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Antimicrobial
Susceptibility
Testing Protocols


Edited by
Richard Schwalbe
Lynn Steele-Moore
Avery C. Goodwin
Antimicrobial
Susceptibility
Testing Protocols
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Library of Congress Cataloging-in-Publication Data
Antimicrobial susceptibility testing protocols / editors, Richard Schwalbe, Lynn Steele-Moore, and
Avery C. Goodwin.
p. ; cm.
Includes bibliographical references and index.
ISBN-13: 978-0-8247-4100-6 (alk. paper)
ISBN-10: 0-8247-4100-5 (alk. paper)
1. Microbial sensitivity tests. 2. Drug resistance in microorganisms. I. Schwalbe, Richard. II.
Steele-Moore, Lynn. III. Goodwin, Avery C.
[DNLM: 1. Microbial Sensitivity Tests methods. 2. Microbial Sensitivity Tests standards.
3. Anti-Bacterial Agents. 4. Antifungal Agents. 5. Drug Resistance, Fungal. 6. Drug Resistance,
Microbial. QW 25.5.M6 A631 2007]
QR69.A57A63 2007
616.9’041 dc22 2007005182
Visit the Taylor & Francis Web site at

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Dedication

This book is dedicated to Dr. Richard Steven Schwalbe,
who passed away September 1, 1998, before our textbook was completed.
All of the contributors to

Antimicrobial Susceptibility Testing Protocols

respectfully and lovingly present our work as a token of our admiration
to Rick and in the hope that through our efforts we may carry on his
passion and love of susceptibility testing and microbiology.

l’chaim

my friend I miss you.



Preface

Recent reports in the lay press describing bacterial resistance to multiple antibiotics serve to
emphasize the importance of accurate susceptibility testing. The clinical microbiology laboratory
is often a sentinel for detection of drug-resistant microorganisms. Timely notification of suscepti-
bility results to clinicians can result in initiation or alteration of antimicrobial chemotherapy and
thus improve patient care. Standardized protocols require continual scrutiny to detect emerging
phenotypic resistance patterns.
The aim of

Susceptibility Testing Protocols

is twofold: one is to present a comprehensive, up-
to-date procedural manual that can be used by a wide variety of laboratory workers. The second
objective is to delineate the role of the clinical microbiology laboratory in integrated patient care.
Many protocols that are presented are an extrapolation of procedures approved by the Clinical and
Laboratory Standards Institute (CLSI). New procedures that are (at present) nonstandardized are
also described.
The first section of this manual addresses the basic susceptibility disciplines that are already
in place in many clinical microbiology laboratories. These include disk diffusion, macro- and
microbroth dilution, agar dilution, and the gradient method. Step-by-step protocols are provided.
Emphasis is placed on optimizing procedures for detection of resistant microorganisms. A chapter
is devoted to automated susceptibility testing, introducing the systems that are currently available
for purchase, including recent laboratory evaluations, and presents an algorithm that can be followed
by laboratory workers who are considering purchasing a new automated system.
The second section is devoted to descriptions of susceptibility protocols that may be performed
by a subset of laboratories, whether as reference centers or as part of a research protocol. Specialized
protocols such as surveillance procedures for detection of antibiotic-resistant bacteria, serum bac-
tericidal assays, time-kill curves, population analysis, and synergy testing are discussed. Emphasis
in this section is on clear descriptions of methods leading to reproducible results.
The final section of this manual includes a series of chapters designed to be used as reference
sources. Additional chapters focus on antibiotic development and design, use of an antibiogram,
and the interactions of the clinical microbiology laboratory with ancillary areas such as hospital
pharmacy, infectious disease personnel, and infection control. A table of antibiotic classes and
common “bug–drug” susceptibilities are also included.
This manual is directed to personnel engaged in the laboratory disciplines that perform

in vitro

susceptibility testing, including clinical microbiology, food and agriculture microbiology, pharma-
ceuticals research, and other applied and basic research environments. It is meant to be used as a

bench manual. References are supplied at the end of each chapter to provide additional sources of
information to those individuals wishing to pursue a specific topic in greater detail.

Antibacterial Susceptibility Testing Protocols

differs from other available sources of information
by its scope. Its aim is to combine an updated series of laboratory-based techniques and charts
within the context of the role of clinical microbiology in modern medicine.
We would like to acknowledge Debbi Reader Covey for her secretarial assistance and unwa-
vering support. Most of all, we would like to thank Dr. Richard S. Schwalbe for many, many things
and for the great person he was.

Lynn Steele-Moore
Avery C. Goodwin, Ph.D.



Editors

Rick Schwalbe

was the director of clinical microbiology at the University of Maryland Medical
Systems and the director of clinical microbiology and virology at the Veteran’s Administration
Medical Center in Baltimore, Maryland. He was associate professor of pathology at the University
of Maryland School of Medicine. At the time of his death he was president of the Maryland ASM.
He served on the editoral board of

Antimicrobial Agents and Chemotherapy

and had over 100

publications.

