Antimicrobial Therapy in Veterinary Medicine

Antimicrobial
Therapy in
Veterinary Medicine

Fifth Edition

Antimicrobial
Therapy in
Veterinary Medicine

Fifth Edition

Editors

Steeve Giguère

DVM, PhD, DACVIM
Professor, Large Animal Internal Medicine
Marguerite Hodgson Chair in Equine Studies
College of Veterinary Medicine
University of Georgia

John F. Prescott

MA, VetMB, PhD
Professor
Department of Pathobiology
University of Guelph

Patricia M. Dowling

DVM, MS, DACVIM, DACVCP
Professor, Veterinary Clinical Pharmacology
Veterinary Biomedical Sciences
University of Saskatchewan

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Contents

Contributors ix
Preface xiii
Important Notice xv
Abbreviations xvii

SECTION I GENERAL PRINCIPLES OF ANTIMICROBIAL THERAPY 1
1 3
2 Antimicrobial Drug Action and Interaction: An Introduction 11
3 Steeve Giguère 21
4 41
5 Antimicrobial Susceptibility Testing Methods and Interpretation of Results 79
6 Joseph E. Rubin 105
7 117
Antimicrobial Resistance and Its Epidemiology
Patrick Boerlin and David G. White 133
135
Principles of Antimicrobial Drug Bioavailability and Disposition 153
J. Desmond Baggot and Steeve Giguère 175
189
The Pharmacodynamics of Antimicrobial Agents 199
Marilyn N. Martinez, Pierre-Louis Toutain, and John Turnidge 211
233
Principles of Antimicrobial Drug Selection and Use
Steeve Giguère v

Antimicrobial Stewardship in Animals
J. Scott Weese, Stephen W. Page, and John F. Prescott

SECTION II CLASSES OF ANTIMICROBIAL AGENTS

8 Beta-lactam Antibiotics: Penam Penicillins
John F. Prescott

9 Beta-lactam Antibiotics: Cephalosporins
John F. Prescott

10 Other Beta-lactam Antibiotics: Beta-lactamase Inhibitors, Carbapenems, and Monobactams
John F. Prescott

11 Peptide Antibiotics: Polymyxins, Glycopeptides, Bacitracin, and Fosfomycin
Patricia M. Dowling

12 Lincosamides, Pleuromutilins, and Streptogramins
Steeve Giguère

13 Macrolides, Azalides, and Ketolides
Steeve Giguère

14 Aminoglycosides and Aminocyclitols
Patricia M. Dowling

vi Contents 257
269
15 Tetracyclines 279
Jérôme R.E. del Castillo 295
315
16 Chloramphenicol, Thiamphenicol, and Florfenicol 333
Patricia M. Dowling
357
17 Sulfonamides, Diaminopyrimidines, and Their Combinations 359
John F. Prescott 379
395
18 Fluoroquinolones 421
Steeve Giguère and Patricia M. Dowling 431
443
19 Miscellaneous Antimicrobials: Ionophores, Nitrofurans, Nitroimidazoles, Rifamycins, and Others
Patricia M. Dowling 455
457
20 Antifungal Chemotherapy 473
Steeve Giguère 495
519
SECTION III SPECIAL CONSIDERATIONS 529
541
21 Prophylactic Use of Antimicrobial Agents, and Antimicrobial Chemotherapy for the Neutropenic Patient
Steeve Giguère, Anthony C.G. Abrams-Ogg, and Stephen A. Kruth

22 Performance Uses of Antimicrobial Agents and Non-antimicrobial Alternatives
Thomas R. Shryock and Stephen W. Page

23 Antimicrobial Therapy of Selected Organ Systems
Patricia M. Dowling

24 Antimicrobial Therapy of Selected Bacterial Infections
Steeve Giguère

25 Antimicrobial Drug Residues in Foods of Animal Origin
Patricia M. Dowling

26 Regulation of Antimicrobial Use in Animals
Karolina Törneke and Christopher Boland

SECTION IV ANTIMICROBIAL DRUG USE IN SELECTED ANIMAL SPECIES

27 Antimicrobial Drug Use in Horses
Steeve Giguère and Tiago Afonso

28 Antimicrobial Drug Use in Dogs and Cats
Jane E. Sykes

29 Antimicrobial Drug Use in Cattle
Michael D. Apley and Johann F. Coetzee

30 Antimicrobial Drug Use in Mastitis
Sarah Wagner and Ron Erskine

31 Antimicrobial Drug Use in Sheep and Goats
Chris R. Clark

32 Antimicrobial Drug Use in New World Camelids
Christopher K. Cebra and Margaret L. Cebra

33 Antimicrobial Drug Use in Swine Contents vii
34 David G.S. Burch
35 553
36 Antimicrobial Drug Use in Poultry 569
37 Charles L. Hofacre, Jenny A. Fricke, and Tom Inglis 589
38 601
39 Antimicrobial Drug Use in Companion Birds 623
Index Keven Flammer 637
645
Antimicrobial Drug Use in Rabbits, Rodents, and Ferrets 663
Colette L. Wheler

Antimicrobial Drug Use in Reptiles
Ramiro Isaza and Elliott R. Jacobson

Antimicrobial Drug Use in Zoological Animals
Ellen Wiedner and Robert P. Hunter

Antimicrobial Drug Use in Aquaculture
Renate Reimschuessel, Ron A. Miller, and Charles M. Gieseker

Contributors

Chapter numbers are in parentheses. Christopher Boland (26)
Director, Capstone Consultants
Anthony C.G. Abrams-Ogg (21) Advisor to Agricultural Compounds and
Associate Professor Veterinary Medicines Group
Department of Clinical Studies New Zealand Ministry for Primary Industries
Ontario Veterinary College Wellington, New Zealand
University of Guelph
Ontario, Canada David G.S. Burch (33)
Director
Tiago Afonso (27) Octagon Services Ltd.
Graduate Research Assistant The Round House, The Friary, Old Windsor
College of Veterinary Medicine Berkshire, United Kingdom
University of Georgia
Athens, Georgia Christopher K. Cebra (32)
Professor
Michael D. Apley (29) Department of Clinical Sciences
Professor and Section Head College of Veterinary Medicine
Department of Clinical Sciences Oregon State University
Kansas State University Corvallis, Oregon
College of Veterinary Medicine
Manhattan, Kansas Margaret L. Cebra (32)
Private Consultant
J. Desmond Baggot (4) 33766 SE Terra Circle
75 Morehampton Square Corvallis, Oregon
Dublin 4, Ireland
Chris R. Clark (31)
Patrick Boerlin (3) Assistant Professor, Large Animal Medicine
Associate Professor Department of Large Animal Clinical Sciences
Department of Pathobiology Western College of Veterinary Medicine
Ontario Veterinary College University of Saskatchewan
University of Guelph Saskatoon, Saskatchewan, Canada
Ontario, Canada
ix

x Contributors

Johann F. Coetzee (29) Charles M. Gieseker (39)
Associate Professor, CYCADS Office of Research
Section Leader Center for Veterinary Medicine
Veterinary Diagnostic & Production U.S. Food and Drug Administration
Animal Medicine Laurel, Maryland
College of Veterinary Medicine
Iowa State University Steeve Giguère (1, 4, 6, 12, 13, 18,
Ames, Iowa 20, 21, 24, 27)
Professor, Large Animal
Jérôme R.E. del Castillo (15) Internal Medicine
Associate Professor of Veterinary Pharmacology Marguerite Hodgson Chair
and Toxicology in Equine Studies
Département de Biomédecine Vétérinaire, College of Veterinary Medicine
Université de Montréal University of Georgia
St-Hyacinthe, Québec, Canada Athens, Georgia

Patricia M. Dowling (11, 14, 16, 18, Charles L. Hofacre (34)
19, 23, 25) Professor
Professor, Veterinary Population Health
Clinical Pharmacology College of Veterinary Medicine
Veterinary Biomedical Sciences University of Georgia
University of Saskatchewan Athens, Georgia
Saskatoon, Saskatchewan, Canada
Robert P. Hunter (38)
Ron Erskine (30) Principal Consultant
Professor Emerging Markets Regulatory
Large Animal Clinical Sciences Elanco Animal Health
College of Veterinary Medicine Greenfield, Indiana
East Lansing, Michigan
Tom Inglis (34)
Keven Flammer (35) Poultry Health Services Ltd.
Professor of Avian Medicine Airdrie, Alberta, Canada
Clinical Sciences
College of Veterinary Medicine Ramiro Isaza (37)
North Carolina State University Associate Professor
Raleigh, North Carolina Department of Small Animal
Clinical Sciences
Jenny A. Fricke (34) College of Veterinary Medicine
Associate Veterinarian University of Florida
Poultry Health Services Ltd. Gainesville, Florida
Airdrie, Alberta, Canada

Elliott R. Jacobson (37) Contributors xi
Emeritus Professor
Department of Small Animal Renate Reimschuessel (39)
Clinical Sciences Director
College of Veterinary Medicine Veterinary Laboratory Response Network
University of Florida Center for Veterinary Medicine
Gainesville, Florida U.S. Food and Drug Administration
Laurel, Maryland
Stephen A. Kruth (21)
Professor Emeritus Joseph E. Rubin (2)
Department of Clinical Studies Assistant Professor
Ontario Veterinary College Department of Veterinary Microbiology
University of Guelph University of Saskatchewan
Ontario, Canada Saskatoon, Saskatchewan, Canada

Marilyn N. Martinez (5) Thomas R. Shryock (22)
Senior Research Scientist Senior Research Advisor—Microbiology
Office of New Animal Drug Evaluation Global Regulatory Affairs
Center for Veterinary Medicine Elanco Animal Health
U.S. Food and Drug Administration Greenfield, Indiana
Rockville, Maryland
Jane E. Sykes (28)
Ron A. Miller (39) Professor
Regulatory Review Microbiologist Department of Medicine & Epidemiology
Office of New Animal Drug Evaluation University of California, Davis
Center for Veterinary Medicine Davis, California
U.S. Food and Drug Administration
Rockville, Maryland Karolina Törneke (26)
Senior Expert
Stephen W. Page (7, 22) Medical Products Agency
Director Uppsala, Sweden
Advanced Veterinary Therapeutics
Newtown NSW, Australia Pierre-Louis Toutain (5)
Professor, Emeritus
John F. Prescott (7, 8, Ecole Nationale Veterinaire de Toulouse
9, 10, 17) France
Professor
Department of Pathobiology John Turnidge (5)
University of Guelph Clinical Professor
Ontario, Canada Pathology and Paediatrics, Faculty of
Health Sciences
University of Adelaide
Australia

xii Contributors David G. White (3)
Director, Office of Research
Sarah Wagner (30) Center for Veterinary Medicine
Associate Professor U.S. Food and Drug Administration
Department of Animal Sciences Laurel, Maryland
North Dakota State University
Fargo, North Dakota Ellen Wiedner (38)
Clinical Lecturer in Zoo and
J. Scott Weese (7) Wildlife Medicine
Professor College of Veterinary Medicine
Pathobiology University of Florida
Ontario Veterinary College Gainesville, Florida
University of Guelph
Ontario, Canada

Colette L. Wheler (36)
Saskatchewan Poultry Extension Veterinarian
Department of Veterinary Pathology, Western
College of Veterinary Medicine
University of Saskatchewan
Saskatoon, Saskatchewan, Canada

Preface

The field of anti-infective therapy has expanded agents, and antimicrobial drug residues in foods of
considerably since the first edition of Antimicrobial animal origin have been revised extensively against
Therapy in Veterinary Medicine was published in 1988. the  background of new regulations and the extensive
The fifth edition is a completely updated and considera- re-examination in many countries of the use of antimi-
bly expanded version of the previous edition, with the crobial agents as growth promoters or in the prevention
same aim of providing a comprehensive source for this of disease in animals. The final section addresses the
crucial topic in veterinary medicine. Everyone working specific principles of antimicrobial therapy in multiple
with antimicrobial drugs is aware of the continuing veterinary species. A chapter on antimicrobial therapy
threat of resistance and of the important role that each of in zoological animals has been added to this edition to
us plays in trying to preserve the efficacy of these drugs. reflect the increase in popularity of these species.

The book is divided into four sections. The first pro- Two members of the previous editorial team (J.D.
vides general principles of antimicrobial therapy and Baggot and R.D. Walker) have retired. We thank them
includes a new chapter on antimicrobial stewardship. for their outstanding contributions over the years and
The second section describes each class of antimicrobial we wish them the best in their new endeavors. The fifth
agents, revised to include not only the most up-to-date edition welcomes 13 new contributors. We are grateful
information on antimicrobial agents specific to veteri- to all the contributors for the care and effort they have
nary species but also newly developed drugs not yet put into their chapters. We thank the staff of Wiley
used in veterinary medicine. The third section deals Blackwell Publishing, particularly Susan Engelken and
with special considerations. It includes chapters on pro- Erica Judisch, for their help, patience, and support of
phylactic and metaphylactic use of antimicrobial agents, this book. We encourage readers to send us comments
antimicrobial chemotherapy for the neutropenic patient, or suggestions for improvements so that future editions
and approach to therapy of selected bacterial pathogens can be improved.
and organ systems. Chapters on regulations of antibiotic
use in animals, performance uses of antimicrobial Steeve Giguère, John Prescott, and Patricia Dowling

xiii

Important Notice

The indications and dosages of all drugs in this book are use in the diseases and dosages recommended. In
the recommendations of the authors and do not always addition, while every effort has been made to check the
agree with those given on package inserts prepared by contents of this book, errors may have been missed. The
pharmaceutical manufacturers in different countries. package insert for each drug product should therefore
The medications described do not necessarily have the be consulted for use, route of administration, dosage,
specific approval of national regulatory authorities, and (for food animals) withdrawal period, as approved
including the U.S. Food and Drug Administration, for by the reader’s national regulatory authorities.

xv

Abbreviations

Abbreviations used in this book include:

MIC minimum inhibitory concentration

MBC minimum bactericidal concentration

PO per os, oral administration

IM intramuscular administration

IV intravenous administration

SC subcutaneous administration

SID single daily administration

BID twice-daily administration (every 12 hours)

TID 3 times daily administration (every 8 hours)

QID 4 times daily administration (every 6 hours)

q 6 h, q 8 h, q 12 h, etc. Every 6, 8, 12 hours, etc.

