m45-a2: methods for antimicrobial dilution and disk ... · available, changes will be incorporated...

M45-A2

Vol. 30 No. 18 Replaces M45-A

Vol. 26 No. 19

Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second Edition

This document provides guidance to clinical microbiology laboratories for standardized susceptibility testing of infrequently isolated or fastidious bacteria that are not presently included in CLSI documents M02 or M07. The tabular information in this document presents the most current information for drug selection, interpretation, and quality control for the infrequently isolated or fastidious bacterial pathogens included in this guideline. A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.

Product Name: Infobase 2011 - Release Date: January 2011

This document is protected by international copyright laws.

Clinical and Laboratory Standards Institute Advancing Quality in Health Care Testing Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) is an international, interdisciplinary, nonprofit, standards-developing, and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the health care community. It is recognized worldwide for the application of its unique consensus process in the development of standards and guidelines for patient testing and related health care issues. Our process is based on the principle that consensus is an effective and cost-effective way to improve patient testing and health care services.

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Most documents are subject to two levels of consensus—“proposed” and “approved.” Depending on the need for field evaluation or data collection, documents may also be made available for review at an intermediate consensus level.

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The comments of users are essential to the consensus process. Anyone may submit a comment, and all comments are addressed, according to the consensus process, by the committee that wrote the document. All comments, including those that result in a change to the document when published at the next consensus level and those that do not result in a change, are addressed by the committee in an appendix to the document. Readers are strongly encouraged to comment in any form and at any time on any document. Address comments to Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, PA 19087, USA.

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M45-A2 ISBN 1-56238-732-4

Volume 30 Number 18 ISSN 0273-3099

Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second Edition James H. Jorgensen, PhD Patrick McDermott, PhD Janet A. Hindler, MCLS, MT(ASCP) Jean B. Patel, PhD, D(ABMM) Kathryn Bernard, MS Paul C. Schreckenberger, PhD, D(ABMM) Diane M. Citron, M(ASCP) John D. Turnidge, MD Franklin R. Cockerill, III, MD David F. Welch, PhD, D(ABMM) Thomas R. Fritsche, PhD, MD Guido Funke, MD Henry Heine, PhD

Abstract If the susceptibility of a bacterial pathogen to antimicrobial agents cannot be predicted based on the identity of the organism alone, in vitro antimicrobial susceptibility testing of the organism isolated may be indicated. Susceptibility testing is particularly necessary in those situations in which the etiological agent belongs to a bacterial species for which resistance to commonly used antimicrobial agents has been documented, or could arise. A variety of laboratory techniques can be used to measure the in vitro susceptibility of bacteria to antimicrobial agents. Clinical and Laboratory Standards Institute document M45-A2—Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second Edition describes the standard microdilution and agar disk diffusion methods. It also includes a series of procedures designed to standardize test performance. The performance, applications, and limitations of the current CLSI-recommended methods are described. The tabular information in this document presents the most current information for drug selection, interpretation, and quality control for the infrequently isolated or fastidious bacterial pathogens included in this guideline. As more information becomes available, changes will be incorporated into future revisions of this document. Clinical and Laboratory Standards Institute (CLSI). Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second Edition. CLSI document M45-A2 (ISBN 1-56238-732-4). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2010.

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in the CLSI catalog and posted on our website at www.clsi.org. If your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: [email protected]; Website: www.clsi.org



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Copyright ©2010 Clinical and Laboratory Standards Institute. Except as stated below, neither this publication nor any portion thereof may be adapted, copied, or otherwise reproduced, by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission from Clinical and Laboratory Standards Institute (“CLSI”). CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of this publication for use in its laboratory procedure manual at a single site. To request permission to use this publication in any other manner, contact the Executive Vice President, Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. Suggested Citation CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second Edition. CLSI document M45-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. Proposed Guideline October 2005 Approved Guideline May 2006 Approved Guideline—Second Edition August 2010 ISBN 1-56238-732-4 ISSN 0273-3099



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Committee Membership Area Committee on Microbiology

Subcommittee on Antimicrobial Susceptibility Testing Franklin R. Cockerill, III, MD Chairholder Mayo Clinic College of Medicine Rochester, Minnesota, USA Matthew A. Wikler, MD, MBA, FIDSA Vice-Chairholder Institute for One World Health San Diego, California, USA Karen Bush, PhD Indiana University Bloomington, Indiana, USA Michael N. Dudley, PharmD, FIDSA Mpex Pharmaceuticals San Diego, California, USA

George M. Eliopoulos, MD Beth Israel Deaconess Medical Center Boston, Massachusetts, USA Dwight J. Hardy, PhD University of Rochester Medical Center Rochester, New York, USA David W. Hecht, MD Loyola University Medical Center Maywood, Illinois, USA Janet A. Hindler, MCLS, MT(ASCP) UCLA Medical Center Los Angeles, California, USA Jean B. Patel, PhD, D(ABMM) Centers for Disease Control and Prevention Atlanta, Georgia, USA

Mair Powell, MD, FRCP, FRCPath MHRA London, United Kingdom Richard B. Thomson, Jr., PhD Evanston Hospital and The University of Chicago School of Medicine Evanston, Illinois, USA John D. Turnidge, MD Women’s and Children’s Hospital North Adelaide, Australia Melvin P. Weinstein, MD Robert Wood Johnson Medical School New Brunswick, New Jersey, USA Barbara L. Zimmer, PhD Siemens Healthcare Diagnostics West Sacramento, California, USA

John H. Rex, MD, FACP Chairholder AstraZeneca Cheshire, United Kingdom Mary Jane Ferraro, PhD, MPH Vice-Chairholder Massachusetts General Hospital Boston, Massachusetts, USA Nancy L. Anderson, MMSc, MT(ASCP) Centers for Disease Control and Prevention Atlanta, Georgia, USA Barbara Ann Body, PhD, D(ABMM) Laboratory Corporation of America Burlington, North Carolina, USA Betty (Betz) A. Forbes, PhD, D(ABMM) Medical College of Virginia Campus Richmond, Virginia, USA Thomas R. Fritsche, MD, PhD Marshfield Clinic Marshfield, Wisconsin, USA Freddie Mae Poole, MS, MT FDA Center for Devices and Radiological Health Rockville, Maryland, USA

Fred C. Tenover, PhD, D(ABMM) Cepheid Sunnyvale, California, USA John D. Turnidge, II, MD Women’s and Children’s Hospital North Adelaide, Australia Advisors Donald R. Callihan, PhD BD Diagnostic Systems Sparks, Maryland, USA James H. Jorgensen, PhD University of Texas Health Science Center San Antonio, Texas, USA Jean B. Patel, PhD, D(ABMM) Centers for Disease Control and Prevention Atlanta, Georgia, USA Michael A. Pfaller, MD University of Iowa College of Medicine Iowa City, Iowa, USA Thomas R. Shryock, PhD Elanco Animal Health Greenfield, Indiana, USA

Jana M. Swenson, MMSc Centers for Disease Control and Prevention Atlanta, Georgia, USA Jeffrey L. Watts, PhD, RM(AAM) Pfizer Animal Health Richland, Michigan, USA Melvin P. Weinstein, MD Robert Wood Johnson Medical School New Brunswick, New Jersey, USA Nancy Wengenack, PhD Mayo Clinic Rochester, Minnesota, USA Matthew A. Wikler, MD, MBA, FIDSA Institute for One World Health San Francisco, California, USA Michael L. Wilson, MD Denver Health Medical Center Denver, Colorado, USA Gail L. Woods, MD Central Arkansas Veterans Healthcare System Little Rock, Arkansas, USA Barbara L. Zimmer, PhD Siemens Healthcare Diagnostics West Sacramento, California, USA



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Advisors Paul G. Ambrose, PharmD, FIDSA ICPD/Orway Research Institute Albany, New York, USA Patricia A. Bradford, PhD Novartis Institutes for Biomedical Research Cambridge, Massachusetts, USA Steven D. Brown, PhD The Clinical Microbiology Institute Wilsonville, Oregon, USA Karen Carroll, MD Johns Hopkins Medical Institutions Baltimore, Maryland, USA Edward M. Cox, Jr., MD, MPH FDA Center for Drug Evaluation and Research Rockville, Maryland, USA William A. Craig, MD Wm. S. Middleton Memorial VA Hospital Madison, Wisconsin, USA Cynthia L. Fowler, MD bioMérieux, Inc. Durham, North Carolina, USA

Yoichi Hirakata, MD, FJSIM, PhD Tohoku University Graduate School of Medicine Sendai, Japan Ronald N. Jones, MD JMI Laboratories North Liberty, Iowa, USA Gunnar Kahlmeter, MD, PhD ESCMID Växjö, Sweden Frederic J. Marsik, PhD, ABMM FDA Center for Drug Evaluation and Research Silver Spring, Maryland, USA Linda A. Miller, PhD GlaxoSmithKline Collegeville, Pennsylvania, USA Harriette L. Nadler, PhD DJA Global Pharmaceuticals, Inc. Chadds Ford, Pennsylvania, USA Freddie Mae Poole, MS, MT FDA Center for Devices and Radiological Health Rockville, Maryland, USA

Sandra S. Richter, MD, D(ABMM) University of Iowa Carver College of Medicine Iowa City, Iowa, USA Flavia Rossi, MD University of Sao Paulo Sao Paulo, Brazil Dale A. Schwab, PhD, D(ABMM) Quest Diagnostics, Nichols Institute San Juan Capistrano, California, USA Jana M. Swenson, MMSc Centers for Disease Control and Prevention Atlanta, Georgia, USA Fred C. Tenover, PhD, D(ABMM) Cepheid Sunnyvale, California, USA Joseph G. Toerner, MD, MPH FDA Center for Drug Evaluation and Research Silver Spring, Maryland, USA Hui Wang, PhD Peking Union Medical College Hospital Beijing, China

Working Group on Fastidious Organisms James H. Jorgensen, PhD Chairholder University of Texas Health Science Center San Antonio, Texas, USA Janet A. Hindler, MCLS, MT(ASCP) Vice-Chairholder UCLA Medical Center Los Angeles, California, USA Kathryn Bernard, MS Public Health Agency of Canada Winnipeg, Manitoba, Canada Diane M. Citron, M(ASCP) R.M. Alden Research Laboratory Culver City, California, USA Franklin R. Cockerill, III, MD Mayo Clinic College of Medicine Rochester, Minnesota, USA Thomas R. Fritsche, MD, PhD Marshfield Clinic Marshfield, Wisconsin, USA

Guido Funke, MD Gartner & Colleagues Laboratories Weingarten, Germany Henry Heine, PhD Ordway Research Institute, Inc. Albany, New York, USA Patrick McDermott, PhD FDA Center for Veterinary Medicine Laurel, Maryland, USA Jean B. Patel, PhD, D(ABMM) Centers for Disease Control and Prevention Atlanta, Georgia, USA Paul C. Schreckenberger, PhD, D(ABMM), F(AAM) Loyola University Medical Center Maywood, Illinois, USA John D. Turnidge, MD SA Pathology at Women’s and Children’s Hospital North Adelaide, Australia

David F. Welch, PhD, D(ABMM) University of Texas Southwestern Medical Center Dallas, Texas, USA Advisors Paul G. Ambrose, PharmD, FIDSA ICPD/Ordway Research Latham, New York, USA Anton F. Ehrhardt, PhD Cubist Pharmaceuticals, Inc. Lexington, Massachusetts, USA Frederic J. Marsik, PhD, ABMM FDA Center for Drug Evaluation and Research Silver Spring, Maryland, USA Jana M. Swenson, MMSc Centers for Disease Control and Prevention Atlanta, Georgia, USA



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Advisors (Continued) Fred C. Tenover, PhD, D(ABMM) Cepheid Sunnyvale, California, USA Mary K. York, PhD, ABMM MKY Microbiology Consulting Walnut Creek, California, USA Staff Clinical and Laboratory Standards Institute Wayne, Pennsylvania, USA Lois M. Schmidt, DA Vice President, Standards Development Tracy A. Dooley, BS, MLT(ASCP) Staff Liaison Melissa A. Lewis, ELS Editorial Manager



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Contents

Abstract .................................................................................................................................................... i

Committee Membership ........................................................................................................................ iii

Foreword ................................................................................................................................................ xi

1 Scope .......................................................................................................................................... 1

2 Introduction ................................................................................................................................ 1

2.1 Resistance Mechanisms in Gram-Positive Rods ........................................................... 2 2.2 Resistance in Infrequently Isolated or Fastidious Gram-Positive Cocci ....................... 2 2.3 Infrequently Isolated Nonfastidious Gram-Negative Rods ........................................... 2 2.4 Fastidious Gram-Negative Rods ................................................................................... 2 2.5 Moraxella catarrhalis ................................................................................................... 3 2.6 Potential Bacterial Agents of Bioterrorism ................................................................... 3 2.7 The Development of Interpretive Criteria or Breakpoints ............................................ 3

3 Standard Precautions .................................................................................................................. 4

4 Terminology ............................................................................................................................... 4

4.1 A Note on Terminology ................................................................................................ 4 4.2 Definitions .................................................................................................................... 4 4.3 Abbreviations and Acronyms ....................................................................................... 5

5 Indications for Performing Susceptibility Tests ......................................................................... 5

6 Methods for Dilution Antimicrobial Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria ..................................................................................................................... 6

6.1 Selection of Antimicrobial Agents ................................................................................ 6 6.2 Antimicrobial Agents .................................................................................................... 6 6.3 Interpretive Categories .................................................................................................. 7

7 Methods for Antimicrobial Disk Diffusion Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria ..................................................................................................................... 7

8 Therapy-Related Comments ...................................................................................................... 7

9 Quality Control .......................................................................................................................... 7

9.1 Minimum Laboratory Requirements for Testing Infrequently Isolated or Fastidious Bacteria ......................................................................................................................... 8

10 Detection of Resistance to Some β-Lactams by a Direct β-Lactamase Test ............................. 8

Table 1. Abiotrophia spp. and Granulicatella spp. (Formerly Known as Nutritionally Deficient or Nutritionally Variant Streptococci)—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing ............................................................................................................................ 10

Table 2. Aeromonas spp. (Includes Members of A. caviae Complex, A. hydrophila Complex, and A. veronii Complex) and Plesiomonas shigelloides—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing ........................................................... 12



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Contents (Continued)

Table 3. Bacillus spp. (Not B. anthracis)—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing .................................................................................................... 14

Table 4. Campylobacter jejuni/coli—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing ............................................................................................ 16

Table 5. Corynebacterium spp. (Including C. diphtheriae) and Coryneformsa—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing ............................................. 18

Table 6. Erysipelothrix rhusiopathiae—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing .................................................................................................... 20

Table 7. HACEK Group: Aggregatibacter spp. (formerly Haemophilus aphrophilus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans), Cardiobacterium spp., Eikenella corrodens, and Kingella spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing .................................................................................................... 22

Table 8. Helicobacter pylori—Interpretive Criteria for Agar Dilution Susceptibility Testing ............. 24

Table 9. Lactobacillus spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing ............................................................................................................................ 26

Table 10. Leuconostoc spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing ............................................................................................................................ 28

Table 11. Listeria monocytogenes—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing ............................................................................................................................ 29

Table 12. Moraxella catarrhalis—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing ............................................................................................ 30

Table 13. Pasteurella spp.—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing ................................................................................................... 32

Table 14. Pediococcus spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing ............................................................................................................................ 34

Table 15. Vibrio spp. (including V. cholerae)—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing ..................................................................... 36

Table 16. Potential Bacterial Agents of Bioterrorism: Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Brucella spp.— Interpretive Criteria for Broth Microdilution Susceptibility Testing .................................................... 38

Table 17. Summary of Testing Conditions and Quality Control Recommendations for Infrequently Isolated or Fastidious Bacteria .............................................................................................................. 42

Table 18. MIC: Quality Control Ranges for Nonfastidious Organisms (Unsupplemented Cation- Adjusted Mueller-Hinton Medium) ...................................................................................................... 44

Table 18A. MIC: Quality Control Ranges for Broth Microdilution Methods (Cation-Adjusted Mueller-Hinton Broth With Lysed Horse Blood [2.5% to 5% v/v]) ..................................................... 45



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Contents (Continued)

Table 18B. MIC: Quality Control Ranges for Campylobacter jejuni (Broth Microdilution Method) (Cation-Adjusted Mueller-Hinton Broth With Lysed Horse Blood [2.5% to 5% v/v]) ........................ 46

Table 18C. MIC: Quality Control Ranges for Agar Dilution Methods (Mueller-Hinton Agar With Aged [≥ 2-Week-Old] Sheep Blood) .................................................................................................... 47

Table 18D. MIC: Quality Control Ranges for Broth Microdilution Method (Cation-Adjusted Mueller-Hinton Broth + 2% Defined Growth Supplementa) ................................................................ 47

Table 18E. MIC: Quality Control Ranges for Broth Microdilution Methods (Brucella Broth Without Supplements Adjusted to pH 7.1 ± 0.1) .................................................................................. 48

Table 19. Disk Diffusion: Quality Control Ranges for Nonfastidious Organisms (Unsupplemented Mueller-Hinton Medium) ...................................................................................................................... 49

Table 19A. Disk Diffusion: Quality Control Ranges for Fastidious Organisms (Mueller-Hinton Medium With 5% Sheep Blood) ........................................................................................................... 50

Glossary I (Part 1). β-Lactams: Class and Subclass Designation and Generic Name ........................... 51

Glossary I (Part 2). Non–β-lactams: Class and Subclass Designation and Generic Name ................... 52

Glossary II. Abbreviations/Routes of Administration/Drug Class for Antimicrobial Agents Listed in M100-S20 ............................................................................................................................................. 53

References ............................................................................................................................................. 56

Additional References ........................................................................................................................... 58

Summary of Consensus Comments and Subcommittee Responses ...................................................... 71

Summary of Delegate Comments and Subcommittee Responses ......................................................... 72

The Quality Management System Approach ........................................................................................ 76

Related CLSI Reference Materials ....................................................................................................... 77



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Foreword This document was developed for the purpose of providing guidance to clinical or public health microbiology laboratories regarding the performance of standardized susceptibility testing, when needed, of infrequently isolated or fastidious bacteria that are not presently included in CLSI documents M021 or M07.2 Helicobacter pylori, Vibrio cholerae, and several potential agents of bioterrorism were moved from CLSI documents M02,1 M07,2 and M1003 to this document because they are fastidious or infrequently encountered in most microbiology laboratories. Some of the organisms included herein are aerobic gram-negative bacilli that are not members of the family Enterobacteriaceae but may be tested by the standard CLSI broth microdilution or disk diffusion methods in the same manner as the much more common Enterobacteriaceae isolates. Some aerobic gram-positive cocci and bacilli that are encountered periodically by clinical laboratories can likewise be tested reliably by the standard CLSI minimal inhibitory concentration (MIC) or disk diffusion test methods in a manner analogous to Staphylococcus or Enterococcus spp. In addition, several genera of fastidious gram-positive and gram-negative bacteria can be tested in the same manner as the streptococci, using blood-supplemented Mueller-Hinton media. For the purpose of this document, the term fastidious is used to describe bacteria that require media supplemented with blood or blood components and that possibly need an atmosphere other than ambient air (eg, with 5% CO2) for acceptable growth. Because the standard CLSI media, reagents, and procedures can be used to test the organisms included in this guideline, the quality control procedures, strains, and acceptable zone diameter and MIC limits that have been established through previous rigorous studies can be used for tests with the less common organisms that are included in this document. The working group used a thorough search of the published literature in conjunction with the clinical experience of the members to apply or adapt interpretive criteria or breakpoints from other organisms that could best be applied to the interpretation of tests of the less common organisms in this document. Users of the guideline should be aware that the very extensive microbiological, clinical, and pharmacodynamic databases normally employed for setting breakpoints by CLSI did not exist for the collection of “orphan” organisms described in this document.

Interpretive breakpoints for cefazolin and for ertapenem, imipenem, and meropenem for the Enterobacteriaceae were voted to be changed by the Subcommittee on Antimicrobial Susceptibility Testing subsequent to final approval of this edition of M45. It is anticipated that those modified breakpoints will be adopted for use with Aeromonas spp. and Vibrio spp. in the next edition of M45 or possibly in a future supplement.

It is important for users of M45-A2 to recognize that commercial susceptibility testing devices are not addressed in this guideline. The methods described herein are generic reference procedures that can be used for routine susceptibility testing by clinical laboratories, or that can be used by clinical laboratories to evaluate commercial devices for possible routine use. Results generated by reference methods, such as those contained in CLSI documents, may be used by regulatory authorities to evaluate the performance of commercial systems as part of the approval process. Clearance by a regulatory authority indicates that the commercial susceptibility testing device provides susceptibility results that are substantially equivalent to results generated using the reference methods for the organisms and antimicrobial agents described in the manufacturer’s approved package insert. Some laboratories could find that a commercial dilution, antibiotic gradient, colorimetric, turbidimetric, fluorometric, or other method is suitable for selective or routine use. Key Words Agar dilution, antimicrobial agent, antimicrobial susceptibility, broth dilution, disk diffusion, microdilution, minimal inhibitory concentration (MIC), susceptibility testing



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Updated Information in This Edition Below is a summary of the changes in this document, which supersede the information presented in the previous edition of M45. The list includes “major” changes that appear for the first time in this edition of M45, or were modified since publication of M45-A. Other minor or editorial changes that were made to the general formatting are not listed here. Foreword Added paragraph from current edition of M100 related to using commercial systems vs CLSI reference methods (p. xi). Section 1, Scope Noted that testing of Haemophilus included in M100 relates to H. influenzae and H. parainfluenzae only (p. 1). Section 2, Introduction Updated HACEK group designation for Aggregatibacter (formerly Actinobacillus) (p. 1). Section 4.2, Definitions Added definitions for susceptible, intermediate, resistant, and nonsusceptible (p. 4). Section 4.3, Abbreviations and Acronyms Added a list of abbreviations and acronyms used in the document (p. 5). Section 6.3, Interpretive Categories Deleted only “S” criteria box because the definition of “nonsusceptible” is now in the list of definitions. Section 9, Quality Control Expanded statement about selecting quality control strains (p. 7). Tables 1 through 19A General Modified therapy (Rx) comment for rifampin (Tables 3, 5, 7, and 12) (pp. 14, 18, 22, and 30). Table 2, Aeromonas spp. and Plesiomonas shigelloides Clarified incubation conditions for disk diffusion and MIC testing (p. 12). Added new (revised) breakpoints for cefazolin, cefotaxime, ceftazidime, ceftriaxone, and aztreonam. Also added dosage regimens on which the new breakpoints are based (p. 12). Table 3, Bacillus spp. (not B. anthracis) Added fluoroquinolones to the agents to consider for primary testing box (p. 14). Table 4, Campylobacter jejuni/coli Clarified incubation conditions for disk diffusion and MIC testing (p. 16). Added azithromycin and clarithromycin to the examples indicating macrolide resistance (p. 16). Table 5, Corynebacterium spp. Modified vancomycin breakpoints (p. 18). Clarified scope of recommendations that apply to Corynebacterium spp. and other coryneforms including the genera Arcanobacterium, Brevibacterium, Cellulomonas, Dermabacter, Leifsonia, Microbacterium, Oerskovia, Rothia, and Turicella (p. 19).



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Table 7, HACEK Group Updated HACEK group designation for Aggregatibacter (formerly Actinobacillus) (p. 22). Table 8, Helicobacter pylori Imported table from M100-S20 (p. 24). Table 9, Lactobacillus spp. Added breakpoints for daptomycin and linezolid (p. 26). Added intermediate and resistant breakpoints for imipenem (p. 26). Modified breakpoints for clindamycin and vancomycin (p. 26). Added comment to ampicillin regarding combined therapy with a penicillin and an aminoglycoside for treatment of serious infections (p. 26). Updated Derivation of Interpretive Criteria section (p. 27). Table 10, Leuconostoc spp. Added comment regarding combined therapy with a penicillin and an aminoglycoside for treatment of serious infections (p. 28). Table 12, Moraxella catarrhalis Modified MIC breakpoints for amoxicillin-clavulanic acid (“S” only), azithromycin, clarithromycin, and erythromycin (p. 30). Added disk diffusion breakpoints for amoxicillin-clavulanic acid, azithromycin, clarithromycin, erythromycin, tetracycline, and trimethoprim-sulfamethoxazole (p. 30). Updated Derivation of Interpretive Criteria section (p. 31). Table 13, Pasteurella spp. Clarified incubation conditions for disk diffusion and MIC testing (p. 32). Table 15, Vibrio spp. Imported table from M100-S20 (p. 36). Clarified incubation conditions for disk diffusion and MIC testing (p. 36). Listed agents to consider for primary testing for Vibrio spp. and V. cholerae (p. 36). Added MIC breakpoints for azithromycin for V. cholerae (p. 37). Updated Derivation of Interpretive Criteria section (p. 37). Table 16, Potential Bacterial Agents of Bioterrorism Imported table from M100-S20 (p. 38). Added agents to consider for primary testing for Brucella and Francisella (p. 38). Table 17, Summary of Testing Conditions and Quality Control Added Helicobacter pylori, V. cholerae, and potential agents of bioterrorism (pp. 42 and 43). Quality Control Tables Table 18 Added ranges for ampicillin and piperacillin for E. coli ATCC® 35218 (p. 44). Table 18A Added ranges for amoxicillin for E. coli ATCC® 35218 (p. 45). Table 18B Imported Campylobacter quality control from M100-S20 (p. 46).



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Table 18C Imported Helicobacter quality control from M100-S20 (p. 47). Table 18D Imported potential bacterial agents of bioterrorism quality control from M100-S20 (p. 47). Table 19 Added ranges for azithromycin, clarithromycin, and erythromycin for S. aureus ATCC® 25923 (p. 49). Glossaries Glossary I – Added new antimicrobial subclass for ceftaroline and ceftobiprole (p. 51). Glossaries I and II – Added razupenem to carbapenem subclass (p. 51).

Added sulopenem to penem subclass (p. 51). Added linopristin-flopristin to streptogramin class (p. 52). Added mupirocin to pseudomonic acid class (p. 52).



