular, quality control tests should confirm that excess surface moisture is not produced and that plates are not over-dried.
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2. Selection of control organisms |
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2.1 The performance of the tests should be monitored by the use of appropriate control strains. The control strains listed (Tables 2 and 3) include susceptible strains that have been chosen to monitor test performance and resistant strains that can be used to confirm that the method will detect phenotypically resistant isolates.
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2.2 Store control strains at –70°C on beads in glycerol broth. Non-fastidious organisms may be stored at –20°C. Two vials of each control strain should be stored, one for an ‘in-use’ supply, the other for archiving.
2.3 Every week subculture a bead from the ‘in-use’ vial on to appropriate non-selective media and check for purity. From this pure culture, prepare one subculture on each of the following 5 days. For fastidious organisms that will not survive on plates for 5/6 days, subculture the strain daily for no more than 6 days.
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3. Preparation of inoculum |
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The inoculum should give semi-confluent growth of colonies after overnight incubation. Use of an inoculum that yields semi-confluent growth has the advantage that an incorrect inoculum can easily be observed. A denser inoculum will result in reduced zones of inhibition and a lighter inoculum will have the opposite effect. The following methods reliably give semi-confluent growth with most isolates.
NB. Other methods of obtaining semi-confluent growth may be used if they are 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% v/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 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 as indicated 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 
0.5 McFarland standard.
3.1.3 Inoculum preparation by the direct colony suspension method (the method of choice for fastidious organisms, i.e. Haemophilus spp., Neisseria gonorrhoeae, Neisseria meningitidis, Moraxella catarrhalis, Streptococcus pneumoniae, 
- and β-haemolytic streptococci, Clostridium perfringens, Bacteroides fragilis, Bacteroides thetaiotaomicron, Campylobacter spp., Pasteurella multocida and coryneform organisms).
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
3.1.5 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 4 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 four or five when possible) in 3 mL of 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 5 select the volume to transfer (with the appropriate fixed volume micropipette) to 5 mL of 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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3.4 Direct susceptibility testing
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.
3.4.1 Direct susceptibility testing of urines
- 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.
- 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.
3.4.2.2 Gram-negative bacilli
Using a venting needle, place one drop in 5 mL of sterile water and use this to inoculate ISA or equivalent agar.
3.4.2.2.1 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.
3.4.2.2.2 Staphylococci and enterococci
Using a venting needle, place three drops in 5 mL of sterile water and use this to inoculate ISA or equivalent agar.
3.4.2.2.3 Pneumococci, ‘viridans’ streptococci and diptheroids
Using a venting needle, place one drop in the centre of an ISA 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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4. Inoculation of agar plates |
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4.1 Use the adjusted suspension within 15 min to inoculate plates by dipping a sterile cotton-wool swab into the suspension and remove the excess liquid by turning the swab against the side of the container. 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 extended times before the discs are applied the organism may begin to grow, resulting in reduced zones of inhibition. Discs should therefore be applied to the surface of the agar within 15 min of inoculation.
4.2 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.
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5. Antimicrobial discs |
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5.1 Disc contents
Disc contents are given in Tables 7
–30.
5.2 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.
5.2.1 Store discs in sealed containers with a desiccant and protected from light (this is particularly important for some light-susceptible agents such as metronidazole, chloramphenicol and the quinolones).
5.2.2 Store stocks at –20°C except for drugs known to be unstable at this temperature (refer to the disc manufacturer's instructions on disc storage). If this is not possible, store discs at <8°C.
5.2.3 Store working supplies of discs at <8°C.
5.2.4 To prevent condensation, allow discs to warm to room temperature before opening containers.
5.2.5 Store disc dispensers in sealed containers with an indicating desiccant.
5.2.6 Discard any discs on the expiry date shown on the side of the container.
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.
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6. Incubation |
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6.1 Timing
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.
Conditions of incubation for different organisms are summarized in Table 6. Stacking plates too high in the incubator may affect results owing to uneven heating of plates. The efficiency of heating of plates depends on the incubator and the racking system used. Control of incubation, including height of plate stacking, should therefore be part of the laboratory's quality assurance programme.
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7. Measuring zones and interpretation |
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7.1 Acceptable inoculum density
The inoculum should give semi-confluent growth of colonies on the susceptibility plate, within the range illustrated in Figure 1.
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7.2 Measuring zones
7.2.1 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.
7.2.2 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.
7.2.3 Colonies growing within the zone of inhibition should be subcultured and identified and the test repeated if necessary.
7.2.4 When using cefoxitin for the detection of methicillin/oxacillin/cefoxitin resistance in S. 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 (see Figure 2).
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7.2.5 Confirm that the zone of inhibition for the control strain falls within the acceptable ranges in Tables 26
–30 before interpreting the test. 7.3 A template can also be used for interpreting zone diameters (Figure 3). 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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Funding |
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The Working Party on Susceptibility Testing is funded by the British Society for Antimicrobial Chemotherapy.
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Transparency declarations |
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None to declare.
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Appendix 1. Testing antimicrobial susceptibility to co-trimoxazole |
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Breakpoints for testing susceptibility to co-trimoxazole are provided. However, the following recommendations from the UK Committee on the Safety of Medicines (CSM) should be noted.