For example, a dosage of “10 mg/kg TID IM” means 10 milligrams of the drug per kilogram of body weight,
administered every 8 hours intramuscularly.

xvii

Section I

General Principles of Antimicrobial Therapy

Antimicrobial Drug Action and 1
Interaction: An Introduction

Steeve Giguère

Antimicrobial drugs exploit differences in structure or The marked structural and biochemical differences
biochemical function between host and parasite. Modern between prokaryotic and eukaryotic cells give antimi-
chemotherapy is traced to Paul Ehrlich, a pupil of Robert crobial agents greater opportunities for selective toxicity
Koch, who devoted his career to discovering agents that against bacteria than against other microorganisms such
possessed selective toxicity so that they might act as so- as fungi, which are nucleated like mammalian cells, or
called “magic bullets” in the fight against infectious dis- viruses, which require their host’s genetic material for
eases. The remarkable efficacy of modern antimicrobial replication. Nevertheless, in recent years increasingly
drugs still retains a sense of the miraculous. Sulfonamides, effective antifungal and antiviral drugs have been intro-
the first clinically successful broad-spectrum antibacte- duced into clinical practice.
rial agents, were produced in Germany in 1935.
Important milestones in the development of antibacterial
However, it was the discovery of the antibiotic peni- drugs are shown in Figure 1.1. The therapeutic use of these
cillin, a fungal metabolite, by Fleming in 1929, and its agents in veterinary medicine has usually followed their use
subsequent development by Chain and Florey during in human medicine because of the enormous costs of devel-
World War II, that led to the antibiotic revolution. opment. However, some antibacterial drugs have been
Within a few years of the introduction of penicillin, developed specifically for animal health and production
many other antibiotics were described. This was (e.g., tylosin, tiamulin, tilmicosin, ceftiofur, tulathromycin,
followed by the development of semisynthetic and gamithromycin, tildipirosin). Figure 1.1 highlights the rela-
synthetic (e.g., sulfonamides and fluoroquinolones) tionship between antibiotic use and the development of
antimicrobial agents, which has resulted in an increas- resistance in many target microorganisms.
ingly powerful and effective array of compounds used to
treat infectious diseases. In relation to this, the term Spectrum of Activity of Antimicrobial Drugs
antibiotic has been defined as a low molecular weight
substance produced by a microorganism that at low Antimicrobial drugs may be classified in a variety of
concentrations inhibits or kills other microorganisms. ways, based on four basic features.
In contrast, the word antimicrobial has a broader defini-
tion than antibiotic and includes any substance of natu- Class of Microorganism
ral, semisynthetic, or synthetic origin that kills or
inhibits the growth of a microorganism but causes little Antiviral and antifungal drugs generally are active only
or no damage to the host. In many instances, antimicro- against viruses and fungi, respectively. However, some imi-
bial agent is used synonymously with antibiotic. dazole antifungal agents have activity against staphylococci

Antimicrobial Therapy in Veterinary Medicine, Fifth Edition. Edited by Steeve Giguère, John F. Prescott and Patricia M. Dowling.
© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

3

Human infectious diseases 8 Antibacterial agents
1930
Serious infections respond to sulfonamide Penicillin discovered
Florey demonstrates penicillin's effectiveness 2
4 First sulfonamide released
Penicillin-resistant infections 6
become clinically significant 8 Streptomycin, first aminoglycoside
'40 Chloramphenicol
Gentamicin-resistant Pseudomonas and 2 Chlortetracycline, first tetracycline
methicillin-resistant staphylococcal infections 4
6 Erythromycin, first macrolide
become clinically significant 8
Beginning in early 1970s, increasing '50 Vancomycin
trend of nosocomial infections due to 2
4 Methicillin, penicillinase-resistant penicillin
opportunistic pathogens 6
Ampicillin-resistant infections become frequent 8 Gentamicin, antipseudomonal penicillin
'60 Ampicillin
AIDS-related bacterial infections 2 Cephalothin, first cephalosporin
4
Expansion of methicillin-resistant staphylococcal 6 Amikacin, aminoglycoside for
infections 8 gentamicin-resistant strains
'70 Carbenicillin, first antipseudomonal beta-lactam
Vancomycin-resistant enterococci 2
Multidrug-resistant Mycobacterium tuberculosis 4 Cefoxitin, expanded-spectrum cephalosporin
Penicillin-resistant Streptococcus pneumoniae 6 Cefaclor, oral cephalosporin with improved activity
8 Cefotaxime, antipseudomonal cephalosporin
Spread of extended-spectrum '80 Clavulanic-acid-amoxicillin, broad beta-lactamase inhibitor
beta-lactamases among Gram-negatives 2 Imipenem-cilastatin
4 Norfloxacin, newer quinolone for urinary tract infections
Multidrug-resistant Pseudomonas, 6 Aztreonam, first monobactam
Acinetobacter baumanii, and S. pneumoniae 8 Newer fluoroquinolone for systemic use
'90 Improved macrolides
2
4 Oral extended-spectrum cephalosporins
6 Effective antiviral drugs for HIV
8 Quinupristin-dalfopristin
2000 Linezolid, first approved oxazolidinone
2 Broader-spectrum fluoroquinolones
4 Telithromycin, first ketolide
6 Tigecycline, first glycylcycline
8 Retapamulin, first pleuromutilin (topical)
10 Doripenem
Telavancin, semi-synthetic derivative of vancomycin
Ceftaroline

Figure 1.1. Milestones in human infectious disease and their relationship to development of antibacterial drugs. Modified
and reproduced with permission from Kammer, 1982.

4

Chapter 1. Antimicrobial Drug Action and Interaction 5

Table 1.1. Spectrum of activity of common antibacterial drugs.

Class of Microorganism

Drug Bacteria Fungi Mycoplasma Rickettsia Chlamydia Protozoa

Aminoglycosides + − + − − −
Beta-lactams + − − − − −
Chloramphenicol + − + + + −
Fluoroquinolones + − + + + −
Glycylcyclines + + + + +/−
Lincosamides + − + − − +/−
Macrolides + − + − + +/−
Oxazolidinones + − + − − −
Pleuromutilins + − + − + −
Tetracyclines + − + + + +/−
Streptogramins + − + − + +/−
Sulfonamides + − + − + +
Trimethoprim + − − − − +

+/–: Activity against some protozoa.

and nocardioform bacteria. Antibacterial agents are aminoglycosides) and others are usually bacteriostatic
described as narrow-spectrum if they inhibit only bacteria (e.g., chloramphenicol, tetracyclines), but this distinction
or broad-spectrum if they also inhibit mycoplasma, rickett- is an approximation, depending on both the drug con-
sia, and chlamydia. The spectrum of activity of common centration at the site of infection and the microorganism
antibacterial agents is shown in Table 1.1. involved. For example, benzyl penicillin is bactericidal
at usual therapeutic concentrations and bacteriostatic at
Antibacterial Activity low concentrations.

Some antibacterial drugs are also considered narrow- Time- or Concentration-Dependent Activity
spectrum in that they inhibit only Gram-positive or
Gram-negative bacteria, whereas broad-spectrum drugs Antimicrobial agents are often classified as exerting
inhibit both Gram-positive and Gram-negative bacteria. either time-dependent or concentration-dependent
However, this distinction is not always absolute, as some activity depending on their pharmacodynamic prop-
agents may be primarily active against Gram-positive bac- erties. The pharmacodynamic properties of a drug
teria but will also inhibit some Gram-negatives (Table 1.2). address the relationship between drug concentration
and antimicrobial activity (chapter 5). Drug pharma-
Bacteriostatic or Bactericidal Activity cokinetic features, such as serum concentrations over
time and area under the serum concentration-time
The minimum inhibitory concentration (MIC) is the curve (AUC), when integrated with MIC values, can
lowest concentration of an antimicrobial agent required predict the probability of bacterial eradication and
to prevent the growth of the pathogen. In contrast, the clinical success. These pharmacokinetic and pharma-
minimum bactericidal concentration (MBC) is the low- codynamic relationships are also important in pre-
est concentration of an antimicrobial agent required to venting the selection and spread of resistant strains.
kill the pathogen. Antimicrobials are usually regarded as The most significant factor determining the efficacy
bactericidal if the MBC is no more than 4 times the of beta-lactams, some macrolides, tetracyclines, tri-
MIC. Under certain clinical conditions this distinction methoprim-sulfonamide combinations, and chloram-
is important, but it is not absolute. In other words, phenicol is the length of time that serum concentrations
some  drugs are often bactericidal (e.g., beta-lactams,

6 Section I. General Principles of Antimicrobial Therapy

Table 1.2. Antibacterial activity of selected antibiotics.

Aerobic Bacteria Anaerobic Bacteria

Spectrum Gram + Gram – Gram + Gram – Examples

Very broad ++ ++ Carbapenems; chloramphenicol;
third-generation
Intermediately broad + + + (+) fluoroquinolones; glycylcyclines
Narrow
+ (+) + (+) Third- and fourth-generation
cephalosporins
(+) (+) (+) (+)
+ +/− + (+) Second-generation
cephalosporins
+ − + (+)
Tetracyclines
+ +/– + (+) Ampicillin; amoxicillin;
+/− + −−
(+) + −− first-generation cephalosporins
Penicillin; lincosamides;
(+) (+) −−
− − ++ glycopeptides; streptogramins;
+ − (+) (+) oxazolidinones
Macrolides
Monobactams; aminoglycosides
Second-generation
fluoroquinolones
Trimethoprim-sulfa
Nitroimidazoles
Rifamycin

+: Excellent activity.
(+): Moderate activity.
+/−: Limited activity.
−: No or negligible activity.

exceed the MIC of a given pathogen. Increasing the of efficacy for these drugs is the 24-hour area under
concentration of the drug several-fold above the MIC the serum concentration versus time curve (AUC)/
does not significantly increase the rate of microbial MIC ratio. Glycopeptides, rifampin, and, to some
killing. Rather, it is the length of time that bacteria are extent, fluoroquinolones fall within this category
exposed to concentrations of these drugs above the (chapter 5).
MIC that dictates their rate of killing. Optimal dosing
of such antimicrobial agents involves frequent admin- Mechanisms of Action of Antimicrobial
istration. Other antimicrobial agents such as the ami- Drugs
noglycosides, fluoroquinolones, and metronidazole
exert concentration-dependent killing characteristics. Antibacterial Drugs
Their rate of killing increases as the drug concentra-
tion increases above the MIC for the pathogen and it is Figure 1.2 summarizes the diverse sites of action of the
not necessary or even beneficial to maintain drug lev- antibacterial drugs. Their mechanisms of action fall into
els above the MIC between doses. Thus, optimal dos- four categories: inhibition of cell wall synthesis, damage
ing of aminoglycosides and fluoroquinolones involves to cell membrane function, inhibition of nucleic acid syn-
administration of high doses at long dosing intervals. thesis or function, and inhibition of protein synthesis.
Some drugs exert characteristics of both time- and
concentration-dependent activity. The best predictor Antibacterial drugs that affect cell wall synthesis
(beta-lactam antibiotics, bacitracin, glycopeptides) or

Chapter 1. Antimicrobial Drug Action and Interaction 7

Nitroimidazoles, Cell wall Beta-lactam
nitrofurans Cell membrane antibiotics,
glycopeptides,
Sulfonamides, Purine bacitracin
trimethoprim synthesis

DNA Fluoroquinolones Polyenes
Novobiocin

Transfer Rifampin Ribosome
RNA Messenger New
RNA
protein
Amino acids 30S

50S

Tetracyclines,
aminoglycosides

Oxazolidinones Chloramphenicol

Lincosamides,
macrolides,
streptogramins

Figure 1.2. Sites of action of commonly used antibacterial drugs that affect virtually all important processes in a bacterial
cell. Modified and reproduced with permission after Aharonowitz and Cohen, 1981.

inhibit protein synthesis (aminoglycosides, chloram- cell  membrane function (polymyxins) or nucleic acid
phenicol, lincosamides, glycylcyclines, macrolides, function (fluoroquinolones, nitroimidazoles, nitro-
oxazolidinones, streptogramins, pleuromutilins, tetra- furans, rifampin), although the development of fluoro-
cyclines) are more numerous than those that affect quinolones has been a major advance in antimicrobial

8 Section I. General Principles of Antimicrobial Therapy

therapy. Agents that affect intermediate metabolism drugs that are currently available. However, even if we
(sulfonamides, trimethoprim) have greater selective improve these practices, resistant bacteria will continue
toxicity than those that affect nucleic acid synthesis. to develop and new drugs will be needed.

Searching for New Antibacterial Drugs The approaches in the search for novel antibiotics
include further development of analogs of existing
Infection caused by antibiotic-resistant bacteria has agents; identifying novel targets based on a biotech-
been an increasingly growing concern in the last decade. nological approach, including use of information
The speed with which some bacteria develop resistance obtained from bacterial genome sequencing and gene
considerably outpaces the slow development of new cloning; screening of natural products from plants and
antimicrobial drugs. Since 1980, the number of antimi- microorganisms from unusual ecological niches other
crobial agents approved for use in people in the United than soil; development of antibacterial peptide mole-
States has fallen steadily (Figure  1.3). Several factors cules derived from phagocytic cells of many species;
such as complex regulatory requirements, challenges in screening for novel antimicrobials using combinato-
drug discovery, and the high cost of drug development rial chemical libraries; development of synthetic
coupled with the low rate of return on investment anti- antibacterial drugs with novel activities, such as oxa-
biotics provide compared with drugs for the treatment zolidinones; development of new antibiotic classes
of chronic conditions all contribute to driving pharma- that were abandoned early in the antibiotic revolution
ceutical companies out of the antimicrobial drug mar- because there were existing drug classes with similar
ket. This has left limited treatment options for infections activities; development of “chimeramycins” by labora-
caused by methicillin-resistant staphylococci and van- tory recombination of genes encoding antibiotics of
comycin-resistant enterococci. The picture is even different classes; and combination of antibacterial
bleaker for infections cause by some Gram-negative drugs with iron-binding chemicals targeting bacterial
bacteria such as Pseudomonas aeruginosa, Acinetobacter iron uptake mechanisms.
baumanii, and extended-spectrum beta-lactamase
(ESBL)-resistant E. coli, Klebsiella spp., and Enterobacter Antifungal Drugs
spp., which are occasionally resistant to all the antimi-
crobial agents on the market. Judicious use of the antibi- Most currently used systemic antifungal drugs dam-
otics currently available and better infection control age cell membrane function by binding ergosterols
practices might help prolong the effectiveness of the that are unique to the fungal cell membrane (polyenes,
azoles; chapter 20). The increase in the number of

Number of new antibacterial agents18
16
14
12
10

8
6
4
2
0

1980–1984 1985–1989 1990–1994 1995–1999 2000–2004 2005–2009 2010–2012

Figure 1.3. New antimicrobial agents approved for use in people in the United States since 1980.

Chapter 1. Antimicrobial Drug Action and Interaction 9

HIV-infected individuals and of people undergoing detect all such interactions. Although the techniques to
organ or bone marrow transplants has resulted in quantify and detect interactions are relatively crude, the
increased numbers of immunosuppressed individuals observed interactions occur clinically.
in many societies. The susceptibility of these people to
fungal infections has renewed interest in the discovery The two methods commonly used, the checkerboard
and development of new antifungal agents. The focus and the killing curve methods, measure two different
of antifungal drug development has shifted to cell wall effects (growth inhibition and killing, respectively) and
structures unique to fungi (1,3-β-D-glucan synthase have sometimes shown poor clinical and laboratory cor-
inhibitors, chitin synthase inhibitors, mannoprotein relation. In the absence of simple methods for detecting
binders; Figure 20.1). synergism or antagonism, the following general guide-
lines may be used.