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CLSI Subcommittee on Antimicrobial Susceptibility Testing Mission Statement The CLSI Subcommittee on Antimicrobial Susceptibility Testing is composed of representatives from the professions, government, and industry, including microbiology laboratories, government agencies, health care providers and educators, and pharmaceutical and diagnostic microbiology industries. Using the CLSI voluntary consensus process, the subcommittee develops standards and guidelines that promote accurate antimicrobial susceptibility testing and appropriate reporting. The mission of the CLSI Subcommittee on Antimicrobial Susceptibility Testing is to: • Develop standard reference methods for antimicrobial susceptibility tests. • Provide quality control parameters for standard test methods. • Establish interpretive criteria for the results of standard antimicrobial susceptibility tests. • Provide suggestions for testing and reporting strategies that are clinically relevant and cost-effective. • Continually refine standards and optimize the detection of emerging resistance mechanisms through

the development of new or revised methods, interpretive criteria, and quality control parameters. • Educate users through multimedia communication of standards and guidelines. • Foster a dialogue with users of these methods and those who apply them. The ultimate purpose of the subcommittee’s mission is to provide useful information to enable laboratories to assist the clinician in the selection of appropriate antimicrobial therapy for patient care. The standards and guidelines are meant to be comprehensive and to include all antimicrobial agents for which the data meet established CLSI guidelines. The values that guide this mission are quality, accuracy, fairness, timeliness, teamwork, consensus, and trust.



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©Clinical and Laboratory Standards Institute. All rights reserved. 1

Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second

Edition 1 Scope CLSI documents M02,1 M07,2 and M1003 describe standardized methods and interpretive criteria for antimicrobial susceptibility testing of common aerobic bacteria, including some fastidious organisms. However, there are a number of less frequently encountered or fastidious bacteria that are not addressed in those CLSI documents. Some are organisms that may cause serious infections, infections associated with trauma and environmental contamination, or device-associated infections in immunocompromised or postsurgical patients. These latter organisms are addressed in this guideline with the goal of providing recommendations for clinical microbiology laboratories on how and when to determine the susceptibility of these diverse organisms. This edition of the guideline also includes some fastidious or unusual organisms, including those potentially associated with bioterrorism, which were previously included in CLSI document M100.3 2 Introduction Organisms that previously lacked defined methods for susceptibility testing and interpretive criteria included various coryneform bacteria, Bacillus spp. (other than B. anthracis), Abiotrophia and Granulicatella spp., several genera of gram-positive bacteria with intrinsic glycopeptide resistance (eg, Erysipelothrix spp., Lactobacillus spp., Leuconostoc spp., and Pediococcus spp.), as well as several species of fastidious gram-negative bacteria (eg, HACEK group organisms and Pasteurella spp.). In addition, more detailed guidance for test performance and interpretation were needed, especially breakpoints for Listeria spp., Aeromonas spp., Plesiomonas sp., Vibrio spp., Moraxella catarrhalis, and Campylobacter spp. The lack of test methods or interpretive criteria made it difficult to assess the frequency of acquired resistance in these less frequently isolated or fastidious organisms and discouraged the testing of individual patient isolates by clinical laboratories. However, concerns had been raised that resistance exists in some of these organisms, and that laboratories should be prepared to test them when appropriate.4,5

Because infections due to organisms addressed in M45 occur less frequently than many of the organisms presently covered in CLSI documents M021 and M07,2 and because of the fact that many of the antimicrobial agents of interest have been marketed for a number of years, it is not reasonable to expect the intensive CLSI document M236 specified studies to be conducted on this special group of organisms. Instead, the goal of this document is to recommend test conditions and interpretive criteria based on a careful review of published microbiological data (distributions of minimal inhibitory concentrations [MICs]), limited animal model studies, the extant clinical literature regarding therapy for these organisms, and in a few instances, a review of existing pharmacokinetic data on the drugs of interest. In some cases, limited in vitro studies were performed. This edition of M45 includes several potential bacterial agents of bioterrorism that could be encountered initially by clinical microbiology laboratories and that should be forwarded to appropriate reference or public health laboratories for identification, confirmation, and possible susceptibility testing. The procedures included in this document are intended for use by those reference or public health laboratories. It is hoped that this CLSI guideline will assist clinical microbiology laboratories in determining an approach for testing these unusual organisms that is relevant to their individual practice settings.



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2.1 Resistance Mechanisms in Gram-Positive Rods Bacillus cereus and B. thuringiensis have long been noted as producers of a potent broad-spectrum β-lactamase that affects penicillins and cephalosporins.7 However, these related species are often susceptible to several other drug classes including vancomycin, aminoglycosides, macrolides, and quinolones that might be used to treat ocular or wound infections. Among the Corynebacterium spp., C. jeikeium and C. urealyticum are often multidrug resistant, including resistance to β-lactams, macrolides, and aminoglycosides.8 Even C. diphtheriae may be macrolide and rifampin resistant, whereas C. pseudodiphtheriticum and C. striatum may possess erm genes and be resistant to macrolides and lincosamides.8 Furthermore, some strains of C. striatum are said to be resistant to tetracyclines and quinolones.8 The related bacilli Arcanobacterium and Arthrobacter have been reported to be resistant to aminoglycosides and quinolones,8 and Brevibacterium spp. may demonstrate reduced β-lactam susceptibility. Turicella spp. may be macrolide and clindamycin resistant.8

Erysipelothrix rhusiopathiae and most Lactobacillus spp. isolates are intrinsically resistant to vancomycin; and the uncommon species Microbacterium resistans and Leifsonia aquatica have been reported to have diminished vancomycin susceptibility.9,10 2.2 Resistance in Infrequently Isolated or Fastidious Gram-Positive Cocci Leuconostoc and Pediococcus are intrinsically resistant to vancomycin but are usually susceptible to β-lactams, chloramphenicol, and aminoglycosides, although Leuconostoc can be resistant to carbapenems and cephalosporins.11 Abiotrophia and Granulicatella (formerly nutritionally deficient streptococci) may demonstrate diminished susceptibility to penicillin, resulting in greater difficulty in treatment of patients with endocarditis, and in one case, fluoroquinolone resistance was reported in an isolate from an immunosuppressed patient.12,13 2.3 Infrequently Isolated Nonfastidious Gram-Negative Rods There are a few nonfastidious gram-negative rods that are not addressed in CLSI documents M02,1 M07,2 and M1003 but that are capable of being grown using unsupplemented Mueller-Hinton medium. Aeromonas spp. may produce as many as three different β-lactamases, including a carbapenemase, that result in resistance to ampicillin but variable susceptibility to cephalosporins.14 The significance of the carbapenemase produced by some strains is not fully understood.15 Although Plesiomonas shigelloides is now regarded as a member of the Enterobacteriaceae,16 the genus has not been included in most prior studies to derive interpretive criteria for CLSI dilution and disk diffusion procedures, as described in CLSI documents M02,1 M07,2 and M100.3 Most of the clinically significant noncholera Vibrio spp. as well as V. cholerae can be grown in standard Mueller-Hinton medium. Susceptibilities of Vibrio spp. have been shown to vary by species, particularly with regard to the older penicillins, cephalosporins, and sulfonamides.17 2.4 Fastidious Gram-Negative Rods The HACEK (ie, Aggregatibacter [formerly Haemophilus aphrophilus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans], Cardiobacterium, Eikenella, and Kingella) group of fastidious gram-negative bacilli has long been recognized as causative agents of infective endocarditis.18 Aggregatibacter aphrophilus is the species most often associated with endocarditis or brain abscess. Aggregatibacter actinomycetemcomitans may be resistant to penicillins, macrolides, and aminoglycosides.19 Cardiobacterium hominis, Eikenella corrodens, Kingella spp., Capnocytophaga spp., and Pasteurella spp. may also produce β-lactamases that may be inhibited by clavulanic acid.20-23 Both fluoroquinolone and macrolide resistance have been reported in C. jejuni, C. coli, and C. fetus.24 There are several recommended regimens for the empiric treatment of H. pylori gastric infections that include two



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or more antibiotics combined with a proton pump inhibitor. Relapses or treatment failures can occur when macrolide, metronidazole, or fluoroquinolone resistance occurs.25 Thus, laboratories may be asked to culture and perform susceptibility testing of clarithromycin on H. pylori isolates from apparent treatment failures. 2.5 Moraxella catarrhalis Moraxellae are intrinsically resistant to trimethoprim alone. The majority of M. catarrhalis strains produce one of two β-lactamases (BRO-1, or less commonly, BRO-2), rendering them resistant to penicillin and ampicillin.26 Acquired resistance in M. catarrhalis to tetracyclines and trimethoprim-sulfamethoxazole has been reported in some isolates; resistance to macrolides is very rare. 2.6 Potential Bacterial Agents of Bioterrorism Extreme Caution: In the United States, notify public health officials of all isolates presumptively identified as B. anthracis, Y. pestis, B. mallei, B. pseudomallei, F. tularensis, Brucella abortus, B. melitensis, or B. suis. All of these bacterial species are identified as select agents according to the Select Agent Rule (www.cdc.gov/od/sap)27 because they could pose a severe threat to public health and safety. Entities that possess, use, or transfer these select agents must be registered with the US Departments of Health and Human Services and Agriculture. Clinical laboratories that are not registered should transfer these agents to a registered reference or public health laboratory in accordance with the Select Agent Rule. For laboratories outside the United States, check local public health requirements for handling and reporting of potential bacterial agents of bioterrorism. In addition to conforming to the Select Agent Rule in the United States or other local public health requirements for laboratories outside the United States, the confirmation of isolates of these bacteria may require specialized testing that is available only in reference or public health laboratories. Recommended precautions for testing involve the use of Biosafety Level 2 (BSL-2) practices, containment equipment, and facilities for activities using clinical materials and diagnostic quantities of infectious cultures. Use Biosafety Level 3 (BSL-3) practices, containment equipment, and facilities for work involving production quantities or concentrations of cultures and for activities with a high potential for aerosol production. If BSL-2 or BSL-3 facilities are not available, forward isolates to a reference or public health laboratory with a minimum of BSL-2 facilities for susceptibility testing. Some potential bacterial agents of bioterrorism can be tested by methods used for nonfastidious organisms. Others require modification of the standard methods in order to be tested. Broth dilution tests for the following organisms are described in this document: • Bacillus anthracis, Burkholderia mallei, B. pseudomallei, and Yersinia pestis, using cation-adjusted

Mueller-Hinton broth (CAMHB) • Francisella tularensis, using CAMHB supplemented with 2% defined growth supplement • Brucella spp., using unsupplemented Brucella broth 2.7 The Development of Interpretive Criteria or Breakpoints To establish MIC interpretive criteria or breakpoints for new antimicrobial agents, to modify existing breakpoints, or to establish breakpoints for organisms that have not previously existed in the standards, the Subcommittee on Antimicrobial Susceptibility Testing has employed an intensive analysis of MIC ranges of a particular drug with isolates that lack known resistance mechanisms, as well as with strains that contain known resistance mechanisms that affect the activity of the particular drug class. In addition, clinical and bacteriological response data collected during large clinical trials of new agents, as well as pharmacokinetic and pharmacodynamic simulations, are considered. The process of integrating these four types of data is outlined in detail in CLSI document M23.6 Notably, however, when establishing or



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reestablishing breakpoints for older drugs or for organisms that have previously lacked breakpoints, large prospectively collected clinical data are often not available. This guideline was developed to assist clinical microbiology laboratories in developing a strategy for susceptibility testing of infrequently encountered or fastidious organisms when circumstances indicate that testing of individual isolates would be helpful for clinical management, or for surveys of resistance for public health or research purposes. The testing methods and interpretive breakpoints included in this guideline were proposed based on an exhaustive search of the medical literature, and the experience of the working group members. In many cases, breakpoints were adapted from those for other, related organisms with approved CLSI breakpoints (see CLSI document M1003) because of the similarities between MIC distributions and types of infections that the organisms caused. The derivation of the breakpoint is noted in the table for each organism. As stated, the large databases requested in CLSI document M236 have not been available for assessment with the “orphan” organisms included in this guideline. 3 Standard Precautions Because it is often impossible to know what isolates or specimens might be infectious, all patient and laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard precautions are guidelines that combine the major features of “universal precautions and body substance isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus are more comprehensive than universal precautions, which are intended to apply only to transmission of blood-borne pathogens. Standard and universal precaution guidelines are available from the US Centers for Disease Control and Prevention.28 For specific precautions for preventing the laboratory transmission of all known infectious agents from laboratory instruments and materials and for recommendations for the management of exposure to all known infectious diseases, refer to CLSI document M29.29 Extreme caution must be exercised in testing the potential agents of bioterrorism listed in this document. Testing should be undertaken only in a registered reference or public health reference laboratory equipped to safely handle those agents. 4 Terminology 4.1 A Note on Terminology CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the global metrological community have evolved differently in the United States, Europe, and elsewhere; that these differences are reflected in CLSI, International Organization for Standardization (ISO), and European Committee for Standardization (CEN) documents; and that legally required use of terms, regional usage, and different consensus timelines are all important considerations in the harmonization process. In light of this, CLSI’s consensus process for development and revision of standards and guidelines focuses on harmonization of terms to facilitate the global application of standards and guidelines. 4.2 Definitions antimicrobial susceptibility test interpretive category – a classification based on an in vitro response of an organism to an antimicrobial agent at levels corresponding to blood or tissue levels attainable with usually prescribed doses of that agent. 1) susceptible – a category that implies that isolates are inhibited by the usually achievable

concentrations of antimicrobial agent when the recommended dosage is used for the site of infection.



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2) intermediate – a category that includes isolates with antimicrobial agent MICs that approach usually attainable blood and tissue levels and for which response rates may be lower than for susceptible isolates. The intermediate category implies clinical efficacy in body sites where the drugs are physiologically concentrated (eg, quinolones and β-lactams in urine) or when a higher than normal dosage of a drug can be used (eg, β-lactams). This category also includes a buffer zone, which should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations, especially for drugs with narrow pharmacotoxicity margins.

3) resistant – a category that implies that isolates are not inhibited by the usually achievable

concentrations of the agent with normal dosage schedules and/or that demonstrate zone diameters that fall in the range where specific microbial resistance mechanisms (eg, β-lactamases) are likely, and clinical efficacy of the agent against the isolate has not been reliably shown in treatment studies.

4) nonsusceptible – a category used for isolates for which only a susceptible interpretive criterion has

been designated because of the absence or rare occurrence of resistant strains. Isolates that have MICs above or zone diameters below the value indicated for the susceptible breakpoint should be reported as nonsusceptible; NOTE 1: An isolate that is interpreted as nonsusceptible does not necessarily mean that the isolate has a resistance mechanism. It is possible that isolates with MICs above the susceptible breakpoint that lack resistance mechanisms may be encountered within the wild-type distribution subsequent to the time the susceptible only breakpoint is set; NOTE 2: For strains yielding results in the “nonsusceptible” category, organism identification and antimicrobial susceptibility test results should be confirmed.

minimal inhibitory concentration (MIC) – the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in an agar or broth dilution susceptibility test. quality control (QC) – in microbiology, the operational techniques that are used to ensure accuracy and reproducibility. 4.3 Abbreviations and Acronyms

ATCC American Type Culture Collection BAP blood agar plate BMHA Mueller-Hinton agar with 5% sheep blood BSL biosafety level CAMHB cation-adjusted Mueller-Hinton broth ESBL extended-spectrum β-lactamase FDA US Food and Drug Administration LHB lysed horse blood MHA Mueller-Hinton agar MIC minimal inhibitory concentration QC quality control 5 Indications for Performing Susceptibility Tests Susceptibility testing is indicated for an 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. Certain organisms included in this guideline comprise part of the normal microbiota of human skin and mucous membranes (eg, Corynebacterium spp., Abiotrophia and Granulicatella spp., Lactobacillus spp., Pediococcus spp., Leuconostoc spp.) or represent environmental organisms (Bacillus spp.). Susceptibility testing of these organisms should not be performed on isolates from nonsterile or superficial sources. The



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need for susceptibility testing of Aeromonas and Vibrio spp. isolates recovered from feces is controversial. Testing should be undertaken only in consultation with an infectious disease–trained physician or other expert clinicians who can assist in determining if susceptibility testing is needed in the management of a specific patient, and in the interpretation of any results generated. Generally, testing of these organisms should be limited to isolates recovered from normally sterile sites (eg, blood, cerebrospinal fluid, joint or bone specimens, prosthetic devices, or long-term indwelling catheters), serious wound infections (Aeromonas and Vibrio spp.), and refractory or persistent diarrhea due to Campylobacter jejuni/coli or gastritis due to H. pylori. When the nature of the infection is not clear and the specimen contains mixed growth of normal flora in which the organisms probably bear little relationship to the infectious process, susceptibility tests are often unnecessary, and the results may be misleading. Many times, therapy of individual patients infected with the organisms included in this guideline will be empiric, based on the fact that a genus or species is very likely susceptible to a highly effective drug such that susceptibility testing would not be required. 6 Methods for Dilution Antimicrobial Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria This document employs the standard broth microdilution technique and agar dilution technique (Helicobacter pylori only). Both techniques are taken from CLSI document M07,2 based largely on information gathered from the International Collaborative Study on Antibiotic Sensitivity Testing.30 Although the broth microdilution method is primarily a standard reference method, it may be sufficiently practical to warrant its use in both clinical and research laboratories. The details of performing the broth microdilution procedure are not repeated in this document. Instead, readers should refer directly to CLSI document M072 for procedural details. However, key elements of the recommended methods are highlighted in each organism table for easy reference. 6.1 Selection of Antimicrobial Agents To make routine susceptibility testing relevant and practical, the number of agents tested should be limited. In each organism table in this document, the consensus primary antimicrobial agents for testing are highlighted in a box labeled “Agents to Consider for Primary Testing.” Interpretive criteria are provided for the primary agents and several potentially useful alternatives for each organism (see Tables 1 to 16). This does not imply that testing of every agent in each table should be undertaken. In consultation with the clinicians caring for the patient, a priority list of critical drugs for a specific patient’s isolate can be developed. 6.2 Antimicrobial Agents A comprehensive review of antimicrobial agent classes, including those in this document, is found in CLSI document M072 and is not repeated here. 6.2.1 Broth Microdilution Procedure The broth microdilution procedure employing CAMHB, CAMHB supplemented with lysed horse blood (LHB) (2.5% to 5% v/v) or defined growth supplement, or Brucella broth are used for the various fastidious organisms, as indicated in the individual tables and described in detail in CLSI document M07.2 Consult CLSI document M072 for the details of medium preparation, drug dilutions, inoculum preparation, incubation, and reading of MIC end points. 6.2.2 Agar Dilution Procedure The agar dilution procedure using Mueller-Hinton agar (MHA) supplemented with 5% v/v aged (> 2-week-old) sheep blood is recommended only for testing H. pylori.



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6.3 Interpretive Categories In addition to the MICs generated with one or more antimicrobial agents on a particular isolate, an interpretive category of susceptible, intermediate, resistant, or nonsusceptible can be assigned based on the tables included in this document. Definitions of those categories are found in CLSI document M072 and in Section 4.2 of this document. 7 Methods for Antimicrobial Disk Diffusion Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria In many clinical microbiology laboratories, the agar disk diffusion method is used for testing common, rapidly growing bacterial pathogens and sometimes for fastidious species, as indicated earlier. In laboratories that routinely perform dilution testing or use an automated susceptibility testing device, the disk diffusion test may represent a convenient method for testing the infrequently isolated organisms described in this guideline. The standardized disk diffusion testing method recommended by the CLSI Subcommittee on Antimicrobial Susceptibility Testing is described in CLSI document M02.1 Disk diffusion testing interpretive criteria are provided where possible in M45. It is best suited to testing the rapidly growing pathogens (including Aeromonas spp. and Vibrio spp.) and modified for testing some fastidious organisms, such as Campylobacter jejuni/coli, Moraxella catarrhalis, and Pasteurella spp. Adequate studies have not been conducted to recommend reproducible disk diffusion breakpoints for many of the organisms in this guideline. For those organisms, only the broth microdilution test or agar dilution (Helicobacter pylori) should be performed. Such organisms should not be tested by the disk diffusion method because the results cannot be interpreted reliably. The preparation of MHA including supplementation with 5% defibrinated sheep blood for fastidious organisms is described in CLSI document M02.1 8 Therapy-Related Comments Some of the comments in the tables relate to therapy concerns. These are denoted with an Rx symbol. It may be appropriate to include some of these comments (or modification thereof) on the patient report. An example would be inclusion of a comment that “Rifampin resistance may emerge during therapy if rifampin is administered alone.” 9 Quality Control An effective quality control (QC) program is designed to monitor the accuracy of a susceptibility test procedure, the performance of reagents and equipment, and the performance of persons who conduct the tests. These goals are best realized with the use of standard reference strains selected for their genetic stability and for their usefulness in the particular method. A detailed approach to performance of routine QC testing and maintenance of QC strains is outlined in CLSI documents M021 and M07.2 The latest editions of those documents should be consulted for the recommended QC procedures. Rather than attempting to identify numerous new QC reference strains that correspond to the many genera and species included in this guideline, the standard control strains recommended in those documents are applicable to QC of the media, antimicrobial agents, and procedures recommended in this document for infrequently isolated or fastidious bacteria. For that reason, an abbreviated list of acceptable limits of MICs and zone diameters was extracted from CLSI documents M021 and M072 (and the accompanying M100 supplement) for inclusion in Tables 18, 18A, 18B, 18C, 18D, 18E, 19, and 19A of this document. Other control values may be extracted from M100,3 if needed, to provide on-scale MICs for individual test panels.



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9.1 Minimum Laboratory Requirements for Testing Infrequently Isolated or Fastidious Bacteria Most MIC and disk diffusion tests described in M45 should be performed only in select situations by qualified laboratorians experienced with the recommended procedures. Criteria that can be used to determine qualifications include the following: • Performs antimicrobial susceptibility testing at least once per week using a CLSI broth microdilution

reference method, or an FDA-cleared commercial microdilution method or other approved methods that may be used by laboratories outside the United States with visual interpretations of MICs, and that may be adapted and validated for the testing conditions described in this guideline.

• Performs antimicrobial susceptibility testing at least once per week using a CLSI disk diffusion

reference method, if Aeromonas spp., Plesiomonas sp., Vibrio spp., Campylobacter jejuni/coli, Moraxella catarrhalis, or Pasteurella spp. will be tested by disk diffusion.

• Possesses the most current editions of CLSI documents (eg, M02,1 M07,2 and M1003). • Is part of a clinical microbiology laboratory coordinated by a doctoral-level clinical microbiologist,

infectious diseases physician, or pathologist with expertise in antimicrobial susceptibility testing. • Possesses appropriate biosafety facilities and legal authority to work with potential agents of

bioterrorism, if these organisms are being tested.

10 Detection of Resistance to Some β-Lactams by a Direct β-Lactamase Test Testing for β-lactamase activity using a chromogenic, cephalosporin-based method such as nitrocefin may yield clinically relevant information earlier than the results of an MIC test. A positive β-lactamase test result predicts resistance to penicillin, ampicillin, and amoxicillin among Aggregatibacter spp., Cardiobacterium hominis, Eikenella corrodens, Kingella spp., M. catarrhalis, and Pasteurella spp. A negative β-lactamase test result does not rule out resistance due to other mechanisms. Do not test organisms other than those listed above because the results may not be predictive of susceptibility or resistance to the β-lactams most often used for therapy.



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Table 1. Abiotrophia spp. and Granulicatella spp. (Formerly Known as Nutritionally Deficient or Nutritionally Variant Streptococci)—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments (1) Growth characteristics on routine media: very fastidious; requires cysteine or pyridoxal for growth. Some strains may grow marginally on enriched

chocolate agar or anaerobe agar formulations supplemented with added cysteine; CO2; 20 to 24 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than

“susceptible.” For strains yielding results suggestive of a “nonsusceptible” category, organism identification and antimicrobial susceptibility test results should be confirmed. Subsequently, the isolates should be saved and submitted to a reference laboratory for confirmation.

Antimicrobial

Class

Antimicrobial Agent

MIC (μg/mL) Interpretive Criteria

Comments S I R PENICILLINS

Penicillin ≤ 0.12 0.25–2 ≥ 4 Ampicillin ≤ 0.25 0.5–4 ≥ 8

CEPHEMS Cefepime ≤ 1 2 ≥ 4 Cefotaxime ≤ 1 2 ≥ 4 Ceftriaxone ≤ 1 2 ≥ 4 CARBAPENEMS Imipenem ≤ 0.5 1 ≥ 2 Meropenem ≤ 0.5 1 ≥ 2 GLYCOPEPTIDES

Vancomycin ≤ 1 – – See comment (2).

Testing Conditions Medium: CAMHB with LHB (2.5% to 5% v/v) and 0.001% (ie,

1 µg/mL) pyridoxal hydrochloride Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: 35 °C; ambient air; 20 to 24 hours

Minimal QC Recommendations Streptococcus pneumoniae ATCC®a 49619

Agents to Consider for Primary Testing Penicillin Cefotaxime or ceftriaxone Vancomycin

10 ©Clinical and Laboratory Standards Institute. All rights reserved.

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Table 1. Abiotrophia spp. and Granulicatella spp. (Continued)

Footnote a. ATCC is a registered trademark of the American Type Culture Collection.

Supplemental Information

Resistance: Abiotrophia spp. and Granulicatella spp. may demonstrate diminished susceptibility to penicillin, resulting in greater difficulty in treatment of patients with endocarditis; in one case, fluoroquinolone resistance was reported in an isolate from an immunosuppressed patient (see reference 11 under Additional References). Reasons for Testing/Not Testing: For isolates from respiratory sources or wounds, testing is usually not necessary. Testing of isolates from normally sterile sources (blood cultures, deep tissue, implanted prosthetic devices) may be warranted, especially in immunodeficient patients. Derivation of Interpretive Criteria: Interpretive criteria are adapted from those for Streptococcus spp., as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints include references 2, 11, and 15 under Additional References. Testing Notes: Many laboratories cannot readily distinguish Abiotrophia spp. from Granulicatella spp. or determine species level identification. The requirement for cysteine or pyridoxal in the medium or satellite growth is characteristic for both Abiotrophia and Granulicatella.

