4 International Baytril Symposium Proceedings of the

Transcription

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Proceedings of the
4th International Baytril® Symposium
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Bayer Animal Health GmbH
51368 Leverkusen
Germany
www.animalhealth.bayerhealthcare.com
Impressum
Publisher
Coordination Dr. Joy Olsen
Global Veterinary Services
E-mail:
joy.olsen@bayerhealthcare.com
Cover image
Eye of Science
Nicole Ottawa & Oliver Meckes GbR
Reutlingen
www.eyeofscience.com
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Proceedings of the 4th International Baytril® Symposium
Introduction
Two decades ago, the introduction of Baytril® significantly changed the science and art
of treating infectious diseases in small and exotic animals. The challenges associated with
a wide range of infections, including the most severe ones, could be rapidly and more
successfully met for the benefit of our four-legged patients.Today, Baytril continues to be
a firmly established cornerstone and therapeutic standard in veterinary medicine.
This success is rooted in the combination of two basic factors: the outstanding properties
of Baytril as an antiinfective, and the clinical experience of veterinarians around the
globe, acquired over time through innumerable treatments. When tackling infections,
veterinary health professionals still trust that Baytril will work rapidly to restore the health
of their patients, easing suffering and sometimes even saving lives.
Veterinary medicine is continually evolving and offering ever more advanced and high
quality care. Bayer Animal Health has strived to support these advancements not only by
bringing pivotal products such as Baytril to the market, but also through actively supporting progressive clinical research and continuing education, helping to provide timely,
relevant information and improve the management of infectious disease in companion
animal practice.
Symposia such as this 4th International Baytril Symposium underscore our commitment
to the science behind our products and to the veterinary profession. It is fitting that here
in Florence, Italy, a city rich in art, culture, and history, we reflect on the current state of
the art in antimicrobial therapy for a variety of infections in our small animal patients.As
the birthplace of Leonardi da Vinci and the Renaissance, Florence spawned masterpieces
from artists such as Raphael, Botticelli, da Vinci, and Michelangelo, which remain singular
benchmarks of art & culture today.
We would like to express our sincere gratitude to our international panel of speakers for
sharing their collective knowledge, insight and expertise in this forum. Special thanks and
acknowledgement also go to Dr. Ralf Mueller, our symposium chairman, for guiding us
through a program we trust is interesting for all.
Ralf Ebert, DVM
Joy Olsen, DVM
Leverkusen, Germany
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Preface
Medicine is the art and science of healing. It encompasses a range of health care practices
which have evolved to both maintain and restore health through the prevention and treatment of illness. Medical and spiritual healers have been part of all cultures over many
thousands of years. Shamans in various European and American Indian cultures, mudangs
in Korea, ngakpas in Tibet, kahunas in Hawaii, and medicinal doctors in Western civilisations have tried to help injured and sick people. Contemporary medicine applies health
science, biomedical research, and medical technology to diagnose and treat injury and
disease, typically through medication, surgery, or some other form of therapy. One of the
most prominent threats to health both in times past and nowadays is bacterial infection.
Infection may occur through accidental or surgical trauma, a compromised immune
system, or a variety of other causes. Only comparatively recently did it become possible
to treat such bacterial infections with specifically targeted antimicrobial compounds that
show a high enough efficacy and a concurrently low toxicity to be useful in human and
veterinary medicine.
Enrofloxacin (Baytril®) was the first fluoroquinolone to be introduced to veterinary medicine 20 years ago and few medications have had a similar impact on treatment in small
animal practice. Bayer has conducted and supported high quality research on this antibiotic not only during the years of introducing Baytril to the veterinary profession but until
this day.
On this 4th International Baytril symposium, researchers from many European and North
American countries present scientific information on enrofloxacin, underscoring the continuing importance of this antibiotic in veterinary medicine. As chairperson of this symposium I would like to thank the distinguished researchers for the time and effort they
have put into the wide range of cutting edge information in both their manuscripts and
presentations about enrofloxacin and its use in veterinary medicine. This symposium is
now a well-established tradition and will provide competent, up-to-date and clinically
relevant information, in line with the last three symposia.
I would also like to acknowledge Bayer Animal Health for its support of veterinary medicine over the years and its relentless commitment to both research and evidence-based
clinical medicine which ultimately benefits our patients. Such ongoing high quality
research allows us to practice veterinary medicine at the highest possible level. For close
to 100 years, Bayer has been a conductor or partner in scientific research and the veterinary community appreciates this continued commitment to high quality medicine.