Antibacterial Drug Interactions: Synergism, Synergism of Antibacterial Combinations
Antagonism, and Indifference
Antimicrobial combinations are frequently synergistic if
Knowledge of the different mechanisms of action of they involve (1) sequential inhibition of successive steps
antimicrobials provides some ability to predict their in metabolism (e.g., trimethoprim-sulfonamide); (2)
interaction when they are used in combination. It was sequential inhibition of cell wall synthesis (e.g., mecilli-
clear from the early days of their use that combinations nam-ampicillin); (3) facilitation of drug entry of one
of antibacterials might give antagonistic rather than antibiotic by another (e.g., beta-lactam-aminoglycoside);
additive or synergistic effects. Concerns regarding (4) inhibition of inactivating enzymes (e.g., amoxicillin-
combinations include the difficulty in defining syner- clavulanic acid); and (5) prevention of emergence of
gism and antagonism, particularly their method of resistant populations (e.g., macrolide-rifampin).
determination in vitro; the difficulty of predicting the
effect of a combination against a particular organism; Antagonism of Antibacterial Combinations
and the uncertainty of the clinical relevance of in vitro
findings. The clinical use of antimicrobial drug combi- To some extent the definition of antagonism as it
nations is described in chapter 6. Antimicrobial com- relates to antibacterial combinations reflects a labora-
binations are used most frequently to provide tory artifact. However, there have been only a few well-
broad-spectrum empiric coverage in the treatment of documented clinical situations where antagonism is
patients that are critically ill. With the availability clinically important. Antagonism may occur if anti-
of  broad-spectrum antibacterial drugs, combinations bacterial combinations involve (1) inhibition of bacte-
of  these drugs are less commonly used, except for ricidal activity such as treatment of meningitis in
specific purposes. which a bacteriostatic drug prevents the bactericidal
activity of another; (2) competition for drug-binding
An antibacterial combination is additive or indifferent sites such as macrolide-chloramphenicol combinations
if the combined effects of the drugs equal the sum of (of uncertain clinical significance); (3) inhibition of
their independent activities measured separately; syner- cell permeability mechanisms such as chlorampheni-
gistic if the combined effects are significantly greater col-aminoglycoside combinations (of uncertain clini-
than the independent effects; and antagonistic if the cal significance); and (4) induction of beta-lactamases
combined effects are significantly less than their inde- by beta-lactam drugs such as imipenem and cefoxitin
pendent effects. Synergism and antagonism are not combined with older beta-lactam drugs that are beta-
absolute characteristics. Such interactions are often hard lactamase unstable.
to predict, vary with bacterial species and strains, and
may occur only over a narrow range of concentrations The impressive complexity of the interactions of
or ratios of drug components. Because antimicrobial antibiotics, the fact that such effects may vary depend-
drugs may interact with each other in many different ing of the bacterial species, and the uncertainty of the
ways, it is apparent that no single in vitro method will applicability of in vitro findings to clinical settings make
predicting the effects of some combinations hazardous.
For example, the same combination may cause both
antagonism and synergism in different strains of the

10 Section I. General Principles of Antimicrobial Therapy

same bacterial species. Laboratory determinations are Bibliography
really required but may give conflicting results depend-
ing on the test used. Knowledge of the mechanism of Aharonowitz Y, Cohen G. 1981. The microbiological produc-
action is probably the best approach to predicting the tion of pharmaceuticals. Sci Am 245:141.
outcome of the interaction in the absence of other
guidelines. Boucher HW, et al. 2009. Bad bugs, no drugs: no ESKAPE!
An update from the Infectious Diseases Society of America.
In general, the use of combinations should be Clin Infect Dis 48:1.
avoided, because the toxicity of the antibiotics will be
at least additive and may be synergistic, because Bryskier A. 2005. In pursuit of new antibiotics. In: Bryskier A
the  ready availability of broad-spectrum bactericidal (ed). Antimicrobial Agents: Antibacterials and Antifungals.
drugs  has made their use largely unnecessary, and Washington, DC: ASM Press.
because they may be more likely to lead to bacterial
superinfection. There are, however, well-established Cantón R, et al. 2011. Emergence and spread of antibiotic
circumstances, discussed in chapter 6, in which resistance following exposure to antibiotics. FEMS
combinations of drugs are more effective and often less Microbiol Rev 35:977.
toxic than drugs administered alone.
Kammer RB. 1982. Milestones in antimicrobial therapy. In:
Morin RB, Gorman M (eds). Chemistry and Biology of
Beta-Lactam Antibiotics, vol. 3. Orlando: Academic Press.

Pillai SK, et al. 2005. Antimicrobial combinations. In: Lorian
V (ed). Antibiotics in Laboratory Medicine, 5th ed.
Philadelphia: Lippincott Williams and Wilkins.

Antimicrobial Susceptibility Testing 2
Methods and Interpretation of Results

Joseph E. Rubin

The veterinary diagnostic microbiology laboratory plays predictability of the susceptibility patterns of the most
a key role in the practice of evidence-based antimicro- likely pathogen(s). For example, susceptibility testing is
bial therapy by providing culture and susceptibility not indicated in horses with “strangles,” as S. equi
information to practitioners. Before the introduction of is uniformly susceptible to penicillin (Erol et al., 2012).
antimicrobials, we were largely powerless to treat inva- Similarly, culture and susceptibility testing is not
sive infections. The antimicrobial age began with the required for first time, uncomplicated urinary tract
familiar story of the discovery of penicillin in 1928 by infections in dogs, as empiric amoxicillin therapy is
Alexander Fleming. By the early 1940s that Penicillium advocated (Pressler et al., 2010).
notatum extract was successfully used against infections
caused by organisms ranging from Staphylococcus Early methods used to assess the susceptibility of
aureus to Neisseria gonorrhoeae (Aronson, 1992; organisms to antimicrobials were developed by indi-
Bryskier, 2005). Unfortunately, the evolutionary power vidual labs and lacked standardization; the first effort
of bacteria resulted in the rapid emergence of anti- to standardize susceptibility testing was published in
microbial resistance. Susceptibility testing is now vital 1971 (Ericsson et al., 1971). National standards organi-
to effective therapeutic decision making. zations responsible for guidelines for conducting and
interpreting antimicrobial susceptibility tests were
Although veterinary laboratories utilize many of the subsequently formed. In the United States, the Clinical
same basic microbiological techniques as human diag- and Laboratory Standards Institute (CLSI) formed in
nostic labs, they face some unique challenges. These the late 1960s as the National Committee for Clinical
challenges include the difficulty in cultivation of fasti- Laboratory Standards (NCCLS) and was tasked with
dious veterinary-specific organisms, selection of species- developing a standard for disk diffusion antimicrobial
customized antimicrobial panels for susceptibility susceptibility testing (Barry, 2007). While standardiza-
testing, and considerations of drug withdrawal times tion of methods yields more comparable data between
and food safety. labs, heterogeneity in interpretive criteria persists
(see Table 2.1). In 1997, the European Committee on
In the clinical setting, the goal of antimicrobial sus- Antimicrobial Susceptibility Testing (EUCAST) was
ceptibility testing is to help clinicians choose optimal formed to harmonize both testing methods and inter-
antimicrobial therapy. The decision to undertake cul- pretive criteria throughout Europe. In North America,
ture and susceptibility testing depends on the site of the CLSI methodologies are used for both human and
infection, state of the patient (otherwise healthy vs. veterinary diagnostics. The CLSI standards are availa-
critically ill), prior history of infections and antimicro- ble for purchase on their website (www.clsi.org), while
bial use, co-morbidities and underlying disease, and the

Antimicrobial Therapy in Veterinary Medicine, Fifth Edition. Edited by Steeve Giguère, John F. Prescott and Patricia M. Dowling.
© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

11

12 Section I. General Principles of Antimicrobial Therapy

Table 2.1. Test factors leading to spurious results.

Factor Artificially Resistant Artificially Susceptible

Expired reagents Mueller-Hinton agar used for diffusion-based testing that has dried out, may not Degraded drugs
be thick enough allowing the drug to diffuse out further leading to larger
Inoculum density zones of inhibition Too light an inoculum
Incubation period Inadequate incubation period
Incubation Too dense an inoculum Above 35°C methicillin resistance
Prolonged incubation period
temperature may not be expressed in MRSA
Medium Decreased divalent cations Increased divalent cations
pH too high or too low
Incubation atmosphere pH too high or too low
Endpoint definition Depending on drug, CO2 atmosphere may increase or decrease zone diameter or MIC
For the sulfonamides, endpoints are defined by 80% reduction in growth
Failure to identify
Mixed culture compared to control
Accurate identification of organism is required to interpret susceptibility test results
The phenotype of the more resistant organism may dominate

EUCAST publishes their guidelines free of charge on (A)
their website (www.eucast.org).

Antimicrobial Susceptibility Testing (B)
Methods

Antimicrobial susceptibility tests yield either categorical (C)
(susceptible, intermediate, or resistant) or quantitative
(minimum inhibitory concentration [MIC]) data that
can be categorically interpreted. Testing methods can
be  divided into two distinct categories, diffusion and
dilution based.

Diffusion-Based Methods (D)

Two types of diffusion tests are available that yield either (E)
categorical (disk diffusion) or quantitative (gradient
strip) susceptibility data. These tests are based on the (F)
inhibition of bacterial growth by antimicrobial diffusing
from a source disk or strip through solid media Figure 2.1. Disk diffusion: The results of the disk diffusion
(Figure 2.2). The size of the inhibitory zone is a function test can be influenced by the depth of the medium (A and B,
of the rate of drug diffusion, thickness of the media, increase in zone of inhibition; C, decrease in zone of inhibi-
concentration of drug in the disk, and the susceptibility tion) or the quality of the inoculum (D, false increase in zone
of the organism, making method standardization neces- of inhibition; E, false decrease in zone of inhibition; F, mixed
sary for interpretive criteria to be applied (Figure 2.1). culture, false decrease in zone of inhibition).

Disk diffusion testing is conducted on 4-mm
thick  Mueller-Hinton agar plates using antimicrobial
impregnated filter paper discs (CLSI, 2006a,b). Room-
temperature plates are inoculated with a lawn of bacteria
drawn from a McFarland 0.5 (approximately 108 CFU/
ml) suspension using a sterile swab. Plates are allowed to

Chapter 2. Antimicrobial Susceptibility Testing Methods and Interpretation of Results 13

dry for up to 15 minutes before the disk is applied and Diffusion-based tests are technically simple to per-
are then incubated at 35°C at room atmosphere. After form and versatile, allowing customization of test panels
up to 24 hours the zone of inhibition is measured to bacterial and patient species and type of infection.
(Figure 2.2A). Owing to differences in antimicrobial dif- While disk diffusion tests are less inexpensive than gra-
fusion rate, amount of drug included in disks, and phar- dient tests, they only provide categorical information
macodynamic interactions, the size of the inhibitory (susceptible, intermediate, or resistant).
zone corresponding to resistance breakpoints is unique
to each drug organism combination. The relative Dilution-Based Methods
clinical  appropriateness of different antimicrobials can
therefore not be determined by simply comparing Dilutional susceptibility testing can be done using either
inhibitory zone diameters. broth or agar media and yields quantitative (MIC) data.
Doubling dilutions of antimicrobial (. . . 0.12 μ g/ml,
Gradient tests (e.g., Etest) are conducted in the same 0.25 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml . . .) are tested.
way as disk tests. These strips contain a gradient of anti- An antimicrobial free control plate or broth must always
microbial from low to high concentrations corresponding be included. The lowest concentration without bacterial
to printed MIC values on the back of the strip. Following growth defines the MIC, except for the sulfonamides
incubation, the apex of the teardrop zone of inhibition and trimethoprim, where an 80% reduction in growth
indicates the MIC of the organism (Figure 2.2B). compared to the control constitutes inhibition.

(A) (B) (C)

2 μg/ml 4 μg/ml 8 μg/ml

Disk diffusion Antimicrobial 2 μg/ml, 4 μg/ml, 8 μg/ml
(Bauer-Kirby procedure) gradient method Etest® Agar dilution

(D) (E)

Broth microdilution Broth macrodilution

Figure 2.2. Antimicrobial susceptibility testing methods.

14 Section I. General Principles of Antimicrobial Therapy

For agar media dilution, Mueller-Hinton agar plates Interpretation of Susceptibility Test Results
are prepared incorporating doubling dilutions of anti-
microbial. Antimicrobial stock solutions at 10 times the Categorical interpretation of antimicrobial suscepti-
test concentration are prepared using the solvents and bility test results requires the development of clinical
diluents recommended by the CLSI (CLSI, 2006a,b). The resistance breakpoints. Resistance breakpoints are
mass of antimicrobial required is determined by the fol- designed to predict clinical outcomes: susceptible = high
lowing equation: probability of success following treatment, resist-
ant = low probability of success following treatment. For
Mass = [(Volume ml)(Concentration mg / ml)]/ an antimicrobial to be effective clinically, it must reach a
potency sufficiently high concentration at the site of infection to
inhibit growth or kill the organism. Resistance break-
To prepare media, antimicrobial stock solution is points are therefore related to achievable drug concen-
added in a 1:9 ratio to molten Mueller-Hinton agar no trations in target tissues. Because drug concentrations
hotter than 50°C, and poured into sterile petri dishes. vary in different body sites or fluids, pharmacokinetic
Separate plates are prepared for each antimicrobial con- studies are required to determine if therapeutic concen-
centration test. Plates must not be stored for more than trations are reached in target tissues. Resistance break-
7 days prior to use and for some drugs (e.g., imipenem), points are also specific to animal species, dosing regimen
they must be prepared fresh on the day of use (CLSI, (dose, route of administration, and frequency), disease,
2006a,b). Room-temperature plates are inoculated and target pathogen. When any of these factors are
with  approximately 104 CFU using either a multi-spot altered (e.g., drug given orally instead of injected), the
replicator or manually by pipette. To prevent discrete predictive value of resistance breakpoints for clinical
samples from mixing, plates are left on the bench top for outcomes cannot be relied upon. Veterinary-specific
up to 30 minutes for the bacterial spots to be absorbed resistance breakpoints are published by the CLSI. The
prior to incubation. Plates are incubated in room air CLSI human guidelines, EUCAST, and the British
at  35°C for 16–20 hours and examined for growth Society for Antimicrobial Chemotherapy (BSAC) are
(Figure  2.2C). Because this technique is very labor resources that may be useful when species-specific crite-
intensive, its use is mainly limited to research. ria are not available. However, extrapolation of non-
approved breakpoints should be done with extreme
For broth dilution, Mueller-Hinton broths contain- caution. The lack of validated veterinary-specific resist-
ing doubling dilutions of antimicrobial are prepared. As ance breakpoints is an important limitation for veteri-
in agar dilution, antimicrobial stock solutions at 10 narians trying to practice evidence-based medicine. As
times the final concentration are prepared and added to an example, there are no validated breakpoints for any
test medium in a 1:9 ratio. Each antimicrobial concen- pathogens causing enteric disease in veterinary species
tration is dispensed into separate vials and inoculated (Table 2.2).
with bacteria to yield a final concentration of 5 ×
105 CFU/mL. A McFarland 0.5 inoculum is typically Furthermore, when antimicrobials are used in food
made in either sterile water or saline and then aliquoted animals, the prescribing veterinarian is responsible for
into the Mueller-Hinton broth to yield the final concen- the prevention of violative drug residues. Expert-
tration. Growth is evidenced by turbidity and the MIC mediated advice regarding drug withdrawal periods is
is defined by the lowest concentration where growth is available from food animal residue avoidance databases.
not seen. In the United States, practitioners can contact www.
farad.org and in Canada, www.cgfarad.usask.ca.
Commercially prepared microdilution plates
(Figure 2.2D) allow a large number of bacterial isolates Because it is conceptually simple to think of an iso-
to be tested efficiently without the need to prepare, store, late’s susceptibility categorically (susceptible, intermedi-
and incubate large volumes of media in house. The ate, or resistant), it is tempting to classify an isolate as
efficiency of the microdilution method comes with susceptible or resistant even when no validated break-
increased costs for consumables. (Figure 2.2E). points exist. It is essential to remember that resistance
breakpoints are designed to be clinically predictive,

Chapter 2. Antimicrobial Susceptibility Testing Methods and Interpretation of Results 15

Table 2.2. Drugs with veterinary-specific CLSI resistance breakpoints.