Antimicrobial

Class

Antimicrobial Agent


Comments S I R MACROLIDES

Erythromycin ≤ 0.25 0.5 ≥ 1 QUINOLONES

Ciprofloxacin ≤ 1 2 ≥ 4 Gatifloxacin ≤ 1 2 ≥ 4 Levofloxacin ≤ 2 4 ≥ 8

PHENICOLS Chloramphenicol ≤ 4 – ≥ 8

LINCOSAMIDES Clindamycin ≤ 0.25 0.5 ≥ 1

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Table 2. Aeromonas spp. (Includes Members of A. caviae Complex, A. hydrophila Complex, and A. veronii Complex) and Plesiomonas shigelloides—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing

General Comments (1) Growth characteristics on routine media: nonfastidious; grows well on a blood agar plate (BAP); ambient air; 16 to 20 hours.

Antimicrobial

Class

Antimicrobial Agent

Disk

Content

Zone Diameter (mm) Interpretive Criteria

MIC (μg/mL) Interpretive Criteria Comments

S I R S I RPENICILLINS AND β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS

Amoxicillin-clavulanic acid

20/10 μg ≥ 18 14–17 ≤ 13 ≤ 8/4 16/8 ≥ 32/16

Ampicillin-sulbactam 10/10 μg ≥ 15 12–14 ≤ 11 ≤ 8/4 16/8 ≥ 32/16 Piperacillin-

tazobactam 100/10 μg ≥ 21 18–20 ≤ 17 ≤ 16/4 32/4–64/4 ≥ 128/4

CEPHEMS Cefazolin 30 μg – – – ≤ 1 2 ≥ 4 Interpretive criteria are based on a dosage

regimen of at least 1 g every 8 h. Cefepime 30 μg ≥ 18 15–17 ≤ 14 ≤ 8 16 ≥ 32 Cefotaxime 30 μg ≥ 26 23–25 ≤ 22 ≤ 1 2 ≥ 4 Interpretive criteria are based on a dosage

regimen of 1 g every 8 h. Cefoxitin 30 μg ≥ 18 15–17 ≤ 14 ≤ 8 16 ≥ 32 Ceftazidime 30 μg ≥ 21 18–20 ≤ 17 ≤ 4 8 ≥ 16 Interpretive criteria are based on a dosage

regimen of 1 g every 8 h. Ceftriaxone 30 μg ≥ 23 20–22 ≤ 19 ≤ 1 2 ≥ 4 Interpretive criteria are based on a dosage

regimen of 1 g every 24 h. Cefuroxime sodium

(parenteral) 30 μg ≥ 18 15–17 ≤ 14 ≤ 8 16 ≥ 32 Interpretive criteria are based on a dosage

regimen of 1.5 g every 8 h. CARBAPENEMS

Ertapenem 10 μg ≥ 19 16–18 ≤ 15 ≤ 2 4 ≥ 8 Imipenem 10 μg ≥ 16 14–15 ≤ 13 ≤ 4 8 ≥ 16 Meropenem 10 μg ≥ 16 14–15 ≤ 13 ≤ 4 8 ≥ 16

Testing Conditions Medium: CAMHB for microdilution, MHA for disk diffusion testing Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: 35 °C; ambient air; Disk diffusion: 16 to 18 hours Broth microdilution method: 16 to 20 hours

Minimal QC Recommendations Escherichia coli ATCC 25922® Escherichia coli ATCC 35218® (for β- lactam/β-lactamase inhibitor combinations)

Agents to Consider for Primary Testing Amoxicillin-clavulanic acid 3rd- or 4th-generation cephalosporins Fluoroquinolones Trimethoprim-sulfamethoxazole


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Table 2. Aeromonas spp. and Plesiomonas shigelloides (Continued)

Supplemental Information Aeromonas spp. There are currently 17 valid species in the genus Aeromonas; however, only five (A. caviae, A. hydrophila, A. jandaei, A. schubertii, and A. veronii bv. sobria) are currently recognized as human pathogens causing a variety of clinical infections including gastroenteritis, cellulitis, and bacteremia. New syndromes attributed to this genus include hemolytic uremic syndrome, burn-associated sepsis, and a variety of respiratory tract infections, including epiglottitis. The three Aeromonas spp. predominantly recovered from clinical material are A. hydrophila, A. caviae, and A. veronii bv. sobria. Therefore, most of the published data on susceptibility testing are limited to these three species. Plesiomonas sp. The genus Plesiomonas with its only species, P. shigelloides, has recently been placed in the family Enterobacteriaceae. However, because of its phenotypic similarity to Aeromonas spp., as well as its similar disease spectrum, it is included with Aeromonas in these susceptibility testing guidelines. Resistance: Aeromonas spp. are uniformly resistant to ampicillin; however, susceptibility to amoxicillin-clavulanic acid and cefazolin differs among species. Aeromonas strains may possess multiple, distinct, inducible β-lactamases, and like other genera with inducible β-lactamases, resistance may emerge during therapy with a β-lactam. Plesiomonas sp. is resistant to penicillins due to the production of penicillinases. There are conflicting data regarding resistance of P. shigelloides to ampicillin and other penicillin drugs, and this may be related to test medium and inoculum effect. As a result, testing of P. shigelloides to ampicillin is not recommended in this guideline. Reasons for Testing/Not Testing: Testing is most often limited to isolates from extraintestinal sites. Derivation of Interpretive Criteria: Interpretive criteria are adapted from those for Enterobacteriaceae, as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints for Aeromonas spp. include references 18 and 22 to 27; and for Plesiomonas shigelloides, include references 30 to 34 under Additional References.

Antimicrobial

Class

Antimicrobial

Agent

Disk Content



S I R S I RMONOBACTAMS

Aztreonam 30 μg ≥ 21 18–20 ≤ 17 ≤ 4 8 ≥ 16 Interpretive criteria are based on a dosage regimen of 1 g every 8 h.

AMINOGLYCOSIDES Amikacin 30 μg ≥ 17 15–16 ≤ 14 ≤ 16 32 ≥ 64 Gentamicin 10 μg ≥ 15 13–14 ≤ 12 ≤ 4 8 ≥ 16

TETRACYCLINES Tetracycline 30 μg ≥ 15 12–14 ≤ 11 ≤ 4 8 ≥ 16

QUINOLONES Ciprofloxacin 5 μg ≥ 21 16–20 ≤ 15 ≤ 1 2 ≥ 4 Levofloxacin 5 μg ≥ 17 14–16 ≤ 13 ≤ 2 4 ≥ 8

FOLATE PATHWAY INHIBITORS Trimethoprim-

sulfamethoxazole 1.25/23.75 μg ≥ 16 11–15 ≤ 10 ≤ 2/38 – ≥ 4/76

PHENICOLS Chloramphenicol 30 μg ≥ 18 13–17 ≤ 12 ≤ 8 16 ≥ 32

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Table 3. Bacillus spp. (Not B. anthracis)—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments

(1) Growth characteristics on routine media: nonfastidious; grows well on BAP; ambient air; 16 to 20 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than


Antimicrobial

Class

Antimicrobial Agent



Penicillin ≤ 0.12 – ≥ 0.25 Ampicillin ≤ 0.25 – ≥ 0.5

CEPHEMS Cefazolin ≤ 8 16 ≥ 32 Cefotaxime ≤ 8 16–32 ≥ 64 Ceftazidime ≤ 8 16 ≥ 32 Ceftriaxone ≤ 8 16–32 ≥ 64

CARBAPENEMS Imipenem ≤ 4 8 ≥ 16

GLYCOPEPTIDES Vancomycin ≤ 4 – – See comment (2).

AMINOGLYCOSIDES Amikacin ≤ 16 32 ≥ 64 Gentamicin ≤ 4 8 ≥ 16

MACROLIDES Erythromycin ≤ 0.5 1–4 ≥ 8

Testing Conditions Medium: CAMHB Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland

standard Incubation: 35 °C; ambient air; 16 to 20 hours

Minimal QC Recommendations Staphylococcus aureus ATCC® 29213

Agents to Consider for Primary Testing Vancomycin Fluoroquinolones Clindamycin

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Table 3. Bacillus spp. (Continued)

Antimicrobial

Class

Antimicrobial Agent


Comments S I R TETRACYCLINES

Tetracycline ≤ 4 8 ≥ 16 QUINOLONES

Ciprofloxacin ≤ 1 2 ≥ 4 Levofloxacin ≤ 2 4 ≥ 8

LINCOSAMIDES Clindamycin ≤ 0.5 1–2 ≥ 4

FOLATE PATHWAY INHIBITORS Trimethoprim-sulfamethoxazole ≤ 2/38 – ≥ 4/76

PHENICOLS Chloramphenicol ≤ 8 16 ≥ 32

ANSAMYCINS Rifampin ≤ 1 2 ≥ 4 (3) Rx: Rifampin resistance may emerge during therapy if rifampin is

administered alone.


Resistance: Bacillus cereus isolates are generally resistant to penicillins and cephalosporins. Reasons for Testing/Not Testing: Bacillus spp. are frequently encountered as contaminating bacteria in cultures. Testing of isolates from normally sterile sources (blood cultures, deep tissue, implanted prosthetic devices) may be warranted, especially in immunodeficient patients.

Derivation of Interpretive Criteria: Interpretive criteria are adapted from those for Staphylococcus spp., as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints include references 35 to 38 under Additional References. Testing Notes: Although many Bacillus spp. produce β-lactamase, β-lactamase testing of this genus is unreliable. Recommendations for disk diffusion of Bacillus spp. cannot be made at this time because limited data exist for disk diffusion testing of this genus.

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Table 4. Campylobacter jejuni/coli—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing

General Comments (1) Growth characteristics on routine media: fastidious; grows on media such as MHA supplemented with 5% sheep blood; requires a microaerobic atmosphere (10% CO2, 5% O2, and 85% N2); 36 to 37 °C for 48 hours or 42 °C for 24 hours.

Antimicrobial

Class

Antimicrobial

Agent

Disk

Content



Comments S I R S I R MACROLIDE

Erythromycin 15 μg – – 6 ≤ 8 16 ≥ 32 (2) A disk diffusion zone of 6 mm (growth up to the edge of a 6-mm disk) indicates macrolide (eg, erythromycin, azithromycin, clarithromycin) resistance. Appearance of any zone of inhibition would require MIC determination for accurate categorization of susceptibility.

QUINOLONE Ciprofloxacin 5 μg – – 6 ≤ 1 2 ≥ 4 (3) A disk diffusion zone of 6 mm (growth up to the edge

of a 6-mm disk) indicates ciprofloxacin resistance. Appearance of any zone of inhibition would require MIC determination for accurate categorization of susceptibility.

TETRACYCLINES Tetracycline – – – – ≤ 4 8 ≥ 16 Doxycycline – – – – ≤ 2 4 ≥ 8

Testing Conditions Medium: CAMHB with LHB (2.5% to 5% v/v) for microdilution, Mueller-Hinton agar with

5% sheep blood (BMHA) for disk diffusion testing Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: Disk diffusion: 36 to 37 °C for 48 hours; 42 °C for 24 hours Broth microdilution method: 36 to 37 °C for 48 hours; 42 °C for 24 hours (incubation at less than 36 °C or greater than 42 °C may not yield satisfactory

growth). Microaerobic atmosphere equivalent to 10% CO2, 5% O2, and 85% N2. Use of

a compressed gas incubator is preferable; however, acceptable performance may be achieved using microaerobic gas-generating sachets. Sealed plastic bags or pouches do not result in reproducible data and are not recommended.

Note: Agar dilution testing is described in CLSI document M31 (see additional

reference 42).

Minimal QC Recommendations Microdilution: Campylobacter jejuni ATCC® 33560, 36 to 37 °C for 48 hours or 42 °C for 24 hours Disk Diffusion: Staphylococcus aureus ATCC® 25923, MHA, 35 to 37 °C for 16 to 18 hours in ambient air

Agents to Consider for Primary Testing Erythromycin Ciprofloxacin


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Table 4. Campylobacter jejuni/coli (Continued)

Supplemental Information Resistance: Resistance among Campylobacter jejuni/coli is known to occur with erythromycin (0% to 11%) but is more problematic with fluoroquinolones and is highly variable from country to country, with rates of 10% to as high as 40% being reported. Emergence of resistance to ciprofloxacin may occur while the patient is on therapy. Strains resistant to both macrolides and fluoroquinolones have been reported. Reasons for Testing/Not Testing: Testing may be useful for epidemiological purposes or for management of patients with prolonged or severe symptoms. Derivation of Interpretive Criteria: Interpretive criteria are adapted from those for Enterobacteriaceae (ciprofloxacin and tetracycline), as published in CLSI document M100.3 Interpretive criteria for erythromycin and doxycycline are based on population distributions following testing of 150 strains of wild-type Campylobacter jejuni/coli at 36 to 37 °C, using a microaerobic atmosphere for 48 hours. The erythromycin and ciprofloxacin disk diffusion screening breakpoints were developed by testing a group of 417 susceptible or resistant Campylobacter jejuni/coli isolates. These studies were conducted by two members of the CLSI M45 working group. Key citations used in derivation of interpretive breakpoints include references 49, 54, and 58 under Additional References.

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Table 5. Corynebacterium spp. (Including C. diphtheriae) and Coryneformsa—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments

(1) Growth characteristics on routine media: often fastidious; requires blood-supplemented media for adequate growth; ambient air; 20 to 24 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than


Antimicrobial

Class

Antimicrobial Agent


Comments S I R PENICILLINS (3) Interpretive criteria may not apply to meningitis.

Penicillin ≤ 1 2 ≥ 4 CEPHEMS See comment (3).

Cefepime ≤ 1 2 ≥ 4 Cefotaxime ≤ 1 2 ≥ 4 Ceftriaxone ≤ 1 2 ≥ 4

CARBAPENEMS See comment (3). Imipenem ≤ 4 8 ≥ 16 Meropenem ≤ 4 8 ≥ 16

GLYCOPEPTIDES Vancomycin ≤ 2 – – See comment (2).

LIPOPEPTIDES Daptomycin ≤ 1 – – See comment (2).

Testing Conditions Medium: CAMHB with LHB (2.5% to 5% v/v). If testing daptomycin, the

medium should contain 50 µg/mL calcium. Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: 35 °C; ambient air; 24 to 48 hours (see Testing Notes)

Minimal QC Recommendations Streptococcus pneumoniae ATCC® 49619 Escherichia coli ATCC® 25922 for gentamicin

Agents to Consider for Primary Testing Penicillin Vancomycin Gentamicin Erythromycin


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Table 5. Corynebacterium spp. (Continued)

Antimicrobial

Class

Antimicrobial Agent


Comments S I R AMINOGLYCOSIDES

Gentamicin ≤ 4 8 ≥ 16 MACROLIDES

Erythromycin ≤ 0.5 1 ≥ 2 QUINOLONES

Ciprofloxacin ≤ 1 2 ≥ 4 TETRACYCLINES

Doxycycline ≤ 4 8 ≥ 16 Tetracycline ≤ 4 8 ≥ 16

LINCOSAMIDES Clindamycin ≤ 0.5 1–2 ≥ 4

FOLATE PATHWAY INHIBITORS Trimethoprim-sulfamethoxazole ≤ 2/38 – ≥ 4/76

ANSAMYCINS Rifampin ≤ 1 2 ≥ 4 (4) Rx: Rifampin resistance may emerge during therapy if rifampin is

administered alone. STREPTOGRAMINS

Quinupristin-dalfopristin ≤ 1 2 ≥ 4 OXAZOLIDINONES

Linezolid ≤ 2 – – See comment (2). aCoryneforms include the genera Arcanobacterium, Brevibacterium, Cellulomonas, Dermabacter, Leifsonia, Microbacterium, Oerskovia, Rothia, and Turicella.


Resistance: Some species of Corynebacterium may exhibit resistance to multiple drug classes. Reasons for Testing/Not Testing: Testing of isolates from normally sterile sources (blood cultures, deep tissue, implanted prosthetic devices) may be warranted, especially in immunodeficient patients. Derivation of Interpretive Criteria: Interpretive criteria for penicillin and erythromycin are based primarily on MIC distributions following testing of a large number of isolates. Cephalosporin interpretive criteria are adapted from those for Streptococcus spp.; linezolid interpretive criteria are adapted from those for Enterococcus spp.; and remaining interpretive criteria are adapted from those for Staphylococcus spp., as published in CLSI document M100.3 In addition to the references listed at the end of this document, data from three large organism collections tested by two of the working group members were used to derive the interpretive breakpoints. Testing Notes: Resistant results can be reported at 24 hours. Isolates demonstrating susceptible results for β-lactams should be reincubated and results reported at 48 hours.

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Table 6. Erysipelothrix rhusiopathiae—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments (1) Growth characteristics on routine media: fastidious; may take one to three days for colonies to grow on BAP or chocolate agar; ambient air. At 24 hours,

growth may be pinpoint colonies. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than


Antimicrobial

Class

Antimicrobial Agent



Penicillin ≤ 0.12 – – See comment (2). Ampicillin ≤ 0.25 – – See comment (2).

CEPHEMS Cefepime ≤ 1 – – See comment (2). Cefotaxime ≤ 1 – – See comment (2). Ceftriaxone ≤ 1 – – See comment (2).

CARBAPENEMS Imipenem ≤ 0.5 – – See comment (2). Meropenem ≤ 0.5 – – See comment (2).

MACROLIDES Erythromycin ≤ 0.25 0.5 ≥ 1

QUINOLONES Ciprofloxacin ≤ 1 – – See comment (2). Gatifloxacin ≤ 1 – – See comment (2). Levofloxacin ≤ 2 – – See comment (2).

LINCOSAMIDES Clindamycin ≤ 0.25 0.5 ≥ 1

Testing Conditions Medium: CAMHB with LHB (2.5% to 5% v/v) Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland


Minimal QC Recommendations Streptococcus pneumoniae ATCC® 49619

Agents to Consider for Primary Testing Penicillin or ampicillin


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Table 6. Erysipelothrix rhusiopathiae (Continued)

Supplemental Information Resistance: Erysipelothrix rhusiopathiae is considered intrinsically resistant to vancomycin; routine testing of vancomycin is not necessary. No resistance has been described for β-lactams and fluoroquinolones. Reasons for Testing/Not Testing: Although antimicrobial susceptibility testing is not required, it is important to identify this organism promptly because of its potentially fulminant nature when causing endocarditis and the fact that it is intrinsically resistant to vancomycin, which is the therapy often used empirically for gram-positive organisms. For patients with penicillin allergy, testing of erythromycin and clindamycin may be warranted. Derivation of Interpretive Criteria: Interpretive criteria for ciprofloxacin are adapted from those for Staphylococcus spp., as published in CLSI document M100.3 Interpretive criteria for all other antimicrobial agents are adapted from those for Streptococcus spp., as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints include references 84 and 86 to 88 under Additional References.

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Table 7. HACEK Group: Aggregatibacter spp. (formerly Haemophilus aphrophilus, H. paraphrophilus, H. segnis, and Actinobacillus actinomycetemcomitans), Cardiobacterium spp., Eikenella corrodens, and Kingella spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments (1) Growth characteristics on routine media: very fastidious; most will grow on BAP or chocolate agar in CO2; 24 to 48 hours; some strains may not grow well

in supplemented broths (CAMHB + 2.5% to 5% LHB). See Testing Notes.

(2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than “susceptible.” For strains yielding results suggestive of a “nonsusceptible” category, organism identification and antimicrobial susceptibility test results should be confirmed. Subsequently, the isolates should be saved and submitted to a reference laboratory for confirmation.

Antimicrobial

Class

Antimicrobial Agent


Comments S I R PENICILLINS AND β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS

Ampicillin ≤ 1 2 ≥ 4 Ampicillin-sulbactam ≤ 2/1 – ≥ 4/2 Amoxicillin-clavulanic acid ≤ 4/2 – ≥ 8/4 Penicillin ≤ 1 2 ≥ 4

CEPHEMS Ceftriaxone ≤ 2 – – See comment (2). Cefotaxime ≤ 2 – – See comment (2).

CARBAPENEMS

Imipenem (Aggregatibacter spp.) ≤ 4 8 ≥ 16 Meropenem (Aggregatibacter spp.) ≤ 4 8 ≥ 16 Imipenem (other organisms) ≤ 0.5 1 ≥ 2 Meropenem (other organisms) ≤ 0.5 1 ≥ 2

Testing Conditions Medium: CAMHB with LHB (2.5% to 5% v/v) Inoculum: Direct colony suspension, equivalent to a 0.5

McFarland standard Incubation: 35 °C; 5% CO2; 24 to 48 hours

Minimal QC Recommendations Streptococcus pneumoniae ATCC® 49619 Escherichia coli ATCC® 35218 (for β-lactam/β-lactamase inhibitor combinations)

Agents to Consider for Primary Testing Ampicillin Amoxicillin-clavulanic acid Ceftriaxone or cefotaxime Imipenem Ciprofloxacin or levofloxacin Trimethoprim-sulfamethoxazole


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Table 7. HACEK Group (Continued)

Antimicrobial

Class

Antimicrobial Agent


Comments S I R MACROLIDES

Azithromycin ≤ 4 – – See comment (2). Clarithromycin ≤ 8 16 ≥ 32

QUINOLONES Ciprofloxacin ≤ 1 2 ≥ 4 Levofloxacin ≤ 2 4 ≥ 8

TETRACYCLINES Tetracycline ≤ 2 4 ≥ 8


ANSAMYCINS Rifampin ≤ 1 2 ≥ 4 (3) Rx: Rifampin resistance may emerge during therapy if rifampin

is administered alone. FOLATE PATHWAY INHIBITORS

Trimethoprim-sulfamethoxazole ≤ 0.5/9.5 1/19–2/38 ≥ 4/76

Supplemental Information Resistance: β-lactamase production among members of the HACEK group is well documented, and β-lactamase-producing isolates are ampicillin resistant. Some isolates of Aggregatibacter spp. may be resistant to ampicillin due to mechanisms other than β-lactamase production. Reasons for Testing/Not Testing: Testing of isolates from normally sterile sources (blood cultures, deep tissue, implanted prosthetic devices) may be warranted, especially in immunodeficient patients or those patients unable to tolerate empiric β-lactam therapy. For isolates of Eikenella from bite wound infections, testing may not be necessary when using amoxicillin-clavulanate, considering the high probability of susceptibility to amoxicillin-clavulanic acid. Derivation of Interpretive Criteria: Interpretive criteria for penicillin are based primarily on MIC distributions. Chloramphenicol interpretive criteria are adapted from those for Streptococcus spp.; all others are adapted from those for Haemophilus influenzae. Key citations used in derivation of interpretive breakpoints include references 94, 101, 105, 107,109, and 113 under Additional References. Testing Notes: Routine performance of a β-lactamase test is recommended for all HACEK isolates. Antimicrobial susceptibility testing may be difficult, given the slow growth and fastidiousness of some members in the HACEK group. Do not attempt to interpret results of testing for isolates that produce insufficient growth in growth-control wells.

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Table 8. Helicobacter pylori—Interpretive Criteria for Agar Dilution Susceptibility Testing

Test/Report Group

Antimicrobial Agent

MIC Interpretive Standard(µg/mL)

Comments S I R A Clarithromycin ≤ 0.25 0.5 ≥ 1.0 (1) These breakpoints presume that clarithromycin will be used in an

approved regimen that includes a proton-pump inhibitor and possibly one or more additional antimicrobial agents.

Testing Conditions Medium: Agar dilution: MHA and aged (≥ 2-week-old) sheep blood (5%

v/v) Inoculum: A saline suspension equivalent to a 2.0 McFarland standard

(containing 1 × 107 to 1 × 108 CFU/mL), to be prepared from a 72-hour-old subculture from a BAP. The inoculum (1 to 3 μL per spot) is replicated directly onto the antimicrobial agent-containing agar dilution plates.

Incubation: 35 ± 2 °C; 72 hours; microaerobic atmosphere produced by a gas-generating system suitable for campylobacters

Minimal QC Recommendations (See Table 18C for acceptable QC ranges.) Helicobacter pylori ATCC® 43504


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Table 9. Lactobacillus spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments (1) Growth characteristics on routine media: often fastidious; requires blood-supplemented media for adequate growth; 5% CO2; 24 to 48 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than


Antimicrobial

Class

Antimicrobial Agent



Penicillin ≤ 8 – – See comment (2). (3) Therapy of serious infections such as endocarditis often involves combined therapy with a penicillin and an aminoglycoside.

Ampicillin ≤ 8 – –

CARBAPENEMS Imipenem ≤ 0.5 1 ≥ 2 See comment (2).

AMINOGLYCOSIDES Gentamicin ≤ 4 8 ≥ 16 (4) For combined therapy.

See comment (3). GLYCOPEPTIDES

Vancomycin ≤ 2 4–8 ≥ 16 LIPOPEPTIDES

Daptomycin ≤ 4 – – See comment (2). MACROLIDES

Erythromycin ≤ 0.5 1–4 ≥ 8 LINCOSAMIDES

Clindamycin ≤ 0.5 1 ≥ 2 OXAZOLIDINONES

Linezolid ≤ 4 – – See comment (2).


standard Incubation: 35 °C; 5% CO2; 24 to 48 hours


Agents to Consider for Primary Testing Penicillin or ampicillin Gentamicin (for combined therapy)

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Table 9. Lactobacillus spp. (Continued)


Resistance: Many species of Lactobacillus spp. that grow well in ambient air are intrinsically resistant to vancomycin. Reasons for Testing/Not Testing: Testing of isolates from normally sterile body sources (blood cultures, deep tissue) may be warranted. Derivation of Interpretive Criteria: Interpretive criteria for gentamicin, linezolid, and vancomycin are adapted from those for Staphylococcus spp., as published in CLSI document M100.3 Interpretive criteria for clindamycin and imipenem are based on MIC distributions following testing of a large number of isolates and data presented in various publications listed below. Interpretive criteria for all other antimicrobial agents are adapted from those for Enterococcus spp., as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints include references 117, 119, 120, and 127 under Additional References.