Dr. Ralf Mueller
DVM, PhD, DACVD, FACVSc, DECVD
Faculty of Veterinary Medicine
Ludwig Maximilian University Munich, Germany
Chairman
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Contents
6
Baytril®: Historical Impact and Milestones of Veterinary Medicine’s
Most Successful Antimicrobial
Dr. David P. Aucoin
16
Diagnosis and Management of Bacterial Urinary Tract Infections
in Dogs and Cats
Dr. Jodi L. Westropp
24
Bacterial Pathogens of the Respiratory Tract in Dogs
and Antimicrobial Therapy
Dr. Andreas Moritz
36
Update on Clinical Management of Pyoderma
Dr. Antonella Vercelli
44
Bacterial Diseases and Antimicrobial Therapy in Exotics –
Overview on the Use of Enrofloxacin
Dr. Norin Chai
62
Use of Enrofloxacin in Cats with Resistant Mycoplasma spp. Infections
Dr. Mike Lappin
FRIDAY 19th JUNE
68 Unique Pharmacologic Characteristics of Baytril®
Dr. Joy Olsen
74
Pharmacokinetics – What You Need to Know
Dr. Gert Daube, Sandra Mensinger, Dr. Bernd Stephan
80
Baytril® Resistance Monitoring – Susceptibility Status
after More Than 20 Years
Dr. Hans-Robert Hehnen, Dr. Sonja M. Friederichs, Julia C. Heimbach,
Dr. Anno de Jong, Dr. Bernd Stephan
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Clinical Efficacy, Rapid Bactericidal Action and Low Potential
for Resistance Selection of Baytril®
Dr. Joseph M. Blondeau
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Baytril®: Historical Impact and Milestones of
Veterinary Medicine’s Most Successful Antimicrobial
Historical perspective
The developmental history of anti-infectives and
Bayer is often not appreciated by today’s physicians and veterinarians. From the first sulfonamide (prontosil) to the latest 3rd generation
fluoroquinolone, Bayer has led the way. Nothing
is more demonstrative of the leadership role Bayer
has played in anti-infectives than in the development of its most successful antimicrobials, ciprofloxacin in human medicine and enrofloxacin for
veterinary use. These two antimicrobials introduced over 20 years ago have altered therapy like
no others and continue today to be the gold standards other drugs are measured against.
O
Nalidixic Acid
COOH
N
CH3
N
CH2CH3
Norfloxacin
The family of quinolone anti-infectives were discovered serendipitously at Bayer while working
on the anti-malarial compound chloroquine.
Nalidixic acid was restricted to urinary tract infections due to poor systemic absorption but was
very active against most Gram-negative bacteria,
including Pseudomonas species. Since then, Bayer
is responsible for the synthesis of thousands of
compounds from the basic quinolone structure
and, using its chemistry expertise, demonstrated
the role of structure-activity relationships in developing viable drug candidates.
The introduction of a simple halogen molecule,
fluorine, became the preferred structure in a
newer generation of quinolones called fluoroquinolones (Fig. 1). In the late 1980s, norfloxacin
became the first widely used 2nd generation
fluoroquinolone in North America and was my
first introduction to this new class of antimicrobials. However, it wasn’t until the release of enrofloxacin, the first veterinary fluoroquinolone
released in North America, that there was a
change in the approach and treatment of infectious disease.
C2H5
HN
N
N
COOH
F
O
O
Enrofloxacin
O
F
OH
N
N
Figure 1 Quinolone chemical structures.
6
N
Until Baytril® was released in North America in
the late 1980s, the selection of oral antimicrobials
was restricted to amoxicillin, amoxicillin-clavulanic acid (Synulox, Clavamox), cefadroxil, and
potentiated sulfonamides (Tribrissen). All other
oral antimicrobials used were not approved in
companion animals, but out of need used quite
often. These included cephalexin, doxycycline,
and chloramphenicol. Few if any clinical studies
were available on these drugs and efficacy and
toxicity were anecdotal at best. Moreover, none of
these drugs had efficacy against the resistant
Gram-negative bacteria that were becoming
common in chronic urinary tract infections or
against the Pseudomonas aeruginosa infections in
chronic otitis externa. Clinicians like myself were
relegated to using aminoglycosides, gentamicin
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David P. Aucoin, DVM, Dip ACVCP
VCA Antech
Santa Monica, CA, USA
and amikacin. Much of my early years as a clinical pharmacologist were spent determining safe
dosing strategies with these drugs involving costly
and expensive serum drug monitoring. Those
days ended with the advent of Baytril.
Baytril and its metabolites are eliminated into the
urine where high concentrations and tissue penetration make it the drug of choice in urinary
tract infections.