Drug Animal Species/Pathogens

Gentamicin Canine (Enterobacteriaceae, Pseudomonas aeruginosa)
Spectinomycin Equine (Enterobacteriaceae, Pseudomonas aeruginosa, Actinobacillus spp.)
Ampicillin Bovine (respiratory disease—Mannheimia haemolytica, Pasteurella multocida, Histophilus somni)
Canine (skin and soft tissue infections—Staphylococcus pseudintermedius, Streptococcus canis; other
Penicillin-novobiocin
Cefpodoxime infections—Escherichia coli )
Ceftiofur Equine (respiratory disease—Streptococcus equi subsp. zooepidemicus)
Bovine (mastitis—Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis)
Danofloxacin Canine (wounds and abscesses—Staphylococcus aureus, Staphylococcus pseudintermedius, Streptococcus canis,
Enrofloxacin
Escherichia coli, Pasteurella multocida, Proteus mirabilis)
Difloxacin Bovine (respiratory disease—Mannheimia haemolytica, Pasteurella multocida, Histophilus somni; mastitis—
Marbofloxacin
Orbifloxacin Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli )
Clindamycin Porcine (respiratory disease—Actinobacillus pleuropneumoniae, Pasteurella multocida, Salmonella cholerasuis,
Pirlimycin
Tilmicosin Streptococcus suis)
Tulathromycin Equine (respiratory disease—Streptococcus equi subsp. zooepidemicus)
Florfenicol Bovine (respiratory disease—Mannheimia haemolytica, Pasteurella multocida)

Tiamulin Feline (dermal)
Oxytetracycline Canine (dermal, respiratory, and UTI—Enterobacteriaceae, Staphylococcus spp.)
Chickens and turkeys (Pasteurella multocida, Escherichia coli )
Bovine (respiratory disease—Mannheimia haemolytica, Pasteurella multocida, Histophilus somni)
Canine (dermal and UTI—Enterobacteriaceae, Staphylococcus spp.)

Feline (dermal)
Canine (dermal and UTI—Enterobacteriaceae, Staphylococcus spp.)

Feline (dermal)
Canine (dermal and UTI—Enterobacteriaceae, Staphylococcus spp.)
Canine (skin and soft tissue infections—Staphylococcus spp.)
Bovine (mastitis—Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis )
Bovine (respiratory disease—Mannheimia haemolytica)
Porcine (respiratory disease—Actinobacillus pleuropneumoniae, Pasteurella multocida )
Bovine (respiratory disease—Mannheimia haemolytica, Pasteurella multocida, Histophilus somni )
Bovine (respiratory disease—Mannheimia haemolytica, Pasteurella multocida, Histophilus somni )
Porcine (respiratory disease—Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida,

Streptococcus suis, Salmonella cholerasuis)
Porcine (respiratory disease—Actinobacillus pleuropneumoniae)
Bovine (respiratory disease—Mannheimia haemolytica, Pasteurella multocida, Histophilus somni )
Porcine (respiratory disease—Actinobacillus pleuropneumoniae, Pasteurella multocida, Streptococcus suis )

viewing antimicrobial susceptibility through the lens of and be clinically resistant or have an MIC above the
the patient by incorporating pharmacokinetic informa- epidemiological cut-off while remaining susceptible
tion. In contrast, epidemiological cut-offs describe anti- (Figure 2.3). While epidemiological cut-offs are invalu-
microbial susceptibility from the perspective of the able research tools, they do not incorporate pharma-
organism. Isolates with MICs above the epidemiological cokinetic data and should not be used to guide therapy
cut-off have acquired resistance mechanisms that make of patients.
them less susceptible to an antimicrobial than wild-type
organisms of the same species. Epidemiological cut-offs In practice, the application of antimicrobial suscepti-
are established by evaluating the MIC distributions of bility test results is reduced to susceptible = good treat-
large isolate collections. An organism can have an MIC ment choice and resistant = bad treatment choice, rather
below the epidemiological cut-off for a particular drug than a thorough analysis of the susceptibility profile.
Interpretive reading is a more biological approach that

16 Section I. General Principles of Antimicrobial Therapy

Ciprofloxacin MIC distribution for E. coli Gentamicin MIC distribution for P. aeruginosa
(source: EUCAST)
8000 10000 (source: EUCAST)

6000 7500

4000 Clinical breakpoint 5000 Epidemiological cut-off
Epidemiological cut-off Clinical breakpoint
2000 2500

0 0

0.002
0.004
0.008
0.016
0.032
0.064
0.0125

0.25
0.5
1
2
4
8
16
32
64
128
256
512

0.002
0.004
0.008
0.016
0.032
0.064
0.0125

0.25
0.5
1
2
4
8
16
32
64
128
256
512

2000 Ampicillin MIC distribution for P. mirabilis
(source: EUCAST)

1500

1000 Breakpoint and cut-off

500

0 0.002
0.004
0.008
0.016
0.032
0.064
0.0125

0.25
0.5
1
2
4
8
16
32
64
128
256
512

Figure 2.3. Comparison of clinical resistance breakpoints and epidemiological cut-off values from EUCAST databases. Each
histogram depicts the number of isolates (y axis) with each MIC (x axis). Epidemiological cut-offs are higher (E. coli and cipro-
floxacin), lower (P. aeruginosa and gentamicin), or the same (P. mirabilis and ampicillin) as clinical resistance breakpoints.

incorporates knowledge of intrinsic drug resistance, only  oxacillin resistance reliably predicts mecA in
indicator drugs, exceptional resistance phenotypes, and S.  pseudintermedius (CLSI, 2008a,b; Papich, 2010). In
consideration of antimicrobial selection pressure. For Enterobacteriaceae, a combination of ceftazidime and
example, “Enterococcus spp.” may be commonly reported cefotaxime with and without clavulanic acid is used to
by diagnostic labs, but identification at the species level detect ESBLs; a greater than or equal to eight-fold
(e.g., Enterococcus faecium vs. Enterococcus faecalis) is increase in susceptibility (decrease in the MIC) in the
necessary for interpretive reading. For an excellent clavulanic acid potentiated cephalosporins indicates the
review of interpretive reading, see Livermore (2001). presence of ESBL and therefore clinical resistance to all
Interpretive reading is used to detect specific resistance penicillins, cephalosporins, and aztreonam (CLSI,
phenotypes such as methicillin resistance or the produc- 2008a,b; Table 2.3).
tion of extended-spectrum beta-lactamases (ESBLs).
Some of these tests are organism specific and use across Knowledge of intrinsic resistance is invaluable when
species or genera may not yield reliable results. For interpreting susceptibility reports. Resistance should
example, the CLSI recommends that either cefoxitin always be assumed for certain drug-organism combina-
or  oxacillin resistance may be used as indicators of tions (e.g., cephalosporins and enterococci). Because in
mecA mediated methicillin resistance in S. aureus, while vitro resistance expression may not be reflective of drug-
organism interactions in vivo, isolates should be reported

Chapter 2. Antimicrobial Susceptibility Testing Methods and Interpretation of Results 17

Table 2.3. Failure of in vitro tests to predict in vivo outcomes.

Factor Positive Outcomes Negative Outcomes

Patient/Disease Factors Pharmacokinetic High urine drug concentrations Failure of drugs to penetrate sequestered sites such as the CNS or prostate
Pharmacodynamic Drug interactions decreasing absorption or increasing elimination
Organism/Test Factors No infection Failure of aminoglycosides in acidic or anaerobic environments
Disease/pathology Self-limiting infection Failure of folate synthesis inhibitors in purulent environments (excessive

Therapeutic Utilization of localized therapy, high PABA in environment)
concentrations overcoming Predisposing disease or underlying pathology such as atopy, diabetes, or
Resistance low-level resistance
Organism lifestyle neoplasia
Organism identification Different dose, dosing frequency, Indwelling medical device
Antimicrobial route of administration than label Different dose, dosing frequency, route of administration than label
susceptibility test Poor owner compliance
Misidentified organism
False positive culture Development of resistance in vivo
Incorrectly performed or reported Biofilm formation
Intracellular infections
test Misidentified organism
Mixed infection
Incorrectly performed or reported test
Inducible resistance

as resistant irrespective of in vitro test results where Table 2.4. Intrinsic resistance phenotypes of importance
intrinsic resistance is recognized. A detailed description to veterinary medicine.
of intrinsic resistance phenotypes is published by
EUCAST and is available at www.eucast.org/expert_ Organism Intrinsic Resistance Phenotypes
rules/. Some commonly encountered veterinary patho-
gens with intrinsic resistance to antimicrobials are Enterobacteriaceae Benzylpenicillin, macrolides, lincosamides,
included in Table 2.4. streptogramins, and rifampin
Klebsiella spp.
An appreciation of exceptional (unexpected) resistance Proteus mirabilis Ampicillin and ticarcillin
phenotypes allows unusual isolates or test results to be Proteus vulgaris Tetracycline and nitrofurantoin
identified and investigated further. Vancomycin-resistant Ampicilin, cefazolin, tetracycline, and
staphylococci, penicillin-resistant group A streptococci, Acinetobacter
and metronidazole-resistant anaerobes are all exceptional baumannii nitrofurantoin
phenotypes that should be confirmed before starting anti- Pseudomonas Ampicillin, amoxicillin + clavulanic acid,
microbial therapy. While such results can be due to the aeruginosa
emergence of resistance, it is more likely that these results cefazolin, and trimethoprim
reflect errors in reporting, testing, isolate identification, Enterococcus faecalis Ampicillin, amoxicillin + clavulanic acid,
or testing mixed cultures isolation (Livermore et al.,
2001). The CLSI M100 document as well as the EUCAST Enterococcus faecium piperacillin, cefazolin, chloramphenicol,
expert rules describe exceptional phenotypes (CLSI, trimethiprim + sulphonamide, and
2008b; Leclerq et al., 2008). Enterococcus galinarum tetracycline
Cephalosporins, aminoglycosides (low-level
Bacterial resistance mechanisms often predictably resistance), erythromycin, clindamycin,
confer resistance to multiple antimicrobials such that sulfonamides
resistance to one may indicate resistance to others. Cephalosporins, aminoglycosides (low-level
resistance), erythromycin, sulfonamides
Cephalosporins, aminoglycosides (low-level
resistance), erythromycin, clindamycin,
sulphonamides, and vancomycin

Data from EUCAST expert rules.

18 Section I. General Principles of Antimicrobial Therapy

By testing indicator drugs, susceptibility test results can Figure2.4. InducibleclindamycinresistanceStaphylococcus
be extrapolated to a broader panel of antimicrobials aureus displaying typical “D-zone” of inhibition associated
than could practically be tested. For example, oxacillin with inducible clindamycin resistance (top), and clindamycin
resistance in staphylococci indicates methicillin resist- susceptibility with erythromycin resistance (bottom).
ance and therefore resistance to all beta-lactams without
having to specifically test other beta-lactams. For
Enterobacteriaceae, cephalothin test results are predic-
tive for cephalexin and cefadroxil but not for ceftiofur
or  cefovecin. For β-hemolytic streptococci, penicillin
susceptibility is predictive of ampicillin, amoxicillin,
amoxicillin/clavulanic acid, and a number of cephalo-
sporins. See the CLSI guidelines for other examples.

Minimizing the selective pressure for antimicrobial
resistance should always be considered when selecting
therapy. While antimicrobial resistance follows usage,
certain bug-drug combinations are more likely to select
for resistance or promote mutational resistance than
others and should be avoided when possible. For exam-
ple, staphylococci readily develop resistance to rifampin,
while the fluoroquinolones and cephalosporins are
known to select for methicillin-resistant isolates
(Dancer, 2008; Livermore et al., 2001). Among Gram-
negative bacteria, there is evidence to suggest that the
fluoroquinolones and extended-spectrum cephalospor-
ins are more potent selectors of resistance than the
aminoglycosides, and that the third-generation cephalo-
sporins select for resistance more so than beta-lactamase
inhibitor potentiated penicillins (Peterson, 2005). See
chapter 3 for a discussion of the epidemiology of anti-
microbial resistance.

Other Susceptibility Testing Methods

Inducible resistance phenotypes pose unique diagnostic indicates resistance induction (Figure 2.4). It is recom-
challenges; standard diffusion or dilution testing meth- mended that staphylococci and streptococci appearing
ods may fail to detect resistance. Interpretive reading to be clindamycin susceptible but erythromycin resist-
can play a key role in identifying those phenotypes. For ant should be tested for inducible clindamycin resist-
example, inducible clindamycin resistance should be ance using the D-test. Inducibly clindamycin resistant
suspected in staphylococci and streptococci appearing isolates should always be reported as resistant, as in vivo
to be resistant to erythromycin but susceptible to clinda- induction of resistance following clindamycin therapy
mycin. Resistance can be elicited in inducible isolates can lead to treatment failure (Levin et al., 2005). Recent
using the “D-test,” a double disk test where erythromy- studies have documented inducible clindamycin resist-
cin and clindamycin disks are placed adjacently in an ance in both Staphylcoccus aureus and Staphylococcus
otherwise standard disk diffusion test. Blunting of the pseudintermedius isolated from animals (Rubin et al.,
inhibitory zone surrounding the clindamycin disk 2011a,b).
(resulting in a “D” shape) in the presence of erythromycin

Chapter 2. Antimicrobial Susceptibility Testing Methods and Interpretation of Results 19

Selective media have been designed to quickly iden- incubated overnight and those plates with between 20
tify particular antimicrobial-resistant organisms from and 200 colonies are counted and recorded; higher or
clinical samples. A detailed description of screening lower counts are not reliable. Preliminary analysis
media for extended-spectrum beta-lactamases in includes visual inspection of bacterial counts plotted on
Enterobacteriaceae, methicillin (oxacillin) resistance in a log10 scale. A ≥ 3 log decrease in counts after 24 hours
staphylococci, and high-level aminoglycoside and van- incubation indicates bactericidal activity (CLSI, 1999).
comycin resistance in enterococci is published by the See chapters 4 and 5 for discussions of pharmacokinet-
CLSI (CLSI, 2008a,b). ics and the selection of antimicrobial therapy.