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Table 10. Leuconostoc spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments (1) Growth characteristics on routine media: often fastidious; requires blood-supplemented media for adequate growth; ambient air; 20 to 24 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than


Antimicrobial

Class

Antimicrobial Agent



Penicillin ≤ 8 – – See comment (2). (3) Therapy of serious infections such as endocarditis often involves combined therapy with a penicillin and an aminoglycoside.

Ampicillin ≤ 8 – –

AMINOGLYCOSIDES Gentamicin ≤ 4 8 ≥ 16 (4) For combined therapy.

See comment (3). TETRACYCLINES

Minocycline ≤ 4 8 ≥ 16 PHENICOLS

Chloramphenicol ≤ 8 16 ≥ 32


Resistance: Leuconostoc spp. are considered intrinsically resistant to vancomycin; routine testing of vancomycin is not necessary. Reasons for Testing/Not Testing: Testing of isolates from normally sterile body sources (blood cultures, deep tissue) may be warranted. Derivation of Interpretive Criteria: Interpretive criteria for gentamicin are adapted from those for Staphylococcus spp., as published in CLSI document M100.3 Interpretive criteria for all other antimicrobial agents are adapted from those for Enterococcus spp., as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints include references 134, 135, and 141 under Additional References.





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Table 11. Listeria monocytogenes—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments

(1) Growth characteristics on routine media: often fastidious; requires blood-supplemented media for adequate growth; ambient air; 20 to 24 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than


Antimicrobial Class

Antimicrobial Agent


Comments S I RPENICILLINS

Penicillin ≤ 2 – – See comment (2). Ampicillin ≤ 2 – – See comment (2).

FOLATE PATHWAY INHIBITORS Trimethoprim-sulfamethoxazole ≤ 0.5/9.5 1/19–2/38 ≥ 4/76


Resistance: Listeria monocytogenes is intrinsically resistant to cephalosporins. Reasons for Testing/Not Testing: Resistance to ampicillin or penicillin has not been described. Testing may be limited to suspected treatment failures or for patients with a penicillin allergy. Derivation of Interpretive Criteria: Interpretive criteria for penicillin and ampicillin were previously published in CLSI document M100-S15. Interpretive criteria for trimethoprim-sulfamethoxazole are adapted from those for Streptococcus spp., as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints include references 147 and 151 under Additional References.

Testing Conditions Medium: CAMHB with LHB (2.5% to 5% v/v) Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland standard Incubation: 35 °C; ambient air; 20 to 24 hours

Minimal QC Recommendations Streptococcus pneumoniae ATCC® 49619

Agents to Consider for Primary Testing Penicillin or ampicillin Trimethoprim-sulfamethoxazole

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Table 12. Moraxella catarrhalis—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing

General Comments (1) Growth characteristics on routine media: nonfastidious; grows well on BAP; ambient air; 16 to 20 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than


Antimicrobial

Class

Antimicrobial Agent

Disk Content



Comments S I R S I RPENICILLINS AND β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS

Amoxicillin-clavulanic acid 20/10 μg ≥ 24 – ≤ 23 ≤ 4/4 ≥ 8/4 CEPHEMS

Cefaclor – – – – ≤ 8 16 ≥ 32 Cefuroxime (oral) – – – – ≤ 4 8 ≥ 16 Cefotaxime – – – – ≤ 2 – – See comment (2). Ceftazidime – – – – ≤ 2 –- – See comment (2). Ceftriaxone – – – – ≤ 2 – – See comment (2).

MACROLIDES Azithromycin 15 μg ≥ 26 – – ≤ 0.25 – – See comment (2). Clarithromycin 15 μg ≥ 24 – – ≤ 1 – – See comment (2). Erythromycin 15 μg ≥ 21 – – ≤ 2 – – See comment (2).

QUINOLONES Ciprofloxacin – – – – ≤ 1 – – See comment (2). Levofloxacin – – – – ≤ 2 – – See comment (2).

Testing Conditions Medium: CAMHB; MHA for disk diffusion Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland

standard Incubation: 35 °C

Disk diffusion: 5% CO2, 20 to 24 hours Broth microdilution method: ambient air; 20 to 24 hours

Minimal QC Recommendations Staphylococcus aureus ATCC® 29213 (MIC)Escherichia coli ATCC® 35218 (for β-lactam/β-lactamase inhibitor combinations) Staphylococcus aureus ATCC® 25923 for disk diffusion)

Agents to Consider for Primary Testing Amoxicillin-clavulanic acid Cefaclor or cefuroxime Trimethoprim-sulfamethoxazole

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Table 12. Moraxella catarrhalis (Continued)

Antimicrobial Class Antimicrobial Agent Disk Content



Comments S I R S I RTETRACYCLINES

Tetracycline 30 μg ≥ 29 25–28 ≤ 24 ≤ 2 4 ≥ 8 LINCOSAMIDE

Clindamycin – – – – ≤ 0.5 1–2 ≥ 4 FOLATE PATHWAY INHIBITORS

Trimethoprim-sulfamethoxazole

1.25/23.75 μg ≥ 13 11–12 ≤ 10 ≤ 0.5/9.5 1/19–2/38

≥ 4/76

PHENICOLS Chloramphenicol – – – – ≤ 2 4 ≥ 8

ANSAMYCINS Rifampin – – – – ≤ 1 2 ≥ 4 (3) Rx: Rifampin resistance may

emerge during therapy if rifampin is administered alone.


Resistance: Most strains of Moraxella catarrhalis produce β-lactamase and are resistant to ampicillin and amoxicillin. Reasons for Testing/Not Testing: Testing is not recommended routinely. However, testing may be useful for epidemiological purposes or for management of patients with prolonged or severe infections. Derivation of Interpretive Criteria: Interpretive criteria are adapted from those for Haemophilus spp., as published in CLSI document M1003; however, for macrolides, interpretive criteria were derived from a large organism collection tested by working group members. Testing Notes: If desired, β-lactamase testing can be performed using chromogenic cephalosporin methods such as nitrocefin.

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Table 13. Pasteurella spp.—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing

General Comments (1) Growth characteristics on routine media: often fastidious; requires blood-supplemented media for adequate growth; ambient air; 20 to 24 hours. (2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories

other than “susceptible.” For strains yielding results suggestive of a “nonsusceptible” category, organism identification and antimicrobial susceptibility test results should be confirmed. Subsequently, the isolates should be saved and submitted to a reference laboratory for confirmation.

Antimicrobial

Class

Antimicrobial

Agent

Disk Content




Amoxicillin – – – – ≤ 0.5 – –a See comment (2). Ampicillin 10 μg ≥ 27 – – ≤ 0.5 – –a See comment (2). Penicillin 10 units ≥ 25 – – ≤ 0.5 – –a See comment (2). Amoxicillin-

clavulanic acid 20/10 μg ≥ 27 – – ≤ 0.5/0.25 – –a See comment (2).

CEPHALOSPORINS Ceftriaxone 30 μg ≥ 34 – – ≤ 0.12 – – See comment (2).

QUINOLONES Moxifloxacin 5 μg ≥ 28 – – ≤ 0.06 – – See comment (2). Levofloxacin 5 μg ≥ 28 – – ≤ 0.06 – – See comment (2).

TETRACYCLINES Tetracycline 30 μg ≥ 23 – – ≤ 1 – – See comment (2). Doxycycline 30 μg ≥ 23 – – ≤ 0.5 – – See comment (2).

Testing Conditions Medium: CAMHB with LHB (2.5% to 5%) for broth microdilution; BMHA for disk diffusion (see Testing Notes) Inoculum: Direct colony suspension, equivalent to a 0.5 McFarland

standard Incubation: 35 °C; ambient air Disk diffusion: 16 to 18 hours Broth microdilution method: 18 to 24 hours

Minimal QC Recommendations Streptococcus pneumoniae ATCC® 49619 Escherichia coli ATCC® 35218 (for β-lactam/β-lactamase inhibitor combinations) Staphylococcus aureus ATCC® 25923 for disk diffusion (amoxicillin-clavulanic acid and doxycycline)

Agents to Consider for Primary Testing Penicillins β-lactam/β-lactamase inhibitor combinations Cephalosporins Tetracyclines Macrolides Fluoroquinolones Trimethoprim-sulfamethoxazole

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Table 13. Pasteurella spp. (Continued)

Antimicrobial

Class

Antimicrobial

Agent

Disk

Content



S I R S I RMACROLIDES

Erythromycin 15 μg ≥ 27 25–26 ≤ 24 ≤ 0.5 1 ≥ 2 Azithromycin 15 μg ≥ 20 – – ≤ 1 – – See comment (2).

OTHERS Chloramphenicol 30 μg ≥ 28 – – ≤ 2 – – See comment (2). Trimethoprim-

sulfamethoxazole 1.25/23.75 μg ≥ 24 – – ≤ 0.5/9.5 – – See comment (2).


Resistance: aRare isolates of Pasteurella spp. have been encountered that produce β-lactamase and have ampicillin, amoxicillin, and penicillin MICs > 0.5 µg/mL. Reasons for Testing/Not Testing: For isolates from bite wounds, routine testing is usually not necessary. Multiple organisms are often present in these specimens; therefore, empiric therapy directed toward these organisms is generally effective for P. multocida, as well. Testing of isolates from normally sterile sources (blood cultures, deep tissue, implanted prosthetic devices) and also respiratory specimens may be warranted, especially in immunodeficient patients. Derivation of Interpretive Criteria: Interpretive criteria are based primarily on MIC distributions obtained by testing large numbers of isolates recovered from human-animal bite infections. Key citations used in derivation of interpretive breakpoints include references 173 and 180 under Additional References. Testing Notes: Testing for β-lactamase production using a chromogenic cephalosporin test is recommended for isolates recovered from respiratory or normally sterile sources. β-lactamase–positive isolates are resistant to ampicillin, amoxicillin, and penicillin. For β-lactamase–producing strains, ampicillin MIC may be > 4 μg/mL and the MIC is reduced with addition of a β-lactamase inhibitor. Some isolates may require incubation in 5% CO2 for disk diffusion tests, and these should be tested only by broth microdilution. Testing of S. aureus ATCC® 25923 using BMHA has been shown to produce zones within the acceptable limits noted in CLSI document M100,3 Table 3, for the antimicrobial agents listed in this table.


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Table 14. Pediococcus spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing

General Comments

(1) Growth characteristics on routine media: often fastidious; requires blood-supplemented media for adequate growth; ambient air; 20 to 24 hours.

(2) For some organism/antimicrobial combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than “susceptible.” For strains yielding results suggestive of a “nonsusceptible” category, organism identification and antimicrobial susceptibility test results should be confirmed. Subsequently, the isolates should be saved and submitted to a reference laboratory for confirmation.

Antimicrobial Class

Antimicrobial Agent


Comments S I RPENICILLINS

Penicillin ≤ 8 – – See comment (2). Ampicillin ≤ 8 – – (3) Therapy of serious infections such as endocarditis often involves

combined therapy with a penicillin and an aminoglycoside. CARBAPENEMS

Imipenem ≤ 0.5 – – See comment (2). AMINOGLYCOSIDES

Gentamicin ≤ 4 8 ≥ 16 (4) For combined therapy. See comment (3).



Resistance: Pediococcus spp. are considered intrinsically resistant to vancomycin; routine testing of vancomycin is not necessary. Reasons for Testing/Not Testing: Testing of isolates from normally sterile sources (blood cultures, deep tissue, implanted prosthetic devices) may be warranted, especially in immunodeficient patients. Derivation of Interpretive Criteria: Interpretive criteria for gentamicin are adapted from those for Staphylococcus spp., as published in CLSI document M100.3 Interpretive criteria for all other antimicrobial agents are adapted from those for Enterococcus spp., as published in CLSI document M100.3 Key citations used in derivation of interpretive breakpoints include references 185, 187, 193, and 195 under Additional References.





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Table 15. Vibrio spp. (including V. cholerae)—Information and Interpretive Criteria for Broth Microdilution and Disk Diffusion Susceptibility Testing

General Comments (1) Growth characteristics on routine media: halophilic; grows well on BAP; ambient air; 20 to 24 hours.

Antimicrobial

Class

Antimicrobial Agent

Disk

Diffusion



Comments


Ampicillin 10 μg ≥ 17 14–16 ≤ 13 ≤ 8 16 ≥ 32 (2) Class representative for ampicillin and amoxicillin.

Amoxicillin-clavulanic acid

20/10 μg ≥ 18 14–17 ≤ 13 ≤ 8/4 16/8 ≥ 32/16 (3) For Vibrio spp. other than V. cholerae.

Ampicillin-sulbactam 10/10 μg ≥ 15 12–14 ≤ 11 ≤ 8/4 16/8 ≥ 32/16 See comment (3). Piperacillin 100 μg ≥ 21 18–20 ≤ 17 ≤ 16 32–64 ≥ 128 See comment (3). Piperacillin-

tazobactam 100/10 μg ≥ 21 18–20 ≤ 17 ≤ 16/4 32/4–64/4 ≥ 128/4 See comment (3).

CEPHEMS See comment (3). Cefazolin 30 μg – – – ≤ 1 2 ≥ 4 Breakpoints are based on a dosage regimen

of 1 g every 8 h. Cefepime 30 μg ≥ 18 15–17 ≤ 14 ≤ 8 16 ≥ 32 Cefotaxime 30 μg ≥ 26 23–25 ≤ 22 ≤ 1 2 ≥ 4 Breakpoints are based on a dosage regimen

of 1 g every 8 h. Cefoxitin 30 μg ≥ 18 15–17 ≤ 14 ≤ 8 16 ≥ 32 Ceftazidime 30 μg ≥ 21 18–20 ≤ 17 ≤ 4 8 ≥ 16 Breakpoints are based on a dosage regimen

of 1 g every 8 h. Cefuroxime sodium

(parenteral) 30 μg ≥ 18 15–17 ≤ 14 ≤ 8 16 ≥ 32 Breakpoints are based on a dosage regimen

of 1.5 g every 8 h. CARBAPENEMS See comment (3).

Imipenem 10 μg ≥ 16 14–15 ≤ 13 ≤ 4 8 ≥ 16 Meropenem 10 μg ≥ 16 14–15 ≤ 13 ≤ 4 8 ≥ 16

Testing Conditions Medium: CAMHB for microdilution; MHA for disk diffusion Inoculum: Growth method or direct colony suspension, equivalent to a 0.5

McFarland standard. Prepare inoculum in 0.85% NaCl (normal saline).

Incubation: 35 ± 2 °C; ambient air; Disk diffusion: 16 to 18 hours Broth microdilution method: 16 to 20 hours

Minimal QC Recommendations Escherichia coli ATCC® 25922 Escherichia coli ATCC® 35218 (for β-lactam/β-lactamase inhibitor combinations)

Agents to Consider for Primary Testing For Vibrio spp.: Cefotaxime Ceftazidime Tetracycline Fluoroquinolones For V. cholerae: Ampicillin Azithromycin Doxycycline Tetracycline Trimethoprim-sulfamethoxazole Sulfonamides Chloramphenicol

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Table 15. Vibrio spp. (Continued)

Antimicrobial Class

Antimicrobial

Agent

Disk Content



S I R S I R MACROLIDES Azithromycin – – – – ≤ 2 – – (4) For V. cholerae only. AMINOGLYCOSIDES See comment (3).

Amikacin 30 μg ≥ 17 15–16 ≤ 14 ≤ 16 32 ≥ 64 Gentamicin 10 μg ≥ 15 13–14 ≤ 12 ≤ 4 8 ≥ 16

TETRACYCLINES (5) When testing V. cholera, tetracycline results can be used to predict the likely susceptibility of isolates to doxycycline; doxycycline disk diffusion tests should not be used because there is poor correlation with MIC test results.

Tetracycline 30 μg ≥ 15 12–14 ≤ 11 ≤ 4 8 ≥ 16 Doxycycline – – – – ≤ 4 8 ≥ 16 (6) For V. cholerae only.

QUINOLONES See comment (3). Ciprofloxacin 5 μg ≥ 21 16–20 ≤ 15 ≤ 1 2 ≥ 4 Levofloxacin 5 μg ≥ 17 14–16 ≤ 13 ≤ 2 4 ≥ 8 Ofloxacin 5 μg ≥ 16 13–15 ≤ 12 ≤ 2 4 ≥ 8

FOLATE PATHWAY INHIBITORS Trimethoprim-

sulfamethoxazole 1.25/23.75 μg ≥ 16 11–15 ≤ 10 ≤ 2/38 – ≥ 4/76

Sulfonamides 250 μg or 300 μg

≥ 17 13–16 ≤ 12 ≤ 256 – ≥ 512 (7) Sulfisoxazole can be used to represent any of the current available sulfonamide preparations. (8) For V. cholerae only.

PHENICOLS Chloramphenicol 30 μg ≥ 18 13–17 ≤ 12 ≤ 8 16 ≥ 32 (9) Use with caution for disk diffusion

as the disk diffusion test may misclassify many organisms (higher minor error rate). (10) Not routinely reported on isolates from the urinary tract. (11) For V. cholerae only.

Supplemental Information Resistance: Halophilic Vibrio spp. are usually resistant to sulfonamides, penicillins, and older cephalosporins (cephalothin, cefuroxime). Reasons for Testing/Not Testing: Testing is most often limited to isolates from extraintestinal sites. Derivation of Interpretive Criteria: With the exception of azithromycin, interpretive criteria are adapted from those for Enterobacteriaceae, as published in CLSI document M1003; azithromycin susceptible criterion adapted from Staphylococcus spp. Key citations used in derivation of interpretive breakpoints include references 197, 200, 202 to 206, 208, and 209 under Additional References. Testing Notes: Inoculum suspension should be prepared in 0.85% NaCl (normal saline). This will allow most isolates of Vibrio spp. to grow satisfactorily on MHA and CAMHB without adding supplemental NaCl to these test media.

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Table 16. Potential Bacterial Agents of Bioterrorism: Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Brucella spp.—Interpretive Criteria for Broth Microdilution Susceptibility Testing

Agents to Consider for Primary Testing

aOrganisms that are susceptible to penicillin are also considered susceptible to amoxicillin.

Bacillus

anthracis Yersinia pestis

Burkholderia

mallei Burkholderia pseudomallei

Francisella tularensis Brucella spp.

Penicillina Gentamicin Ceftazidime Amoxicillin-clavulanic acid

Gentamicin Streptomycin

Gentamicin Streptomycin

Doxycycline Tetracycline

Streptomycin

Imipenem

Ceftazidime Doxycycline Tetracycline


Ciprofloxacin Doxycycline Tetracycline


Imipenem

Ciprofloxacin or levofloxacin

Trimethoprim- sulfamethoxazole

Ciprofloxacin


Chloramphenicol



Chloramphenicol

Testing Conditions Medium: Broth microdilution: unsupplemented Brucella broth pH adjusted

to 7.1 ± 0.1 for Brucella spp.; CAMHB + 2% defined growth supplement for F. tularensis; CAMHB for all other organisms

Inoculum: Growth method or direct colony suspension in CAMHB, equivalent to a 0.5 McFarland standard; for F. tularensis, prepare inoculum as a direct colony suspension from a chocolate agar plate

Incubation: 35 ± 2 °C; ambient air; 16 to 20 hours; for Y. pestis, incubate 24 hours and if unacceptable growth in the control well, reincubate an additional 24 hours; for F. tularensis and Brucella spp., incubate 48 hours (see comment 8).

Minimal QC Recommendations (See Table 3 [CAMHB], Table 3C [CAMHB + 2% defined growth supplement], and Table 3D [Brucella-broth] for acceptable QC ranges.) Escherichia coli ATCC® 25922 (all organisms) Escherichia coli ATCC® 35218 (for amoxicillin-clavulanic acid and B. pseudomallei) Staphylococcus aureus ATCC® 29213 (for B. anthracis and F. tularensis) Pseudomonas aeruginosa ATCC® 27853 (for B. mallei/pseudomallei and F. tularensis) Streptococcus pneumoniae ATCC® 49619 (for Brucella spp. only)

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Table 16. Potential Bacterial Agents of Bioterrorism (Continued)

General Comments

Important: For complete information on safety precautions, see Biosafety in Microbiological and Biomedical Laboratories. 5th ed. Washington, DC: US Government Printing Office; 2007. http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm. Accessed 6 January 2010.

(1) Extreme Caution: Notify public health officials of all isolates presumptively identified as B. anthracis, Y. pestis, B. mallei, B. pseudomallei, Brucella spp.,

or F. tularensis. Confirmation of isolates of these bacteria may require specialized testing that is available only in reference or public health laboratories. (2) Recommended precautions: Use BSL-2 practices, containment equipment, and facilities for activities using clinical materials and diagnostic quantities of

infectious cultures. Use BSL-3 practices, containment equipment, and facilities for work involving production quantities or concentrations of cultures and for activities with a high potential for aerosol production. If BSL-2 or BSL-3 facilities are not available, forward isolates to a reference or public health laboratory with a minimum of BSL-2 facilities for susceptibility testing.

(3) Interpretive criteria are based on microorganism MIC population distributions, pharmacokinetics, and pharmacodynamics of the antimicrobial agents, and/or

animal model data. (4) Test method and interpretive criteria for B. anthracis do not apply to other Bacillus spp. (5) WARNING: For Y. pestis, studies have demonstrated that although β-lactam antimicrobial agents may appear active in vitro, they lack efficacy in animal

models of infection. Y. pestis should be reported as resistant to these antimicrobial agents. Rx: Retrospective clinical data suggest that β-lactam antimicrobial agents are not effective clinically.

(6) The recommended medium for testing F. tularensis consists of CAMHB to which a 2% defined growth supplement (25.9 g L-cysteine HCl, 1.1 g L-cystine, 1 g

adenine, 0.03 g guanine HCl, 0.01 g vitamin B12, 0.1 g cocarboxylase, 0.25 g NAD, 10 g L-glutamine, 0.02 g ferric nitrate, 100 g glucose, 3 mg thiamine HCl, and 13 mg p-aminobenzoic acid [in 1 L H2O]) is added after autoclaving. The pH of the medium should be adjusted to 7.1 ± 0.1.

(7) For some organism/antimicrobial agent combinations, the absence or rare occurrence of resistant strains precludes defining any results categories other than

“susceptible.” For strains yielding results suggestive of a “nonsusceptible” category, organism identification and antimicrobial susceptibility test results should be confirmed.

(8) Incubation in 5% CO2 may be required for growth of some strains of Brucella spp., especially B. abortus. Incubation of broth MIC tests in CO2 may increase

the MIC of aminoglycosides and decrease the MIC of tetracyclines, usually by one doubling dilution.

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Organism Group

Antimicrobial Agent

MIC Interpretive Standard (µg/mL)

Comments S I R PENICILLINS B. anthracis Penicillin ≤ 0.12 – ≥ 0.25 (9) Class representative for amoxicillin.

(10) B. anthracis strains may contain inducible β-lactamases. In vitro penicillinase induction studies suggest that penicillin MICs may increase during therapy. This is supported by reduced response rates to penicillin in animal treatment studies of B. anthracis infection. However, β-lactamase testing of clinical isolates of B. anthracis is unreliable and should not be performed. If MIC susceptibility testing using CLSI methods indicates that B. anthracis isolates are susceptible to penicillin, amoxicillin may still be considered for prophylactic use in children and pregnant women. (References: MMWR 21 October 2001 and websites: www.cdc.gov or www.bt.cdc.gov/agent/anthrax/exposure/index.asp)

β-LACTAM/β-LACTAMASE INHIBITOR COMBINATIONS B. pseudomallei Amoxicillin-clavulanic acid ≤ 8/4 16/8 ≥ 32/16 CEPHEMS (PARENTERAL) (Including cephalosporins I, II, III, and IV. Please refer to Glossary I.) B. mallei B. pseudomallei

Ceftazidime ≤ 8 16 ≥ 32

CARBAPENEMS B. mallei B. pseudomallei

Imipenem ≤ 4 8 ≥ 16

AMINOGLYCOSIDES Y. pestis Gentamicin ≤ 4 8 ≥ 16 Streptomycin ≤ 4 8 ≥ 16 F. tularensis Gentamicin ≤ 4 – – See comment (7). Brucella spp. Streptomycin ≤ 8 – – See comments (7) and (8).

(11) The streptomycin-susceptible breakpoint is ≤ 16 μg/mL when the test is incubated in CO2 and ≤ 8 μg/mL when incubated in air.

TETRACYCLINES (12) Organisms that are susceptible to tetracycline are also considered susceptible to doxycycline. B. anthracis Brucella spp.

Tetracycline ≤ 1 – – See comments (7) and (8). Doxycycline

≤ 1 – – See comments (7) and (8).

B. mallei B. pseudomallei Y. pestis

Tetracycline

≤ 4 8 ≥ 16

Doxycycline ≤ 4 8 ≥ 16

F. tularensis Tetracycline ≤ 4 – – See comment (7). Doxycycline ≤ 4 – – See comment (7).

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Organism Group

Antimicrobial Agent

MIC Interpretive Standard (µg/mL)

Comments S I R FLUOROQUINOLONES

B. anthracis Ciprofloxacin ≤ 0.25 – – See comment (7). Levofloxacin ≤ 0.25 – – See comment (7).

Y. pestis Ciprofloxacin ≤ 0.25 – – See comment (7). Levofloxacin ≤ 0.25 – – See comment (7).

F. tularensis Ciprofloxacin ≤ 0.5 – – See comment (7). Levofloxacin ≤ 0.5 – – See comment (7).

FOLATE PATHWAY INHIBITORS B. pseudomallei Y. pestis

Trimethoprim-sulfamethoxazole ≤ 2/38 – ≥ 4/76

Brucella spp. Trimethoprim-sulfamethoxazole ≤ 2/38 – – See comments (7) and (8). PHENICOLS Y. pestis Chloramphenicol ≤ 8 16 ≥ 32 F. tularensis Chloramphenicol ≤ 8 – – See comment (7).

