Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Tenth Edition.

76 Pages·1999·0.55 MB·English

by  CLSI Clinical and Laboratory Standards Institute

M02-A10

Vol. 29 No. 1

Replaces M02-A9
January 2009
Vol. 26 No. 1
Performance Standards for Antimicrobial
Disk Susceptibility Tests; Approved
Standard—Tenth Edition

This document contains the current Clinical and Laboratory Standards Institute-
recommended methods for disk susceptibility testing, criteria for quality control testing,
and updated tables for interpretive zone diameters.
A standard for global application developed through the Clinical and Laboratory
Standards Institute consensus process.

Licensed to: Alejandra Corso
This document is protected by copyright. CLSI order # 60040, id # 478160, Downloaded on 1/13/2009.
Clinical and Laboratory Standards Institute
Advancing Quality in Health Care Testing

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M02-A10
ISBN 1-56238-688-3
Volume 29 Number 1 ISSN 0273-3099
Performance Standards for Antimicrobial Disk Susceptibility Tests;
Approved Standard—Tenth Edition

Matthew A. Wikler, MD, MBA, FIDSA Janet F. Hindler, MCLS, MT(ASCP)
Franklin R. Cockerill, III, MD Jean B. Patel, PhD, D(ABMM)
Karen Bush, PhD Mair Powell, MD, FRCP, FRCPath
Michael N. Dudley, PharmD John D. Turnidge, MD
George M. Eliopoulos, MD Melvin P. Weinstein, MD
Dwight J. Hardy, PhD Barbara L. Zimmer, PhD
David W. Hecht, MD

Mary Jane Ferraro, PhD, MPH
Jana M. Swenson, MMSc

Abstract

Susceptibility testing is indicated for any organism that contributes to an infectious process warranting antimicrobial
chemotherapy, if its susceptibility cannot be reliably predicted from knowledge of the organism’s identity. Susceptibility tests are
most often indicated when the causative organism is thought to belong to a species capable of exhibiting resistance to commonly
used antimicrobial agents.

A variety of laboratory methods can be used to measure the in vitro susceptibility of bacteria to antimicrobial agents. In many
clinical microbiology laboratories, an agar disk diffusion method is used routinely for testing common, rapidly growing, and
certain fastidious bacterial pathogens. Clinical and Laboratory Standards Institute document M02-A10—Performance Standards
for Antimicrobial Disk Susceptibility Tests; Approved Standard—Tenth Edition includes a series of procedures to standardize the
way disk diffusion tests are performed. The performance, applications, and limitations of the current CLSI-recommended
methods are also described.

The supplemental information (M100 tables) presented with this standard represents the most current information for drug
selection, interpretation, and quality control using the procedures standardized in M02. These tables, as in previous years, have
been updated and should replace tables published in earlier years. Changes in the tables since the previous edition (M100-S18)
appear in boldface type and are also summarized in the front of the document.

Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Disk Susceptibility Tests;
Approved Standard—Tenth Edition. CLSI document M02-A10 (ISBN 1-56238-688-3). Clinical and Laboratory Standards
Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2009.
Number 1 M02-A10

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two
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Suggested Citation

(CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Tenth
Edition. CLSI document M02-A10. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.)

Proposed Standard Approved Standard—Sixth Edition
July 1975 January 1997
Tentative Standard Approved Standard—Seventh Edition
October 1979 January 2000
Approved Standard Approved Standard—Eighth Edition
December 1984 January 2003
Tentative Standard—Fourth Edition Approved Standard—Ninth Edition
November 1988 January 2006
Approved Standard—Fourth Edition Approved Standard—Tenth Edition
April 1990 January 2009
Approved Standard—Fifth Edition
December 1993