Lynn Steele-Moore

was the manager of Dr. William J. Holloway’s Infectious Disease Laboratory
at Christiana Care Health Services in Wilmington, Delaware, for 22 years. She currently is employed
by the U.S. Food and Drug Administration in Silver Spring, Maryland. Lynn and the late Dr. Richard
Schwalbe collaborated on many projects, this text being one of them. Unfortunately, Dr. Schwalbe
passed away before the text was completed. However, his contributions can be seen throughout the
text with chapters written by many of his friends. We all lovingly dedicate this text to our beloved
friend, Rick Schwalbe.

Avery C. Goodwin

worked at GlaxoSmithKline on the development of antimicrobial drugs. He is
currently employed by the U.S. Food and Drug Administration in Silver Spring, Maryland, as a
microbiology reviewer in the Division of Anti-Infective and Ophthalmology Products.



Contributors List

Arthur L. Barry

The Clinical Microbiology Institute
Wilsonville, Oregon

Donna Berg

Christiana Care Health Services

Wilmington, Delaware

Barbara A. Brown-Elliott

The University of Texas Health Center
Tyler, Texas

Cassandra B. Calderón

Select Specialty Hospital
Youngstown, Ohio

Emilia Cantón

La Fe University Hospital
Valencia, Spain

Samuel Cohen

ARUP Laboratories
Salt Lake City, Utah

James D. Dick

Johns Hopkins Medical Institutions
Baltimore, Maryland

Michael Dowzicky

Wyeth Pharmaceuticals

Collegeville, Pennsylvania

Ana Espinel-Ingroff

VCU Medical Center
Richmond, Virginia

Ann Hanlon

Johns Hopkins Medical Institutions
Baltimore, Maryland

Estrella Martin-Mazuelos

Valme University Hospital
Barcelona, Spain

Sophie Michaud

Faculté de Médecine de l'Université de
Sherbrooke
Sherbrooke, Québec, Canada

Harriette Nadler

Downington, Pennsylvania

Barbara G. Painter

Pharma Microbiology Consult, LLC

Chattanooga, Tennessee

Donald H. Pitkin

Consultant
Mohrsville, Pennsylvania

John H. Powers

National Institute of Health
Bethesda, Maryland

Sadaf Qaiyumi

U.S. Food and Drug Administration
Laurel, Maryland

Nancy T. Rector

BD Diagnostics
Sparks, Maryland

Darcie E. Roe-Carpenter

Barton Memorial Hospital
South Lake Tahoe, California

Beulah Perdue Sabundayo

The Johns Hopkins University School of

Medicine
Baltimore, Maryland

Sheryl Stuckey

Holy Cross Hospital
Silver Spring, Maryland

Merwyn Taylor

Johns Hopkins Medical Institutions
Baltimore, Maryland


John Thomas

West Virginia University Schools of Medicine
and Dentistry
Morgantown, West Virginia

Punam Verma

Virginia Mason Medical Center
Seattle, Washington

Audrey Wanger

The University of Texas Medical School
Houston, Texas

Richard J. Wallace, Jr.

The University of Texas Health Center
Tyler, Texas

C. Douglas Webb

Consultant, Anti Infectives
Tybee Island, Georgia


Table of Contents

Chapter 1

An Overview of the Clinical and Laboratory Standards Institute (CLSI)
and Its Impact on Antimicrobial Susceptibility Tests 1

Arthur L. Barry

Chapter 2

Antimicrobial Classifications: Drugs for Bugs 7

Cassandra B. Calderón and Beulah Perdue Sabundayo

Chapter 3

Disk Diffusion Test and Gradient Methodologies 53

Audrey Wanger

Chapter 4

Macro- and Microdilution Methods of Antimicrobial Susceptibility Testing 75

Sadaf Qaiyumi

Chapter 5

Automated Systems: An Overview 81

Sheryl Stuckey

Chapter 6

Agar Dilution Susceptibility Testing 91

Ann Hanlon, Merwyn Taylor, and James D. Dick

Chapter 7

Susceptibility-Testing Protocols for Antibiograms and Preventive Surveillance:
A Continuum of Data Collection, Analysis, and Presentation 105

John G. Thomas and Nancy T. Rector

Chapter 8

Anaerobe Antimicrobial Susceptibility Testing 139

Darcie E. Roe-Carpenter

Chapter 9

Antifungal Susceptibility Testing of Yeasts 173

Ana Espinel-Ingroff and Emilia Cantón

Chapter 10

Antifungal Susceptibility Testing of Filamentous Fungi 209

Ana Espinel-Ingroff and Emilia Cantón

Chapter 11

Susceptibility Testing of Mycobacteria 243

Barbara A. Brown-Elliott, Samuel Cohen, and Richard J. Wallace, Jr.