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Table 17. Summary of Testing Conditions and Quality Control Recommendations for Infrequently Isolated or Fastidious Bacteria

Table No.

Organism/Organism Group

Broth Microdilution MIC Test Medium

Broth Microdilution MIC Incubation Conditions

Disk DiffusionTest Medium/Incubation

Conditions

Quality Control 1 Abiotrophia spp.,

Granulicatella spp. CAMHB-LHB (2.5%–5% v/v) + 0.001% pyridoxal HCl

35 °C; ambient air; 20–24 h NAa S. pneumoniae ATCC® 49619

2 Aeromonas hydrophila complex, Plesiomonas shigelloides

CAMHB

35 °C; ambient air; 16–20 h MHA (unsupplemented)/35 °C; ambient air; 16–18 h)

E. coli ATCC® 25922 E. coli ATCC® 35218b

3 Bacillus spp. (not B. anthracis)

CAMHB

35 °C; ambient air; 16–20 h

NA S. aureus ATCC® 29213

4 Campylobacter jejuni/coli

CAMHB-LHB (2.5%–5% v/v)

36–37 °C/48 h or 42 °C/24 h; 10% CO2, 5% O2, 85% N2 (microaerobic)

MHA with 5% sheep blood/36–37 °C/48 h or 42 °C/24 h; 10% CO2, 5% O2, 85% N2 (microaerobic)

C. jejuni ATCC® 33560 for microdilution S. aureus ATCC® 25923, MHA/35–37 °C for 16–18 h in ambient air for disk diffusion

5 Corynebacterium spp. CAMHB-LHB (2.5%–5% v/v)

35 °C; ambient air; 24–48 h NA S. pneumoniae ATCC® 49619 E. coli ATCC® 25922 for gentamicin

6 Erysipelothrix rhusiopathiae


35 °C; ambient air; 20–24 h NA S. pneumoniae ATCC® 49619

7 HACEK group CAMHB-LHB (2.5%–5% v/v)

35 °C; 5% CO2; 24–48 h NA S. pneumoniae ATCC® 49619 E. coli ATCC® 35218b

8 Helicobacter pylori

Agar dilution: MHA and aged (≥ 2-week-old) sheep blood (5% v/v)

35 ± 2 °C; 72 h; 10% CO2, 5% O2, 85% N2 (microaerobic)

NA Helicobacter pylori ATCC® 43504

9 Lactobacillus spp. CAMHB-LHB (2.5%–5% v/v)

35 °C; 5% CO2; 24 to 48 hours NA S. pneumoniae ATCC® 49619 E. coli ATCC® 25922 for gentamicin

10 Leuconostoc spp. CAMHB-LHB (2.5%–5% v/v)


11 Listeria monocytogenes CAMHB-LHB (2.5%–5% v/v)

35 °C; ambient air; 20–24 h NA S. pneumoniae ATCC® 49619

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Table 17. (Continued)

Table No.

Organism/Organism Group

Broth Microdilution MIC Test Medium

Broth Microdilution MIC Incubation Conditions

Disk Diffusion Test Medium/Incubation

Conditions

Quality Control 12 Moraxella catarrhalis CAMHB

35 °C; ambient air; 20–24 h

MHA (unsupplemented)/35 °C; 5% CO2; 20–24 h)

S. aureus ATCC® 29213 E. coli ATCC® 35218b

13 Pasteurella spp.


35 °C; ambient air; 18–24 h MHA with 5% sheep blood/35 °C; ambient air; 16–18 h

S. pneumoniae ATCC® 49619 E. coli ATCC® 35218b S. aureus ATCC® 25923 (for disk diffusion with selected drugs)

14 Pediococcus spp. CAMHB-LHB (2.5%–5% v/v)


15 Vibrio spp. (including V. cholerae)

CAMHBc 35 °C; ambient air; 16–20 h MHA (unsupplemented)/35 °C; ambient air; 16–18 h)c

E. coli ATCC® 25922 E. coli ATCC® 35218b

Potential Bacterial Agents of Bioterrorism 16 Bacillus anthracis CAMHB 35 °C; ambient air; 16–20 h NA E. coli ATCC® 25922

S. aureus ATCC® 29213 16 Brucella spp. Unsupplemented Brucella

broth pH adjusted to 7.1 ± 0.1

35 °C; ambient air; 48 h NA E. coli ATCC® 25922 S. pneumoniae ATCC® 49619

16 Burkholderia mallei CAMHB 35 °C; ambient air; 16–20 h NA E. coli ATCC® 25922 Pseudomonas aeruginosa ATCC® 27853

16 Burkholderia pseudomallei

CAMHB 35 °C; ambient air; 16–20 h NA E. coli ATCC® 25922 E. coli ATCC® 35218b P. aeruginosa ATCC® 27853

16 Francisella tularensis CAMHB + 2% defined growth supplement

35 °C; ambient air; 48 h NA E. coli ATCC® 25922 S. aureus ATCC® 29213 P. aeruginosa ATCC® 27853

16 Yersinia pestis

CAMHB 35 ± 2 °C; ambient air; 24 h, and if unacceptable growth in the control well, reincubate an additional 24 h

NA E. coli ATCC® 25922

Footnotes

a NA, not applicable b E. coli ATCC® 35218 is used for QC when testing β-lactam/β-lactamase inhibitor combination drugs. c Prepare inoculum in 0.85% NaCl (normal saline). Abbreviations: CAMHB (cation-adjusted Mueller-Hinton broth); LHB (lysed horse blood); MHA (Mueller-Hinton agar)

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Table 18. MIC: Quality Control Ranges for Nonfastidious Organisms (Unsupplemented Cation-Adjusted Mueller-Hinton Medium)

Antimicrobial Agent

Staphylococcus aureus

ATCC® 29213

Escherichia coli

ATCC® 25922

Escherichia coli

ATCC® 35218a Amikacin 1–4 0.5–4 – Amoxicillin-clavulanic acid 0.12/0.06–0.5/0.25 2/1–8/4 4/2–16/8 Ampicillin 0.5–2 2–8 > 32 Ampicillin-sulbactam – 2/1–8/4 8/4–32/16 Azithromycin 0.5–2 – – Aztreonam – 0.06–0.25 – Cefaclor 1–4 1–4 – Cefazolin 0.25–1 1–4 – Cefepime 1–4 0.015–0.12 – Cefotaxime 1–4 0.03–0.12 – Cefoxitin 1–4 2–8 – Cefpodoxime 1–8 0.25–1 – Cefprozil 0.25–1 1–4 – Ceftazidime 4–16 0.06–0.5 – Ceftriaxone 1–8 0.03–0.12 – Cefuroxime 0.5–2 2–8 – Cephalothin 0.12–0.5 4–16 – Chloramphenicol 2–16 2–8 – Ciprofloxacin 0.12–0.5 0.004–0.015 – Clarithromycin 0.12–0.5 – – Clindamycin 0.06–0.25 – – Ertapenem 0.06–0.25 0.004–0.015 – Erythromycin 0.25–1 – – Gentamicin 0.12–1 0.25–1 – Imipenem 0.015–0.06 0.06–0.25 – Levofloxacin 0.06–0.5 0.008–0.06 – Meropenem 0.03–0.12 0.008–0.06 – Ofloxacin 0.12–1 0.015–0.12 –

Penicillin 0.25–2 – – Piperacillin 1–4 1–4 > 64 Piperacillin-tazobactam 0.25/4–2/4 1/4–4/4 0.5/4–2/4 Rifampin 0.004–0.015 4–16 – Tetracycline 0.12–1 0.5–2 – Trimethoprim-sulfamethoxazole ≤ 0.5/9.5 ≤ 0.5/9.5 – Vancomycin 0.5–2 – –

Footnote a. Because this strain may lose its plasmid, careful organism maintenance is required; refer to CLSI

document M07-A8,2 Section 16.4.



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Table 18A. MIC: Quality Control Ranges for Broth Microdilution Methods (Cation-Adjusted Mueller-Hinton Broth With Lysed Horse Blood [2.5% to 5% v/v])

Antimicrobial Agent

Streptococcus pneumoniae ATCC® 49619

Escherichia coli ATCC®

25922

Escherichia coli ATCC®

35218 Amoxicillin 0.03–0.12 – ≥ 256 Amoxicillin-clavulanic acid 0.03/0.015–0.12/0.06 – 4–16a

Ampicillin 0.06–0.25 – – Ampicillin-sulbactam – – 8–32a

Azithromycin 0.06–0.25 – – Cefepime 0.03–0.25 – – Cefotaxime 0.03–0.12 – – Ceftriaxone 0.03–0.12 – – Chloramphenicol 2–8 – – Ciprofloxacin 0.25–1a – – Clarithromycin 0.03–0.12 – – Clindamycin 0.03–0.12 – – Daptomycinb 0.06–0.5 – – Doxycycline 0.015–0.12 – – Erythromycin 0.03–0.12 – – Gatifloxacin 0.12–0.5 – – Gentamicin – 0.25–1a – Imipenem 0.03–0.12 – – Levofloxacin 0.5–2 – – Linezolid 0.5–2 – – Meropenem 0.06–0.25 – – Minocycline – 0.25–1a – Moxifloxacin 0.06–0.25 – – Penicillin 0.25–1 – – Quinupristin-dalfopristin 0.25–1 – – Rifampin 0.015–0.06 – – Tetracycline 0.12–0.5 – – Trimethoprim-sulfamethoxazole 0.12/2.4–1/19 – – Vancomycin 0.12–0.5 – –

Footnotes

a. These QC ranges were validated for tests performed in CAMHB with LHB and were not established

by the studies outlined in CLSI document M23.6 The validation studies were conducted in at least three laboratories using multiple lots of media.

b. QC ranges reflect MICs obtained when Mueller-Hinton broth is supplemented with calcium to a final

concentration of 50 μg/mL. Agar dilution has not been validated for daptomycin.



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Table 18B. MIC: Quality Control Ranges for Campylobacter jejuni (Broth Microdilution Method) (Cation-Adjusted Mueller-Hinton Broth With Lysed Horse Blood [2.5% to 5% v/v])

Antimicrobial Agent

Campylobacter jejuni ATCC® 33560

36–37 °C/48 hours 42 °C/24 hours Azithromycin 0.03–0.25 0.03–0.12 Ciprofloxacin 0.06–0.25 0.03–0.12 Doxycycline 0.12–0.5 0.12–0.5 Erythromycin 0.5–2 0.25–2 Gentamicin 0.5–2 0.25–2 Levofloxacin 0.06–0.25 0.03–0.25 Meropenem 0.008–0.03 0.008–0.03 Tetracycline 0.25–2 0.25–1

NOTE 1: These MICs were obtained in several reference laboratories by broth microdilution. If four or

fewer concentrations are tested, QC may be more difficult. NOTE 2: For four-dilution ranges, results at the extremes of the acceptable range or ranges should be

suspect. Verify control validity with data from other control strains.



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Table 18C. MIC: Quality Control Ranges for Agar Dilution Methods (Mueller-Hinton Agar With Aged [≥ 2-Week-Old] Sheep Blood)

Antimicrobial Agent

Helicobacter pylori

ATCC® 43504 Amoxicillin 0.015–0.12 Clarithromycin 0.015–0.12 Metronidazole 64–256 Telithromycin 0.06–0.5 Tetracycline 0.12–1

Table 18D. MIC: Quality Control Ranges for Broth Microdilution Method (Cation-Adjusted Mueller-Hinton Broth + 2% Defined Growth Supplementa)

Antimicrobial Agent


ATCC® 29213

Escherichia coli

ATCC® 25922 Pseudomonas aeruginosa

ATCC® 27853 24 hours 48 hours 24 hours 48 hours 24 hours 48 hours

Chloramphenicol 4–16 4–32 2–8 4–16 – – Ciprofloxacin 0.25–1 0.25–1 0.004–0.015 0.004–0.03 0.12–1 0.25–1 Doxycycline 0.12–1 0.25–2 1–4 1–8 4–32 4–32 Gentamicin 0.25–1 0.25–1 0.25–2 0.25–2 0.5–2 0.5–4 Levofloxacin 0.12–0.5 0.12–0.5 0.008–0.03 0.008–0.06 0.5–2 0.5–4 Nalidixic acid – – 1–8 2–8 – – Streptomycin 8–32 8–64 8–32 8–32 32–128 32–256 Tetracycline 0.25–2 0.5–4 1–4 2–8 8–32 8–64 Trimethoprim- sulfamethoxazole

≤ 0.25/4.75 ≤ 0.25/4.75 ≤ 0.5/9.5 ≤ 0.5/9.5 – –

NOTE: Francisella tularensis MIC results read after 24 hours of incubation should use 24-hour QC ranges; results read after 48 hours should use only the 48-hour QC ranges.

Footnote

a. Add 2% defined growth supplement (25.9 g L-cysteine HCl, 1.1 g L-cystine, 1 g adenine, 0.03 g guanine HCl, 0.01 g vitamin B12, 0.1 g cocarboxylase, 0.25 g NAD, 10 g L-glutamine, 0.02 g ferric nitrate, 100 g glucose, 3 mg thiamine HCl, and 13 mg p-aminobenzoic acid [in 1 L H2O]) to CAMHB after autoclaving. The pH of medium should be adjusted to 7.1 ± 0.1.



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Table 18E. MIC: Quality Control Ranges for Broth Microdilution Methods (Brucella Broth Without Supplements Adjusted to pH 7.1 ± 0.1)

Antimicrobial Agent

Escherichia coli ATCC® 25922

Staphylococcus aureus ATCC® 29213


24 hours 48 hours 24 hours 48 hours 24 hours 48 hoursAzithromycin – – 2–8 2–16 0.25–1 0.25–1 Chloramphenicol 2–8 4–16 4–16 4–16 1–8 2–8 Ciprofloxacin – – 0.25–1 0.25–1 0.25–1 0.25–2 Doxycycline 0.5–2 1–4 0.12–0.5 0.12–0.5 0.03–0.12 0.03–0.25 Gentamicin 1–8 1–8 – – – – Levofloxacin – – 0.06–0.5 0.12–0.5 0.25–1 0.25–2 Rifampin 4–16 4–16 – – 0.008–0.03 0.008–0.06 Streptomycin 4–32 4–32 8–64 8–64 16–64 16–128 Tetracycline 0.5–2 0.5–4 0.12–1 0.25–1 0.03–0.25 0.06–0.5 Trimethoprim- sulfamethoxazole

– – – – 0.5/9.5–2/38 0.5/9.5–2/38



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Table 19. Disk Diffusion: Quality Control Ranges for Nonfastidious Organisms (Unsupplemented Mueller-Hinton Medium)

Antimicrobial Agent Disk Content

Escherichia coli

ATCC® 25922


ATCC® 25923

Escherichia coli

ATCC® 35218a Amikacin 30 μg 19–26 20–26 – Amoxicillin-clavulanic acid

20/10 μg 18–24 28–36 17–22

Ampicillin 10 μg 16–22 27–35 6 Ampicillin-sulbactam 10/10 μg 19–24 29–37 13–19 Azithromycin 15 μg – 21–26 – Aztreonam 30 μg 28–36 – – Cefazolin 30 μg 21–27 29–35 – Cefepime 30 μg 31–37 23–29 – Cefotaxime 30 μg 29–35 25–31 – Cefoxitin 30 μg 23–29 23–29 – Ceftazidime 30 μg 25–32 16–20 – Ceftriaxone 30 μg 29–35 22–28 – Cefuroxime 30 μg 20–26 27–35 – Cephalothin 30 μg 15–21 29–37 – Chloramphenicol 30 μg 21–27 19–26 – Ciprofloxacin 5 μg 30–40 22–30 – Clarithromycin 15 μg – 26–32 – Doxycycline 30 μg 18–24 23–29 – Ertapenem 10 μg 29–36 24–31 – Erythromycin 15 μg – 22–30 – Gentamicin 10 μg 19–26 19–27 – Imipenem 10 μg 26–32 – – Levofloxacin 5 μg 29–37 25–30 – Meropenem 10 μg 28–34 29–37 – Ofloxacin 5 μg 29–33 24–28 – Piperacillin 100 μg 24–30 – 12–18 Piperacillin-tazobactam 100/10 μg 24–30 27–36 24–30 Tetracycline 30 μg 18–25 24–30 – Trimethoprim- sulfamethoxazoleb

1.25/23.75 μg 23–29 24–32 –

Footnotes a. Careful organism maintenance is required; refer to CLSI document M02-A10,1 Section 15.4. b. These agents can be affected by excess levels of thymidine and thymine. See CLSI document M02-

A10,1 Section 7.1.3 for guidance, should a problem with QC occur.



Number 18 M45-A2


Table 19A. Disk Diffusion: Quality Control Ranges for Fastidious Organisms (Mueller-Hinton Medium With 5% Sheep Blood)

Antimicrobial

Agent

Disk Content


Ampicillin 10 μg 30–36 Azithromycin 15 μg 19–25 Ceftriaxone 30 μg 30–35 Chloramphenicol 30 μg 23–27 Erythromycin 15 μg 25–30 Levofloxacin 5 μg 20–25 Moxifloxacin 5 μg 25–31 Penicillin 10 units 24–30 Tetracycline 30 μg 27–31 Trimethoprim- sulfamethoxazole

1.25/23.75 μg 20–28



Volume 30 M45-A2


Glossary I (Part 1). β-Lactams: Class and Subclass Designation and Generic Name Antimicrobial Class Antimicrobial Subclass Agents Included; Generic Names

Penicillinse Penicillina Penicillin Aminopenicillina Amoxicillin

Ampicillin Ureidopenicillina Azlocillin

Mezlocillin Piperacillin

Carboxypenicillina Carbenicillin Ticarcillin

Penicillinase-stable penicillinsb

Cloxacillin Dicloxacillin Methicillin Nafcillin Oxacillin

Aminopenicillin Mecillinam β-Lactam/β-lactamase inhibitor combinations

Amoxicillin-clavulanic acid Ampicillin-sulbactam Piperacillin-tazobactam Ticarcillin-clavulanic acid

Cephems (parenteral) Cephalosporin Ic,e Cefazolin Cephalothin Cephapirin Cephradine

Cephalosporin IIc,e Cefamandole Cefonicid Cefuroxime (parenteral)

Cephalosporin IIIc,e Cefoperazone Cefotaxime Ceftazidime Ceftizoxime Ceftriaxone

Cephalosporin IVc,e Cefepime Cephalosporins with anti-MRSA activity Ceftaroline

Ceftobiprole Cephamycind Cefmetazole

Cefotetan Cefoxitin

Oxacephem Moxalactam Cephems (oral) Cephalosporine Cefaclor

Cefadroxil Cefdinir Cefditoren Cefetamet Cefixime Cefpodoxime Cefprozil Ceftibuten Cefuroxime (oral) Cephalexin Cephradine

Carbacephem Loracarbef Monobactamse Aztreonam Penems Carbapenem Doripenem

Ertapenem Imipenem Meropenem

Razupenem Penem Faropenem

Sulopenem a Penicillinase-labile; hydrolyzed by staphylococcal penicillinase. b Not hydrolyzed by staphylococcal penicillinase. c Cephalosporin I, II, III, and IV are sometimes referred to as 1st-, 2nd-, 3rd, and 4th-generation cephalosporins, respectively.

Cephalosporin III and IV are also referred to as “extended-spectrum cephalosporins.” This does not imply activity against extended-spectrum β-lactamase (ESBL)-producing gram-negative bacteria.

d Although often referred to as a second-generation cephalosporin, cephamycins are not included with the other cephalosporins with regard to reporting of ESBL-producing strains.

e For all confirmed ESBL-producing strains, the test interpretation should be reported as resistant for this antimicrobial class or subclass.



Number 18 M45-A2


Glossary I (Part 2). Non–β-lactams: Class and Subclass Designation and Generic Name Antimicrobial Class Antimicrobial Subclass Agents Included; Generic Names

Aminocyclitols Spectinomycin Trospectinomycin

Aminoglycosides Amikacin Gentamicin Kanamycin Netilmicin Streptomycin Tobramycin

Ansamycins Rifampin Quinolones Quinolone Cinoxacin

Garenoxacin Nalidixic acid

Fluoroquinolone Ciprofloxacin Clinafloxacin Enoxacin Fleroxacin Gatifloxacin Gemifloxacin Grepafloxacin Levofloxacin Lomefloxacin Moxifloxacin Norfloxacin Ofloxacin Sparfloxacin Trovafloxacin

Folate pathway inhibitors Iclaprim Sulfonamides Trimethoprim Trimethoprim-sulfamethoxazole

Fosfomycins Fosfomycin Ketolides Telithromycin Lincosamides Clindamycin Lipopeptides Daptomycin

Polymyxins Colistin Polymyxin B

Macrolides Azithromycin Clarithromycin Dirithromycin Erythromycin

Nitrofurans Nitrofurantoin Nitroimidazoles Metronidazole Oxazolidinones Linezolid Glycopeptides Glycopeptide Vancomycin

Lipoglycopeptide Dalbavancin Oritavancin Teicoplanin Telavancin

Phenicols Chloramphenicol Streptogramins Linopristin-flopristin

Quinupristin-dalfopristin Tetracyclines Doxycycline

Minocycline Tetracycline

Glycylcyclines Tigecycline Pseudomonic acid Mupirocin



Volume 30 M45-A2


Glossary II. Abbreviations/Routes of Administration/Drug Class for Antimicrobial Agents Listed in M100-S20

Antimicrobial Agent Agent Abbreviationa Routes of Administrationb Drug Class PO IM IV Topical Amikacin AN, AK, Ak,

AMI, AMK X X Aminoglycoside

Amoxicillin AMX, Amx, AMOX, AC

X Penicillin

Amoxicillin-clavulanic acid AMC, Amc, A/C, AUG, Aug, XL, AML

X β-Lactam/β-lactamase Inhibitor

Ampicillin AM, Am, AMP X X X Penicillin Ampicillin-sulbactam SAM, A/S,

AMS, AB X β-Lactam/β-lactamase

Inhibitor Azithromycin AZM, Azi, AZI, AZ X X Macrolide Azlocillin AZ, Az, AZL X X Penicillin Aztreonam ATM, AZT, Azt, AT,

AZM X Monobactam

Carbenicillin (indanyl salt) Carbenicillin

CB, Cb, BAR X

X

X

Penicillin

Cefaclor CEC, CCL, Cfr, FAC, CF

X Cephem

Cefadroxil CFR, FAD X Cephem Cefamandole MA, CM, Cfm, FAM X X Cephem Cefazolin CZ, CFZ, Cfz, FAZ, KZ X X Cephem Cefdinir CDR, Cdn, DIN, CD,

CFD X Cephem

Cefditoren CDN X Cephem Cefepime FEP, Cpe, PM, CPM X X Cephem Cefetamet CAT, FET X Cephem Cefixime CFM, FIX, Cfe, IX X Cephem Cefmetazole CMZ, CMZS, CMT X X Cephem Cefonicid CID, Cfc, FON, CPO X X Cephem Cefoperazone CFP, Cfp, CPZ, PER,

FOP, CP X X Cephem

Cefotaxime CTX, TAX, Cft, FOT, CT X X Cephem Cefotetan CTT, CTN, Ctn, CTE,

TANS, CN X X Cephem

Cefoxitin FOX, CX, Cfx, FX X X Cephem Cefpodoxime CPD, Cpd, POD, PX X Cephem Cefprozil CPR, CPZ, FP X Cephem Ceftaroline CPT X Cephem Ceftazidime CAZ, Caz, TAZ, TZ X X Cephem Ceftibuten CTB, TIB, CB X Cephem Ceftizoxime ZOX, CZX, CZ, Cz,

CTZ, TIZ X X Cephem

Ceftobiprole BPR X Cephem Ceftriaxone CRO, CTR, FRX, Cax,

AXO, TX X X Cephem

Cefuroxime (oral) Cefuroxime (parenteral)

CXM, CFX, ROX, Crm, FUR, XM

X

X

X

Cephem

Cephalexin CN, LEX, CFL X Cephem Cephalothin CF, Cf, CR, CL, CEP,

CE, KF X Cephem



Number 18 M45-A2


Glossary II. (Continued) Antimicrobial Agent Agent Abbreviationa Routes of Administrationb Drug Class PO IM IV Topical Cephapirin CP, HAP X X Cephem Cephradine RAD, CH X Cephem Chloramphenicol C, CHL, CL X X Phenicol Cinoxacin CIN, Cn X Quinolone Ciprofloxacin CIP, Cp, CI X X Fluoroquinolone Clarithromycin CLR, CLM,

CLA, Cla, CH X Macrolide

Clinafloxacin CFN, CLX, LF X X Fluoroquinolone Clindamycin CC, CM, CD, Cd, CLI, DA X X X Lincosamide Colistin CL, CS, CT X Lipopeptide Dalbavancin DAL X Glycopeptide Daptomycin DAP X Lipopeptide Dicloxacillin DX, DIC X Penicillin Dirithromycin DTM, DT X Macrolide Doripenem DOR X Carbapenem Ertapenem ETP X X Carbapenem Erythromycin E, ERY, EM X X Macrolide Faropenem FAR, FARO X Penem Fleroxacin FLE, Fle, FLX, FO X X Fluoroquinolone Fosfomycin FOS, FF, FO, FM X Fosfomycin Garenoxacin GRN X X Quinolone Gatifloxacin GAT X X Fluoroquinolone Gemifloxacin GEM X Fluoroquinolone Gentamicin Gentamicin synergy

GM, Gm, CN, GEN GM500, HLG, Gms

X X Aminoglycoside

Grepafloxacin GRX, Grx, GRE, GP X Fluoroquinolone Iclaprim ICL X Folate pathway inhibitor Imipenem IPM, IMI, Imp, IP X Carbapenem Kanamycin K, KAN, HLK, KM X X Aminoglycoside Levofloxacin LVX, Lvx,

LEV, LEVO, LE X X Fluoroquinolone

Linezolid LNZ, LZ, LZD X X Oxazolidinone Linopristin-flopristin LFE X Streptogramin Lomefloxacin LOM, Lmf X Fluoroquinolone Loracarbef LOR, Lor, LO X Cephem Mecillinam MEC X Penicillin Meropenem MEM, Mer, MERO, MRP,

MP X Carbapenem

Methicillin DP, MET, ME, SC X X Penicillin Mezlocillin MZ, Mz, MEZ X X Penicillin Minocycline MI, MIN, Min, MN, MNO,

MC, MH X X Tetracycline

Moxalactam MOX X X Cephem Moxifloxacin MXF X X Fluoroquinolone Mupirocin MUP, MOP, MU X Pseudomonic acid Nafcillin NF, NAF, Naf X X Penicillin Nalidixic acid NA, NAL X Quinolone Netilmicin NET, Nt, NC X X Aminoglycoside Nitrofurantoin F/M, FD, Fd, FT,

NIT, NI, F X Nitrofurantoin

Norfloxacin NOR, Nxn, NX X Fluoroquinolone Ofloxacin OFX, OFL, Ofl, OF X X X Fluoroquinolone Oritavancin ORI X Lipoglycopeptide Oxacillin OX, Ox, OXS, OXA X X X Penicillin



Volume 30 M45-A2


Glossary II. (Continued) Antimicrobial Agent Agent Abbreviationa Routes of Administrationb Drug Class

PO IM IV Penicillin P, PEN, PV X X X Penicillin Piperacillin PIP, PI, PP, Pi X X Penicillin Piperacillin-tazobactam TZP, PTZ, P/T, PTc X β-Lactam/β-

lactamase inhibitor combination

Polymyxin B PB X Lipopeptide Quinupristin-dalfopristin SYN, Syn, QDA, RP X Streptogramin Razupenem RZM X Carbapenem Rifampin RA, RIF, Rif, RI, RD X X Ansamycin Sparfloxacin SPX, Sfx, SPA, SO X Fluoroquinolone Spectinomycin SPT, SPE, SC X X Aminocyclitol Streptomycin Streptomycin synergy

S, STR, StS, SM,

ST2000, HLS

X X Aminoglycoside

Sulfonamides SSS, S3 X X Folate pathway antagonist (some PO only)

Sulopenem SLP, SULO X X Penem Teicoplanin TEC, TPN, Tei,

TEI, TP, TPL X X Glycopeptide

Telavancin TLV X Glycopeptide Telithromycin TEL X Ketolide Tetracycline TE, Te, TET, TC X X Tetracycline Ticarcillin TIC, TC, TI, Ti X X Penicillin Ticarcillin-clavulanic acid TIM, Tim, T/C, TCC, TLc X β-Lactam/β-

lactamase inhibitor Tigecycline TGC X Glycylcycline Tobramycin NN, TM, TO, To, TOB X X Aminoglycoside Trimethoprim TMP, T, TR, W X Folate pathway

inhibitor Trimethoprim- sulfamethoxazole

SXT, SxT, T/S, TS, COT X X Folate pathway inhibitor

Trospectinomycin X X Aminocyclitol Trovafloxacin TVA, Tva, TRV, TV X X Fluoroquinolone Vancomycin VA, Va, VAN X X Glycopeptide a Abbreviations assigned to one or more diagnostic products in the United States. If no diagnostic product is

available, abbreviation is that of the manufacturer. b As available in the United States. PO per OS (oral) IM intramuscular IV intravenous



Number 18 M45-A2


References 1 CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Tenth Edition. CLSI document M02-A10.