Antimicrobial resistance can also be identified by Summary
testing for the products of resistance genes. For example,
the nitrocefin test utilizes a cephalosporin (nitrocefin) Antimicrobials are some of the most commonly used
that turns to red from yellow when hydrolyzed by most drugs in veterinary medicine and have improved the
beta-lactamases. However, this reaction is non-specific; health of food and companion animals alike. When
the susceptibility of nitrocefin to hydrolysis means that properly performed and carefully analyzed, antimicro-
narrow- or broad-spectrum beta-lactamases yield the bial susceptibility testing is an invaluable component of
same positive result. Additionally, as the presence or evidence-based treatment of infectious disease. In the
absence of beta-lactamase does not preclude other clinical setting, results should always be interpreted in
resistance mechanisms, interpretation of these results the context of the patient. By considering the pharma-
in  the context of susceptibility testing is therefore cokinetic/pharmacodynamic properties of the antimi-
essential. crobials in conjunction with interpretive reading of in
vitro susceptibility test results, clinical success can be
A latex agglutination test targeting PBP2a, the peni- maximized.
cillin-binding protein conferring methicillin resistance,
is available. This test can be done on primary cultures, While categorical susceptibility data can provide vital
identifying methicillin resistance before the complete information to clinicians, MIC data are superior for
antimicrobial susceptibility profile can be determined, allowing pharmacokinetic principles to be applied
saving 1 day in the diagnostic process. directly. For example, it may be rational to use antimi-
crobials that reach high concentrations in the urine,
For some investigations, MICs insufficiently describe despite susceptibility reports indicating resistance cor-
pharmacodynamic interactions. Time kill assays define related to achievable plasma concentrations. The reader
the effects of antimicrobials on an organism over time, is referred to chapters 5 and 6 for discussion of pharma-
rather than at the single end point with MIC testing. A cokinetics and the principles of antimicrobial selection.
time kill curve is performed by growing a bacterial cul-
ture in broth containing a known concentration of anti- Bibliography
microbial and evaluating changes in the concentration
of viable organisms over time (CFU/ml) using colony Aronson JK. 1992. Penicillin. Eur J Clin Pharmacol 42:1.
counts. Although the time points selected depend on the Barry AL. 2007. An overview of the Clinical and Laboratory
research question, time zero, 4 hours, 8 hours, 12 hours,
24 hours, and 48 hours is a good base model. At time Standards Institute (CLSI) and its impact on antimicrobial
zero, broths are inoculated to a known organism con- susceptibility tests. In: Schwalbe R, Steele-Moore L,
centration (e.g., 105 CFU/ml). Colony counts are per- Goodwin AC (eds). Antimicrobial Susceptibility Testing
formed on serial ten-fold dilutions of 100 μL broth Protocols. Boca Raton, FL: CRC Press.
aliquots. The first dilution, 10−1, is made by plating out Bryskier A. 2005. Penicillins. In: Bryskier A (ed).
100 μL of broth directly. The next dilution, 10−2, is made Antimicrobial Agents: Antibacterials and Antifungals.
by diluting 100 μL broth in 900 μL of saline; the third Washington, DC: ASM Press, p. 113.
dilution is made by diluting 100 μL of 10−2 in 900 μL of CLSI. 1999. Methods for Determining Bactericidal Activity
saline, and so on. Depending on the organism being of Antimicrobial Agents. M26-A. Wayne, PA: Clinical and
tested and the expected concentration of bacteria, dilu- Laboratory Standard Institute.
tions from 10−1 to 10−8 should be sufficient. Plates are

20 Section I. General Principles of Antimicrobial Therapy

CLSI. 2006a. Methods for Dilution Antimicrobial Levin TP, et al. 2005. Potential clindamycin resistance
Susceptibility Tests for Bacteria That Grow Aerobically; in  clindamycin-susceptible, erythromycin-resistant
Approved Standard. M7-A7. Wayne, PA: Clinical and Staphylococcus aureus: report of a clinical failure. Antimicrob
Laboratory Standard Institute. Agents Chemother 49:1222.

CLSI. 2006b. Performance Standards for Antimicrobial Disk Livermore DM, et al. 2001. Interpretative reading: recogniz-
Susceptibility Tests; Approved Standard M2-A9. Wayne, ing the unusual and inferring resistance mechanisms from
PA: Clinical and Laboratory Standards Institute. resistance phenotypes. J Antimicrob Chemother 48 Suppl
1:87.
CLSI. 2008a. Performance Standards for Antimicrobial Disk
and Dilution Susceptibility Tests for Bacteria Isolated from Papich MG. 2010. Proposed changes to Clinical Laboratory
Animals. M31-A3. Wayne, PA: Clinical and Laboratory Standards Institute interpretive criteria for methicillin-
Standards Institute. resistant Staphylococcus pseudintermedius isolated from
dogs. J Vet Diagn Invest 22:160.
CLSI. 2008b. Performance Standards for Antimicrobial
Susceptibility Testing. M100-S18. Wayne, PA: Clinical and Peterson LR. 2005. Squeezing the antibiotic balloon: the
Laboratory Standards Institute. impact of antimicrobial classes on emerging resistance.
Clin Microbiol Infect 11 Suppl 5:4.
Dancer SJ. 2008. The effect of antibiotics on methicillin-resistant
Staphylococcus aureus. J Antimicrob Chemother 61:246. Pressler B, et al. 2010. Urinary Tract Infections. In: Ettinger
SJ, Feldman EC (eds). Textbook of Veterinary Internal
Ericsson HM, et al. 1971. Antibiotic sensitivity testing. Medicine. St. Louis: Saunders Elsevier.
Report of an international collaborative study. Acta Pathol
Microbiol Scand B Microbiol Immunol 217 Suppl 217:1. Rubin JE, et al. 2011a. Antimicrobial susceptibility of
Staphylococcus aureus and Staphylococcus pseudinterme-
Erol E, et al. 2012. Beta-hemolytic Streptococcus spp. from dius isolated from various veterinary species. Can Vet J
horses: a retrospective study (2000–2010). J Vet Diagn 52:153.
Invest 24:142.
Rubin JE, et al. 2011b. Antimicrobial susceptibility of canine
Leclerq R, et al. 2008. Expert rules in antimicrobial suscepti- and human Staphylococcus aureus collected in Saskatoon,
bility testing. European Committee on Antimicrobial Canada. Zoonoses Public Health 58:454.
Susceptibility Testing.

Antimicrobial Resistance 3
and Its Epidemiology

Patrick Boerlin and David G. White

Introduction dilemma is the observation that the introduction of new
classes or modifications of older classes of antimicrobials
Since the discovery of penicillin in the late 1920s, hun- over the past 7 decades has been matched, slowly but
dreds of antimicrobial agents have been developed for surely, by the systematic emergence of new bacterial
anti-infective therapy. Antimicrobials have become indis- resistance mechanisms. Antimicrobial resistance mecha-
pensable in decreasing morbidity and mortality associ- nisms have been reported for all known antibiotics cur-
ated with a host of infectious diseases and, since their rently available for clinical use in human and veterinary
introduction into veterinary medicine, animal health and medicine. Therefore, successful sustainable management
productivity have improved significantly (National of current antimicrobials (Prescott, 2008; Doron and
Research Council, Institute of Medicine, 1998). The emer- Davidson, 2011; Ewers et al., 2011) and the continued
gence of antimicrobial resistance was not an unexpected development of new ones and of alternatives to antimi-
phenomenon and was predicted by Alexander Fleming, crobial drugs are vital to protecting animal and human
who warned in his Nobel Prize lecture in 1945 against the health against infectious microbial pathogens.
misuse of penicillin. However, loss of efficacy through the
emergence, dissemination, and persistence of bacterial Resistance Mechanisms
antimicrobial resistance in many bacterial pathogens
(defined as the ability of a microorganism to withstand A large variety of antimicrobial resistance mechanisms
the effect of a normally active concentration of an antimi- have been identified in bacteria, and several different
crobial agent) has become a general problem and a seri- mechanisms can frequently be responsible for resistance to
ous threat to the treatment of infectious diseases in both a single antimicrobial agent in a given bacterial species.
human and veterinary medicine (Salyers and Amiable- The manually curated Antibiotic Resistance Genes
Cuevas, 1997; Witte, 1998; Marshall and Levy, 2011). Database (ARDB) lists the existence of more than 23,000
potential resistance genes from available bacterial genome
Infections caused by resistant bacteria are more fre- sequences (Liu and Pop, 2009). Antimicrobial resistance
quently associated with higher morbidity and mortality mechanisms can be classified into four major categories
than those caused by susceptible pathogens (Helms et al., (Figure 3.1): (1) the antimicrobial agent can be prevented
2002; Travers and Barza, 2002; Varma et al., 2005). In from reaching its target by reducing its penetration into the
areas of concentrated use, such as hospitals, this has led to bacterial cell; (2) the antimicrobial agent can be expelled
lengthened hospital stays, increased health care costs, out of the cell by general or specific efflux pumps; (3) the
and, in extreme cases, to untreatable infections (Maragakis antimicrobial agent can be inactivated by modification or
et al., 2008; Shorr, 2009). Contributing to this growing

Antimicrobial Therapy in Veterinary Medicine, Fifth Edition. Edited by Steeve Giguère, John F. Prescott and Patricia M. Dowling.
© This chapter is public domain. Published 2013 by John Wiley & Sons, Inc.

21

22 Section I. General Principles of Antimicrobial Therapy

Reduced permeability Antimicrobial agent modification

Active efflux Target modification

Figure 3.1. The four major mechanisms of antimicrobial resistance. Reduced permeability can be due to either lack of perme-
ability of the outer membrane (e.g., down-regulation of porins in Gram-negatives) or of the cell membrane (e.g., lack of ami-
noglycoside active transport under anaerobic conditions).Active efflux can pump antimicrobial agents back into the periplasmic
space (as with the TetA tetracyclines efflux pump in Enterobacteriaceae) or directly in the outer milieu (as for the RND multidrug
efflux transporters).Antimicrobial agent modification by bacterial enzymes can take place either after the agent has penetrated
into the cell (e.g., acetylation of chloramphenicol by CAT enzymes), in the periplasmic space (e.g., splitting of the beta-lactam
ring by beta-lactamases in Enterobacteriaceae), or even outside of the bacterial cell (e.g., beta-lactamase produced by
Staphylococcus aureus), before the agent has reached its target on the surface of the bacterium. Target modification has been
described for both surface-exposed (e.g., peptidoglycan modification in vancomycin-resistant enterococci) and intracellular
targets (e.g., macrolide resistance due to ribosomal methylation in Gram-positive bacteria).

degradation, either before or after penetrating the cell; and Intrinsic resistance is natural to all the members of a
(4) the antimicrobial target can be modified or protected specific bacterial taxonomic group, such as a bacterial
by another molecule preventing access of the antibiotic to genus, species, or subspecies. This type of resistance is
its target, so that the antimicrobial cannot act on it any- most often through structural or biochemical character-
more. Alternatively, the antimicrobial agent target can be istics inherent to the native microorganism. For exam-
rendered dispensable by the acquisition or activation of an ple, many Gram-negative bacteria are naturally resistant
alternate pathway by the microorganism. A few examples to the activity of macrolides since these chemicals are
of each one of these resistance mechanisms are listed in too large to traverse the cell wall and to gain access to
Table 3.1 and more systematic information can be found in their cytoplasmic target. Other examples of innate
the following chapters of this book. resistance include the general reduced activity of amino-
glycosides against anaerobes, because of the lack of ami-
Types of Antimicrobial Resistance noglycoside penetration into the cells under anaerobic
conditions, and polymyxin resistance among Gram-
In the context of antimicrobial resistance, bacteria dis- positive bacteria because of the lack of phosphati-
play three fundamental phenotypes: susceptibility, dylethanolamine in their cytoplasmic membrane. A few
intrinsic resistance, or acquired resistance. examples of intrinsic resistance phenotypes for major
bacterial taxa are presented in Table 3.2. These intrinsic

Chapter 3. Antimicrobial Resistance and Its Epidemiology 23

Table 3.1. Examples of resistance mechanisms (note that this is by far not a comprehensive list of all the resistance
mechanisms known for each category of antimicrobials listed).

Antimicrobial Agent Resistance Mechanism Examples of Genetic Determinant

Tetracycline 2. Inducible efflux of tetracycline in E. coli and other tet(A), tet(B), tet(C)
Enterobacteriaceae
Chloramphenicol Tet(O), tet(M)
Beta-lactams 4. Ribosomal protection in Gram-positive bacteria cmlA, floR
Oxacillin, methicillin 2. Efflux in Enterobacteriaceae catA
Imipenem 3. Acetylation in Enterobacteriaceae blaTEM, blaCTX-M, blaCMY, blaNDM, blaZ
Aminoglycosides 3. Beta-lactamases in Enterobacteriaceae, and Staphylococcus
Streptomycin mecA
Macrolides, lincosamides, streptogramins aureus
Macrolides, streptogramins 4. Alternate penicillin-binding proteins in Staphylococcus Mutations
Fluoroquinolones
aureus Numerous genes with a broad variety
Sulfonamides 1. Decreased porin formation in Enterobacter aerogenes and of specificities
Trimethoprim
Klebsiella spp. Mutations
3. Phosphorylation, adenylation, and acetylation of
ermA, ermB, ermC
aminoglycosides in Gram-negative and –positive bacteria vga(A), msr(A)
4. Modification of ribosomal proteins or of 16S rRNA in qepA
Mutations in gyrA, gyrB, parC, parE
Mycobacterium spp. Diverse qnr genes
4. Methylation of ribosomal RNA in Gram-positive organisms sul1, sul2, sul3
2. Staphylococcus spp.
2. Active efflux Diverse dfr genes
4. DNA topoisomeases with low affinity to quinolones
4. Target protection
4. Bypass of blocked pathway through additional resistant

dihydropteroate synthase in Gram-negative bacteria
4. Bypass of blocked pathway through additional resistant

dihydrofolate reductase

Table 3.2. Examples of intrinsic resistance phenotypes.

Organism Intrinsic Resistance(s)

Most Gram-negative bacteria Penicillin G, oxacillin, macrolides, lincosamides, streptogramins, glycopeptides, bacitracin
(Enterobacteriaceae Pseudomonas
spp., or Campylobacter spp.) Ampicillin
Ampicillin, cephalosporins I, polymyxins
Klebsiella spp. Tetracycline, polymyxins
Proteus vulgaris Ampicillin, amoxicillin-clavulanate, cephalosporins I, polymyxins
Proteus mirabilis Ampicillin, amoxicillin-clavulanate, cephalosporins I, cefoxitin
Serratia marcescens Ampicillin, cephalosporins I and II, ceftriaxone, kanamycine, tetracycline, chloramphenicol,
Enterobacter spp.
Pseudomonas aeruginosa trimethoprim, quinolones
(Streptomycin, kanamycin), macrolides
Haemophilus spp. Cephalosporins I, trimethoprim
Campylobacter jejuni and Campylobacter coli Polymyxins, quinolones
Most Gram-positive bacteria Aminoglycosides (low level)
Streptococcus spp. Oxacillin, cephalosporins, aminoglycosides (low level), sulfonamides (in vivo), trimethoprim (in vivo)
Enterococcus spp. Oxacillin, cephalosporins, lincosamides
Listeria monocytogenes Cephalosporins, sulfonamides, trimethoprim
Bacillus anthracis Aminoglycosides
Anaerobes (including Clostridium spp.)

Adapted from the Communiqué 2005 of the Comité de l’Antibiogramme de la Société Française de Microbiologie.

24 Section I. General Principles of Antimicrobial Therapy

(A) Bimodal distribution of MICs
100

Number of isolates 75

50

25

0
≤4 8 16 32 64 128 256 512 1024 ≥2048

Microgram sulfisoxazole/mL

(B) Multimodal distribution of MICs
6

Number of isolates 4

2

0 2 4 8 16 32 64 128 256 ≥512
≤0.25 0.5 1 Microgram tetracycline/mL

Figure 3.2. Examples of bimodal and multimodal distribution of minimal inhibitory concentrations. (A) Bimodal distribution
of MICs for sulfonamides in a sample of commensal Escherichia coli isolates from swine and cattle. Susceptible isolates are in
white and isolates with a resistance determinant are in black. Note the clear separation between the two groups. (B) Multimodal
distribution of MICs for tetracycline in a sample of E. coli from a variety of origins. Fully susceptible isolates without any resist-
ance determinant are in white. Isolates with a tet(C), tet(A), and tet(B) are in increasingly dark shades of gray. Note that
depending on the respective frequency of each tetracycline resistance determinant, modes may or may not be clearly visible.

resistances should generally be known by clinicians and Acquisition of resistance usually leads to discrete jumps
other users of antimicrobial agents so as to avoid inap- in the MIC of an organism and hence to clear bi- or
propriate and ineffective therapeutic treatments. The polymodal distributions of MICs (Figure 3.2). However,
European Committee on Antimicrobial Susceptibility in some instances such as for fluoroquinolone antimi-
Testing (EUCAST) provides a very useful interactive list crobials, acquisition of resistance (elevated MICs) may
of antimicrobial susceptibility tables for a variety of be a progressive phenomenon, through successive accu-
organism/antimicrobial combinations on its website mulation of multiple genetic modifications blurring the
(http://mic.eucast.org/Eucast2/). minimal changes in MIC provided by each modification
into a smooth continuous MIC distribution curve, since
Antimicrobial resistance can also be acquired, such mutations occur in particular topoisomerase genes in a
as  when a normally susceptible organism develops step-wise manner (Hopkins et al., 2005; Table 3.3).
resistance through some type of genetic modification.