Chapter 12

Methods for Determining Bactericidal Activity and Antimicrobial Interactions:
Synergy Testing, Time-Kill Curves, and Population Analysis 275

Punam Verma

Chapter 13

Serum Bactericidal Testing 299

Harriette Nadler and Michael Dowzicky

Chapter 14

Bioassay Methods for Antimicrobial and Antifungal Agents 313

Donald H. Pitkin and Estrella Martin-Mazuelos

Chapter 15

Molecular Methods for Bacterial Strain Typing 341

Sophie Michaud and Donna Berg

Chapter 16

Pharmacy and Microbiology: Interactive Synergy 363

Beulah Perdue Sabundayo and Cassandra B. Calderón

Chapter 17

Interactions between Clinicians and the Microbiology Laboratory 371

John H. Powers

Chapter 18

Clinical Microbiology in the Development of New Antimicrobial Agents 377

C. Douglas Webb and Barbara G. Painter

Index

393


1

1

An Overview of the
Clinical and Laboratory
Standards Institute (CLSI) and
Its Impact on Antimicrobial
Susceptibility Tests

Arthur L. Barry

CONTENTS

1.1 Historical Perspective 1
1.2 Disk Diffusion Susceptibility Tests 2
1.3 Other CLSI Microbiology Documents 3
1.4 Quality Control Limits 4
1.5 Role of the U.S. Food and Drug Administration (FDA) 5
References 6

1.1 HISTORICAL PERSPECTIVE

In the middle of the twentieth century, medical technologists and clinical pathologists were expected
to maintain expertise in all aspects of the service laboratory; most were poorly trained in diagnostic
microbiology. With the widespread use of antimicrobial chemotherapy, the nature of infectious
diseases gradually changed and that created new challenges to those concerned with the diagnosis
and treatment of infectious diseases. Consequently, clinical laboratories were being asked to perform
increasingly sophisticated procedures. To help clinical pathology laboratories meet those new
challenges, a number of industries were developed to provide supplies and equipment that labora-
torians could no longer make or obtain for themselves. Standardization of methodology was essential
for such commercial endeavors to be successful. Performance standards were needed in order for
each laboratory to judge the quality of different products. Regulatory agencies were also being
asked to monitor the quality of products being sold to diagnostic laboratories. Government agencies
were being forced to provide standards for judging the quality of different reagents and equipment;
for obvious reasons, laboratory professionals wanted to be involved in writing such standards.
In the mid-1960s a group of interested individuals began discussions that led to the concept of
an independent organization that could prepare standards that would be acceptable to everyone
using them. This became known as the National Committee for Clinical Laboratory Standards
(NCCLS), now called the Clinical and Laboratory Standards Institute (CLSI). The name change
(effective January 2005) was felt to be a more accurate representation of the organization. Clinical
laboratory standards were to be prepared by committees composed of experts from academia,


2

Antimicrobial Susceptibility Testing Protocols

government, and industry. Once a standard was written and approved by the CLSI council, it was
to be published as a proposed standard, and all interested individuals were asked to make written

comments or suggestions. After one year of peer review, the subcommittee was asked to respond
to all comments and to make appropriate changes or to explain in writing why some suggestions
were not accepted. By this process, each standard would be a true consensus document. After the
initial review process, each document would be advanced to the status of an approved standard.
When important technical changes are needed, the document should be revised and then go through
the consensus review process again. Each document is reviewed every three years and either
discontinued or revised. In that way, CLSI standards are living documents that are updated as our
understanding of the subject improves.
The CLSI was established in 1967–1968, and a small office was established in Los Angeles,
California. A part-time secretary helped to solicit individuals who would volunteer to formulate
committees that could address specific issues concerning clinical laboratory medicine. Once the
first few standards were published and accepted by clinical laboratories, the CLSI support staff was
expanded and was eventually moved to Villanova, PA and later to Wayne, PA. CLSI documents
are now accepted throughout the world, and regulatory agents often cite CLSI standards as the
accepted state of the art.

1.2 DISK DIFFUSION SUSCEPTIBILITY TESTS

The CLSI began by looking for specific areas that would be most benefited by such consensus
documents. In the area of microbiology, the antimicrobial disk diffusion susceptibility test was a
natural subject. Just before that time, there was a major effort to standardize antimicrobial suscep-
tibility tests on an international level, through the World Health Organization. Sherris and Ericsson
coordinated collaborative studies the results of which were published in 1971 [1]. Their extensive
labors helped to standardize broth dilution and agar dilution antimicrobial susceptibility tests;
microdilution susceptibility tests were not available at that time. Disk diffusion tests had been
carefully standardized for use in the Scandinavian countries by Ericsson [2] and for use in the
United States by Bauer et al. [3]. With those two methods, many procedural details were carefully
controlled and well-defined. A variety of other methods had been advocated, but there was little
effort to control important variables such as the inoculum density, agar medium, incubation con-
ditions, etc. The standardized disk tests of Ericsson [2] and of Bauer et al. [3] both involve measuring