Wayne, PA: Clinical and Laboratory Standards Institute; 2009. 2 CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Eighth Edition.

CLSI document M07-A8. Wayne, PA: Clinical and Laboratory Standards Institute; 2009. 3 CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement. CLSI document M100-S20.

Wayne, PA: Clinical and Laboratory Standards Institute; 2010. 4 Hindler JF, Patel JB. Susceptibility test methods: fastidious bacteria. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, eds.

Manual of Clinical Microbiology. 9th ed. Washington, DC: ASM Press; 2007:1193-1213. 5 Jorgensen JH. The need for susceptibility testing guidelines for fastidious or less-frequently isolated bacteria. Minireview. J Clin Microbiol.

2004;42(2):493-496. 6 CLSI. Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved Guideline—Third Edition. CLSI

document M23-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2008. 7 Andrews JM, Wise R. Susceptibility testing of Bacillus species. J Antimicrob Chemother. 2002;49(6):1040-1042. 8 Funke G, Pünter V, von Graevenitz A. Antimicrobial susceptibility patterns of some recently established coryneform bacteria. Antimicrob

Agents Chemother. 1996;40(12):2874-2878. 9 Funke G, Bernard KA. Coryneform gram-positive rods. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of

Clinical Microbiology. 9th ed. Washington, DC: ASM Press; 2007:485-514. 10 Funke G, Lawson PA, Nolte FS, Weiss N, Collins MD. Aureobacterium resistens spp. nov., exhibiting vancomycin resistance and

teicoplanin susceptibility. FEMS Microbiol Lett. 1998;158(1):89-93. 11 Deye G, Lewis J, Patterson J, Jorgensen J. A case of Leuconostoc ventriculitis with resistance to carbapenem antibiotics. Clin Infect Dis.

2003;37(6):869-870. 12 Murray CK, Walter EA, Crawford S, McElmeel ML, Jorgensen JH. Abiotrophia bacteremia in a patient with neutropenic fever and

antimicrobial susceptibility testing of Abiotrophia isolates. Clin Infect Dis. 2001;32(10):e140-e142. 13 Zheng X, Freeman AF, Villafranca J, et al. Antimicrobial susceptibilities of invasive pediatric Abiotrophia and Granulicatella isolates. J Clin

Microbiol. 2004;42(9):4323-4326. 14 Rossolini GM, Walsh T, Amicosante G. The Aeromonas metallo-β-lactamases: genetics, enzymology, and contribution to drug resistance.

Microb Drug Resist. 1996;2(2):245-251. 15 Hayes MV, Thomson CJ, Amyes SGB. The “hidden” carbapenemase of Aeromonas hydrophila. J Antimicrob Chemother. 1996;37(1):33-

44. 16 Abbott SL. Klebsiella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and other Enterobacteriaceae. In: Murray PR, Baron EJ,

Jorgensen JH, Landry ML, Pfaller MA, eds. Manual of Clinical Microbiology. 9th ed. Washington, DC: ASM Press; 2007:698-715. 17 Abbott SL, Janda JM, Johnson JA, Farmer JJ III. Vibrio and related organisms. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller

MA, eds. Manual of Clinical Microbiology. 9th ed. Washington, DC: ASM Press; 2007:723-733. 18 Das M, Badley AD, Cockerill FR, Steckelberg JM, Wilson WR. Infective endocarditis caused by HACEK microorganisms. Ann Rev Med.

1997;48:25-33. 19 Kugler KC, Biedenback DJ, Jones RN. Determination of the antimicrobial activity of 29 clinically important compounds tested against

fastidious HACEK group organisms. Diagn Microbiol Infect Dis. 1999;34(1):73-76. 20 Gomez-Garces JL, Alos JI, Sanchez J, Cogollos R. Bacteremia by multidrug-resistant Capnocytophaga sputigena. J Clin Microbiol.

1994;32(4):1067-1069. 21 Lu PL, Hsueh PR, Hung CC, Teng LJ, Jang TN, Luh KT. Infective endocarditis complicated with progressive heart failure due to beta-

lactamase-producing Cardiobacterium hominis. J Clin Microbiol. 2000;38(5):2015-2017. 22 Paul K, Patel SS. Eikenella corrodens infections in children and adolescents: case reports and review of the literature. Clin Infect Dis.

2001;33(1):54-61. 23 Sordillo EM, Rendel M, Sood R, Belinfanti J, Murray O, Brook D. Septicemia due to beta-lactamase-positive Kingella kingae. Clin Infect

Dis. 1993;17(4):818-819. 24 Engberg J, Aarestrup FM, Taylor DE, Gerner-Schmidt P, Nachamkin I. Quinolone and macrolide resistance in Campylobacter jejuni and C.

coli resistance mechanisms and trends in human isolates. Emerg Infect Dis. 2001;7(3):24-34.



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25 Mégraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev. 2007;20(2):280-322. 26 Wallace RJ, Steingrube VA, Nash DR, et al. BRO Beta-lactamases of Branhamella catarrhalis and Moraxella subgenus Moraxella,

including evidence for chromosomal beta-lactamase transfer by conjugation in B. catarrhalis, M. nonliquefaciens, and M. lacunata. Antimicrob Agents Chemother. 1989;33(11):1845-1854.

27 CDC. National Select Agent Registry. www.cdc.gov/od/sap. Accessed 9 February 2010. 28 Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings 2007.

http://www.cdc.gov/ncidod/dhqp/gl_isolation.html. Accessed 9 February 2010. 29 CLSI. Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—Third Edition. CLSI document

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1971;217(suppl B):1-90.



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Additional References General Abramowicz M, ed. Choice of antibacterial drugs. Treat Guidel Med Lett. 2004;2(19):13-26. Baddour LM, Wilson WR, Bayer AS, et al. Infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association (endorsed by the Infectious Diseases Society of America). Circulation. 2005;111:e394-e434. Gilbert DN, Moellering RC, Eliopoulos GM, Sande MA. The Sanford Guide to Antimicrobial Therapy. Hyde Park, VT: Antimicrobial Therapy, Inc; 2005. Mandell GL, Douglas RG, Dolin R, Bennett JE. Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases. 6th ed. Philadelphia, PA: Churchill Livingstone; 2005. Table 1. Abiotrophia spp. and Granulicatella spp. 1. Biermann C, Fries G, Jehnichen P, Bhakdi S, Husmann M. Isolation of Abiotrophia adjacens from a brain

abscess which developed in a patient after neurosurgery. J Clin Microbiol. 1999;37(3):769-771. 2. Bouvet A, Cremieux AC, Contrepois A, Vallois JM, Lamesch C, Carbon C. Comparison of penicillin and

vancomycin, individually and in combination with gentamicin and amikacin, in the treatment of experimental endocarditis induced by nutritionally variant streptococci. Antimicrob Agents Chemother. 1985;28(5):607-611.

3. Chang HH, Lu CY, Hsueh PR, Wu MH, Wang JK, Huang LM. Endocarditis caused by Abiotrophia defectiva

in children. Pediatr Infect Dis J. 2002;21(7):697-700. 4. Christensen JJ, Facklam RR. Granulicatella and Abiotrophia species from human clinical specimens. J Clin

Microbiol. 2001;39(10):3520-3523. 5. Collins MD, Lawson PA. The genus Abiotrophia is not monophyletic: proposal of Granulicatella gen. nov.,

Granulicatella adiacens comb. nov, Granulicatella elegans comb. nov., and Granulicatella balaenopterae comb. nov. Int J Syst Evol Microbiol. 2000;50(Pt 1):365-369.

6. del Pozo JL, Garcia-Quetglas E, Hernaez S, et al. Granulicatella adiacens breast implant-associated infection.

Diagn Microbiol Infect Dis. 2008;61(11):58-60. 7. Heath CH, Bowen SF, McCarthy JS, Dwyer B. Vertebral osteomyelitis and discitis associated with

Abiotrophia adiacens (nutritionally variant streptococcus) infection. Aust N Z J Med. 1998;28(5):663. 8. Kiernan TJ, O’Flaherty NO, Gilmore R, et al. Abiotrophia defectiva endocarditis and associated

hemophagocytic syndrome—a first case report and review of the literature. Int J Infect Dis. 2008;12(5):478-482.

9. Lin C-H, Hsu R-B. Infective endocarditis caused by nutritionally variant streptococci. Am J Med Sci.

2007;334(4):235-239.

10. Michelow IC, McCracken GH Jr, Luckett PM, Krisher K. Abiotrophia spp. brain abscess in a child with Down’s syndrome. Pediatr Infect Dis J. 2000;19(8):760-763.

11. Murray CK, Walter EA, Crawford S, McElmeel ML, Jorgensen JH. Abiotrophia bacteremia in a patient with

neutropenic fever and antimicrobial susceptibility testing of Abiotrophia isolates. Clin Infect Dis. 2001;32(10):e140-e142.



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12. Namdari H, Kintner K, Jackson BA, et al. Abiotrophia species as a cause of endophthalmitis following cataract extraction. J Clin Microbiol. 1999;37(5):1564-1566.

13. Poyart C, Quesne G, Acar P, Berche P, Trieu-Cuot P. Characterization of the Tn916-like transposon TN3872

in a strain of Abiotrophia defectiva (Streptococcus defectivus) causing sequential episodes of endocarditis in a child. Antimicrob Agents Chemother. 2000;44(3):790-793.

14. Stein DS, Libertin CR. Time kill curve analysis of vancomycin and rifampin alone and in combination

against nine strains of nutritionally deficient streptococci. Diagn Microbiol Infect Dis. 1988;10(3):139-144. 15. Tuohy M, Procop GW, Washington JA. Antimicrobial susceptibility of Abiotrophia adiacens and

Abiotrophia defectiva. Diagn Microbiol Infect Dis. 2000;38(3):189-191.

16. Yemisen M, Koksal F, Mete B, et al. Abiotrophia defectiva: A rare cause of infective endocarditis. Scand J Infect Dis. 2006;38(10):939-941.

Table 2. Aeromonas spp. 17. Bakken JS, Sanders CC, Clark RB, Hori M. Beta-Lactam resistance in Aeromonas spp. caused by inducible

beta-lactamases active against penicillins, cephalosporins, and carbapenems. Antimicrob Agents Chemother. 1988; 32(9):1314-1319.

18. Burgos A, Quindos G, Martinez R, Rojo P, Cisterna R. In vitro susceptibility of Aeromonas caviae,

Aeromonas hydrophila and Aeromonas sobria to fifteen antibacterial agents. Eur J Clin Microbiol Infect Dis. 1990;9(6):413-417.

19. Clark NM, Chenoweth CE. Aeromonas infection of the hepatobiliary system: report of 15 cases and review of

the literature. Clin Infect Dis. 2003;37(4):506-513. 20. Janda JM, Abbott SL. Evolving concepts regarding the genus Aeromonas: an expanding panorama of species,

disease presentations, and unanswered questions. Clin Infect Dis. 1998;27(2):332-344. 21. Jones BL, Wilcox MH. Aeromonas infections and their treatment. J Antimicrob Chemother. 1995;35(4):453-

461. 22. Kämpfer P, Christmann C, Swings J, Huys G. In vitro susceptibilities of Aeromonas genomic species to 69

antimicrobial agents. Syst Appl Microbiol. 1999;22(4):662-669. 23. Ko WC, Yu KW, Liu CY, Huang CT, Leu HS, Chuang YC. Increasing antibiotic resistance in clinical

isolates of Aeromonas strains in Taiwan. Antimicrob Agents Chemother. 1996;40(5):1260-1262. 24. Koehler JM, Ashdown LR. In vitro susceptibilities of tropical strains of Aeromonas species from Queensland,

Australia, to 22 antimicrobial agents. Antimicrob Agents Chemother. 1993;37(4):905-907. 25. Overman TL, Janda JM. Antimicrobial susceptibility patterns of Aeromonas jandaei, A. schubertii, A. trota,

and A. veronii biotype veronii. J Clin Microbiol. 1999;37(3):706-708. 26. Vila J, Marco F, Soler L, Chacon M, Figueras MJ. In vitro antimicrobial susceptibility of clinical isolates of

Aeromonas caviae, Aeromonas hydrophila, and Aeromonas veronii biotype sobria. J Antimicrob Chemother. 2002;49(4):701-702.

27. Vila J, Ruiz J, Gallardo F, et al. Aeromonas spp. and traveler’s diarrhea: clinical features and antimicrobial

resistance. Emerg Infect Dis. 2003;9(5):552-555. Plesiomonas shigelloides 28. Avison MB, Bennett PM, Walsh TR. Beta-lactamase expression in Plesiomonas shigelloides. J Antimicrob

Chemother. 2000;45(6):877-880.



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29. Brenden RA, Miller MA, Janda JM. Clinical disease spectrum and pathogenic factors associated with Plesiomonas shigelloides infections in humans. Rev Infect Dis. 1988;10(2):303-316.

30. Clark RB, Lister PD, Arneson-Rotert L, Janda JM. In vitro susceptibilities to Plesiomonas shigelloides to 24

antibiotics and antibiotic-beta-lactamase-inhibitor combinations. Antimicrob Agents Chemother. 1990;34(1):159-160.

31. González-Rey C, Svenson SB, Bravo L, et al. Serotypes and anti-microbial susceptibility of Plesiomonas

shigelloides isolates from humans, animals and aquatic environments in different countries. Comp Immunol Microbiol Infect Dis. 2004;27(2):129-139.

32. Kain KC, Kelly MT. Antimicrobial susceptibility of Plesiomonas shigelloides from patients with diarrhea.

Antimicrob Agents Chemother. 1989;33(9):1609-1610. 33. Stock I, Wiedemann B. Natural antimicrobial susceptibilities of Plesiomonas shigelloides strains. J

Antimicrob Chemother. 2001;48(6):803-811.

34. Stock I, Wiedemann B. Beta-lactam-susceptibility patterns of Plesiomonas shigelloides strains: importance of inoculum and medium. Scand J Infect Dis. 2001;33(9):692-696.

Table 3. Bacillus spp. (not B. anthracis) 35. Andrews JM, Wise R. Susceptibility testing of Bacillus species. J Antimicrob Chemother. 2002;499(6):1040-

1042. 36. Citron DM, Appleman MD. In vitro activities of daptomycin, ciprofloxacin, and other antimicrobial agents

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78. Riegel P, Ruimy R, Christen R, Monteil H. Species identities and antimicrobial susceptibilities of corynebacteria isolated from various clinical sources. Eur J Clin Microbiol Infect Dis. 1996;15(8):657-662.

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112. Vogt K, Klefisch F, Hahn H, Schmutzler H. Antibacterial efficacy of ciprofloxacin in a case of endocarditis due to Cardiobacterium hominis. Zentralbl Bakteriol. 1994;281(1):80-84.

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Table 9. Lactobacillus spp. 114. Barrett MS, Jones RN. In vitro activity of quinupristin/dalfopristin (RP 59500) against a large collection of

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128. Vay C, Cittadini R, Barberis C, et al. Antimicrobial susceptibility of non-enterococcal intrinsic glycopeptide-resistant gram-positive organisms. Diagn Microbiol Infect Dis. 2007;57(2):183-188.

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135. de la Maza L, Ruoff KL, Ferraro MJ. In vitro activities of daptomycin and other antimicrobial agents against

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145. Davis JA, Jackson CR. Comparative antimicrobial susceptibility of Listeria monocytogenes, L. innocua, and L. welshimeri. Microb Drug Resist. 2009;15(1):27-32.

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165. Meza A, Verghese A, Berk SL. Moraxella catarrhalis. In: Yu V, Weber R, Raoult D, eds. Antimicrobial

Therapy and Vaccines. 2nd ed. New York, NY: Apple Tree Productions; 2002:437-448. 166. Reynolds R, Shackcloth J, Felmingham D, McGowan A, on behalf of the BSAC Extended Working Party on

Respiratory Resistance Surveillance. Comparison of BSAC agar dilution and NCCLS broth microdilution MIC methods for the in vitro susceptibility testing of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis: the BSAC Respiratory Resistance Surveillance Programme. J Antimicrob Chemother. 2003;52(6):925-930.

167. Shah SS, Ruth A, Coffin SE. Infection due to Moraxella osloensis: case report and review of the literature.

Clin Infect Dis. 2000;30(1):179-181. 168. Verduin CM, Hol C, Fleer A, van Dijk H, van Belkum A. Moraxella catarrhalis: from emerging to

established pathogen. Clin Microbiol Rev. 2002;15(1):125-144. 169. Wallace RJ, Steingrube VA, Nash DR, et al. BRO beta-lactamases of Branhamella catarrhalis and Moraxella

subgenus Moraxella, including evidence for chromosomal beta-lactamase transfer by conjugation in B. catarrhalis, M. nonliquefaciens, and M. lacunata. Antimicrob Agents Chemother. 1989;33(11):1845-1854.

Table 13. Pasteurella spp. 170. Al-Sabah S, Goldberg P, Qureshi ST. Pasteurella multocida septic shock following liver transplantation

treated with drotrecogin alpha (activated). Transpl Infect Dis. 2007;9(3):233-236. 171. Andrews JM, Jevons G, Brenwald N, Fraise A; BSAC Working Party on Sensitivity Testing. Susceptibility

testing Pasteurella multocida by BSAC standardized methodology. J Antimicrob Chemother. 2004;54(5):962-964.

172. Chang K, Siu LK, Chen YH, et al. Fatal Pasteurella multocida septicemia and necrotizing fasciitis related

with wound licked by a domestic dog. Scand J Infect Dis. 2007;39(2):167-170.

173. Citron DM, Warren YA, Fernandez HT, Goldstein MA, Tyrrell KL, Goldstein EJ. Broth microdilution and disk diffusion tests for susceptibility testing of Pasteurella species from human clinical specimens. J Clin Microbiol. 2005;43(5):2485-2488.

174. Fernández-Valencia JA, García S, Prat S. Pasteurella multocida septic shock after a cat scratch in an elderly

otherwise healthy woman: a case report. Am J Emerg Med. 2008;26(3):380.e1-3.

175. Freshwater A. Why your housecat’s trite little bite could cause you quite a fright: a study of domestic felines on the occurrence and antibiotic susceptibility of Pasteurella multocida. Zoonoses Public Health. 2008;55(8-10):507-513.

176. Goldstein EJ, Citron DM, Merriam CV, Warren YA, Tyrrell KL, Fernandez HT. In vitro activities of

garenoxacin (BMS-284756) against 170 clinical isolates of nine Pasteurella species. Antimicrob Agents Chemother. 2002;46(9):3068-3070.



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177. Goldstein EJ, Citron DM, Richwald GA. Lack of in vitro activity of oral forms of certain cephalosporins, erythromycin, and oxacillin against Pasteurella multocida. J Clin Microbiol. 1988;32(2):213-215.

178. Johnson LB, Busuito MJ, Khatib R. Breast implant infection in a cat owner due to Pasteurella multocida. J

Infect. 2000;41(1):110-111. 179. Lion C, Lozniewski A, Rosner V, Weber M. Lung abscess due to beta-lactamase-producing Pasteurella

multocida. Clin Infect Dis. 1999;29(5):1345-1346. 180. Mortensen JE, Giger O, Rodgers GL. In vitro activity of oral antimicrobial agents against clinical isolates of

Pasteurella multocida. Diagn Microbiol Infect Dis. 1998;30(2):99-102. 181. Van Langenhove G, Daelemans R, Zachee P, Lins RL. Pasteurella multocida as a rare cause of peritonitis in

peritoneal dialysis. Nephron. 2000;85(3):283-284.

182. Yokose N, Dan K. Pasteurella multocida sepsis, due to a scratch from a pet cat, in a post-chemotherapy neutropenic patient with non-Hodgkin lymphoma. Int J Hematol. 2007;85(2):146-148.

Table 14. Pediococcus spp. 183. Barrett MS, Jones RN. In vitro activity of quinupristin/dalfopristin (RP 59500) against a large collection of

infrequently isolated or tested species. Diagn Microbiol Infect Dis. 1996;25(3):147-149.

184. Barton LL, Rider ED, Coen RW. Bacteremic infection with Pediococcus: vancomycin-resistant opportunist. Pediatrics. 2001;107(4):775-776.

185. Collins LA, Malanoski GJ, Eliopoulos GM, Wennersten CB, Ferraro MJ, Moellering RC Jr. In vitro activity

of RP59500, an injectable streptogramin antibiotic, against vancomycin-resistant gram-positive organisms. Antimicrob Agents Chemother. 1993;37(3):598-601.

186. Danielsen M, Simpson PJ, O’Connor EB, Ross RP, Stanton C. Susceptibility of Pediococcus spp. to

antimicrobial agents. J Appl Microbiol. 2007;102(2):384-389. 187. de la Maza L, Ruoff KL, Ferraro MJ. In vitro activities of daptomycin and other antimicrobial agents against

vancomycin-resistant gram-positive bacteria. Antimicrob Agents Chemother. 1989;33(8):1383-1384. 188. Heinz M, von Wintzingerode F, Moter A, et al. A case of septicemia with Pediococcus acidilactici after long-

term antibiotic treatment. Eur J Clin Microbiol Infect Dis. 2000;19(12):946-948. 189. Huang YT, Liao CH, Teng LJ, Hsueh PR. Daptomycin susceptibility of unusual gram-positive bacteria:

comparison of results obtained by the Etest and the broth microdilution method. Antimicrob Agents Chemother. 2007;51(4):1570-1572.

190. Klare I, Konstabel C, Werner G, et al. Antimicrobial susceptibilities of Lactobacillus, Pediococcus, and

Lactococcus 3 human isolates and cultures intended for probiotic or nutritional use. J Antimicrob Chemother. 2007;59(5):900-913.

191. Long SS, Pickering LK, Prober CG, eds. Principles and Practice of Pediatric Infectious Diseases.

Philadelphia, PA: Churchill Livingstone; 2002.

192. Suh B. Resolution of persistent Pediococcus bacteremia with daptomycin treatment: case report and review of the literature. Diagn Microbiol Infect Dis. 2010;66(1):111-115.

193. Swenson JM, Facklam RR, Thornsberry C. Antimicrobial susceptibility of vancomycin-resistant

Leuconostoc, Pediococcus, and Lactobacillus species. Antimicrob Agents Chemother. 1990;34(4):543-549. 194. Vay C, Cittadini R, Barberis C, et al. Antimicrobial susceptibility of non-enterococcal intrinsic glycopeptide-

resistant gram-positive organisms. Diagn Microbiol Infect Dis. 2007;57(2):183-188.



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195. Yamane N, Jones RN. In vitro activity of 43 antimicrobial agents tested against ampicillin-resistant enterococci and gram-positive species resistant to vancomycin. Diagn Microbiol Infect Dis. 1991;14(4):337-345.

Table 15. Vibrio spp. 196. Asenjo CW, Ramirez-Rhonda CH. Halophilic Vibrio infections: a review. Bol Asoc Med P R. 1991;83(4):154-

156.