ISBN 1-56238-688-3
ISSN 0273-3099
ii
Volume 29 M02-A10

Committee Membership

Area Committee on Microbiology

Mary Jane Ferraro, PhD, MPH John D. Turnidge, MD Michael A. Pfaller, MD
Chairholder Women’s and Children’s Hospital University of Iowa College of
Massachusetts General Hospital North Adelaide, Australia Medicine
Boston, Massachusetts Iowa City, Iowa
Michael L. Wilson, MD
John H. Rex, MD, FACP Denver Health Medical Center Robert P. Rennie, PhD
Vice-Chairholder Denver, Colorado University of Alberta Hospital
AstraZeneca Edmonton, Alberta, Canada
Cheshire, United Kingdom Advisors
Thomas R. Shryock, PhD
Barbara Ann Body, PhD, Nancy L. Anderson, MMSc, Elanco Animal Health
D(ABMM) MT(ASCP) Greenfield, Indiana
Laboratory Corporation of America Centers for Disease Control and
Burlington, North Carolina Prevention Jana M. Swenson, MMSc
Atlanta, Georgia Centers for Disease Control and
Betty (Betz) A. Forbes, PhD, Prevention
D(ABMM) Ellen Jo Baron, PhD Atlanta, Georgia
Medical College of Virginia Stanford Hospital and Clinics
Campus Palo Alto, California Melvin P. Weinstein, MD
Richmond, Virginia Robert Wood Johnson University
Donald R. Callihan, PhD Hospital
Freddie Mae Poole BD Diagnostic Systems New Brunswick, New Jersey
FDA Center for Devices and Sparks, Maryland
Radiological Health Matthew A. Wikler, MD, MBA,
Rockville, Maryland Lynne S. Garcia, MS FIDSA
LSG & Associates Pacific Beach BioSciences, Inc.
Daniel F. Sahm, PhD Santa Monica, California San Diego, California
Eurofins Medinet
Herndon, Virginia Richard L. Hodinka, PhD Gail L. Woods, MD
Children’s Hospital of Philadelphia Central Arkansas Veterans
Fred C. Tenover, PhD, ABMM Philadelphia, Pennsylvania Healthcare System
Cepheid Little Rock, Arkansas
Sunnyvale, California James H. Jorgensen, PhD
University of Texas Health Science
Center
San Antonio, Texas

Subcommittee on Antimicrobial Susceptibility Testing

Matthew A. Wikler, MD, MBA, George M. Eliopoulos, MD Jean B. Patel, PhD, D(ABMM)
FIDSA Beth Israel Deaconess Medical Centers for Disease Control and
Chairholder Center Prevention
Pacific Beach BioSciences, Inc. Boston, Massachusetts Atlanta, Georgia
San Diego, California
Dwight J. Hardy, PhD Mair Powell, MD, FRCP, FRCPath
Franklin R. Cockerill, III, MD University of Rochester Medical MHRA
Vice-Chairholder Center London, United Kingdom
Mayo Clinic/Mayo Foundation Rochester, New York
Rochester, Minnesota John D. Turnidge, MD
David W. Hecht, MD Women’s and Children’s Hospital
Karen Bush, PhD Loyola University Medical Center North Adelaide, Australia
Johnson & Johnson Pharmaceutical Maywood, Illinois
Research Institute Melvin P. Weinstein, MD
Raritan, New Jersey Janet F. Hindler, MCLS, Robert Wood Johnson Medical
MT(ASCP) School
Michael N. Dudley, PharmD UCLA Medical Center New Brunswick, New Jersey
Mpex Pharmaceuticals Los Angeles, California
San Diego, California