the diameter of each zone of inhibition and comparing that to minimum inhibitory concentrations
(MICs) obtained by agar or broth dilution susceptibility tests.
In the United States, the majority of clinical microbiologists felt that the time required to
standardize inoculum densities and to measure zones of inhibition was too difficult for busy clinical
laboratories. The most popular method utilized one or more disks for each agent (high- or low-
content disks) and simply reported a strain to be susceptible if there was any zone of inhibition
and resistant if it grew up to the disk. That was the method that the U.S. Food and Drug Admin-
istration (FDA) approved for inclusion in the package insert that was provided in each package of
antibiotic disks [4]. A chaotic situation remained because there was no national effort to bring some
degree of standardization to the disk diffusion susceptibility test procedure.
In 1968, I was given the honor to chair a CLSI subcommittee on antimicrobial disk susceptibility
tests. After interviewing a number of opinion leaders, those that had the foresight to understand
the need for standard methodologies were appointed to the subcommittee and serious discussions
of methodologic details pursued. In principle, the method of Bauer et al. [3] was accepted by the
subcommittee, but a few minor changes were added. Four years later, a document was prepared
and given the designation of M2, the second standard written for the Microbiology Area Committee.
That document defined the Kirby-Bauer method and the agar overlay modification of that method
[5]. Quality control guidelines were also included even though quality control was unknown in
clinical microbiology laboratories at that point in time. The M2 document was forwarded to the


Overview of the Clinical and Laboratory Standards Institute

3

CLSI council for review, but it was not approved until 1975. In the interim, the FDA conducted its
own survey and concluded that the methods of Bauer et al. [3] and of Barry, Garcia, and Thrupp
[5] were preferable, and the package inserts for antibiotic disks were rewritten [4]. With the changes
in the FDA’s recommendations and publication of the M2 document, CLSI standards were widely
accepted within the United States and in many other countries. The M2 document has undergone

numerous revisions: it is now in its ninth edition [6].

1.3 OTHER CLSI MICROBIOLOGY DOCUMENTS

Once the M2 document was accepted, other subcommittees were established in order to address
additional issues concerning antimicrobial susceptibility tests. Table 1.1 describes the standards
that have been developed over the years.
H. Frankel and A. Barry cochaired a subcommittee that provided a reference that manufacturers
of dehydrated media could use to help standardize Mueller-Hinton agars (CLSI M6). That ongoing
subcommittee has successfully improved the performance of Mueller-Hinton agar sold in the
United States.
C. Thornsberry chaired a subcommittee to standardize agar and broth dilution tests of aerobic
microorganisms [7]. Broth microdilution methods were being popularized at that time, and the
subcommittee was able to standardize that procedure before it was widely used and before inappro-
priate procedures could be well engrained. Consequently, manufacturers of microdilution trays were
given specific standards that their product should meet, and there have been very few problems with
commercial panels that were subsequently marketed. This document, M7, is now in its seventh edition.

TABLE 1.1
Overview of CLSI Documents Relating to Antimicrobial Susceptibility Tests

CLSI Designation

a

General Subject Covered

M2 Disk diffusion tests
M6 Dehydrated Mueller-Hinton agar
M7 Broth agar dilution tests—aerobes

M11 Anaerobic dilution tests
M21 Serum bactericidal test
M23 Guidelines for new drug reviews
M24 Mycobacteria and nocardia
M26 Bactericidal activity
M27 Antifungal tests of yeasts
M31 Veterinary drugs and pathogens
M32 Mueller-Hinton broth
M33 Antiviral agents
M37 M23 for veterinary agents
M38 Antifungal tests of filamentous fungi
M39 Cumulative susceptibility reports
M42 Disk diffusion—aquatic animals
M44 Disk diffusion—yeasts
M45 Dilution and disk—fastidious bacteria
M49 Broth dilution—aquatic animals
M100 Informational supplements for M2 and M7

a

Each document is assigned a letter (M for microbiology) and a sequential number; this may be followed
by a P (proposed), T (tentative), A (approved) or R (report) and by the number of editions, if more than one.
Refer to CLSI website (www.clsi.org) for an updated list each year.


4

Antimicrobial Susceptibility Testing Protocols

V. Sutter chaired a subcommittee that did the same thing for tests of anaerobic bacteria. Initially,

that subcommittee limited their document to agar dilution tests of nonfastidious anaerobes. They
defined a reference test that was to be used for evaluating other test procedures and anticipated
modifications that would permit testing more fastidious species of anaerobes. That document, M11,
has now been expanded and has undergone seven revisions [8].
C. Stratton developed the first draft of two documents; one dealt with methods for measuring
the bactericidal activity of antibacterial agents and the other concerned the serum bactericidal tests.
J. Jorgensen later brought those documents to the status of approved standards (M21). J. Watts
developed two documents that deal with issues that are specific for veterinary practice. G. Woods
guided the development of a document that defined procedures for susceptibility tests of