197. Bhattacharya MK, Dutta D, Ramamurthy T, Sarkar D, Singharoy A, Bhattacharya SK. Azithromycin in the treatment of cholera in children. Acta Paediatr. 2003;92(6):676-678.

198. Chiang SR, Chuang YC. Vibrio vulnificus infection: clinical manifestations, pathogenesis, and antimicrobial therapy. J Microbiol Immunol Infect. 2003;36(2):81-88.

199. Chien JY, Shih JT, Hsueh PR, Yang PC, Luh KT. Vibrio alginolyticus as the cause of pleural empyema and

bacteremia in an immunocompromised patient. Eur J Clin Microbiol Infect Dis. 2002;21(5):401-403. 200. Chuang YC, Liu JW, Ko WC, Lin KY, Wu JJ, Huang KY. In vitro synergism between cefotaxime and

minocycline against Vibrio vulnificus. Antimicrob Agents Chemother. 1997;41(10):2214-2217. 201. French GL, Woo ML, Hui YW, Chan KY. Antimicrobial susceptibilities of halophilic vibrios. J Antimicrob

Chemother. 1989;24(2):183-194. 202. Gomez JM, Fajardo R, Patino JF, Arias CA. Necrotizing fasciitis due to Vibrio alginolyticus in an

immunocompetent patient. J Clin Microbiol. 2003;41(7):3427-3429. 203. Hsueh PR, Chang JC, Chang SC, Ho SW, Hsieh WC. In vitro antimicrobial susceptibility of Vibrio vulnificus

isolated in Taiwan. Eur J Clin Microbiol Infect Dis. 1995;14(2):151-153.

204. Khan WA, Saha D, Rahman A, Salam MA, Bogaerts J, Bennish ML. Comparison of single-dose azithromycin and 12-dose, 3-day erythromycin for childhood cholera: a randomised, double-blind trial. Lancet. 2002;360(9347):1722-1727.

205. Morris JG Jr, Tenney JH, Drusano GL. In vitro susceptibility of pathogenic Vibrio species to norfloxacin

and six other antimicrobial agents. Antimicrob Agents Chemother. 1985;28(3):442-445. 206. Ottaviani D, Bacchiocchi I, Masini L, et al. Antimicrobial susceptibility of potentially pathogenic halophilic

vibrios isolated from seafood. Int J Antimicrob Agents. 2001;18(2):135-140. 207. Saha D, Karim MM, Khan WA, Ahmed S, Salam MA, Bennish ML. Single-dose azithromycin for the

treatment of cholera in adults. N Engl J Med. 2006;354(23):2452-2462. 208. Sur D, Dutta S, Sarkar BL, et al. Occurrence, significance and molecular epidemiology of cholera outbreaks

in West Bengal. Indian J Med Res. 2007;125(6):772-776. 209. Tang HJ, Chang MC, Ko WC, Huang KY, Lee CL, Chuang YC. In vitro and in vivo activities of newer

fluoroquinolones against Vibrio vulnificus. Antimicrob Agents Chemother. 2002;46(11):3580-3584.

210. Yamamoto T, Nair GB, Albert MJ, Parodi CC, Takeda Y. Survey of in vitro susceptibilities of Vibrio cholerae O1 and O139 to antimicrobial agents. Antimicrob Agents Chemother. 1995;39(1):241-244.

211. Zanetti S, Spanu T, Deriu A, Romano L, Sechi LA, Fadda G. In vitro susceptibility of Vibrio spp. isolated from the environment. Int J Antimicrob Agents. 2001;17(5):407-409.



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Summary of Consensus Comments and Subcommittee Responses M45-A: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline Table 5, Corynebacterium spp. 1. Do the testing and reporting recommendations listed for Corynebacterium species apply to other coryneform

bacteria?

• Yes, these apply to other more commonly encountered genera including Arcanobacterium, Arthrobacter, Brevibacterium, Cellulomonas, Dermabacter, Leifsonia, Microbacterium, Oerskovia, Rothia, and Turicella. This is clarified in M45-A2.

Table 9, Lactobacillus spp. (formerly Table 8)

2. The susceptible only breakpoint of 0.5 µg/mL for imipenem with Lactobacillus spp. seems to be set too low, because we routinely see isolates with higher MICs. This includes some strains that are penicillin susceptible. Will the imipenem breakpoint be changed (raised)?

• The working group reviewed additional data from a multicenter study of Lactobacillus spp. and decided to add intermediate and resistant categories for Lactobacillus spp. with imipenem. However, the original susceptible breakpoint of 0.5 µg/mL was retained. There are species-specific MIC distributions with this genus. The more commonly occurring species L. casei and L. rhamnosus have higher MICs and will often test as intermediate or resistant to imipenem. The less common and more fastidious species, L. fermentum, L. gasseri, and L. jensenii, will usually test as susceptible to imipenem. In reviewing the clinical literature, there were few cases of serious infections treated with imipenem alone, and most articles did not stipulate the imipenem MICs. Therefore, the working group did not feel that there was sufficient evidence to allow the breakpoints to be raised. The working group inserted a new comment that states, “Therapy of serious infections such as endocarditis often involves combined therapy with a penicillin and an aminoglycoside” to appear alongside penicillin and ampicillin.

Clinical and Laboratory Standards Institute consensus procedures include an appeals process that is described in detail in Section 8 of the Administrative Procedures. For further information, contact CLSI or visit our website at www.clsi.org.



Number 18 M45-A2


Summary of Delegate Comments and Subcommittee Responses M45-A2: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline—Second Edition Foreword 1. Second paragraph, third sentence – “US Food and Drug Administration (FDA).” Please clarify if these

documents are or can be used by any regulatory agencies in other countries. The scope does not restrict this document to the United States.

• This document and CLSI reference methods can be used by any regulatory authority, and the text in this

paragraph was revised to reflect this. Section 1, Scope 2. First three paragraphs – Statements about gram-positive and gram-negative organisms should be kept together.

Start a new paragraph at: “Key gram-positive organisms include Corynebacterium spp., Bacillus spp., and several genera that have intrinsic vancomycin resistance.”

Add: “Nonfastidious gram-negative bacteria include Aeromonas spp., Plesiomonas sp., Vibrio spp., and Moraxella catarrhalis.” to the beginning of the (new) third paragraph, then continue with “The fastidious gram-negative bacilli…linezolid.” Move the following to the new second paragraph (to follow the statement “Key gram-positive…vancomycin resistance.”): “Fastidious gram-positive bacteria that may cause endocarditis include Abiotrophia spp. and Granulicatella spp. Acquired antimicrobial resistance mechanisms have been reported in many of these organisms, and the medical literature includes descriptions of susceptibility results derived from use of standard CLSI methods or certain nonstandard procedures.”

• The Scope and Introduction were revised to follow the format of CLSI’s definition of the material to be

included in these sections as outlined in CLSI’s Style Guide for Authors. Some of the original text that is being referred to in this comment was deleted or moved to the Introduction.

3. First paragraph and second paragraph, first three sentences – Indicated text is not the Scope. Relocate to the

Foreword or Introduction. • The Scope and Introduction were revised to follow the format of CLSI’s definition of the material to be

included in these sections, as outlined in CLSI’s Style Guide for Authors. Some of the original text that is being referred to in this comment was deleted or moved to the Introduction.

4. Third paragraph – This paragraph is not part of the Scope. Relocate to the Foreword or Introduction. • The Scope and Introduction were revised to follow the format of CLSI’s definition of the material to be

included in these sections, as outlined in CLSI’s Style Guide for Authors.

Clinical and Laboratory Standards Institute consensus procedures include an appeals process that is described in detail in Section 8 of the Administrative Procedures. For further information, contact CLSI or visit our website at www.clsi.org.



Volume 30 M45-A2


Some of the original text that is being referred to in this comment was deleted or moved to the Introduction.

5. Fifth paragraph, first sentence – This sentence is not part of the Scope. Relocate to the Foreword or

Introduction. • The Scope and Introduction were revised to follow the format of CLSI’s definition of the material to be

included in these sections, as outlined in CLSI’s Style Guide for Authors. Some of the original text that is being referred to in this comment was deleted or moved to the Introduction.

Section 2.6, Potential Bacterial Agents of Bioterrorism 6. Fourth line – The statement is made that entities that have any of these agents “must be registered with the US

Departments of Health and Human Services and Agriculture.” Is this true only for entities within the United States? If so, the statement should be qualified to indicate that. If that is the case, what is required in other countries?

• This statement was clarified to note that this is a US requirement, and additional text was added for

laboratories outside the US to check with their local public health department for handling and reporting potential bacterial agents of bioterrorism.

Section 9.1, Minimum Laboratory Requirements for Testing Infrequently Isolated or Fastidious Bacteria 7. First bullet – Are there any additional criteria for laboratories outside the United States that can be added, since

the FDA statement is US focused? • This sentence was modified to state the use of other approved methods that may be used by laboratories

outside the United States. 8. “FDA-cleared commercial microdilution method” – This phrase restricts the document to methods approved by

US regulations; please include a more general statement to advise readers on suitable commercial methods that would apply outside the United States.

• This sentence was modified to state the use of other approved methods that may be used by laboratories

outside the United States. Tables 9. Boldface type randomly occurs in various tables in the zone diameters, MIC, and comments, as well as

occasionally in the testing conditions box. What is the boldface type supposed to indicate to the user of the document? I finally found a note in Table 8 that indicates “Information in boldface type is considered tentative for one year,” although there is no boldface type in this table except for headings (which I assume are not tentative). If this statement regarding boldface type is meant to apply to several of the tables, then it should be listed on each table to which it applies. Also, when does the “clock” on one year start? It would be better to put a date in when the document is published. What happens when that date is reached? Are the data then not appropriate for use anymore?

• The boldface type is to indicate information that is new since the last edition.

The use of the term “tentative for one year” is taken from the M100 document and was done to allow time for laboratories to try to implement changes. Since M45 is not updated as frequently, the bolding was removed throughout the document and changes since the last edition are provided on the Updated Information page. A sentence was added on the update page stating that this information appears for the first time in this edition of M45 or was modified since publication of M45-A.



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10. The handling of comments is somewhat confusing. In Table 5, for example, there are “general comments,” and then in the first line of the table there is a third comment that seems to be treated the same as the other comments in “See comment #.” If a comment is referred to more than once in a given table, it would be easier to find if it were listed in the “General Comments” rather than buried on a line in the table.

• General comments are usually in the beginning of the table because they may pertain to the table as a

whole or to a few of the individual drugs within the table. Comments within the gray shaded portion of the table apply to all the drugs listed for that drug class.

This is standard formatting in the susceptibility testing documents (eg, M100). However, this issue will be revisited by the AST Subcommittee Text and Table Working Group in June 2010 to make certain this formatting approach is still best.

11. Tables 1, 3, 5, 6, 7, 9, 10, and 11 – There are multiple laboratories that do not have facility or sufficient skill to perform MICs but are still willing to adopt CLSI guidelines. Provide standardized zone diameters for use in the disk diffusion method. This is a need for most of the clinical laboratories, especially in third world countries.

• Since the last edition of M45, the working group added disk diffusion breakpoints for M. catarrhalis based on available data. Should disk diffusion data for other organisms/antimicrobial agents become available, the working group will take these into consideration and, if appropriate, provide additional disk diffusion breakpoints in the next edition of the M45 document.

Table 5, Corynebacterium spp. (Including C. diphtheriae) and Coryneforms—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing 12. There is an “a” in the Table title, but no footnotes to the Table of Contents; remove the “a.” • Footnote “a” appears directly under the table explaining the genera included for Coryneforms. Table 8, Helicobacter pylori—Interpretive Criteria for Agar Dilution Susceptibility Testing 13. Under comments: “FDA-cleared commercial microdilution method.” This phrase restricts the document to

methods approved by US regulations; please include a more general statement to advise readers on suitable commercial methods that would apply outside the United States.

• The reference to the FDA was removed from the comment in Table 8. Table 9, Lactobacillus spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing 14. Gentamicin – It is not clear what the comment “For combined therapy” means. Does this mean the MICs are

only valid if gentamicin is combined with another antimicrobial? Does it make a difference what the other antimicrobial is? Is this supposed to refer to comment 3?

• The medical literature indicates that treatment of serious infections such as endocarditis with some

organisms responds best if an aminoglycoside is added to a second active agent such as a β-lactam or vancomycin in order to achieve synergistic killing of the organism.

The working group added the comment “See Comment (3)” referring the reader to the therapy comment for use of a penicillin and an aminoglycoside for combined therapy.

Table 14, Pediococcus spp.—Information and Interpretive Criteria for Broth Microdilution Susceptibility Testing 15. Comment 3 – What combined therapy is being referred to; gentamicin plus what? • Gentamicin may be combined with penicillin or ampicillin. A comment for combined therapy and the use

of a penicillin and an aminoglycoside was added under penicillins. The user is then referred to this therapy comment for gentamicin.



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Tables 18A and 18B 16. The titles for Tables 18A and 18B are identical, but the tables are not. Could one or both table titles be modified

to help the users of the document more easily find the information they are looking for? • The title of Table 18B was changed to reflect that these QC ranges are specifically for Campylobacter

jejuni. Table 18A, 18D, 18E, 19, 19A, Footnote A 17. 18, footnote a – There is no “a” in the table to go with this footnote. As you look through these five tables, the

placement of this footnote indication is inconsistent. Is it really necessary to keep repeating it in each table? I thought once (the first occurrence) in a document was sufficient. The footnote is shown in Table 1, and although ATCC® is in every table after that, it is not footnoted again until Table 18A, is missing from Tables 18B and C, and then reappears again.

• This was corrected to reflect the reference to ATCC® in the first table (Table 1) and deleted from all

following tables. Table 19, Glossary 1 (Part 1). β-Lactams: Class and Subclass Designation and Generic Name 18. Random bolding – Please identify the significance. • The bolding was removed throughout the document and changes since the last edition are provided on

the Updated Information page. A sentence was added on the update page stating that this information appears for the first time in this edition of M45 or was modified since publication of M45-A.



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The Quality Management System Approach Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the development of standards and guidelines, which facilitates project management; defines a document structure via a template; and provides a process to identify needed documents. The approach is based on the model presented in the most current edition of CLSI document HS01—A Quality Management System Model for Health Care. The quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any health care service’s path of workflow (ie, operational aspects that define how a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The QSEs are as follows: Documents and Records Equipment Information Management Process Improvement Organization Purchasing and Inventory Occurrence Management Customer Service Personnel Process Control Assessments—External

and Internal Facilities and Safety

M45-A2 addresses the QSEs indicated by an “X.” For a description of the other documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following page.

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Adapted from CLSI document HS01—A Quality Management System Model for Health Care. Path of Workflow A path of workflow is the description of the necessary steps to deliver the particular product or service that the organization or entity provides. For example, CLSI document GP26⎯Application of a Quality Management System Model for Laboratory Services defines a clinical laboratory path of workflow, which consists of three sequential processes: preexamination, examination, and postexamination. All clinical laboratories follow these processes to deliver the laboratory’s services, namely quality laboratory information. M45-A2 addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following page.

Preexamination Examination Postexamination

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Adapted from CLSI document HS01—A Quality Management System Model for Health Care.



Volume 30 M45-A2


Related CLSI Reference Materials∗ M02-A10 Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard—Tenth

Edition (2009). 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.

M07-A8 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved

Standard—Eighth Edition (2009). This document addresses reference methods for the determination of minimal inhibitory concentrations (MICs) of aerobic bacteria by broth macrodilution, broth microdilution, and agar dilution.

M23-A3 Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters; Approved

Guideline—Third Edition (2008). This document addresses the required and recommended data needed for the selection of appropriate interpretive criteria and quality control ranges for antimicrobial agents.

M29-A3 Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—

Third Edition (2005). Based on US regulations, this document provides guidance on the risk of transmission of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and materials; and recommendations for the management of exposure to infectious agents.

M31-A3 Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated

From Animals; Approved Standard—Third Edition (2008). This document provides the currently recommended techniques for antimicrobial agent disk and dilution susceptibility testing, criteria for quality control testing, and interpretive criteria for veterinary use.

M100-S20 Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement

(2010). This document provides updated tables for the Clinical and Laboratory Standards Institute antimicrobial susceptibility testing standards M02-A10 and M07-A8.

∗ CLSI documents are continually reviewed and revised through the CLSI consensus process; therefore, readers should refer to the most current editions.



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Sustaining Members Abbott American Association for Clinical Chemistry AstraZeneca Pharmaceuticals BD Beckman Coulter, Inc. bioMérieux, Inc. College of American Pathologists Diagnostica Stago GlaxoSmithKline NIST Ortho-Clinical Diagnostics, Inc. Roche Diagnostics, Inc. Professional Members AAMI American Association for Clinical Chemistry American Association for Laboratory Accreditation American Association for Respiratory Care American Medical Technologists American Society for Clinical Laboratory Science American Society for Clinical Pathology American Society for Microbiology American Type Culture Collection Association of Public Health Laboratories Associazione Microbiologi Clinici Italiani (AMCLI) British Society for Antimicrobial Chemotherapy Canadian Society for Medical Laboratory Science COLA College of American Pathologists College of Medical Laboratory Technologists of Ontario College of Physicians and Surgeons of Saskatchewan Critical Path Institute ESCMID Family Health International Hong Kong Accreditation Service Innovation and Technology Commission International Federation of Biomedical Laboratory Science International Federation of Clinical Chemistry Italian Society of Clinical Biochemistry and Clinical Molecular Biology JCCLS The Joint Commission The Korean Society for Laboratory Medicine National Society for Histotechnology, Inc. Nova Scotia Association of Clinical Laboratory Managers Ontario Medical Association Quality Management Program-Laboratory Service RCPA Quality Assurance Programs PTY Limited SIMeL Sociedad Espanola de Bioquimica Clinica y Patologia Molecular Sociedade Brasileira de Analises Clinicas Sociedade Brasileira de Patologia Clinica World Health Organization Government Members Armed Forces Institute of Pathology BC Centre for Disease Control CAREC Centers for Disease Control and Prevention Centers for Disease Control and Prevention – Ethiopia Centers for Disease Control and Prevention – Namibia Centers for Disease Control and Prevention – Tanzania Centers for Disease Control and Prevention RETRO-CI CDC/PEPFAR Centers for Medicare & Medicaid Services Centers for Medicare & Medicaid Services/CLIA Program Chinese Committee for Clinical Laboratory Standards Chinese Medical Association (CMA)

Danish Institute for Food and Veterinary Research Department of Veterans Affairs DFS/CLIA Certification Diagnostic Accreditation Program FDA Center for Biologics Evaluation and Research FDA Center for Devices and Radiological Health FDA Center for Veterinary Medicine Health Canada Institut Pasteur de Côte D’Ivoire Institute of Tropical Medicine Dept. of Clinical Sciences Instituto Nacional de Saude Laboratoire National de la Sante Publique MA Dept. of Public Health Laboratories Malaria Research Training Center Meuhedet Central Lab Ministry of Health and Social Welfare – Tanzania Namibia Institute of Pathology National Cancer Institute National Center for Disease Control and Public Health National Center of Infectious and Parasitic Diseases (Bulgaria) National Health Laboratory Service (South Africa) National HIV & Retrovirology Lab National Institute of Standards and Technology National Pathology Accreditation Advisory Council (Australia) New Jersey State Department of Health and Senior Services New York State Department of Health Ontario Agency for Health Protection and Promotion Pennsylvania Dept. of Health SA Pathology SAIC Frederick Inc – NCI-Frederick Cancer Research & Development Center Saskatchewan Health-Provincial Laboratory Scientific Institute of Public Health State of Alabama State of Wyoming Public Health Laboratory University of Iowa, Hygienic Lab US Naval Medical Research Unit #3 Virginia Department of Agriculture – Animal Health Laboratories Industry Members 3M Medical Division Abbott Abbott Diabetes Care Abbott Point of Care Inc. Access Genetics Aderans Research AdvaMed Akonni Biosystems Ammirati Regulatory Consulting Anapharm, Inc. AspenBio Pharma, Inc. Astellas Pharma AstraZeneca Pharmaceuticals Axis-Shield PoC AS Bayer Healthcare, LLC Diagnostic Division BD BD Biosciences – San Jose, CA BD Diagnostic Systems BD Vacutainer Systems Beaufort Advisors, LLC Beckman Coulter Cellular Analysis Business Center Beckman Coulter, Inc. Beth Goldstein Consultant (PA) Bioanalyse, Ltd. Bio-Development S.r.l. Biohit Oyj. BioMarker Associates, Inc. Biomedia Laboratories SDN BHD bioMérieux, Inc. (MO) bioMérieux, Inc. (NC) Bio-Rad Laboratories, Inc. – France Bio-Rad Laboratories, Inc. – Irvine, CA Bio-Rad Laboratories, Inc. – Singapore Bio-Reference Laboratories Blaine Healthcare Associates, Inc. BRI Consultants Limited Calloway Laboratories Canon U.S. Life Sciences, Inc.

Cempra Pharmaceuticals, Inc. Cepheid The Clinical Microbiology Institute Compliance Insight, Inc. Constitution Medical Inc Controllab Copan Diagnostics Inc. Crescendo Bioscience Cubist Pharmaceuticals, Inc. Dahl-Chase Pathology Associates PA Diagnostica Stago Docro, Inc. DX Assays Pte Ltd. Eiken Chemical Company, Ltd. Elanco Animal Health Elkin Simson Consulting Services Emika Consulting Enigma Diagnostics, Inc. Eurofins Medinet Gen-Probe GeneNews Genzyme Diagnostics GlaxoSmithKline Greiner Bio-One Inc. Habig Regulatory Consulting HandyLab Inc. Himedia Labs Ltd Icon Laboratories, Inc. Innovotech, Inc. Instrumentation Laboratory (MA) Instrumentation Laboratory (NY) Integrated BioBank IntelligentMDx, Inc. Intuity Medical Japan Assn. of Clinical Reagents Industries Johnson & Johnson Pharmaceutical Research and Development, L.L.C. Kaiser Permanente K.C.J. Enterprises KoreaBIO Krouwer Consulting Lab PMM Laboratory Specialists, Inc. LifeLabs LifeScan, Inc. LipoScience, Inc. Maine Standards Company, LLC Medical Device Consultants, Inc The Medicines Company Merck & Company, Inc. Merial Limited Micromyx, LLC Nanosphere, Inc. Nihon Koden Corporation Nissui Pharmaceutical Co., Ltd. NJK & Associates, Inc. NorDx – Scarborough Campus NovaBiotics (Aberdeen, UK) Novartis Institutes for Biomedical Research OncoMethylome Sciences S.a. Optimer Pharmaceuticals, Inc. Ortho Clinical Diagnostics, Inc. (Rochester, NY) Ortho-McNeil, Inc. Paratek Pharmaceuticals, Inc. PathCare Pathology Laboratory PerkinElmer Genetics, Inc Pfizer Animal Health Pfizer Inc Pfizer Italia Srl Phadia AB Philips Healthcare Incubator PPD ProteoGenix, Inc. Quality Regulatory Solutions QML Pathology Quotient Bioresearch Ltd. Radiometer America, Inc. Roche Diagnostics GmbH Roche Diagnostics, Inc. Roche Molecular Systems Sanofi Pasteur Sarstedt, Inc. Seventh Sense Biosystems Siemens Healthcare Diagnostics (CA) Siemens Healthcare Diagnostics (DE) Siemens Healthcare Diagnostics Products GmbH Soloy Laboratory Consulting Services, Llc SomaLogic Sphere Medical Holding Limited Streck Laboratories, Inc. Super Religare Laboratories Ltd Sysmex America, Inc. (Mundelein, IL) Sysmex Corporation (Japan) TheraDoc Therapeutic Monitoring Services, LLC Theravance Inc. Thermo Fisher Scientific

Thermo Fisher Scientific, Oxoid Products Thermo Fisher Scientific, Remel Transasia Bio-Medicals Limited Trek Diagnostic Systems Tulip Group Ventana Medical Systems Inc. Veracyte, Inc. Vivacta Watson Pharmaceuticals Wellstat Diagnostics, LLC XDX, Inc. Associate Active Members 22 MDSS (KS) 3rd Medical Group 31st Medical Group SGSL 48th Medical Group/MDSS RAF Lakenheath (APO) 55th Medical Group/SGSAL (NE) 579 MDSS/SGSAL (DC) 59th MDW/859th MDTS/MTL Wilford Hall Medical Center (TX) 81st MDSS/SGSAL (MS) 82 MDG/SGSCL Sheppard AFB (TX) Academisch Ziekenhuis-VUB (Belgium) ACL Laboratories (IL) ACL Laboratories (WI) Adams County Hospital (OH) Adena Regional Medical Center Hospital (OH) The AGA Khan University Hospital (Pakistan) Akron Children’s Hospital (OH) Al-Ain Hospital (United Arab Emirates) Al Hada Armed Forces Hospital/TAIF/KSA (Saudi Arabia) Al Noor Hospital (United Arab Emirates) Al Rahba Hospital (United Arab Emirates) Alameda County Medical Center (CA) Albany Medical Center Hospital (NY) Albemarle Hospital (NC) Alberta Health Services (Canada) All Children’s Hospital (FL) Allegiance Health (MI) Alpena General Hospital (MI) Alta Bates Summit Medical Center (CA) American University of Beirut Medical Center (NJ) Anand Diagnostic Laboratory (India) Anne Arundel Medical Center (MD) Antelope Valley Hospital District (CA) APP – Unipath (CO) Appalachian Regional Healthcare System (NC) Arkansas Children’s Hospital (AR) Arkansas Dept of Health, Public Health Laboratory (AR) Arkansas Methodist Medical Center (AR) Artemis Health, Inc. (CA) Asan Medical Center (Korea) Asante Health System (OR) Asiri Group of Hospitals Ltd. (Sri Lanka) Aspen Valley Hospital (CO) Aspirus Wausau Hospital (WI) Associated Regional & University Pathologists (UT) Atlantic City Medical Center (NJ) Atrium Medical Center (OH) Auburn Regional Medical Center (WA) Augusta Health (VA) Aultman Hospital (OH) Avera McKennan Hospital (SD) AZ Sint-Jan (Belgium) Azienda Ospedale Di Lecco (Italy) Azienda Ospedaliera Padova (Italy) Azienda Ospedaliera Verona (Italy) Azienda Policlinico Umberto I Di Roma (Italy) Baptist Hospital for Women (TN) Baptist Hospital of Miami (FL) Baptist Memorial Hospital (MS) Baptist Memorial Hospital East (TN) Barnes-Jewish Hospital (MO) Baton Rouge General (LA) Baxter Regional Medical Center (AR) BayCare Health System (FL) Baylor Health Care System (TX) Bayou Pathology, APMC (LA) Baystate Medical Center (MA) BC Biomedical Laboratories (Canada) Beloit Memorial Hospital (WI) Blanchard Valley Hospital (OH) Blanchfield Army Community Hospital (KY) Blue Ridge Regional Hospital (NC) Bon Secours Health Partners (VA) Bonnyville Health Center (Canada) Boston Medical Center (MA)