Activity
Baytril disposition and activity
Baytril exhibits all of the general properties of
fluoroquinolones and then some. Its oral bioavailability is both rapid and complete. So much so
that the equivalent dose injected IM has the same
peak serum concentrations as a dose given orally.
This quick oral absorption is critical to its activity, which is based on its serum concentration.
The quicker the absorption, the higher the serum
concentration.
Baytril is the most lipophilic of the veterinary
quinolones, which contributes to its extensive
disposition into all body tissues including privileged sites such as the prostate and brain. Studies
specifically looking into tissue distribution show
that tissue concentrations often exceed serum
concentration. Baytril has been shown to accumulate into neutrophils and macrophages where
it acts as a drug delivery vehicle into infected tissues. This single characteristic allows enrofloxacin
to be used in any infection caused by susceptible
bacteria.
Baytril’s most unusual and distinctive feature is
its transformation into its primary metabolite,
ciprofloxacin. This fluoroquinolone continues to
be the gold standard in human medicine after
20 years of use. Over 40 % of enrofloxacin is metabolized in the liver to ciprofloxacin which has
equal or superior efficacy to enrofloxacin. Given
ciprofloxacin’s variable and at times poor oral
bioavailability in companion animals, it turns
out that the best way to get ciprofloxacin into a
patient is by using enrofloxacin.
Baytril was unique due to its unprecedented
activity against resistant Gram-negative bacteria.
As a safe and effective replacement for costly and
toxic aminoglycoside therapy, it quickly became
a drug of choice for many chronic infections.
As with many 2nd generation fluoroquinolones,
Baytril has extensive activity against most aerobic Gram-negative bacteria. Most notable was the
high degree of activity against Enterobacteriaceae
such as E.coli and also Pseudomonas aeruginosa
(Figs. 2, 3). Its broad activity includes many Mycoplasma and Ricketssia spp. Surprisingly it also has
considerable activity against a few key aerobic
Gram-positive bacteria such as staphylococcal
species (Fig. 4). In vitro efficacy, however was not
a simple yes-or-no interpretation. It was dose dependent.
Baytril was the first veterinary antimicrobial that
used the extensive clinical experience to modify
its dosing options to achieve regulatory approval
for what clinicians had been doing for years –
modifying the dose depending on the organism
and site of infection.The flexible dosing option
approved in the United States was the first of its
kind in veterinary medicine and represented a
significant change in antimicrobial therapy.
PK/PD relationships
The relationship between in vitro activity and in
vivo efficacy has been well documented through
clinical trials. No other veterinary antimicrobial
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Baytril®: Historical Impact and Milestones of Veterinary Medicines Most Successful Antimicrobial
David P. Aucoin
has undergone as many published studies in as
diverse a list of species as Baytril. In a PubMed
review, over 675 published articles with studies in
over 20 animal species were found.The most important studies in the 1990s were establishing the
dose-response relationship of fluoroquinolones,
which revolutionized the approach to antimicrobial therapy.
In determining dose and dose frequency, it became apparent that unlike almost all other antimicrobials, fluoroquinolones like enrofloxacin
had a very rapid effect on susceptible bacteria.
The killing curve for these drugs were extremely
fast, on the order of a few minutes. This rapid
killing was seen only with aminoglycosides and,
like them, their efficacy seemed to correlate with
some simple pharmacokinetic factors such as
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60
40
20
12
0
≤ 0.5
1
2
≥4
Figure 2 Percent of E. coli (n = 53,121) susceptible at tested MIC concentrations of enrofloxacin.
MIC
(ANTECH Diagnostics 2008)
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86
79
80
60
44
40
20
0
14
≤ 0.5
1
2
≥4
Figure 3 Percent of P. aeruginosa (n = 18,987) susceptible at tested MIC concentrations of enrofloxacin.
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MIC
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maximum serum concentration/MIC ratio or
drug exposure (represented by the PK factor area
under the curve or AUC).
This simple correlation changed the way antimicrobials were characterized. Drugs were either a
dose-dependent antimicrobial like Baytril or
time-dependent like amoxicillin. Dose dependency meant changing the dose and not the
frequency of dosing. Baytril was the first once-aday oral antibiotic and arguably the most effective.
Indications for use
Baytril, as most drugs approved through the arduous regulatory approval process, limits its label
claims. Clinical use, however, has extensively
broadened its clinical indications. Most notable
has been the use of Baytril in pyoderma. In the
1990s, it was clear that almost all canine pyodermas were being treated by antimicrobials with
excellent activity against Staphylococcus, the major
pathogen involved in these chronic infections.