Chapter 3. Antimicrobial Resistance and Its Epidemiology 25

Table 3.3. Characterization of quinolone-resistant avian pathogenic E. coli (n = 56).a

No. of isolates Mutation inb MIC range (μg/ml)c

GyrA GyrB ParC Nal Orb Enr Cip

40 Ser83-Leu None None 64− >256 0.5–8 0.25–2 0.12–1

7 Asp87-Tyr None None 128 0.5–1 0.25–0.5 0.12–0.25

1 Asp87-Tyr None Ser80-Ile >256 >16 16 8

1 Ser83-Leu; None None 128 1 0.5 0.25

Asp87-Gly

1 Ser83-Leu; None None >256 2 1 0.5

Asp87-Ala

1 Ser83-Leu; None Ser80-Arg >256 8 4 2

Asp87-Gly

2 Ser83-Leu Asp426-Thr None 256 2 0.5 0.25–0.5

1 Ser83-Leu Glu466-Asp None >256 8 2 1

1 Ser83-Leu Glu466-Asp Ser80-Ile >256 >16 8 4

1 Ser83-Leu Glu466-Asp Ser80-Ile >256 >16 8 4

aAdapted from Zhao S, et al. 2005. Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates.
Vet Microbiol 107:215.
bSubstituted amino acids, and the position number; e.g., Ser83-Leu indicates substitution of a leucine for a serine at position 83. Amino acids:
Ser, serine; Asp, aspartic acid; Leu, leucine; Tyr, tyrosine; Glu, glutamic acid; Gly, glycine; I, isoleucine; Arg, arginine; Ala, alanine; Thr, threonine;
None, wild-type. No mutations were identified in parE sequences.
cNal, nalidixic acid; Orb, orbifloxacin; Enr, enrofloxacin; Cip, ciprofloxacin.

Acquired resistance can be manifested as resistance lead to clinically relevant resistance levels. Therefore,
to a single agent, to some but not all agents within a class the use of MIC data rather than categorical classifica-
of antimicrobial agents, to an entire class of antimicro- tion of bacteria into resistant and susceptible is
bial agents, or even to agents of several different classes. encouraged. This would avoid many apparent contra-
In the great majority of cases, a single resistance deter- dictions and compromises between clinicians, micro-
minant encodes resistance to one or several antimicro- biologists, and epidemiologists in setting appropriate
bial agents of a single class of antimicrobials (such as susceptibility and resistance breakpoints. A clear
aminoglycosides, beta-lactams, fluoroquinolones) or of distinction should be made between epidemiological
a group of related classes of antimicrobials such as the cut-off values and clinical breakpoints, based on
macrolide-lincosamide-streptogramin group. However, presence of acquired mechanisms causing decreased
some determinants encode resistance to multiple susceptibility to an antimicrobial or clinical respon-
classes. This is, for example, the case for determinants siveness, respectively (Kahlmeter et al., 2003; Bywater
identified in recent years such as the Cfr rRNA methyl- et al., 2006).
transferase (Long et al., 2006) or the aminoglycoside
acetyltransferase variant Aac(6′)-Ib-cr (Robiczek et al., Acquisition of Antimicrobial Resistance
2006), or when multidrug efflux systems are upregu-
lated, as is the case for the AcrAB-TolC efflux pump sys- Bacterial antibiotic resistance can result from the muta-
tem (Randall and Woodward, 2002). The simultaneous tion of genes involved in normal physiological processes
acquisition of several unrelated genetic resistance deter- and cellular structures, from the acquisition of foreign
minants located on the same mobile genetic element is, resistance genes, or from a combination of these mecha-
however, more common as an explanation of multidrug nisms. Mutations occur continuously but at relatively
resistance. low frequency in bacteria, thus leading to the occasional
random emergence of resistant mutants. However,
As should be clear from the discussion above, the under conditions of stress (including those encountered
acquisition of genetic determinants of resistance is
associated with a variety of MICs and does not always

26 Section I. General Principles of Antimicrobial Therapy

by pathogens when facing host defenses or in the pres- genetic elements that can move around within or
ence of antimicrobials), bacterial populations with between genomes in a cell. These have been divided into
increased mutation frequencies can be encountered four classes: (1) plasmids; (2) transposons; (3) bacterio-
(Couce and Blázquez, 2009). This so-called mutator phage; and (4) self-splicing molecular parasites
state has been suggested to be involved in the rapid (Siefert,  2009). Although there are some examples of
development of resistance in vivo during treatment with bacteriophage-mediated antimicrobial resistance trans-
certain antimicrobials such as fluoroquinolones (Komp fer (Colomer-Lluch et al., 2011), the plethora of exam-
Lindgren et al., 2003). However, for the majority of clini- ples of transferable resistance plasmids found across a
cal isolates, antimicrobial resistance results from acqui- broad variety of bacterial hosts suggest that plasmids and
sition of extrachromosomal resistance genes. conjugation are the major players in the global spread of
antimicrobial resistance genes in bacterial populations.
Foreign DNA can be acquired by bacteria in three dif-
ferent ways (Figure 3.3): (1) uptake of naked DNA pre- Plasmids are extrachromosomal self-replicating
sent in the environment by naturally competent bacteria genetic elements that are not essential to survival but
(called transformation); (2) transfer of DNA from one that typically carry genes that impart some selective
bacterium to another by bacteriophages (transduction); advantage(s) to their host bacterium, such as antimicro-
and (3) transfer of plasmids between bacteria through a bial resistance genes. Despite the apparent efficiency of
mating-like process called conjugation. Recently, the these transfer mechanisms, bacteria possess a large
term mobilome was introduced to describe all mobile variety of strategies to avoid being subverted by foreign

Recipient cell
Conjugation

Transduction Plasmid
Bacteriophage
Transposition

Chromosome

Transformation

Donor cell

Figure 3.3. The three mechanisms of horizontal transfer of genetic material between bacteria. White arrows indicate the
movement of genetic material and recombination events. The bold black line represents an antimicrobial resistance gene (or a
cluster of resistance genes). In the case of transduction, a bacteriophage injects its DNA into a bacterial cell, and in the occur-
rence of a lysogenic phase, this DNA is integrated into the chromosome of the recipient cell. In the case of transformation,
“naked” DNA is taken up by a competent cell and may recombine with homologous sequences in the recipient’s genome. In
the case of conjugation, a plasmid is transferred from a donor bacterium (transfer is coupled with replication and a copy of the
plasmid remains in the donor) to recipient cell in which it can replicate. During its stay in various host bacteria, the plasmid may
have acquired a transposon carrying antimicrobial resistance genes.

Chapter 3. Antimicrobial Resistance and Its Epidemiology 27

DNA, so that numerous obstacles have to be overcome resistance genes together on a single mobile element.
to allow the stabilization and expression of genes in a Another group of mobile elements called ISCR that also
new host (Thomas and Nielsen, 2005). In addition, plas- help mobilize adjacent genetic material by mechanisms
mids compete for the replication and partition machin- different from classical insertion sequences has been
ery within cells and plasmids that make use of similar detected increasingly in relation with integrons (see
systems and cannot survive for long together in the below) and antimicrobial resistance genes (Toleman
same cell. This “incompatibility” has led to the classifi- et  al., 2006). Some bacteria (mainly anaerobes and
cation of plasmids into so-called incompatibility groups, Gram-positive bacteria) can also carry so-called conju-
a system widely used to categorize resistance plasmids gative transposons, which are usually integrated in the
into similarity groups and to study their epidemiology bacterial chromosome but can be excised, subsequently
(Carattoli, 2011). Many studies have shown that anti- behaving like a transferable plasmid, and finally re-
microbial resistance plasmids can be transferred between integrate in the chromosome of their next host. The
bacteria under a wide variety of conditions. This magnitude of resistance development is also explained
includes, for example, the relatively high temperature of by the widespread presence of integrons, particularly
the intestine of birds as well as other conditions and at class 1 integrons (Hall et al., 1999; Cambray et al., 2010).
the lower temperatures encountered in the environ- These DNA elements consist of two conserved segments
ment. Some plasmids can be transferred easily between flanking a central region in which antimicrobial resist-
a variety of bacterial species, for instance between harm- ance “gene cassettes” can be inserted. Multiple gene cas-
less commensal and pathogenic bacteria, thus leading in settes can be arranged in tandem, and more than 140
some cases to the emergence and massive establishment distinct cassettes have been identified to date conferring
of newly resistant pathogen populations in individual resistance to numerous classes of antimicrobial drugs as
animals within days (Poppe et al., 2005). well as to quaternary ammonium compounds (Partridge
et al., 2009). In addition, integrons are usually part of
In addition to moving between bacteria, resistance composite transposons, thus further increasing the
genes can also move within the genome of a single bac- mobility of resistance determinants.
terial cell and hop from the chromosome to a plasmid or
between different plasmids or back to the chromosome, The Origin of Resistance Genes and Their
thus allowing development of a variety of resistance Movement across Bacterial Populations
gene combinations and clusters over time. Transposons
and integrons play a major role in this mobility within a Resistance genes and DNA transfer mechanisms have
genome. Transposons (“jumping genes”) are genetic ele- likely existed long before the introduction of therapeutic
ments that can move from one location on the chromo- antimicrobials into medicine. For example, antimicro-
some to another; the transposase genes required for bial-resistant bacteria and resistance determinants have
such movement are located within the transposon itself. been found in Arctic ice beds estimated to be several
The simplest form of a transposon is an insertion thousand years old (D’Costa et al., 2011). More recently,
sequence (IS) containing only those genes required for molecular characterization of the culturable microbi-
transposition. An advancement on the IS model is seen ome of Lechuguilla Cave, New Mexico (from a region of
in the formation of composite transposons. These con- the cave estimated to be over 4 million years old)
sist of a central region containing genes (passenger revealed the presence of bacteria displaying resistance to
sequences) other than those required for transposition a wide range of structurally different antibiotics (Bhullar
(e.g., antibiotic resistance) flanked on both sides by IS et al., 2012). Resistant microorganisms have also been
that are identical or very similar in sequence. A large found among historic culture collections compiled
number of resistance genes in many different bacterial before the advent of antibiotic drugs as well as from
species are known to occur as part of composite trans- humans or wild animals living in remote geographical
posons (Salyers and Amiable-Cuevas, 1997). settings (Smith, 1967; Bartoloni et al., 2004).

Homologous recombination between similar trans- It is widely believed that antibiotic resistance mecha-
posons within a genome also play an important role in nisms arose within antibiotic-producing microorgan-
clustering passenger sequences such as antimicrobial isms as a way of protecting themselves from the action

28 Section I. General Principles of Antimicrobial Therapy

of their own antibiotic, and some resistance genes are veterinary and human medicine. The complexity of
thought to have originated from these organisms. This movement of microorganisms and of horizontal gene
has been substantiated by the finding of aminoglyco- transfer (HGT) involved in the epidemiology of global
side-modifying enzymes in aminoglycoside-producing resistance is difficult to comprehend. The graphical
organisms that display marked homology to modifying depiction of this complex interaction in Figure 3.4 is the
enzymes found in aminoglycoside-resistant bacteria. A best attempt to date to capture this complexity.
number of antibiotic preparations employed for human
and animal use have been shown to be contaminated On a long-term evolutionary scale, the epidemiology
with chromosomal DNA of the antibiotic-producing of antimicrobial resistance should be regarded as domi-
organism, including identifiable antimicrobial resist- nated by the stochastic or chaotic movement of resist-
ance gene sequences (Webb and Davies, 1993). However, ance genes within a gigantic bacterial genetic pool.
as in the case of synthetic antimicrobials such as tri- However, in the shorter term and on a local scale, this
methoprim and sulfonamides, preexisting genes with unrestricted approach may be too simple and of less
other resistance-unrelated roles might have evolved practical relevance than considering only resistant path-
through adaptive mutations and recombinations to ogens. Because of the complexity of the resistance issue,
function as resistance genes. Indeed, some have sug- numerous strategies to control the rise of antimicrobial
gested that in their original host, antimicrobial resist- resistance at every level have emerged in the scientific
ance genes play a role in detoxification of components and medical communities. As with other complex issues
other than antimicrobials, and in a variety of unrelated that global society faces, no single intervention will be
metabolic functions (Martinez, 2008). A vast reservoir decisive alone, but numerous interventions are needed
of such genes, now dubbed the resistome, is present in that cumulatively may preserve acceptable levels of effi-
the microbiome of various natural environments cacy for current and future antimicrobial drugs (Prescott
(D’Costa et al., 2007; Bhullar et al., 2012), which can be et al., 2012).
transferred to medically relevant bacteria through
genetic exchange (Wright, 2010). The Effects of Antimicrobial Use on the
Spread and Persistence of Resistance
Since resistance genes are frequently located on
mobile genetic elements, they can move between patho- The increased prevalence and dissemination of resist-
gens, as well as between non-pathogenic commensal ance is an outcome of natural selection, the Darwinian
bacteria and pathogens. Thus, the issue of resistance has principal of “survival of the fittest.” In any large popula-
to be considered beyond the veterinary profession and tion of bacteria, a few cells that possess traits that enable
specific pathogens. Indeed, there is growing evidence them to survive in the presence of a toxic substance will
that resistance genes identified in human bacterial path- be present. Susceptible organisms (i.e., those lacking
ogens were originally acquired from environmental, the  advantageous trait) will be eliminated, leaving the
non-pathogenic bacteria via horizontal gene exchange remaining resistant populations behind. With long-term
(Martinez et al., 2011; Davies and Davies, 2010). antimicrobial use in a given environment, the microbial
Resistance genes can spread quickly among bacteria, ecology will change dramatically, with less susceptible
sometimes to unrelated genera. Even if an ingested organisms becoming the predominant population
bacterium resides in the intestine for only a short time, (Salyers and Amabile-Cuevas, 1997; Levy, 1998). When
it has the ability to transfer its resistance genes to the this occurs, resistant commensal and opportunistic bac-
resident microflora, which in turn may serve as reser- teria can quickly become established as dominant com-
voirs of resistance genes for pathogenic bacteria. The ponents of the normal flora of various host species,
inclination to exchange genes raises the concern for the displacing susceptible populations. Changes in antimi-
possible spread of antimicrobial resistance determinants crobial resistance frequency when new antimicrobials
from commensal organisms in animals and humans appear on the market or when restrictions are imple-
to  human pathogens (Witte, 1998; Van den Bogaard mented in the use of existing antimicrobials testify for
and  Stobberingh, 2000). Thus, the epidemiology of the validity of these evolutionary rules. Several examples
antimicrobial resistance goes beyond the boundaries of

Chapter 3. Antimicrobial Resistance and Its Epidemiology 29

AQUACULTURE Sea / Swimming
Lakes

Drinking Rivers and Drinking
water streams water

SOIL Industrial &
household
WILDLIFE antibacterial
chemicals
Commercial
Farm effluents and abattoirs / Sewage
manure spreading processing