Mycobac-
terium

and related species. J. Galgiani and M. Pfaller have chaired a subcommittee that addressed
specific reference methods for testing antifungal agents against yeasts (M27) and against filamentous
fungi (M38). R. Hodinka chaired a subcommittee that dealt with methods for testing antiviral agents
(herpes simplex virus). The reader is referred to Table 1.1 for document numbers.
The subcommittee for disk diffusion susceptibility tests has now been given responsibility for
the documents that dealt with aerobic and anaerobic antibacterial dilution tests of human pathogens.
J. Jorgensen and M. Ferraro chaired this subcommittee while it was learning to handle its new
responsibilities. W. Novick coordinated the preparation of guidelines that defined the type of
information that should be provided by drug manufacturers when applying for inclusion in CLSI
tables [9]. The original subcommittee was given responsibility for maintenance of four documents
(M2, M7, M11, and M23) since these involve evaluation of the same type of data for selecting
interpretive criteria and quality control ranges for each new antibacterial agent. In 1964, Bauer et
al. [3] provided interpretive criteria for tests of 20 antimicrobial agents, and 42 years later, the 2006
CLSI document describes criteria for over 90 agents. With this increase in the number of antimi-
crobial agents, the tables became complex and confusing. J. Hindler and J. Swenson have spear-
headed a major effort to make the tables significantly more user-friendly, and their efforts resulted
in an important improvement. Because new agents may be added to the tables once or twice a year,

revision of the documents every three years is not sufficient. In an attempt to maintain an up-to-
date document, an informational supplement is published once a year in January [10]. This sup-
plement contains only the most recent version of the tables, without the text of each standard.
Consequently, it can be published without the usual delays required by the peer review process.
All new entries in the tables are identified and considered tentative for the first year. If written
complaints are received in the first year, the subcommittee will reconsider the issue causing concern.
Although the Microbiology Area Committee has concentrated on issues concerning antimicro-
bial susceptibility tests, other documents have been prepared and are currently available. Subjects
that have been addressed are as follows: blood-borne parasitic diseases, intestinal parasites, quality
control of commercial media, fetal bovine serum, protection of laboratory workers from infectious
agents, Western blot assay for

B. burgdorferi

, and abbreviated identification of bacteria and yeasts.
New documents are in process, including one that addresses PFGE and other methods for bacterial
strain typing (see Chapter 15). In all cases, the documents are reviewed on a regular schedule and
either withdrawn or revised and updated. Area committees other than microbiology have also been
productive in the number of documents that have been published.

1.4 QUALITY CONTROL LIMITS

The most important contribution that was made by these efforts was the selection of quality control
(QC) strains and the definition of expected MICs and/or zone diameters for each new antimicrobial
agent. The control strains that are currently utilized for different purposes are described in Table
1.2. With these QC strains, laboratories have a way to know whether they are performing the tests
appropriately and to monitor the tests that are done on a regular basis. The reader is advised to
refer to the most current CLSI documents for an up-to-date list as changes do occur.

Overview of the Clinical and Laboratory Standards Institute

5

Every time a new antimicrobial agent is added to the tables, appropriate multilaboratory
collaborative studies must be performed, and the acceptable range of zone diameters or range of
MICs is then defined for each QC strain. In general, this range should include at least 95% of all
test results, i.e., 1 out of 20 determinations might be just outside of the QC range. If a single
determination is out of control by random chance, it should come back into the QC range when
repeated. If it remains out of control, some troubleshooting is needed in order to find the problem.

1.5 ROLE OF THE U.S. FOOD AND DRUG ADMINISTRATION (FDA)

As directed by Congress, the FDA has legal responsibility for determining safety and efficacy of new
anti-infective agents. When a new drug is released for sale in the U.S., the FDA-approved package
insert includes specific guidelines for interpretation of

in vitro

tests and quality control limits for tests
performed according to CLSI procedures. The CLSI subcommittee also defines interpretive criteria
and QC limits for their tests. Because the two groups actually examine slightly different data, it is
not surprising that there are occasional discrepancies between the two. Major efforts are being made
to avoid unintended differences, but some minor discrepancies are unavoidable.
The devices division of the FDA also certifies commercial products and monitors the reliability
of each. The CLSI does

not

approve or disapprove any commercial product; they only standardize

methodology.
The CLSI defines methodologies that can survive the consensus process; they have no regulatory
responsibility, but CLSI documents are often cited by agencies that do have such responsibilities.

TABLE 1.2
Microorganisms Designated for Quality Control of Antimicrobial Susceptibility Tests

Species (ATCC Number) Purpose and Comments

Escherichia coli

(25922) For dilution and disk tests

Escherichia coli

(35218) For

β

-lactam/inhibitor combinations

Klebsiella pneumoniae

(700603) For ESBL screening tests

Pseudomonas aeruginosa

(27853) For dilution and disk tests, especially for aminoglycosides vs.

