JAC Advance Access published online on May 12, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn194
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Original research |
BSAC standardized disc susceptibility testing method (version 7)
Department of Microbiology, City Hospital NHS Trust, 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 14 March 2008; returned 30 March 2008; revised 1 April 2008; accepted 2 April 2008
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Abstract |
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Top
Abstract
IntroductionThe changes that have been made to the previous version of the recommendations (version 6) are as follows: medium and incubation condition for testing Acinetobacter spp. (Tables 1 and 6); use of cefoxitin as an indicator antibiotic for detecting methicillin/oxacillin/cefoxitin resistance in coagulase-negative staphylococci (Tables 1, 6 and 11); MIC breakpoint for co-trimoxazole based on the trimethoprim concentration in a 1:19 combination with sulfamethoxazole (Tables 7, 10, 11, 12, 15, 16 and 19); advice on the use of azithromycin for the treatment of infections with Salmonella typhi (footnote to Table 7); amendment to the recommendation for cefuroxime for the treatment of infections with Proteus mirabilis (footnote Table 7); MIC and zone diameter breakpoints for Stenotrophomonas maltophilia only (Table 10); MIC breakpoints for daptomycin (Tables 11 and 15); clarification for staphylococci that the neomycin zone diameter breakpoints are for topical use only and differentiate the isolates outside the ‘wild-type’ population in Table 11; clarification for β-haemolytic streptococci that the linezolid zone diameter breakpoints relate to an MIC breakpoint of 2 mg/L as no data for the intermediate category are currently available (Table 15); clarification that strains with reduced susceptibility to fluoroquinolones give no zone of inhibition with a 30 µg nalidixic acid disc (Tables 16 and 21); erythromycin is no longer used for therapy of Neisseria gonorrhoeae, but may be tested for epidemiological purposes (Table 17); clarification that the ciprofloxacin zone diameter breakpoint for Neisseria meningitidis relates to the MIC breakpoint of 0.03 mg/L as no data for the intermediate category are currently available; clarification that the ciprofloxacin zone diameter breakpoints for Campylobacter spp. relate to an MIC breakpoint of 0.5 mg/L as no data for the intermediate category are currently available; clarification that for ciprofloxacin and vancomycin zone diameter breakpoints for coryneform organisms relate to an MIC breakpoint of 0.5 and 4 mg/L, respectively, as no data for the intermediate category are currently available; MIC and zone diameter breakpoints for Gram-negative rods isolated from urinary tract infections have been expanded to include Klebsiella spp.; and a definition of coliforms is also included (Table 26).
Key Words: 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
New or altered text compared with version 65 is indicated in bold.
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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 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 optical density 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 optical density 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 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, 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
Adjust the density of the organism suspension prepared, as in Section 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.7 and from this the following simplified procedure has been developed.
3.3.1 Suspend colonies (touch 4–5 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 L 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.
The method suggested 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.
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 Iso-Sensitest 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 Iso-Sensitest 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 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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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 7–26.
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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 (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 27–32 before interpreting the test.
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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 BSAC 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.
Appendix 1. Testing antimicrobial susceptibility to co-trimoxazole
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 generalised 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 2. Efficacy of cefaclor in the treatment of respiratory infections caused by H. influenzae
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 as 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 the 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) indicate 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% to 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.
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).
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% to 10% horse blood.
Allow the plate to dry and apply 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.
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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.
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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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Andrews JM. BSAC standardized disc susceptibility testing method (version 6). J Antimicrob Chemother (2007) 60:20–41.
6 . Brown DFJ. Detection of methicillin/oxacillin resistance in staphylococci. J Antimicrob Chemother (2001) 48(Suppl 1):65–70.[Abstract]
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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.
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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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