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Number 1 M02-A10

Members (continued) Yoichi Hirakata, MD, PhD Dale A. Schwab, PhD, D(ABMM)
Tohoku University Graduate School Quest Diagnostics, Nichols Institute
Barbara L. Zimmer, PhD of Medicine San Juan Capistrano, California
Siemens Healthcare Diagnostics Sendai, Japan
West Sacramento, California Daniel J. Sheehan, PhD
Ronald N. Jones, MD Greenwich, Conneticut
Advisors JMI Laboratories
North Liberty, Iowa Jana M. Swenson, MMSc
Paul G. Ambrose, PharmD, FIDSA Centers for Disease Control and
ICPD/Orway Research Institute Gunnar Kahlmeter, MD, PhD Prevention
Albany, New York ESCMID Atlanta, Georgia
Växjö, Sweden
Patricia A. Bradford, PhD George H. Talbot, MD
Wyeth Research Frederic J. Marsik, PhD, ABMM Talbot Advisors LLC
Pearl River, New York FDA Center for Drug Evaluation Wayne, Pennsylvania
and Research
Steven D. Brown, PhD Rockville, Maryland Fred C. Tenover, PhD, ABMM
The Clinical Microbiology Institute Cepheid
Wilsonville, Oregon John E. McGowan, Jr., MD Sunnyvale, California
Emory University, Rollins School
Karen Carroll, MD of Public Health Richard B. Thomson, Jr., PhD
Johns Hopkins Medical Institutions Atlanta, Georgia Northwestern University, Feinberg
Baltimore, Maryland School of Medicine
Linda A. Miller, PhD Evanston, Illinois
Edward M. Cox, Jr., MD, MPH GlaxoSmithKline
FDA Center for Drug Evaluation Collegeville, Pennsylvania Fredricka Valentine, MS,
and Research MT(ASCP)SM
Rockville, Maryland Janice Pohlman, MD, MPH FDA Center for Devices and
FDA Center for Drug Evaluation & Radiological Health
William A. Craig, MD Research Rockville, Maryland
University of Wisconsin Silver Spring, Maryland
Madison, Wisconsin
Sandra S. Richter, MD, D(ABMM)
Cynthia L. Fowler, MD University of Iowa Carver College
BioMérieux, Inc. of Medicine
Durham, North Carolina Iowa City, Iowa

Lawrence V. Friedrich, PharmD Flavia Rossi, MD
Cubist Pharmaceuticals University of Sao Paulo
Mt. Pleasant, South Carolina Sao Paulo, Brazil

Text and Table Working Group

Janet F. Hindler, MCLS, Susan D. Munro, MT(ASCP)
Jana M. Swenson, MMSc
MT(ASCP) Stanford Hospital and Clinics
Chairholder
UCLA Medical Center Palo Alto, California
Centers for Disease Control and
APHL
Prevention
Los Angeles, California Dale A. Schwab, PhD, D(ABMM)
Atlanta, Georgia
Quest Diagnostics, Nichols Institute

Judy Johnston, MS San Juan Capistrano, California
Donald R. Callihan, PhD
Siemens Healthcare Diagnostics
BD Diagnostic Systems
West Sacramento, California Albert T. Sheldon, Jr., PhD
Sparks, Maryland
Antibiotic & Antiseptic Consultants

Ronald N. Jones, MD Cypress, Texas
Franklin R. Cockerill, III, MD
JMI Laboratories
Mayo Clinic and Mayo College of
North Liberty, Iowa Richard B. Thomson, Jr., PhD
Medicine
Northwestern University Feinberg
Rochester, Minnesota
Dyan Luper, BS, MT(ASCP)SM School of Medicine

BD Diagnostic Systems Evanston, Illinois
Sharon K. Cullen, BS, RAC
Sparks, Maryland
Siemens Healthcare Diagnostics
Mary K. York, PhD, ABMM
W. Sacramento, California
Linda M. Mann, PhD, D(ABMM) MKY Microbiology Consulting

Siemens Healthcare Diagnostics Walnut Creek, California

West Sacramento, California
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Volume 29 M02-A10

Quality Control Working Group

Steve Brown, PhD Michael D. Huband Paul E. Oefinger, PhD, D(ABMM)
Co-Chairholder Pfizer Global R&D Covance Central Laboratory
Institutes for Microbiology Groton, Connecticut Services Inc.
Research Indianapolis, Indiana
Franklin, Tennessee Ronald N. Jones, MD
JMI Laboratories Jean Patel, PhD, D(ABMM)
Sharon K. Cullen, BS, RAC North Liberty, Iowa Centers for Disease Control and
Co-Chairholder Prevention
Siemens Healthcare Diagnostics Ann Macone Atlanta, Georgia
West Sacramento, California Paratek Pharmaceuticals, Inc.
Boston, Massachusetts Robert P. Rennie, PhD
William Brasso University of Alberta Hospital
BD Diagnostic Systems Ross Mulder, MT(ASCP) Edmonton, Canada
Sparks, Maryland BioMérieux, Inc.
Hazelwood, Missouri
Janet F. Hindler, MCLS,
MT(ASCP) Susan D. Munro, MT(ASCP)
UCLA Medical Center Stanford Hospital and Clinics
APHL Palo Alto, California
Los Angeles, California