P. aeruginosa

Enterococcus faecalis

(29212) For dilution tests (vancomycin-S)

Enterococcus faecalis

(51299) Aminoglycoside-resistant control strain

Staphylococcus aureus

(29213) For dilution tests (weak

β

-lactamase producer)

Staphylococcus aureus

(25923) For disk tests (

β

-lactamase negative)

Staphylococcus aureus

(43300) For oxacillin dilution tests (MRSA strain)

Streptococcus pneumoniae

(49619) Penicillin intermediate strains for dilution and disk tests of all streptococci

Neisseria gonorrhoeae

(49226) For dilution and disk tests of gonococci

Haemophilus influenzae

(49247) For dilution and disk tests of

H. influenzae

(

β

-lactamase-negative, ampicillin-R)

Haemophilus influenzae

(49766) Ampicillin-S strain for agents not controlled by 49247

Haemophilus influenzae

(10211) For testing quality of HTM agar or broth

Haemophilus somnus

(70025) For fastidious animal pathogens

Actinobacillus pleuropneumoniae

(27090) For fastidious animal pathogens

Bacteroides fragilis

(25285) For dilution tests of anaerobes

Bacteroides thetaiotaomicron

(29741) For dilution tests of anaerobes

Eubacterium lentum

(43055) For dilution tests of anaerobes

Candida parapsilosis

(22019) For dilution tests of yeasts

Candida krusei

(6258) For dilution tests of yeasts

Aspergillus flavus

(204304) For dilution tests of filamentous fungi

Aspergillus funigatus

(204305) For dilution tests of filamentous fungi

Refer to current CLSI documents for an updated list each year.


6

Antimicrobial Susceptibility Testing Protocols

Many laboratorians feel that they can test only those antimicrobial agents that have interpretive
criteria and QC ranges defined in the CLSI tables. Testing and reporting agents that do not have
interpretive criteria are the responsibility of the chief microbiologist and such decisions should be
made with input from the infectious disease clinicians (see Chapter 17). Clearly, the CLSI has
resolved the chaotic situation that existed in the late 1960s, and ongoing activities promise to
maintain up-to-date information when it is needed. The area committee for microbiology regularly
considers proposals for new projects that may have a substantial impact on the practice of clinical
microbiology within the United States.

REFERENCES

1. Ericsson, H.M. and Sherris, J.C. Antibiotic sensitivity testing. Report of an international collaborative
study,

Acta Pathol. Microbiol. Scand.

,

Sect. B

, Suppl. 217, 1971.

2. Ericsson, H. The paper disc method for determination of bacterial sensitivity to antibiotics,

J. Clin.
Lab. Invest

., 12, 1–15, 1960.
3. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., and Turck, M. Antibiotic susceptibility testing by a single
high content disk method,

Amer. J. Clin. Pathol

., 45, 492–496, 1966.
4. Wright, W.W. FDA actions on antibiotic susceptibility discs, in

Techniques for Antibiotic Susceptibility
Testing

, Balows, A., Ed., Charles C Thomas, Springfield, IL, 1974, 26.
5. Barry, A.L., Garcia, F., and Thrupp, L.D. An improved method for testing the antibiotic susceptibility
of rapid growing pathogens,

Amer. J. Clin. Pathol

., 53, 149–158, 1970.
6. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk suscepti-
bility tests, approved standard, M2-A9, 9th ed., CLSI, Wayne, PA, 2006.
7. Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for
bacteria that grow aerobically, approved standard M7-A7, 7th ed., CLSI, Wayne, PA, 2006.
8. Clinical and Laboratory Standards Institute. Methods for antimicrobial susceptibility testing of anaer-
obic bacteria, approved standard M11-A6, 7th ed., CLSI, Wayne, PA, 2007.

9. Clinical and Laboratory Standards Institute. Development of

in vitro

susceptibility testing criteria and
quality control parameters, approved standard M23-A2, 2nd ed., CLSI, Wayne, PA, 2001.
10. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility
testing, informational supplement M100-S16, 16th ed., CLSI, Wayne, PA, 2006.


7

2

Antimicrobial Classifications:
Drugs for Bugs

Cassandra B. Calderón and Beulah Perdue Sabundayo

CONTENTS

2.1 Introduction 9
2.2 Antibiotics 9
2.2.1 Penicillins 9
2.2.1.1 Background 9
2.2.1.2 Mechanism of Action 12
2.2.2.3 Chemical Structure 12
2.2.2.4 Mechanisms of Resistance 12
2.2.2.5 Classification 12
2.2.2.6 Antimicrobial Activity and Therapeutic Uses 12