Rendering Offal plants

Animal Dead Vegetation,
feeds stock seed crops, fruit

COMPANION SWINE HUMAN
ANIMALS
SHEEP CATTLE

FOOD Meat Handling HOSPITALIZED COMMUNITY
preparation - URBAN
VEAL ANIMALS consumption - RURAL

CALVES POULTRY

OTHER EXTENDED
FARMED CARE
LIVESTOCK
FACILITIES

Direct
contact

Figure 3.4. The ecology of the spread of antimicrobial resistance and of resistance genes. A schematic representation of
resistant bacteria and antimicrobial resistance genes transmission routes across the multiple ecological compartments. This
figure is a further development (Irwin et al., 2008) of an original one by Linton, 1977. Reproduced with permission.

of the rise and fall of antimicrobial resistance as selec- disinfectants (Baker-Austin et al., 2006; Salyers and
tion pressures change are described later in this chapter. Amabile-Cuevas, 1997; Hall et al., 1999) or even with
virulence genes (Boerlin et al., 2005; Da Silva and
The clustering of multiple resistance genes on plas- Mendonça, 2012; Johnson et al., 2010).
mids, transposons, and integrons makes the problem of
antimicrobial resistance challenging. Exposure to one Carrying genetic material associated with resistance
antimicrobial may co-select for bacteria that are also genes when they are not needed represents a burden for
resistant to several unrelated agents (Cantón and Ruiz- bacteria. Therefore, when a bacterial population is not
Garbajosa, 2011). There may also be non-antibiotic under the selective pressure of antimicrobials, suscepti-
selection pressure for bacterial antibiotic resistance ble bacteria not carrying resistance genes may be at an
genes. Although much is only speculative on this subject advantage and the population as a whole is expected to
(Meyer and Cookson, 2010), there is growing evidence slowly revert to a mainly susceptible state. A few exam-
showing that disinfectants and biocide may co-select ples of such a reversion have been described in the past
for  antimicrobial resistance (Yazdankhah et al., 2006; (Aarestrup et al., 2001; Dutil et al., 2010). However, other
Hegstad et al., 2010). Not only can resistance determi- studies have also shown that bacteria may exhibit resist-
nants for antibiotics of a different class aggregate, but ance to antimicrobials despite a lack of specific selective
they may also form clusters with resistance genes for pressures, as has been the case, for example, for chloram-
non-antibiotic substances such as heavy metals and phenicol, glycopeptides, or streptothricin (Werner et al.,

30 Section I. General Principles of Antimicrobial Therapy

2001; Bischoff et al., 2005; Johnsen et al., 2005). The agriculture contributes to antimicrobial-resistant food-
mechanisms behind this persistence are unclear but borne bacterial pathogens. These concerns are not new
likely to be multifactorial. They may include compensa- and in the 1960s led to the release in the United Kingdom
tion for the metabolic load imposed by resistance genes of the Swann Report (Anonymous, 1969), which resulted
by as yet not clearly understood mechanisms (Zhang in changes in antimicrobial use in agriculture. Despite
et al., 2006), regulation of gene expression by the pres- the best efforts to date, there is no agreement regarding
ence/absence of antimicrobials, and plasmid addiction the scale of the impact of antimicrobial use in animals on
systems. However, the real significance of each one of human health. The fundamental and obvious concern
these mechanisms remains unclear. For instance, com- over the agricultural use of antibiotics arises from the
pensation for fitness loss has been shown to play a role in potential that antimicrobials used on the farm select for
the case of resistance mechanisms associated with resistant bacterial strains that are transferred to humans
chromosomal mutations, but its role in the persistence via direct contact and ingestion of contaminated food
of resistance associated with mobile genetic elements is and/or water (Figure 3.4). Numerous cases of transmis-
much less evident. Although plasmid addiction systems sion of resistant bacteria between animals and humans at
may avoid reversion of plasmid carriers to a susceptible risk, such as farmers, abattoir workers, and veterinarians,
state, it is not clear if this is a real advantage for the support these concerns (Hunter et al., 1994; van den
affected bacteria (Mochizuki et al., 2006). When resist- Bogaard et al., 2002; Garcia-Graells et al., 2012). The par-
ance genes are physically linked together or to other allel rise and decrease of resistance to glycopeptides in
selectively advantageous genes, co-selection will lead to animal and human enterococci in some European coun-
the persistence of all the resistance genes as part of the tries after the introduction and subsequent ban of
cluster. Several examples of co-selection are known, such avoparcin (see below) and other antimicrobial growth
as the maintenance of glycopeptide resistance in porcine promoters substantiate these fears. The identification of
enterococci by the use of macrolides, or the persistence fluoroquinolone-resistant Campylobacter and quinu-
and higher frequency of antimicrobial resistance in some pristin/dalfopristin-resistant enterococci from animal
pathogen populations due to linkage between virulence sources or their immediate environment has intensified
and resistance genes (Martinez and Baquero, 2002). this debate (Piddock, 1996; Witte, 1998). Food of animal
origin has recently even been suggested to represent a
Finally, the effects of diverse drug administration potential reservoir of resistant extraintestinal pathogenic
protocols (administration route, timing, dosage) on the E. coli for humans, and uropathogenic E. coli in particular
dynamics and persistence of susceptible and resistant bac- (Manges and Johnson, 2012). Methicillin-resistant
teria and on the spread of resistance genes among bacte- Staphylococcus aureus (MRSA) seems to represent
rial populations at the global and individual level are another resistant zoonotic agent (see below). This sug-
complex and poorly understood (MacLean et al., 2010). gests that, because of their intimate contact with humans,
Every effort should be made to define treatment protocols pets and not just farm animals may represent another
that avoid or minimize the windows for selection of resist- source of resistant bacteria and resistance genes of public
ant bacteria. This is of particular direct concern when low- health relevance (Ewers et al., 2010; Platell et al., 2011).
level resistance mechanisms elevate the mutant selection A historical perspective on the issue of agricultural use
window high enough to allow in vivo selection of fully of antimicrobial drugs and its impact on human health
resistant mutants, as can be the case for fluoroquinolones is available (Prescott, 2006).
(Drlica and Zhao, 2007; Cantón and Morosini, 2011).
Overall, there are clear and compelling data demon-
Antimicrobial Resistance and Public Health strating that the use of antimicrobials in animals can
have negative effects on antimicrobial resistance in bac-
Although most of the bacterial antimicrobial resistance teria and pathogens from humans. Although more
observed in human medicine may be ascribed to use research is needed to quantify the risk associated with
in  human patients, it is being resolutely argued that this use in animals and the fraction of resistance in
antimicrobial use in veterinary medicine and food animal human pathogens attributable to it, this situation clearly
warrants some caution and preventive measures.

Chapter 3. Antimicrobial Resistance and Its Epidemiology 31

Examples of Antimicrobial Resistance in range, S. Typhimurium is also one of the most com-
Veterinary Medicine of Public Health mon  serotypes isolated from human salmonellosis.
Significance Historically this serovar has often been associated with
multiresistance, particularly in relation with phage type
Resistance in Salmonella DT104, but this type may be decreasing in frequency,
Although a large body of science is available on the and a new multiresistant monophasic S. Typhimurium
prevalence of antimicrobial resistance and associated variant is now spreading globally (Butaye et al., 2006;
mechanisms in Salmonella, many aspects related to the Hauser et al., 2010).
emergence, persistence, and dissemination of antimi-
crobial resistance in these pathogens remain unclear. An increase in S. Newport infections was reported by
the CDC in 2000. Many of these strains exhibited a mul-
Salmonella can colonize and cause disease in a tidrug-resistant phenotype (commonly referred to as
variety  of food-producing and non-food-producing S. Newport MDR-AmpC) characterized by resistance to
animals. Although all serotypes may be regarded as nine antimicrobials, including amoxicillin-clavulanic
potential human pathogens, the great majority of infec- acid and ceftiofur. In addition to the characteristic
tions are caused by only a limited number. Resistance in resistance to nine specific antimicrobials, these strains
non-typhoidal Salmonella spp. has become an interna- also exhibited decreased susceptibility to ceftriaxone
tional problem (Threlfall, 2000; Poppe et al., 2001; (MIC 16–32 μg/ml; Zhao et al., 2003). These strains are
Williams, 2001). The levels and extent of resistance vary of particular clinical concern, as they possess plasmid-
and are influenced by antimicrobial use practices in or chromosomally encoded AmpC beta-lactamases
humans and animals, as well as by geographical differ- (e.g., blaCMY) that confer decreased susceptibility to a
ences in the epidemiology of Salmonella. Drug resist- wide range of beta-lactams, including ceftriaxone, the
ance phenotypes have been associated with the use of drug of choice for treating complicated salmonellosis in
antimicrobials in food-producing animals (Piddock, children (Gupta et al., 2003). Slightly later, a similar
1996; Wiuff et al., 2000; Molbak, 2004; Alcaine et al., increase in third-generation cephalosporin resistance
2005), in which resistance profiles generally reflect how related to blaCMY plasmids was observed in S. Heidelberg
long an agent has been in use. Thus, irrespective of in Canada, which was attributed to the use of this class
source (food animals, food, humans), the most frequent of antimicrobials in poultry (Dutil et al., 2010; chapter
resistances are usually to older antimicrobials such as 9). In both cases, MDR-AmpC strains found their way
ampicillin, chloramphenicol, streptomycin, sulfameth- into the food chain and were linked to human food-
oxazole, and tetracycline (Anderson, 1968; Chiappini borne infection (Gupta et al., 2003; Zhao et al., 2003;
et al., 2002; Molbak, 2004; Sun et al., 2005). However, Dutil et al., 2010). Multidrug-resistant Salmonella have
there are increasing reports of Salmonella isolates also been associated with illness in animals and humans
worldwide displaying reduced susceptibility or in equine and companion animal veterinary facilities
resistance to extended-spectrum cephalosporins or (Wright et al., 2005). These latter reports frequently
fluoroquinolones (Threlfall et al., 2000; Zhao et al., describe poor hand-washing practices by employees,
2001; Gupta et al., 2003; Alcaine et al., 2005; Johnson eating in work areas, and previous antimicrobial drug
et al., 2005; Su et al., 2008; chapters 9 and 18). This is therapy in affected humans or animals.
particularly troublesome since these antimicrobial
classes are frequently used to treat Salmonella infections Methicillin-Resistant Staphylococcus aureus
in children and adults, respectively (Angulo et al., 2004; MRSA has emerged as a major nosocomial pathogen in
Alcaine et al., 2005). Treatment will be more difficult human hospitals. This problem had remained limited to
with the recent emergence of carbapenemases in hospital settings, but MRSA is now present in the human
Salmonella (Savard et al., 2011). community too. However, MRSA has been emerging
rapidly in animals in recent years, for reasons that are
Salmonella Typhimurium continues to be one of the not clear (chapter 8), and represents an important exam-
serovars most frequently recovered from food animals ple of both the spread of resistance and the links between
worldwide (Zhao et al., 2005). In the United States, it is resistance in human and animal medicine.
among the top four serovars most frequent in cattle,
swine, chickens, and turkeys. Because of its broad host

32 Section I. General Principles of Antimicrobial Therapy

There are an increasing number of reports on MRSA (Butaye et al., 2003; Dibner and Richards, 2005). Past
colonization and infections in animals (Weese, 2010), studies have shown that this practice is also a potentially
demonstrating spread into animal populations (chapter significant driving force in accelerating the emergence
8). Most early reports of MRSA in animals were from of resistant bacteria that could infect humans (Wegener,
horses and from dogs and cats; MRSA have remained a 2003; Kelly et al., 2004; Dibner and Richards, 2005). The
rarity in cattle despite extensive use of cloxacillin in use of antimicrobial agents for growth promotion is
mastitis treatment. A recent report from Belgium discussed in chapter 22.
(Vanderhaegen et al., 2010) suggests that this situation
may be changing. MRSA isolates were originally recov- Most classes of antimicrobials used in animals have
ered more frequently from horses in relation with noso- analogues used in humans and are therefore capable of
comial surgical wound infections possibly originating selecting for resistance to human medical antibiotics.
from humans (Seguin et al., 1999). Equine MRSA usu- The important exceptions are the ionophores (e.g.,
ally belong to a specific clone that seems to be main- lasalocid, monensin, narasin, salinomycin), the quinox-
tained within equine populations (Weese et al., 2005a,b). alines (e.g., olaquindox), bambermycins (flavophospho-
This clone is also occasionally found in humans, partic- lipol), and avilamycin (Turnidge, 2004). Among the
ularly in horse personnel, but is not  one of the most former group, two classes of antimicrobials that
prevalent human MRSA clones. Investigations suggest have  received particular attention in the scientific
that transmission of MRSA goes in both directions community are the streptogramins (quinupristin/dalfo-
between humans and horses and may be associated with pristin, virginiamycin) and glycopeptides (avoparcin,
clinical disease in both groups. vancomycin).

The epidemiology of MRSA in dogs and cats may be Virginiamycin in feed has been approved since 1975
different since the clones found in dogs and cats, and for food-producing animals for growth promotion and
occasionally transmitted between animals, are the same prevention or control of certain diseases in turkeys,
as those frequently found in nosocomial and commu- swine, cattle, and chickens (Kelly et al., 2004). The
nity infections in humans. In addition, many reports human analogue, Synercid, a mixture of the two strepto-
show that the same MRSA strain from clinical infections gramin antibiotics quinupristin and dalfopristin (QD),
or from healthy carriage can be found in pets and was approved in September 1999 by the U.S. FDA for
humans with close contact (van Duijkeren et al., treatment of bacteremias in humans, particularly against
2004a,b; Rankin et al., 2005). In recent years, the MRSA vancomycin-resistant Enterococcus faecium (VREF) and
ST398 clone has emerged massively in livestock (Smith for the treatment of skin and soft tissue infections caused
and Pearson, 2011). This clone seems to be particularly by Staphylococcus aureus and Streptococcus pyogenes.
frequent in pigs and veal calves (Voss et al., 2005) but has Synercid was considered then to be a last resort of ther-
also been described in poultry, dairy cattle, and other apy for potentially life-threatening bloodstream infec-
species, as well as in meat products. The reasons for the tions caused by VREF. The approval of Synercid focused
emergence of this clone in livestock are not completely increased attention on the use of virginiamycin in ani-
understood. Although people working with livestock mal husbandry; specifically, whether farm use of virgin-
(farm workers, veterinarians) are at higher risk of carry- iamycin resulted in streptogramin resistance in bacteria
ing MRSA ST398, its transmission between humans that could result in impaired Synercid therapy in humans
seems not to be as active as for other MRSA. (Wegener 2003; Kelly et al., 2004). Synercid-resistant
E. faecium (SREF) are common in the poultry production
Antimicrobials in Animal Feeds and Association environment, including samples from litter and trans-
with Resistance in Bacteria of Human Health port containers (McDermott et al., 2005). SREF is also
Significance common on poultry meat products at retail, suggesting
that such meats serve as a continual source of resistant
It has been known for decades that continuous oral strains and/or their resistance genes (McDermott et al.,
administration of low concentrations of antimicrobials 2005). Foodborne strains might transfer plasmidborne
increases feed conversion and weight gain and reduces resistance determinants to human native enterococci in
shipping stress-associated diseases in food animals vivo (Jacobsen et al., 1999), which in turn might donate