Boulder Community Hospital (CO) Boyce & Bynum Pathology Labs (MO) Brantford General Hospital (Canada) Bremerton Naval Hospital (WA) Bridgeport Hospital (CT) Brooke Army Medical Center (TX) The Brooklyn Hospital Center (NY) Broward General Medical Center (FL) Bucyrus Community Hospital (OH) Cadham Provincial Laboratory-MB Health (Canada) Calgary Laboratory Services (Canada) California Department of Public Health (CA) California Pacific Medical Center (CA) Cambridge Health Alliance (MA) Camden Clark Memorial Hospital (WV) Canadian Science Center for Human and Animal Health (Canada) Cape Fear Valley Medical Center Laboratory (NC) Capital Coast Health (New Zealand) Capital Health – Regional Laboratory Services (Canada) Capital Health System Mercer Campus (NJ) Carilion Labs Charlotte (NC) Carl R. Darnall Army Medical Center Department of Pathology (TX) Carolinas Healthcare System (NC) Carpermor S.A. de C.V. (Mexico) Catholic Health Initiatives (KY) Cedars-Sinai Medical Center (CA) Central Baptist Hospital (KY) Centre Hospitalier Anna-Laberge (Canada) Chaleur Regional Hospital (Canada) Chang Gung Memorial Hospital (Taiwan) Changhua Christian Hospital (Taiwan) Charleston Area Medical Center (WV) The Charlotte Hungerford Hospital (CT) Chatham – Kent Health Alliance (Canada) CHC Labs (FL) Chester County Hospital (PA) Children’s Healthcare of Atlanta (GA) Children’s Hospital and Regional Medical Center (WA) Children’s Hospital of Central California (CA) Children’s Hospital of Philadelphia (PA) Children’s Hospital Medical Center (OH) Children’s Hospitals and Clinics (MN) Children’s Hospital & Research Center At Oakland (CA) Children’s Medical Center (OH) Children’s Medical Center (TX) Children’s Memorial Hospital (IL) The Children’s Mercy Hospital (MO) Childrens Hosp.- Kings Daughters (VA) Childrens Hospital Los Angeles (CA) Childrens Hospital of Wisconsin (WI) Christiana Care Health Services (DE) CHU Sainte-Justine (Quebec, Canada) CHU – Saint Pierre (Belgium) CHUM Hopital Saint-Luc (Canada) CHW-St. Mary’s Medical Center (CA) City of Hope National Medical Center (CA) Clarian Health – Clarian Pathology Laboratory (IN) Clearstone Central Laboratories (Canada) Cleveland Clinic (OH) Cleveland Heartlab, LLC (OH) Clinical Hospital Merkur (Croatia) Clinical Labs of Hawaii (HI) Clinton Memorial Hospital (OH) Colchester East Hants Health Authority (Canada) Colegio De Tecnologos Medicos De Puerto (PR) College of Physicians and Surgeons of Alberta (Canada) Collingwood General & Marine Hospital (Canada) Columbia Regional Hospital (MO) Commonwealth of Virginia (DCLS) (VA) Community Hospital (IN) Community Hospital of the Monterey Peninsula (CA) Community Medical Center (NJ) Community Memorial Hospital (WI) Complexe Hospitalier de la Sagamie (Canada) Consultants Laboratory of WI LLC (WI) Contra Costa Regional Medical Center (CA) Cook Children’s Medical Center (TX) The Cooley Dickinson Hospital, Inc. (MA) Corniche Hospital (United Arab Emirates)

Cornwall Community Hospital (Canada) Corona Regional Medical Center (CA) Covance CLS (IN) The Credit Valley Hospital (Canada) Crozer-Chester Medical Center (PA) Cumberland Medical Center (TN) Darwin Library NT Territory Health Services (Australia) David Grant Medical Center (CA) Daviess Community Hospital (IN) Deaconess Hospital Laboratory (IN) Dean Medical Center (WI) DHHS NC State Lab of Public Health (NC) DiagnoSearch Life Sciences Inc. (India) Diagnostic Laboratories (CA) Diagnostic Laboratory Services, Inc. (HI) Diagnostic Services of Manitoba (Canada) Diagnósticos da América S/A (Brazil) Dimensions Healthcare System Prince George’s Hospital Center (MD) DMC University Laboratories (MI) Drake Center (OH) Driscoll Children’s Hospital (TX) DUHS Clinical Laboratories Franklin Site (NC) Dynacare Laboratory (WI) Dynacare NW, Inc – Seattle (WA) DynaLIFE (Canada) E. A. Conway Medical Center (LA) East Georgia Regional Medical Center (GA) East Texas Medical Center-Pittsburg (TX) Eastern Health – Health Sciences Centre (Canada) Eastern Health Pathology (Australia) Easton Hospital (PA) Edward Hospital (IL) Effingham Hospital (GA) Eliza Coffee Memorial Hospital (AL) Elmhurst Hospital Center (NY) Emory University Hospital (GA) Evangelical Community Hospital (PA) Evans Army Community Hospital (CO) Exeter Hospital (NH) Federal Medical Center (MN) Fletcher Allen Health Care (VT) Florida Hospital (FL) Fort Loudoun Medical Center (TN) Fort St. John General Hospital (Canada) Forum Health Northside Medical Center (OH) Fox Chase Cancer Center (PA) Franciscan Skemp Medical Center (WI) Fraser Health Authority Royal Columbian Hospital Site (Canada) Fresenius Medical Care/Spectra East (NJ) Gamma-Dynacare Laboratories (Canada) Garden City Hospital (MI) Garfield Medical Center (CA) Gaston Memorial Hospital (NC) Geisinger Medical Center (Danville, PA) Genesis Healthcare System (OH) George Washington University Hospital (DC) Ghent University Hospital (Belgium) Golden Valley Memorial Hospital (MO) Good Samaritan Hospital (OH) Good Shepherd Medical Center (TX) Grana S.A. (TX) Grand River Hospital (Canada) Grey Bruce Regional Health Center (Canada) Gundersen Lutheran Medical Center (WI) Guthrie Clinic Laboratories (PA) Haga Teaching Hospital (Netherlands) Halton Healthcare Services (Canada) Hamad Medical Corporation (Qatar) Hamilton Regional Laboratory Medicine Program (Canada) Hanover General Hospital (PA) Harford Memorial Hospital (MD) Harris Methodist Fort Worth (TX) Harrison Medical Center (WA) Hartford Hospital (CT) Health Network Lab (PA) Health Sciences Research Institute (Japan) Health Waikato (New Zealand) Heart of Florida Regional Medical Center (FL) Heartland Health (MO) Heidelberg Army Hospital (APO, AE) Helen Hayes Hospital (NY) Hennepin Faculty Association (MN) Henry Ford Hospital (MI) Henry M. Jackson Foundation for the Advancement of Military Medicine- MD (MD) Hi-Desert Medical Center (CA) Highlands Medical Center (AL)

Hoag Memorial Hospital Presbyterian (CA) Holy Cross Hospital (MD) Holy Name Hospital (NJ) Holy Spirit Hospital (PA) Hopital du Haut-Richelieu (Canada) Hôpital Maisonneuve – Rosemont (Montreal, Canada) Hopital Santa Cabrini Ospedale (Canada) Horizon Health Network (Canada) Hospital Albert Einstein (Brazil) The Hospital for Sick Children (Canada) Hospital Sacre-Coeur de Montreal (Canada) Hôtel Dieu Grace Hospital Library (Windsor, ON, Canada) Hôtel-Dieu de Lévis (Canada) Hunter Area Pathology Service (Australia) Hunterdon Medical Center (NJ) IBT Reference Laboratory (KS) Imelda Hospital (Belgium) Indian River Memorial Hospital (FL) Inova Central Laboratory (VA) Institut fur Stand. und Dok. im Med. Lab. (Germany) Institut National de Santé Publique du Quebec Centre de Doc. – INSPQ (Canada) Institute Health Laboratories (PR) Institute of Clinical Pathology and Medical Research (Australia) Institute of Laboratory Medicine Landspitali Univ. Hospital (Iceland) Institute of Medical & Veterinary Science (Australia) Integrated Regional Laboratories South Florida (FL) International Health Management Associates, Inc. (IL) Jackson County Memorial Hospital (OK) Jackson Memorial Hospital (FL) Jackson Purchase Medical Center (KY) Jessa Ziekenhuis VZW (Belgium) JHP Pharmaceuticals (NJ) John C. Lincoln Hospital – N.MT. (AZ) John F. Kennedy Medical Center (NJ) John H. Stroger, Jr. Hospital of Cook County (IL) John Muir Health (CA) Johns Hopkins Medical Institutions (MD) Johns Hopkins University (MD) Johnson City Medical Center Hospital (TN) JPS Health Network (TX) Kaiser Permanente (MD) Kaiser Permanente (OH) Kaiser Permanente Medical Care (CA) Kaleida Health Center for Laboratory Medicine (NY) Kantonsspital Aarau AG (Switzerland) Kenora-Rainy River Reg. Lab. Program (Canada) King Abdulaziz Hospital, Al Ahsa Dept. of Pathology & Laboratory Medicine (Saudi Arabia) King Abdulaziz Medical City – Jeddah National Guard Health Affairs (Saudi Arabia) King Fahad National Guard Hospital KAMC – NGHA (Saudi Arabia) King Faisal Specialist Hospital (MD) King Faisal Specialist Hospital & Research Center (Saudi Arabia) King Hussein Cancer Center (Jordan) King’s Daughters Medical Center (KY) Kings County Hospital Center (NY) Kingston General Hospital (Canada) Kootenai Medical Center (ID) Lab Medico Santa Luzia LTDA (Brazil) Labette Health (KS) Laboratory Alliance of Central New York (NY) Laboratory Corporation of America (NJ) LabPlus Auckland District Health Board (New Zealand) LAC/USC Medical Center (CA) Lafayette General Medical Center (LA) Lakeland Regional Medical Center (FL) Lancaster General Hospital (PA) Landstuhl Regional Medical Center (APO, AE) Langley Air Force Base (VA) Laredo Medical Center (TX) LeBonheur Children’s Medical Center (TN) Legacy Laboratory Services (OR) L’Hotel-Dieu de Quebec (Canada) Licking Memorial Hospital (OH) LifeLabs Medical Laboratory Services (Canada) Loma Linda University Medical (CA) Long Beach Memorial Medical Center- LBMMC (CA) Long Island Jewish Medical Center (NY) Louisiana Office of Public Health Laboratory (LA)

Louisiana State University Medical Ctr. (LA) Lourdes Hospital (KY) Lower Columbia Pathologists, P.S. (WA) Lyndon B. Johnson General Hospital (TX) Maccabi Medical Care and Health Fund (Israel) Madigan Army Medical Center (WA) Mafraq Hospital (United Arab Emirates) Magnolia Regional Health Center (MS) Main Line Clinical Laboratories, Inc. (PA) Makerere University Walter Reed Project Makerere University Medical School (Uganda) Marquette General Hospital (MI) Marshfield Clinic (WI) Martha Jefferson Hospital (VA) Martin Luther King, Jr.-Harbor Hospital (CA) Martin Memorial Health Systems (FL) Mary Hitchcock Memorial Hospital (NH) Mary Imogene Bassett Hospital (NY) Mary Washington Hospital (VA) Massachusetts General Hospital (MA) Mater Health Services – Pathology (Australia) Maxwell Air Force Base (AL) Mayo Clinic (MN) Mayo Clinic Florida (FL) MCG Health (GA) MDM-Lab (Saudi Arabia) Meadows Regional Medical Center (GA) Medecin Microbiologiste (Canada) Medical Center Hospital (TX) Medical Center of Louisiana at NO- Charity (LA) Medical Centre Ljubljana (Slovenia) Medical College of Virginia Hospital (VA) Medical Centre Ljubljana (Slovenia) Medical Univ. of South Carolina (SC) Memorial Hermann Healthcare System (TX) Memorial Hospital at Gulfport (MS) Memorial Medical Center (IL) Memorial Medical Center (PA) Memorial Regional Hospital (FL) Mercy Franciscan Mt. Airy (OH) Methodist Dallas Medical Center (TX) Methodist Hospital (Houston, TX) Methodist Hospital (PA) Methodist Hospital (San Antonio, TX) Methodist Hospital – Park Nicollet Health Services (MN) Methodist Hospital Pathology (NE) MetroHealth Medical Center (OH) Metropolitan Hospital Center (NY) Metropolitan Medical Laboratory, PLC (IA) Miami Children's Hospital (FL) The Michener Inst. for Applied Health Sciences (Canada) Mid Michigan Medical Center – Midland (MI) Middelheim General Hospital (Belgium) Middlesex Hospital (CT) MiraVista Diagnostics (IN) Mississippi Baptist Medical Center (MS) Mississippi Public Health Lab (MS) Monongalia General Hospital (WV) Montreal General Hospital (Quebec, Canada) Mt. Carmel Health System (OH) Mt. Sinai Hospital (Canada) Mt. Sinai Hospital – New York (NY) Naples Community Hospital (FL) Nassau County Medical Center (NY) National B Virus Resource Laboratory (GA) National Cancer Center (S. Korea) National Institutes of Health, Clinical Center (MD) National Naval Medical Center (MD) National University Hospital Department of Laboratory Medicine (Singapore) National University of Ireland, Galway (NUIG) (Ireland) Nationwide Children’s Hospital (OH) Nationwide Laboratory Services (FL) Naval Hospital Great Lakes (IL) The Naval Hospital of Jacksonville (FL) Naval Medical Center Portsmouth (VA) NB Department of Health (Canada) The Nebraska Medical Center (NE) New England Baptist Hospital (MA) New Lexington Clinic (KY) New York City Department of Health and Mental Hygiene (NY) New York Presbyterian Hospital (NY) New York University Medical Center (NY) Newark Beth Israel Medical Center (NJ) Nor-Lea General Hospital (NM) North Carolina Baptist Hospital (NC) North District Hospital (Hong Kong, China) North Mississippi Medical Center (MS) North Shore Hospital Laboratory (New Zealand)



North Shore-Long Island Jewish Health System Laboratories (NY) Northridge Hospital Medical Center (CA) Northside Hospital (GA) Northwest Texas Hospital (TX) Northwestern Memorial Hospital (IL) Norton Healthcare (KY) Ochsner Clinic Foundation (LA) Ohio State University Hospitals (OH) Ohio Valley Medical Center (WV) Onze Lieve Vrouwziekenhuis (Belgium) Ordre Professionel des Technologistes Medicaux du Quebec (Quebec, Canada) Orebro University Hospital (Sweden) Orlando Regional Healthcare System (FL) Ospedale Casa Sollievo Della Sofferenza – IRCCS (Italy) The Ottawa Hospital (Canada) Our Lady’s Hospital For Sick Children (Ireland) Our Lady of Lourdes Reg. Medical Ctr. (LA) Palmetto Baptist Medical Center (SC) Parkland Health & Hospital System (TX) Pathlab (IA) Pathology and Cytology Laboratories, Inc. (KY) Pathology Associates Medical Laboratories (WA) Peace River Regional Health Center (FL) Penn State Hershey Medical Center (PA) Pennsylvania Hospital (PA) The Permanente Medical Group (CA) Peterborough Regional Health Centre (Canada) Piedmont Hospital (GA) Pitt County Memorial Hospital (NC) Potomac Hospital (VA) Prairie Lakes Hospital (SD) Premiere Medical Laboratories, P.A. (FL) Presbyterian Hospital – Laboratory (NC) Presbyterian/St. Luke’s Medical Center (CO) Prince County Hospital (Canada) Princess Margaret Hospital (Hong Kong) Providence Alaska Medical Center (AK) Providence Health Care (Canada) Providence Health Services, Regional Laboratory (OR) Providence Medford Medical Center (OR) Provincial Health Services Authority (Vancouver, BC, Canada) Provincial Laboratory for Public Health (Edmonton, AB, Canada) Queen Elizabeth Hospital (Canada) Queen Elizabeth Hospital (China) Queensland Health Pathology Services (Australia) Quest Diagnostics, Incorporated (CA) Quest Diagnostics JV (OH) Quintiles Laboratories, Ltd. (GA) Rady Children’s Hospital San Diego (CA) Ramathibodi Hospital (Thailand) Redington-Fairview General Hospital (ME) Regions Hospital (MN) Reid Hospital & Health Care Services (IN) Renown Regional Medical Center (NV) Rex Healthcare (NC) River Valley Health-Chalmers Regional Hospital (NB) Riverside County Regional Medical Center (CA) Riverside Health System (VA) Riverside Methodist Hospital (OH) Riyadh Armed Forces Hospital, Sulaymainia (Saudi Arabia)

Riyadh National Hospital (Saudi Arabia) Rockford Memorial Hospital (IL) Royal Victoria Hospital (Canada) Sacred Heart Hospital (FL) Sacred Heart Hospital (WI) Sahlgrenska Universitetssjukhuset (Sweden) St. Agnes Healthcare (MD) St. Anthony Hospital (OK) St. Barnabas Medical Center (NJ) St. Christopher’s Hospital for Children (PA) St. Elizabeth Community Hospital (CA) St. Eustache Hospital (Canada) St. Francis Hospital (SC) Saint Francis Hospital & Medical Center (CT) St. Francis Medical Center (NJ) St. John Hospital and Medical Center (MI) St. John’s Hospital (IL) St. John’s Hospital & Health Ctr. (CA) St. John’s Mercy Medical Center (MO) St. John’s Regional Health Center (MO) St. Joseph Hospital (IN) St. Joseph Mercy Hospital (MI) St. Joseph’s Medical Center (CA) St. Joseph’s Regional Medical Center (NJ) St. Jude Children’s Research Hospital (TN) St. Luke’s Hospital (IA) St. Luke’s Hospital (PA) St. Mary Medical Center (CA) St. Mary of Nazareth Hospital (IL) St. Mary’s Hospital (WI) Saint Mary’s Regional Medical Center (NV) St. Tammany Parish Hospital (LA) Saints Memorial Medical Center (MA) Salem Memorial District Hospital (MO) Sampson Regional Medical Center (NC) Samsung Medical Center (Korea) San Francisco General Hospital- University of California San Francisco (CA) Sanford USD Medical Center (SD) Santa Clara Valley Medical Center (CA) SARL Laboratoire Caron (France) Scott & White Memorial Hospital (TX) Seoul National University Hospital (Korea) Seoul St. Mary’s Hospital (Korea) Seton Medical Center (CA) Sheik Kalifa Medical City (UAE) Shiel Medical Laboratory Inc. (NY) Shore Memorial Hospital (NJ) Singapore General Hospital (Singapore) South Bend Medical Foundation (IN) South County Hospital (RI) South Miami Hospital (FL) Southern Community Laboratories (New Zealand) Southern Health Care Network (Australia) Southern Maine Medical Center (ME) Spectrum Health – Blodgett Campus (MI) Stanford Hospital and Clinics (CA) Stanton Territorial Health Authority (Canada) State of Connecticut Department of Public Health (CT) State of Ohio/Corrections Medical Center Laboratory (OH) State of Washington-Public Health Labs (WA) Stevens Memorial Hospital (WA) Stillwater Medical Center (OK) Stony Brook University Hospital (NY)

Stormont-Vail Regional Medical Ctr. (KS) Strong Memorial Hospital (NY) Sudbury Regional Hospital (Canada) Sunbury Community Hospital (PA) Sunnybrook Health Sciences Centre (ON, Canada) Sunrise Hospital and Medical Center (NV) Sutter Roseville Medical Center (CA) Swedish Medical Center (CO) Sydney South West Pathology Service Liverpool Hospital (Australia) T.J. Samson Community Hospital (KY) Taichung Veterans General Hospital (Taiwan) Taipei Veterans General Hospital (Taiwan) Taiwan Society of Laboratory Medicine (Taiwan) Tallaght Hospital (Ireland) Tartu University Clinics (Estonia) Temple Univ. Hospital – Parkinson Pav. (PA) Texas Children’s Hospital (TX) Texas Department of State Health Services (TX) Texas Health Presbyterian Hospital Dallas (TX) Timmins and District Hospital (Canada) Tokyo Metro. Res. Lab of Public Health (Japan) The Toledo Hospital (OH) Ton Yen General Hospital (Taiwan) Touro Infirmary (LA) Tri-City Medical Center (CA) Trident Medical Center (SC) Trinity Medical Center (AL) Tripler Army Medical Center (HI) Tuen Mun Hospital, Hospital Authority (China) Tulane Medical Center Hospital & Clinic (LA) Twin Lakes Regional Medical Center (KY) UCI Medical Center (CA) UCLA Medical Center Clinical Laboratories (CA) UCSD Medical Center (CA) UCSF Medical Center – China Basin (CA) UMC of El Paso- Laboratory (TX) UMC of Southern Nevada (NV) UNC Hospitals (NC) Union Clinical Laboratory (Taiwan) United Christian Hospital (Hong Kong) United Clinical Laboratories (IA) United Medical Center (DC) United States Air Force School of Aerospace Medicine/PHE (TX) Unity HealthCare (IA) Università Campus Bio - Medico Di Roma (Italy) Universitair Ziekenhuis Antwerpen (Belgium) University College Hospital (Ireland) University Hospital (GA) University Hospital Center Sherbrooke (CHUS) (Canada) University Medical Center at Princeton (NJ) University of Alabama Hospital Lab (AL) University of Chicago Hospitals Laboratories (IL) University of Colorado Health Sciences Center (CO) University of Colorado Hospital (CO) University of Illinois Medical Center (IL) University of Iowa Hospitals and Clinics (IA) University of Kentucky Med. Ctr. (KY) University of Maryland Medical System (MD)

University of Medicine & Dentistry of New Jersey (UMDNJ) (NJ) University of MS Medical Center (MS) University of Missouri Hospital (MO) University of MN Medical Center – Fairview (MN) Univ. of Pennsylvania Health System (PA) University of Pittsburgh Medical Center (PA) University of So. Alabama Children’s and Women’s Hospital (AL) University of Texas Health Center (TX) The University of Texas Medical Branch (TX) University of the Ryukyus (Japan) University of Virginia Medical Center (VA) UPMC Bedford Memorial (PA) US Naval Hospital Naples (FPO) USC University Hospital (CA) UZ-KUL Medical Center (Belgium) VA Central Texas Veterans Health Care System (TX) VA (Asheville) Medical Center (NC) VA (Bay Pines) Medical Center (FL) VA (Chillicothe) Medical Center (OH) VA (Cincinnati) Medical Center (OH) VA (Dayton) Medical Center (OH) VA (Durham) Medical Center (NC) VA (Hampton) Medical Center (VA) VA (Indianapolis) Medical Center (IN) VA (San Diego) Medical Center (CA) VA (Tampa) Hospital (FL) Valley Health/Winchester Medical Center (VA) Vancouver Coastal Health Regional Laboratory (BC, Canada) Vancouver Island Health Authority (SI) (Canada) Vanderbilt University Medical Center (TN) Via Christi Regional Medical Center (KS) Virginia Beach General Hospital (VA) Virginia Regional Medical Center (MN) Virtua – West Jersey Hospital (NJ) WakeMed (NC) Walter Reed Army Medical Center (DC) Warren Hospital (NJ) Washington Hospital Center (DC) Waterbury Hospital (CT) Waterford Regional Hospital (Ireland) Wayne Memorial Hospital (NC) Weirton Medical Center (WV) West China Second University Hospital, Sichuan University (China) West Jefferson Medical Center (LA) West Penn Allegheny Health System- Allegheny General Hospital (PA) West Shore Medical Center (MI) West Valley Medical Center Laboratory (ID) Westchester Medical Center (NY) Western Baptist Hospital (KY) Western Healthcare Corporation (Canada) Wheaton Franciscan and Midwest Clinical Laboratories (WI) Wheeling Hospital (WV) Whitehorse General Hospital (Canada) William Beaumont Army Medical Center (TX) William Beaumont Hospital (MI) William Osler Health Centre (Canada) Winchester Hospital (MA) Winn Army Community Hospital (GA) Wishard Health Sciences (IN) Womack Army Medical Center (NC) York Hospital (PA)

OFFICERS BOARD OF DIRECTORS Janet K.A. Nicholson, PhD, President Centers for Disease Control and Prevention Mary Lou Gantzer, PhD, FACB, President-Elect Siemens Healthcare Diagnostics, Inc. Jack Zakowski, PhD, FACB, Secretary Beckman Coulter, Inc. W. Gregory Miller, PhD, Treasurer Virginia Commonwealth University Gerald A. Hoeltge, MD, Immediate Past President Cleveland Clinic Glen Fine, MS, MBA, CAE, Executive Vice President

Maria Carballo Health Canada Russel K. Enns, PhD Cepheid Prof. Naotaka Hamasaki, MD, PhD Nagasaki International University Christopher M. Lehman, MD University of Utah Health Sciences Center Valerie Ng, PhD, MD Alameda County Medical Center/ Highland General Hospital

Luann Ochs, MS BD Diagnostics – TriPath Robert Rej, PhD New York State Department of Health Donald St.Pierre FDA Center for Devices and Radiological Health Michael Thein, PhD Roche Diagnostics GmbH James A. Thomas ASTM International Harriet R. Walsh, MA, MT(ASCP) Centers for Medicare and Medicaid Services



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