2.2.2.7 Adverse Effect Profile 14
2.2.3 Cephalosporins 14
2.2.3.1 Background 14
2.2.3.2 Mechanism of Action 14
2.2.3.3 Chemical Structure 14
2.2.3.4 Mechanisms of Resistance 15
2.2.3.5 Classification 15
2.2.3.6 Antimicrobial Activity and Therapeutic Uses 15
2.2.3.7 Adverse Effect Profile 17
2.2.4 Carbapenems 17
2.2.5 Monobactams 18
2.2.6 Glycopeptides 19
2.2.6.1 Background 19
2.2.6.2 Mechanism of Action 19
2.2.6.3 Mechanisms of Resistance 19
2.2.6.4 Antimicrobial Activity and Therapeutic Uses 19
2.2.6.5 Adverse Effect Profile 20
2.2.7 Streptogramins 21
2.2.8 Cyclic Lipopeptides 21
2.2.8.1 Background 21
2.2.8.2 Mechanism of Action 21
2.2.8.3 Antimicrobial Activity and Therapeutic Uses 21
2.2.8.4 Adverse Effect Profile 21
2.2.9 Oxazolidinones 22
2.2.9.1 Background 22
2.2.9.2 Mechanism of Action 22
2.2.9.3 Antimicrobial Activity and Therapeutic Uses 22
2.2.9.4 Adverse Effect Profile 22

8

Antimicrobial Susceptibility Testing Protocols

2.2.10 Aminoglycosides 22
2.2.10.1 Background 22
2.2.10.2 Mechanism of Action 23
2.2.10.3 Mechanisms of Resistance 23
2.2.10.4 Antimicrobial Activity and Therapeutic Uses 23
2.2.10.5 Dosing 23
2.2.10.6 Adverse Effect Profile 24
2.2.11 Tetracyclines 25
2.2.11.1 Background 25
2.2.11.2 Mechanism of Action 25
2.2.11.3 Mechanisms of Resistance 25
2.2.11.4 Classification 25
2.2.11.5 Antimicrobial Activity and Therapeutic Uses 25
2.2.11.6 Adverse Effect Profile 25
2.2.12 Glycylcyclines 26
2.2.12.1 Background 26
2.2.12.2 Mechanism of Action 26
2.2.12.3 Antimicrobial Activity and Therapeutic Uses 26
2.2.12.4 Adverse Effect Profile 27
2.2.13 Macrolides 27
2.2.13.1 Background 27
2.2.13.2 Mechanism of Action 28
2.2.13.3 Antimicrobial Activity and Therapeutic Uses 28
2.2.13.4 Adverse Effect Profile 29
2.2.14 Ketolides 29
2.2.15 Fluoroquinolones 29

2.2.15.1 Background 29
2.2.15.2 Mechanism of Action 30
2.2.15.3 Antimicrobial Activity and Therapeutic Uses 31
2.2.15.4 Adverse Effect Profile 31
2.2.16 Sulfonamides and Trimethoprim 31
2.2.16.1 Background 31
2.2.16.2 Mechanism of Action 32
2.2.16.3 Antimicrobial Activity and Therapeutic Uses 32
2.2.16.4 Adverse Effect Profile 32
2.3 Antimycobacterial Agents 32
2.3.1 Isoniazid (INH) 32
2.3.2 Rifampin, Rifabutin, and Rifapentine 33
2.3.3 Pyrazinamide (PZA) 34
2.3.4 Ethambutol (ETH) 34
2.3.5 Antimicrobial Activity and Therapeutic Uses 34
2.4 Antiviral Agents 36
2.4.1 Antiretroviral Agents (Fusion Inhibitors, Nucleoside and Nucleotide Reverse
Transcriptase Inhibitors, Nonnucleoside Reverse Transcriptase Inhibitors,
and Protease Inhibitors) 36
2.4.1.1 Mechanisms of Action 36
2.4.1.2 Mechanisms of Resistance 36
2.4.1.3 Classification 36
2.4.1.4 Antiretroviral Activity and Therapeutic Uses 37
2.4.1.5 Adverse Effect Profile 38


Antimicrobial Classifications

9

2.4.2 Antiviral Drugs 40
2.4.2.1 Mechanisms of Action 40
2.4.2.2 Mechanisms of Resistance 40
2.4.2.3 Classification 40
2.4.2.4 Antiviral Effects and Therapeutic Uses 40
2.4.2.5 Adverse Effect Profile 40
2.5 Antifungal Agents 40
2.5.1 Background 40
2.5.2 Mechanisms of Action 43
2.5.3 Mechanisms of Resistance 43
2.5.4 Classification 44
2.5.5 Antifungal Activity and Therapeutic Uses 45
2.5.6 Adverse Effect Profile 47
References 48