Chapter 3. Antimicrobial Resistance and Its Epidemiology 33

these genes to other strains causing human infections. in animals (Heuer et al., 2002) and isolates similar to
The food safety implications prompted the FDA those from animals could be recovered from humans
(http://www.fda.gov/downloads/AnimalVeterinary/ several years after the ban of avoparcin (Hammerum
NewsEvents/CVMUpdates/UCM054722.pdf) and others et  al., 2004; Hammerum, 2012). Thus, antimicrobial
(Cox and Popken, 2004; Kelly et al., 2004) to propose resistance associated with the use of antimicrobial
risk assessment models examining the potential public growth promoters will not vanish as quickly as early
health consequences of virginiamycin use. The potential studies had led us to hope (Johnsen et al., 2011). In addi-
for streptogramin resistance genes to transfer from tion, the global ban of antimicrobial growth promoters
foodborne enterococcal isolates to those causing disease might have undesirable consequences on animal health,
in humans remains difficult to assess, because of com- consequences that remain to be assessed precisely
plex interplays between bacterial specificity for hosts (Casewell et al., 2003). It also increases, at least initially,
and gene transfer (Hammerum et al., 2010). In addition, the use of therapeutic antimicrobials (Grave et al., 2006).
while new resistance genes and new variants thereof As part of the federal strategy for controlling antimicro-
keep emerging and spreading in Gram-positive organ- bial resistance in the United States, the Food and Drug
isms (Witte and Cuny, 2011), a significant proportion of Administration (FDA) in 2012 released Guidance
the streptogramin-resistance determinants from entero- for  Industry #209 “The Judicious Use of Medically
cocci remain unknown in many recent studies. Important Antimicrobial Drugs in Food-Producing
Therefore, estimations of the potential health risks to Animals,” which focuses on two primary principles: (1)
humans resulting from virginiamycin use in animal limiting medically important antimicrobial drugs to
husbandry require further study. uses in food-producing animals that are considered nec-
essary for assuring animal health; and (2) limiting such
Early studies in the 1990s provided evidence in favor drugs to uses in food-producing animals that include
of a causal association between the use of avoparcin and veterinary oversight or consultation (http://www.
the occurrence of VREF on farms in Europe (Bager, f d a . g o v / d o w n l o a d s / A n i m a l Ve t e r i n a r y / G u i d a n c e
1999; Aarestrup et al., 2000). This suggested that food ComplianceEnforcement/GuidanceforIndustry/
animals constitute a potential reservoir of infection for UCM216936.pdf). This guidance, which represents
VREF in humans (Wegener, 2003). In response to con- FDA’s current thinking on this topic, is a very important
tinued pressure from the “major harm” position, the development in the field (chapter 26).
European Union took the “precautionary principle” and
followed the earlier move of Scandinavian countries Surveillance Programs and the Role of
by  suspending the use of the “growth promoter” in Diagnostic Laboratories
feed  antibiotics: avoparcin, bacitracin, virginiamycin,
spiramycin, and tylosin because of their ability to select The seriousness of the antimicrobial resistance threat
for resistance to antimicrobials of human importance has prompted many governments to initiate surveillance
(Turnidge, 2004; chapter 26). The frequency of resist- programs, which include bacteria of animal origin.
ance to vancomycin and to growth promoters in entero- These programs provide a tool to globally assess the
cocci from animal origin generally declined after the extent of the problem, to follow its evolution over time,
ban of antimicrobial growth promoters (Aarestrup et al., and to evaluate the effectiveness of control measures.
2001; Sorum et al., 2004). Interestingly, because of the Such systems include, among others, the National
plasmid-based linkage of glycopeptide and macrolide Antimicrobial Resistance Monitoring System (NARMS)
resistance genes in swine VREF, the decrease of VREF in the United States, the Canadian Integrated Program
frequency in swine isolates after the ban on avoparcin for Antimicrobial Resistance Surveillance (CIPARS) in
was slow until tylosin was also banned as a growth pro- Canada, and the Danish Integrated Antimicrobial
moter (Aarestrup et al., 2001). Some studies have also Resistance Monitoring and Research Program
demonstrated a parallel declining trend in VREF iso- (DANMAP) in Denmark. On the veterinary side, most
lated from food and humans after the ban, thus support- of the national surveillance programs only include bac-
ing the effectiveness of the ban (Klare et al., 1999; teria considered as indicators of the general resistance
Pantosti et al., 1999). However, VREF are still persisting situation (i.e., Escherichia coli and Enterococcus spp.)

34 Section I. General Principles of Antimicrobial Therapy

and zoonotic bacterial agents (Salmonella enterica and Nosocomial Infection and Antimicrobial
Campylobacter spp.). Only a few surveillance programs Resistance in Veterinary Hospitals
obtain antimicrobial susceptibility data from bacterial
pathogens of animals, the most visible being the BfT- Because of the high selection pressure exerted by the
GermVet Monitoring Program in Germany (Schwarz heavy use of antimicrobial agents in human hospitals,
et  al., 2007). Surveillance programs are of particular resistance first emerged as a significant problem in bacte-
interest when, like DANMAP, they include the collection ria associated with nosocomial infections. Veterinary
of data on antimicrobial use and try to link the latter with hospitals and practices, and their intensive care units,
the evolution of resistance. Because of the past problems keep increasing in size. In parallel, companion animal
in lack of standardization of antimicrobial susceptibility medicine is increasingly more sophisticated and inten-
testing, it is encouraging that these national surveillance sive. Consequently, antimicrobial resistance problems
programs use similar (if not identical) methodologies similar to those from human hospitals have appeared in
and provide increasingly comparable data. companion animal practice. Compared, however, to
human medicine, few publications are available on noso-
There is a wealth of information on the prevalence comial infections with multiresistant pathogens in ani-
of  antimicrobial resistance in animal pathogens mals. Nevertheless, what there is shows that the
(Aarestrup, 2006). However, because of the geographi- similarities between veterinary and human hospitals are
cally local and temporarily limited nature of these stud- striking. The heavy use of antimicrobial agents in inten-
ies and their different sampling and susceptibility sive care units is associated with increased antimicrobial
testing methodologies, it is difficult to draw reliable resistance (Ogeer-Gyles et al., 2006a), multidrug resistant
conclusions on the global antimicrobial resistance situ- organisms are widespread in veterinary clinics and hos-
ation in veterinary medicine. Constant efforts are made pital environments (Murphy et al., 2010), and indwelling
by the Clinical and Laboratory Standards Institute devices as well as surgical procedures are “hot spots” for
(CLSI, formerly NCCLS) to develop agreed veterinary nosocomial infections (Ogeer et al., 2006b; Bubenik
standards for susceptibility testing methodologies et al., 2007; Marsh-Ng et al., 2007; Jones et al., 2009).
(chapter 2). However, investigation shows that many
veterinary laboratories do not strictly follow these Besides the problem with MRSA in horses (Anderson
standards. There is a great need for diagnostic laborato- et al., 2009) and companion animals (Wieler et al., 2011)
ries to adhere to standards so as to provide reliable and mentioned above, and increasingly frequent outbreaks
reproducible susceptibility data for clinicians and other in veterinary clinics (van Duijkeren et al., 2010),
users. It should be recognized, however, that most stud- methicillin-resistant Staphylococcus pseudintermedius
ies of antimicrobial resistance in veterinary pathogens (MRSP) is now emerging as a major problem organism
are not based on a representative sample of pathogen in the veterinary world, including in hospital settings
populations but rather on diagnostic laboratory sub- (van Duijkeren et al., 2011; chapter 8). These organisms
missions, so that these reports may overestimate the seem to be resistant to a large number of other antimi-
prevalence of resistance in target pathogen populations. crobials of a variety of classes, making treatment of
Consequently, better-designed studies are needed for MRSP infections even more challenging than treatment
the assessment of the real antimicrobial resistance situ- of MRSA (Steen, 2011). Interestingly, the emergence of
ation in veterinary pathogens at every level, starting MRSP is related to the spread of a very few major clonal
from the farm and all the way up to the global national lineages (Perreten et al., 2010), suggesting the impor-
and international level. tance of infection control as one approach to improving
antimicrobial stewardship (chapter 7).
Susceptibility testing of clinical isolates is a corner-
stone for prudent use of antimicrobials and for an Other multiresistant nosocomial pathogens have
adequate management of single clinical cases (chapters been reported in veterinary hospital and intensive
2 and 7). Unfortunately, microbiological analysis and care units, including Salmonella enterica, E. coli, Acineto-
susceptibility testing are still frequently performed only bacter baumannii, and enterococci, but other resistant
when a problem has not been resolved by empirical pathogens common in human hospitals are also reported
antimicrobial therapy. sporadically.

Chapter 3. Antimicrobial Resistance and Its Epidemiology 35

Multiresistant Salmonella is one of the most regularly and Baquero, 2002), but evidence gathered in molecular
encountered causes of nosocomial infections in veteri- epidemiology studies is accumulating to show that it may
nary hospitals. Equine clinics seem to be particularly be a relatively widespread phenomenon, at least in organ-
prone to such problems (Dargatz and Traub-Dargatz, isms such as E. coli. For instance, tetracycline resistance
2004), and resistance profiles are increasingly problem- genes are frequently linked to enterotoxin genes in enter-
atic (Dallap Schaer et al., 2010). However, multiresistant otoxigenic E. coli, which may explain why tetracycline
Salmonella outbreaks also happen in companion animal resistance is more frequent in ETEC than in commensal
clinics (Wright et al., 2005). As in human hospitals, E. coli populations (Boerlin et al., 2005). Similarly, the
multidrug-resistant Enterobacteriaceae resistant to linkage of chloramphenicol resistance genes to enterotox-
extended-spectrum cephalosporins are increasingly ins genes may partially explain why, despite the ban of
being reported in veterinary nosocomial infections. chloramphenicol approximately 2 decades ago, chloram-
Both AmpC- and ESBL-type beta-lactamases have been phenicol resistance is still widespread in porcine ETEC
described in Salmonella, E. coli (Sanchez et al., 2002), but less frequent in commensal E. coli.
and Klebsiella (Haenni et al., 2011). This may also be a
precursor trend toward the emergence of carbapene- Recent research aimed at characterizing broad host
mases in these organisms (chapter 10). range plasmids recovered from numerous bacterial spe-
cies has shed additional light on potential gene linkage
Acinetobacter baumannii is another often multire- associations. For example, DNA sequencing of multi-
sistant Gram-negative organism of environmental ori- drug resistant plasmids from Salmonella Kentucky
gin causing major nosocomial human hospital infection revealed highly conserved backbones shared with avian
problems. Recent reports suggest that this may also pathogenic E. coli (APEC) virulence plasmids (Fricke
occur in veterinary clinics (Endimiani et al., 2011; et al., 2009). Specifically, the largest plasmid identified
Zordan et al., 2011). Multiresistant A. baumannii strains carried resistance determinants for streptomycin and tetra-
seem to persist better in hospitals under antimicrobial cycline as well as important virulence genes found in
pressure than susceptible organisms. This was the case APEC strains. Given the shared intestinal habitat, it is
in a series of A. baumannii infections in a veterinary likely that S. Kentucky acquired APEC-like plasmids
hospital, in which persistent strains were multiresistant, from commensal and/or pathogenic E. coli strains in the
whereas sporadic ones all presented only few resistances. chicken intestine. These results show that antimicrobial
After eradication of a first multiresistant strain through resistance determinants and APEC virulence factors
hygienic measures, another persistent multiresistant important in avian and possibly human E. coli patho-
strain readily replaced the first (Boerlin et al., 2001). genesis can be encoded by the same plasmid. Under
antimicrobial selection, the propagation of these viru-
Antimicrobial stewardship and clinical use guidelines lence factors within bacterial communities could poten-
are discussed in chapter 7. tially lead to the emergence of new virulent strains from
the commensal microflora of both animals and humans.
Accumulation and Persistence of Antimicrobial
Resistance in Pathogens Do virulence genes accumulate in bacterial popula-
tions because of their genetic linkage with resistance
Resistance gene linkage and co-selection are one of the genes and because of the selection exerted by antimicro-
reasons for the accumulation and persistence of resistance bial use? The extent of genetic linkage and the degree to
in bacterial populations (Bischoff et al., 2005; Johnsen which co-resistance and virulence are related is an
et al., 2005). However, this does not in itself explain why important consideration in assessing risks associated
pathogens are more frequently resistant to antimicrobials with antimicrobial use.
than the normal flora. The most frequently cited explana-
tion for this difference is the higher selection pressure The Control of Antimicrobial Resistance
exerted on pathogens by repeated treatments. Linkage of
resistance and virulence genes on plasmids is likely to be It is doubtful whether new classes of antimicrobial
an additional factor explaining the higher prevalence of agents will be available for veterinary use in the coming
resistance among many pathogens. Such linkages have years. Novel antimicrobials are likely to be restricted to
already been described sporadically in the past (Martinez

36 Section I. General Principles of Antimicrobial Therapy

human medicine and economic considerations will Aarestrup FM, et al. 2001. Effect of abolishment of the use of
limit development of new antimicrobials only for ani- antimicrobial agents for growth promotion on occurrence
mal use. Thus, the antimicrobials available to veterinary of antimicrobial resistance in fecal enterococci from food
medicine will probably remain the same as today. animals in Denmark. Antimicrob Agents Chemother
Therefore, continued efforts should be made to preserve 45:2054.
their efficacy. Many professional associations, govern-
mental agencies worldwide, and international commit- Alcaine SD, et al. 2005. Ceftiofur-resistant Salmonella strains
tees are developing or have provided guidelines for isolated from dairy farms represent multiple widely dis-
responsible and prudent use of antimicrobial agents in tributed subtypes that evolved by independent horizontal
veterinary medicine and agriculture (chapter 7). gene transfer. Antimicrob Agents Chemother 49:4061.
Additionally, economic incentives and the development
of new market segments, such as the production of food Anderson ES. 1968. Drug resistance in Salmonella typhimu-
from organic farms and “antibiotic-free” animals may rium and its implications. Brit Med J 3:333.
reduce the use of antimicrobial agents in animals. The
role of alternatives to antimicrobials such as vaccines, as Anderson ME, et al. 2009. Retrospective multicentre study of
well as pre- and probiotics, also remains to be thor- methicillin-resistant Staphylococcus aureus infections in
oughly assessed and defined. Finally, maintenance and 115 horses. Equine Vet J 41:401.
improvement of good management practices in com-
panion animal medicine as well as in food animal hus- Angulo FJ, et al. 2004. Evidence of an association between
bandry represent cornerstones in the reduction of use of antimicrobial agents in food animals and antimicro-
antimicrobial use and in the control of antimicrobial bial resistance among bacteria isolated from humans and
resistance. the human health consequences of such resistance. J Vet
Med B Infect Dis Vet Pub Health 51:374.
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antimicrobial therapeutic in use. Today, many clinically Medicine. London: HMSO.
important bacteria are characterized by multiple antibi-
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antimicrobial use and misuse. This modern predica- faecium from broilers and pigs following discontinued use
ment of widespread antimicrobial resistance has led rec- of avoparcin. Microb Drug Res 5:53.
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may be lost, unless there is comprehensive and con- Baker-Austin C, et al. 2006. Co-selection of antibiotic and
certed action to combat the present problem and to metal resistance. Trends Microbiol 14:176.
reverse anticipated developments. Resistance is an inev-
itable biological phenomenon: the challenge is to pre- Bartoloni A, et al. 2004. High prevalence of acquired resist-
vent it from continuing to be a persistent and serious ance unrelated to heavy antimicrobial consumption.
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