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Chapter 68 : Susceptibility Test Methods: Dilution and Disk Diffusion Methods

Authors: Jean B. Patel, Fred C. Tenover, John D. Turnidge, James H. Jorgensen
Content Type: Reference
Publication Year: 2011

Category: Clinical Microbiology

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Abstract:

There are a number of methods for antimicrobial susceptibility testing of bacteria, and they are categorized into dilution methods that generate Minimum Inhibitory Concentration (MIC) results and disk diffusion methods that generate zone diameter results. The Clinical and Laboratory Standards Institute (CLSI) reference methods are broth macrodilution, broth microdilution, agar dilution, and disk diffusion. Dilution methods are also readily adaptable to automated test systems. Stock solutions are prepared as discussed in the CLSI document on dilution testing and are the same as those used for agar dilution tests. In fact, the broth microdilution method is now considered the international reference susceptibility testing method. This chapter focuses on in-house preparation and use of broth microdilution panels. Most of the principles and practices discussed in the chapter are pertinent to the broth microdilution method regardless of the source of the antibiotic panels. The dilution scheme for preparing broth microdilution panels is the same as that described for agar and broth macrodilution methods. The definitions of the interpretive categories and the comments concerning the use of the standards for agar and broth macrodilution methods are also applicable to broth microdilution methods. Like full-range dilution testing, breakpoint methods require the use of appropriately adjusted and supplemented Mueller-Hinton broth or agar. In addition, the standard inoculation, incubation, and interpretation procedures recommended for the full-range dilution methods should be followed.

Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68
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Susceptibility Test Methods: Dilution and Disk Diffusion Methods
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Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68
/content/10.1128/9781555816728.chap68.fig68-1
FIGURE 1

Example of the wild-type (WT) MIC distribution data that are used by EUCAST to set the epidemiological cutoff.

10.1128/9781555816728/fig68-1_thmb.gif
10.1128/9781555816728/fig68-1.gif
Image of FIGURE 1
FIGURE 1

Example of the wild-type (WT) MIC distribution data that are used by EUCAST to set the epidemiological cutoff.

Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68
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Tables

Susceptibility Test Methods: Dilution and Disk Diffusion Methods
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Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68
/content/10.1128/9781555816728.chap68.tab68-1
TABLE 1

Antibacterial susceptibility results that may be extrapolated from other test results

10.1128/9781555816728/tab68-1_thmb.gif
10.1128/9781555816728/tab68-1.gif
Generic image for table
TABLE 1

Antibacterial susceptibility results that may be extrapolated from other test results

Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68
/content/10.1128/9781555816728.chap68.tab68-2
TABLE 2

Antimicrobial agents recommended for routine dilution and disk diffusion susceptibility testing

Modified from CLSI document M100-S20 ( ) with permission. Current standards and supplements to them may be obtained from the CLSI, 940 West Valley Rd., Suite 1400, Wayne, PA 19087-1898.

Group A comprises primary drugs to be tested and reported, group B comprises those to be tested as primary drugs but reported selectively, group C comprises supplemental drugs to be reported selectively, and group U comprises drugs to be tested with urinary isolates only

Non- include spp. and other nonfastidious, non-glucose-fermenting gram-negative bacilli but exclude , spp., , , , and , since there are separate listings of suggested drugs to test and report for them.

Those agents marked with an asterisk should be tested only by using an MIC method and not by using disk diffusion.

Results of tests with penicillin apply to other penicillins (e.g., ampicillin, amoxicillin, carboxypenicillins, and ureidopenicillins) against β-lactamase-negative enterococci.

Combination therapy consisting of penicillin, ampicillin, or vancomycin and an aminoglycoside is recommended for serious infections.

Staphylococci resistant to the penicillinase-resistant penicillins should also be considered resistant to penicillins, β-lactam–β-lactamase-inactivating combinations, cephalosporins (except cephalosporins with anti-MRSA activity), and carbapenems.

Cephalothin test results should only be used to represent oral agents, i.e., cefadroxil, cefpodoxime, cephalexin, and loracarbef. Older data which suggest that cephalothin results could predict susceptibility to some other cephalosporins may still be correct, but there are no recent data to confirm this.

Cefoxitin is used as a surrogate for oxacillin for staphylococci.

For use of aminoglycosides to screen enterococci for synergy resistance, see the sections “Breakpoint Susceptibility Tests” and “Resistance Screens.”

Doxycycline or minocycline may be tested on a supplemental basis because of their greater activities against some nonfermentative gram-negative bacilli and staphylococci.

10.1128/9781555816728/tab68-2_thmb.gif
10.1128/9781555816728/tab68-2.gif
Generic image for table
TABLE 2

Antimicrobial agents recommended for routine dilution and disk diffusion susceptibility testing

Modified from CLSI document M100-S20 ( ) with permission. Current standards and supplements to them may be obtained from the CLSI, 940 West Valley Rd., Suite 1400, Wayne, PA 19087-1898.

Group A comprises primary drugs to be tested and reported, group B comprises those to be tested as primary drugs but reported selectively, group C comprises supplemental drugs to be reported selectively, and group U comprises drugs to be tested with urinary isolates only

Non- include spp. and other nonfastidious, non-glucose-fermenting gram-negative bacilli but exclude , spp., , , , and , since there are separate listings of suggested drugs to test and report for them.

Those agents marked with an asterisk should be tested only by using an MIC method and not by using disk diffusion.

Results of tests with penicillin apply to other penicillins (e.g., ampicillin, amoxicillin, carboxypenicillins, and ureidopenicillins) against β-lactamase-negative enterococci.