2.1 INTRODUCTION

Physicians have used drugs for decades to treat infections. However, chemotherapy as a science began
with Paul Ehrlich in the late 1800s. Dr. Ehrlich was a German medical scientist who received the Nobel
Prize for Physiology of Medicine in 1908. He realized that like human and animal cells, certain bacteria
cells colored with certain dyes while others did not. He postulated that it might be possible to make
certain dyes, or chemicals, that would kill bacteria while not harming the host organism. He conducted
hundreds to thousands of experiments testing dyes against various microorganisms. It wasn’t until his
606th experimental compound that he discovered a medically useful drug. This compound, later named

salvarsan

, was arsenic based, and the first treatment for syphilis [1]. In 1889, Vuillemin, a French
bacteriologist, suggested using the word

antiobiosis

, meaning “against life,” to describe the group of
drugs that had action against microorganisms [2]. Selman Waksman, an American microbiologist and
the discoverer of streptomycin, later changed this term to

antibiotic

[3]. Many antibiotics have been
discovered since then (Table 2.1), but the discovery of penicillin may be one of the most important
events in the practice of infectious disease medicine. To date, the U.S. Food and Drug Administration
(FDA) has approved 18 antibiotics derived from penicillin and 25 classified as cephalosporins [4,5].
Traditionally, antimicrobial agents have been classified based on their mechanism of action,
chemical structure, or spectrum of activity. The primary mode of action is the inhibition of vital
steps in the growth or function of the microorganism. These steps include inhibiting bacterial or
fungal cell wall synthesis, inhibiting protein synthesis, inhibiting nucleic acid synthesis, or disrupt-
ing cell membrane function (Table 2.2) [6].
Antibiotics are often described as “bacteriostatic” or “bactericidal” (Table 2.2). The term
bacteriostatic describes a drug that temporarily inhibits the growth of the organism. Once the drug
is removed, the organism will resume growth. The term bactericidal describes a drug that causes
cell death. For infections that cannot be eradicated by host mechanisms (e.g., endocarditis) or for
patients who are immunocompromised (e.g., Acquired Immunodeficiency Syndrome), a bactericidal
drug is often required [7].

2.2 ANTIBIOTICS
2.2.1 P

ENICILLINS

2.2.1.1 Background

Penicillin was first discovered in 1928 by a young Scottish bacteriologist, Alexander Fleming,
while studying staphylococcal bacteria at St. Mary’s Hospital in London [8–10]. Unlike his


10

Antimicrobial Susceptibility Testing Protocols

TABLE 2.1
Origin of Antibiotic Classes

Antibiotic Class Antibiotic(s)
Origin
Discoverer(s)
Year of Discovery

Sulfonamides Sulfanilamide
(not used as an antibiotic)
An azo dye
Paul Gelmo
1908
Prontosil
An azo dye
Gerhard Domagk
1932
Sulfanilamide
(active component of prontosil)
An azo dye
Trefoul, Nitti, and Bovet

1935
Penicillins
Penicillin

Penicillium notatum


Alexander Fleming
Howard Florey and Ernest Chain
1928
Therapeutic usefulness
recorded in 1940
Aminoglycosides Streptomycin

Streptomyces griseus

from soil samples
Selman Waksman
1944
Gentamicin
Micromonospora
Weinstein
1963
Chloramphenicol Chloramphenicol

Streptomyces venezuelae

from soil samples collected
in Venezuela
John Ehrlich, Q.R. Bartz, R.M. Smith,

D.A. Joslyn, and P.R. Burkholder
Published in 1947
Cephalosporins Cephalothin

Cephalosporium acremonium

from water samples
obtained off the Sardinian coast



Giuseppi Brotzu
1948
Tetracyclines Chlortetracycline

Streptomyces aureofaciens

from soil samples collected
in Missouri
Benjamin Duggar
1948
Macrolides
Erythromycin

Streptomyces erythreus

from soil samples collected in
the Philippine Archipelago
James McGuire and R.L. Bunch, R.C.
Anderson, H.E. Boaz, E.H. Flynn,

H.M. Powell, and J.W. Smith.
Recorded in 1952
Glycopeptides Vancomycin

Streptomyces orientalis

from soil samples collected in
Indonesia and India
McCormick
1956
Lincosamides Lincomycin

Streptomyces lincolnensis

from soil samples collected
near Lincoln, Nebraska
1962
Fluoroquinolones Nalidixic acid
A byproduct of chloroquine synthesis
Lesher
1962

Source: Ryan, F.

The Forgotten Plague

, Little, Brown, New York, 1993; Aronson, S.M.

Med Health

, 80(6), 180, 1997; Radetsky, M.

Pediatr Infect Dis J

, 15(9), 811–818, 1996;
Thredlekd, D.S., Ed.,

Drug Facts and Comparisons

, Facts and Comparisons; St. Louis, 2006; McEvoy, G.K., Ed.,

AHFS Drug Information

, American Society of Health-System
Pharmacists, Inc., Bethesda, 2006; Jawetz, E., in

Basic and Clinical Pharmacology

, Katzung, B.G., Ed., Appleton and Lange; Norwalk, CT, 1989, pp. 545–552;
Woods, G.L. and
W
ashington, J.A., in

Principles and Practices of Infectious Diseases

, Mandell, G.L., Bennett, J.E., and Dolin, R., Eds., Churchill Li
vingstone, New York, 1995, pp. 169–199.