‘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 2. Efficacy of cefaclor in the treatment of respiratory infections caused by H. influenzae |
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Concerns have been expressed, particularly by laboratories moving from Stokes' method to the BSAC disc diffusion method, about the interpretation of susceptibility of H. influenzae to cefaclor. When using Stokes' method the majority of isolates appeared susceptible; but with the BSAC disc diffusion method most isolates are now reported resistant. The following comments explain the BSAC rationale for interpretation of cefaclor susceptibility.
Cefaclor pharmacokinetics. Cefaclor is dosed at 250–500 mg orally three times daily: 250 mg three times daily is probably the most common dose but data to confirm this are absent. The expected Cmax for 250 mg is 5–10 mg/L and 10–20 mg/L for 500 mg; the half-life is 1 h; drug concentration in blood is <1 mg/L at 4 h and protein binding is 25–50%. Tissue penetration is similar to other β-lactams.
Cefaclor potency against H. influenzae. Data from the BSAC surveillance programme 2003–04 (n = 899) indicates that the cefaclor MIC range is 0.12–128 mg/L; MIC50 2 mg/L; MIC90 8 mg/L.
Pharmacodynamics. An average patient with an H. influenzae infection will have a free drug time >MIC of 25% with 250 mg dosing and 37% with 500 mg dosing. A conservative time >MIC target for cephalosporins in community practice is 40%–50%, but this is not achieved with cefaclor. Therefore, it is likely that cefaclor will have at best borderline activity against H. influenzae.
Conclusion. The pharmacodynamic data indicate that cefaclor has borderline activity against H. influenzae, even for community use. The outcome of infection will be difficult to predict and susceptibility testing is likely to be of limited value.
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Appendix 3 |
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1. Susceptibility testing of Helicobacter pylori. Disc diffusion methods are not suitable for testing H. pylori as this species is slow growing and results may not be accurate. The recommended method of susceptibility testing is Etest (follow technical guide instructions).
Table A1. Tentative MIC breakpoints for Helicobacter pylori
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Table A2. MIC ranges for wild-type Legionella spp.
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Suspend colonies from a 2–3 day culture on a blood agar plate in sterile distilled water and adjust the density to equal a McFarland 3 standard.
Use a swab dipped in the suspension to inoculate evenly the entire surface of the plate. The medium of choice is Mueller–Hinton agar or Wilkins–Chalgren agar with 5%–10% horse blood.
Allow the plate to dry and apply the Etest strip.
Incubate at 35°C in microaerophilic conditions for 3–5 days.
Read the MIC at the point of complete inhibition of all growth, including hazes and isolated colonies. Tentative interpretative criteria for MICs are given in Table A1.
Table A3. MIC ranges for ‘wild-type’ Listeria spp.
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2. Susceptibility testing of Brucella species. Brucella spp. are Hazard Group 3 pathogens and all work must be done in containment level 3 accommodation. The antimicrobial agents most commonly used for treatment are doxycycline, rifampicin, ciprofloxacin, tetracycline and streptomycin and, from the limited information available, there is little or no resistance to these drugs. Brucella spp. are uncommon isolates and interpretative standards are not available. Since Brucella spp. are highly infectious, susceptibility testing in routine laboratories is not recommended.
3. Susceptibility testing of Legionella species. Legionella spp. are slow growing and have particular growth requirements. Disc diffusion methods for susceptibility testing are unsuitable. Susceptibility should be determined by agar dilution MICs on buffered yeast extract agar with 5% water-lysed horse blood.1 The antimicrobial agents commonly used for treatment are macrolides, rifampicin and fluoroquinolones. Validated MIC breakpoints are not established for Legionella spp. If results for test isolates are within range of the normal wild-type distribution, given in Table A2, clinical susceptibility may be assumed.
4. Susceptibility testing of Listeria spp. For susceptibility testing Listeria spp. an MIC determination is advised on ISA with incubation at 35–37°C in air. If a gradient method is used the test should be undertaken following the manufacturer's instructions. In Table A3 the MIC ranges and cut-offs for ‘wild-type’ strains are shown and these can be used as an aid to interpreting susceptibility.
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References |
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2
Andrews JM, Brown DFJ, Wise R. A survey of antimicrobial susceptibility testing in the United Kingdom. J Antimicrob Chemother (1996) 37:187–8.
3 Andrews JM. The development of the BSAC standardized method of disc diffusion testing. J Antimicrob Chemother (2001) 48(Suppl 1):29–42.[Abstract]
4 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: Antibiotics in Laboratory Medicine.—Lorian V, ed. (1996) Baltimore, MD, USA: Williams and Wilkins. 127–75.
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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.
7 Harbath S, Liassine N, Dharan S, et al. Risk factors for persistent carriage of methicillin-resistant Staphylococcus aureus. Clin Infect Dis (2000) 31:1380–5.[CrossRef][Web of Science][Medline]
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Elliott TS, Foweraker J, Gould FK, et al. Guidelines for the antibiotic treatment of endocarditis in adults: report of the Working Party of the British Society for Antimicrobial Chemotherapy. J Antimicrob Chemother (2004) 54:971–8.
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