Staphylococcal Working Group

Fred C. Tenover, PhD, ABMM Michael N. Dudley, PharmD, Maria M. Traczewski, BS,
Chairholder FIDSA MT(ASCP)
Cepheid Mpex Pharmaceuticals The Clinical Microbiology Institute
Sunnyvale, California San Diego, California Wilsonville, Oregon

Patricia A. Bradford, PhD George M. Eliopoulos, MD Melvin P. Weinstein, MD
Wyeth Research Beth Israel Deaconess Medical Robert Wood Johnson University
Pearl River, New Jersey Center Hospital
Boston, Massachusetts New Brunswick, New Jersey
Karen Bush, PhD
Johnson & Johnson Pharmaceutical Daniel F. Sahm, PhD
Research & Development, L.L.C. Eurofins Medinet
Raritan, New Jersey Herndon, Virginia

William A. Craig, MD Jana Swenson, MMSc
University of Wisconsin Centers for Disease Control and
Madison, Wisconsin Prevention
Atlanta, Georgia

Enterobacteriaceae Working Group

Michael N. Dudley, PharmD, Ronald N. Jones, MD Melvin P. Weinstein, MD
FIDSA JMI Laboratories Robert Wood Johnson University
Chairholder North Liberty, Iowa Hospital
Mpex Pharmaceuticals New Brunswick, New Jersey
San Diego, California David Paterson, MD
University of Pittsburgh Barbara L. Zimmer, PhD
Paul G. Ambrose, PharmD, FIDSA Pittsburgh, Pennsylvania Siemens Healthcare Diagnostics
ICPD/Ordway Research West Sacramento, California
Albany, New York Jana Swenson, MMSc
Centers for Disease Control and
Karen Bush, PhD Prevention
Johnson & Johnson Pharmaceutical Atlanta, Georgia
Research & Development, L.L.C.
Raritan, New Jersey Lauri D. Thrupp, MD
Univ. of California Irvine Medical Ctr.
William A. Craig, MD Orange, California
University of Wisconsin
Madison, Wisconsin

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Number 1 M02-A10

Staff

Clinical and Laboratory Standards Tracy A. Dooley, BS, MLT(ASCP)
Institute Staff Liaison
Wayne, Pennsylvania

Melissa A. Lewis

Lois M. Schmidt, DA Editor
Vice President, Standards
Development and Marketing
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Volume 29 M02-A10