Combination therapy consisting of penicillin, ampicillin, or vancomycin and an aminoglycoside is recommended for serious infections.

Staphylococci resistant to the penicillinase-resistant penicillins should also be considered resistant to penicillins, β-lactam–β-lactamase-inactivating combinations, cephalosporins (except cephalosporins with anti-MRSA activity), and carbapenems.

Cephalothin test results should only be used to represent oral agents, i.e., cefadroxil, cefpodoxime, cephalexin, and loracarbef. Older data which suggest that cephalothin results could predict susceptibility to some other cephalosporins may still be correct, but there are no recent data to confirm this.

Cefoxitin is used as a surrogate for oxacillin for staphylococci.

For use of aminoglycosides to screen enterococci for synergy resistance, see the sections “Breakpoint Susceptibility Tests” and “Resistance Screens.”

Doxycycline or minocycline may be tested on a supplemental basis because of their greater activities against some nonfermentative gram-negative bacilli and staphylococci.

Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68
/content/10.1128/9781555816728.chap68.tab68-3
TABLE 3

Interpretive standards for dilution and disk diffusion susceptibility testing

Adapted from CLSI data ( ) with permission. The interpretive data are valid only if the methodologies in documents M2-A10 ( ) and M7-A8 ( ) are followed. Breakpoints for some agents apply only to certain genera or species. The CLSI frequently updates the interpretive tables through new editions of the standards and supplements to them. Users should refer to the most recent editions. The current standards and supplements to them may be obtained from the CLSI, 940 West Valley Rd., Suite 1400, Wayne, PA 19087-1898.

Penicillin should be used as the class representative for all penicillins (e.g., ampicillin, amoxicillin, mezlocillin, piperacillin, and ticarcillin). Isolates for which MICs are ≤0.03 mg of penicillin per ml generally do not produce β-lactamase, whereas those for which MICs are ≥0.25 mg/ml do and should be regarded as resistant to penicillins. Isolates for which MICs of penicillin are 0.06 or 0.12 mg/ml should be tested for β-lactamase.

Therapy for serious enterococcal infections requires high doses of penicillin or ampicillin in combination with an aminoglycoside. Vancomycin may be substituted for the penicillin in instances of penicillin hypersensitivity or of penicillin or ampicillin resistance.

Oxacillin or methicillin may be tested; however, oxacillin is preferred because of its greater stability in vitro. The results from the testing of oxacillin apply also to other penicillinase-resistant penicillins. Oxacillin-resistant staphylococci should be considered resistant to all penicillins, cephalosporins (except cephalosporins with anti-MRSA activity), carbacephems, carbapenems, and β-lactam–β-lactamase inhibitor combinations. Disk testing with cefoxitin is the most sensitive and specific method for phenotypic detection of A-mediated oxacillin resistance in staphylococci ( ).

For the treatment of urinary tract infections only.

Interpretive criteria are from the Food and Drug Administration drug label.

10.1128/9781555816728/tab68-3_thmb.gif
10.1128/9781555816728/tab68-3.gif
Generic image for table
TABLE 3

Interpretive standards for dilution and disk diffusion susceptibility testing

Adapted from CLSI data ( ) with permission. The interpretive data are valid only if the methodologies in documents M2-A10 ( ) and M7-A8 ( ) are followed. Breakpoints for some agents apply only to certain genera or species. The CLSI frequently updates the interpretive tables through new editions of the standards and supplements to them. Users should refer to the most recent editions. The current standards and supplements to them may be obtained from the CLSI, 940 West Valley Rd., Suite 1400, Wayne, PA 19087-1898.

Penicillin should be used as the class representative for all penicillins (e.g., ampicillin, amoxicillin, mezlocillin, piperacillin, and ticarcillin). Isolates for which MICs are ≤0.03 mg of penicillin per ml generally do not produce β-lactamase, whereas those for which MICs are ≥0.25 mg/ml do and should be regarded as resistant to penicillins. Isolates for which MICs of penicillin are 0.06 or 0.12 mg/ml should be tested for β-lactamase.

Therapy for serious enterococcal infections requires high doses of penicillin or ampicillin in combination with an aminoglycoside. Vancomycin may be substituted for the penicillin in instances of penicillin hypersensitivity or of penicillin or ampicillin resistance.

Oxacillin or methicillin may be tested; however, oxacillin is preferred because of its greater stability in vitro. The results from the testing of oxacillin apply also to other penicillinase-resistant penicillins. Oxacillin-resistant staphylococci should be considered resistant to all penicillins, cephalosporins (except cephalosporins with anti-MRSA activity), carbacephems, carbapenems, and β-lactam–β-lactamase inhibitor combinations. Disk testing with cefoxitin is the most sensitive and specific method for phenotypic detection of A-mediated oxacillin resistance in staphylococci ( ).

For the treatment of urinary tract infections only.

Interpretive criteria are from the Food and Drug Administration drug label.

Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68
/content/10.1128/9781555816728.chap68.tab68-4
TABLE 4

Non-U.S. disk diffusion methods for susceptibility testing

http://www.eucast.org.

http://www.bsac.org.uk.

MH, Mueller-Hinton agar.

10.1128/9781555816728/tab68-4_thmb.gif
10.1128/9781555816728/tab68-4.gif
Generic image for table
TABLE 4

Non-U.S. disk diffusion methods for susceptibility testing

http://www.eucast.org.

http://www.bsac.org.uk.

MH, Mueller-Hinton agar.

Citation: Patel J, Tenover F, Turnidge J, Jorgensen J. 2011. Susceptibility Test Methods: Dilution and Disk Diffusion Methods, p 1122-1143. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch68

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