Baytril has excellent activity against the major
94
100
Staphylococcus species including Staphylococcus
pseudintermedius (formally known as Staphylococcus
intermedius), Staphylococcus schleiferi ssp. coagulans
and ssp. schleiferi and Staphylococcus hyicus). Chronic
pyoderma also harbored Gram-negative bacteria,
which were effectively treated by Baytril, but
not by the 1st generation cephalosporins such as
cephalexin or cefadroxil. Baytril became an
accepted treatment amongst dermatologists for
treatment of refractory canine pyodermas.
Most surprising was the extended use of Baytril
in the treatment of periodontal disease. Most 2nd
generation fluoroquinolones have poor activity
against the major anaerobic bacteria involved in
this disease, especially Porphyromonas spp. However, it was noted that facultative aerobic Gramnegative bacteria were also involved in maintaining an anaerobic environment and their removal
along with those of the anaerobic bacteria was
effective in treatment.
Baytril continues to have strong indication in all
genito-urinary tract infections, especially those of
chronic nature where tissue penetration or penetration into biofilmed colonies is important.
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88
80
60
40
20
5
0
≤ 0.5
1
2
≥4
MIC
Figure 4 Percent of Staphylococcus spp. (n = 14,158) susceptible at tested MIC concentrations of enrofloxacin.
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David P. Aucoin
been common in this disease due the high prevalence of otitis media in these dogs. Unfortunately,
improper dosing continues to be an issue in these
cases and, unlike that in all other infected sites,
the emergence of resistance is higher in these sites
given the same bacteria. However, it would be
wrong to say that Baytril has lost it efficacy
through extensive use. The data supports the
Its good to excellent activity against most
Enterobacteriaceae and staphylococcal species
makes it a drug of choice in these diseases.
Fluoroquinolones remain the mainstay of treatment for otitis externa where multiresistant bacteria, especially Pseudomonas and Proteus species,
have a high prevalence. Systemic treatment has
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86
2002
2003
2004
2005
2006
2007
2008
80
60
40
20
0
Figure 5 Percent of E. coli cultured susceptible to enrofloxacin (MIC ≤ 2 µg/ml) from 2002 through 2008.
A dose of 5 mg/kg once daily is recommended. Data from ANTECH Diagnostics.
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84
86
2002
2003
84
82
2004
2005
84
82
80
79
60
40
20
0
2006
2007
2008
Figure 6 Percent of P. aeruginosa cultured susceptible to enrofloxacin (MIC ≤ 2 µg/ml) 2 µg/ml from 2002 through 2008.
A dose of 10–15 mg/kg once daily is recommended. Data from ANTECH Diagnostics.
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low potential to spread and is preventable with
adequate dosing.We have been collecting data on
antimicrobial susceptibility using the same CLSI
methodology for the past 8 years and enrofloxacin has seen little to no change in its susceptibility to three key pathogens – Staphylococcus spp.,
E. coli, and Pseudomonas aeruginosa.
concept that loss of efficacy absent P. aeruginosa
has been through misuse, especially through inappropriate dosing.
Resistance
The vast majority of clinically relevant mechanisms for resistance to all fluoroquinolones occur
through one of three ways, all mediated through
chromosomal mutation rather than being plasmid-mediated: 1. lack of penetration into bacterium, 2. removal of drug from bacterium
through an efflux pump, 3. change in its binding
site at the type II topoisomerase (DNA gyrase)
and topoisomerase IV. Of these, point mutations
affecting binding to its site of action are most
documented in veterinary isolates. A single base
pair mutation at the binding site can reduce efficacy, but the type of resistance we see for enrofloxacin (> 4 µg/ml MIC) requires a change in
binding in at least 4 sites.
Specifically, looking at E. coli susceptibility, we see
very little migration of MIC values within the
testing range of ≤ 0.5 to 4 µg/ml. E.coli either
is susceptible at ≤ 0.5 µg/ml or is resistant at
≥ 4.0 µg/ml (Fig. 2).This means that the standard
dose of 5 mg/kg should be used in the treatment
of all infections involving E. coli. At the national
level we have not seen any changes to E. coli susceptibility to enrofloxacin over the past 7 years.