Contents

Abstract .................................................................................................................................................... i 
Committee Membership ........................................................................................................................ iii 
Foreword ................................................................................................................................................. x 
Summary of Major Changes in This Document ..................................................................................... x 
Summary of CLSI Processes for Establishing Interpretive Criteria and QC Ranges ......................... xiii 
CLSI Reference Methods vs Commercial Methods and CLSI vs FDA Breakpoints
(interpretive criteria) ............................................................................................................................ xiv 
Subcommittee on Antimicrobial Susceptibility Testing Mission Statement ........................................ xv 
1  Scope .......................................................................................................................................... 1 
2  Introduction ................................................................................................................................ 1 
3  Standard Precautions .................................................................................................................. 1 
4  Terminology ............................................................................................................................... 2 
4.1  Definitions .................................................................................................................... 2 
4.2  Abbreviations/Acronyms .............................................................................................. 3 
5  Indications for Performing Susceptibility Tests ......................................................................... 3 
6  Selection of Antimicrobial Agents for Routine Testing and Reporting ..................................... 4 
6.1  Routine Reports ............................................................................................................ 4 
6.2  Nonproprietary Names .................................................................................................. 4 
6.3  Selection Guidelines ..................................................................................................... 7 
6.4  Suggested Guidelines for Routine and Selective Testing and Reporting ..................... 7 
7  Reagents for the Disk Diffusion Test ......................................................................................... 8 
7.1  Mueller-Hinton Agar .................................................................................................... 8 
7.2  Testing Strains That Fail to Grow Satisfactorily .......................................................... 9 
7.3  Antimicrobial Disks ...................................................................................................... 9 
8  Inoculum Preparation for Disk Diffusion Tests ....................................................................... 10 
8.1  Turbidity Standard for Inoculum Preparation ............................................................. 10 
8.2  Inoculum Preparation .................................................................................................. 10 
9  Procedure for Performing the Disk Diffusion Test .................................................................. 11 
9.1  Inoculation of Test Plates............................................................................................ 11 
9.2  Application of Disks to Inoculated Agar Plates .......................................................... 11 
9.3  Reading Plates and Interpreting Results ..................................................................... 12 
10  Fastidious Organisms ............................................................................................................... 13 
10.1  Haemophilus influenzae and H. parainfluenzae ......................................................... 13 
10.2  Neisseria gonorrhoeae ................................................................................................ 14 
10.3  Neisseria meningitidis ................................................................................................. 14 
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Contents (Continued)

10.4  Streptococcus pneumoniae and Other Streptococcus spp. .......................................... 15 
11  Organisms Requiring Special Consideration ........................................................................... 16 
11.1  Staphylococci .............................................................................................................. 17 
11.2  Enterococci ................................................................................................................. 22 
11.3  β-Lactamase-Mediated Resistance in Gram-Negative Bacilli .................................... 23 
11.4  Streptococcus pneumoniae.......................................................................................... 25 
12  Inducible Clindamycin Resistance ........................................................................................... 25 
13  β-Lactamase Tests ................................................................................................................... 26 
13.1  Purpose ....................................................................................................................... 26 
13.2  Selecting a β-Lactamase Test ..................................................................................... 26 
14  Interpretation of Disk Diffusion Test Results .......................................................................... 27 
14.1  Zone Diameter Interpretive Standards ........................................................................ 27 
14.2  Interpretive Categories ................................................................................................ 27 
15  Quality Control and Quality Assurance Procedures ................................................................ 27 
15.1  Purpose ....................................................................................................................... 27 
15.2  Quality Control Responsibilities ................................................................................. 27 
15.3  Selection of Quality Strains for Quality Control and Quality Assurance ................... 28 
15.4  Storing and Testing Quality Control Strains ............................................................... 29 
15.5  Batch or Lot Quality Control ...................................................................................... 29 
15.6  Zone Diameter Quality Control Limits ....................................................................... 29 
15.7  Frequency of Quality Control Testing ....................................................................... 30 
15.8  Corrective Action ........................................................................................................ 30 
15.9  Reporting Patient Results When Out-of-Control Tests Occur .................................... 32 
15.10  Verification of Patient Test Results ............................................................................ 32 
15.11  Other Control Procedures ........................................................................................... 33 
16  Limitations of Disk Diffusion Methods ................................................................................... 33 
16.1  Application to Various Organism Groups .................................................................. 33 
16.2  Misleading Results ...................................................................................................... 34 
16.3  Emergence of Resistance ............................................................................................ 34 
17  Screening Tests ........................................................................................................................ 34 
References ............................................................................................................................................. 35 
Appendix A. Quality Control Protocol Flow Charts ............................................................................. 37 
Appendix B. Preparation of Media and Reagents ................................................................................. 39 
Appendix C. Conditions for Disk Diffusion Antimicrobial Susceptibility Tests ................................. 42 
Appendix D. Quality Control Strains for Antimicrobial Susceptibility Tests ...................................... 44 
Appendix E. Quality Control Strain Maintenance ................................................................................ 47 
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