The same is also seen for Staphylococcus spp. where,
unlike with E. coli, we do see a significant increase
in activity from ≤ 0.5 µg/ml to 1 µg/ml. In 2008,
the jump was from 88 % to 94 %.This should not
be looked at as a 6 % difference but rather a risk
ratio. At 88 % susceptible 12 out of 100 organisms
are resistant, while at 94 % the number is 6 out of
100. Therefore, using a higher dose decreases the
The frequency of these point mutations is a subject for other presentations, however, it is important to point out that this type of mechanism has
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2002
2003
2004
2005
2006
2007
2008
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60
40
20
0
Figure 7 Percent of Staphylococcal spp. cultured susceptible to enrofloxacin (MIC ≤ 2 µg/ml) 2 µg/ml) from 2002 through 2008.
A dose of 10 mg/kg once daily is recommended. Data from ANTECH Diagnostics.
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risk of failure by half! I currently recommend a
dose of 10 mg/kg once daily. However, there has
not been significant change in enrofloxacin’s activity against Staphylococcus during the past 7 years
(Fig. 6).
We have seen a slight decrease in activity to
P. aeruginosa over the past 7 years (Fig. 7). Enrofloxacin has always shown less potency against
this organism and this factor coupled with the
lack of appropriate dose has contributed to the
increase in resistance. A MIC of 2 µg/ml is considered susceptible but would require a dose of
20 mg/kg to insure clinical efficacy. However,
since the accuracy of any given MIC is ± 1 dilution, I use a maximum of 1 µg/ml as a susceptible breakpoint. At this level, a dose of 10 mg/kg
is sufficient.
Resistance is a concern for all clinicians and
appropriate use of any antimicrobial is needed.
However, in the face of millions of doses of
Baytril used during the past 20 years, it is remarkable how well this antimicrobial has retained
its efficacy and still is considered the gold standard
of all veterinary fluoroquinolones.
Summary
In the space of a few years Baytril changed antimicrobial therapy, offering a once-a-day oral
dose for the vast majority of bacterial infections
in a plethora of animal species. I could only touch
on a few of the major achievements that this drug
and Bayer has contributed to veterinary medicine. The antimicrobial with more firsts than any
other drug in veterinary medicine. It will continue to be a mainstay of anti-infective therapy
for many years to come.
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(Dromaius novaehollandiae). J Zoo Wildl Med; 28: 43–48.
Hwang YH, Kim MS, Song IB et al. (2008). Altered pharmacokinetics of enrofloxacin in experimental models of
hepatic and renal impairment.Vet Res Commun. Published
online December 16, 2008.
Intorre L, Mengozzi G, Bertini S et al. (1997). The plasma
kinetics and tissue distribution of enrofloxacin and its
metabolite ciprofloxacin in the Muscovy duck. Vet Res
Commun; 21: 127–136.
Lewbart G,Vaden S, Deen J et al. (1997). Pharmacokinetics
of enrofloxacin in the red pacu (Colossoma brachypomum)
after intramuscular, oral and bath administration. J Vet Pharmacol Ther; 20: 124–128.
Linnehan RM, Ulrich RW, Ridgway S (1999). Enrofloxacin
serum bioactivity in bottlenose dolphins, Tursiops truncatus,
following oral administration of 5 mg/kg in whole fish. J Vet
Pharmacol Ther; 22: 170–173.
Lucas RJ, Barnett J, Riley P (1999). Treatment of lesions of
osteomyelitis in the hind flippers of six grey seals (Halichoerus grypus).Vet Rec; 145: 547–550.
Jacobson E, Gronwall R, Maxwell L et al. (2005). Plasma
concentrations of enrofloxacin after single-dose oral administration in loggerhead sea turtles (Caretta caretta). J Zoo
Wildl Med; 36: 628–634.
Malbe M, Salonen M, Fang W et al. (1996). Disposition of
enrofloxacin (Baytril®) into the udder after intravenous and
intra-arterial injections into dairy cows. Zentralbl Veterinarmed A; 43: 377–386.
Kaartinen L, Pyorala S, Moilanen M et al. (1997). Pharmacokinetics of enrofloxacin in newborn and one-week-old
calves. J Vet Pharmacol Ther; 20: 479–482.
Martin Barrasa JL, Lupiola Gomez P, Gonzalez Lama Z
et al. (2000). Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa.
J Vet Med B Infect Dis Vet Public Health; 47: 191–196.
Kaartinen L, Salonen M, Alli L et al. (1995). Pharmacokinetics of enrofloxacin after single intravenous, intramuscular and subcutaneous injections in lactating cows. J Vet
Kempf I, Gesbert F, Guittet M et al. (1995). Dose titration
study of enrofloxacin (Baytril®) against respiratory colibacillosis in Muscovy ducks. Avian Dis; 39: 480–488.
Klein H, Hasselschwert D, Handt L et al. (2008). A pharmacokinetic study of enrofloxacin and its active metabolite
ciprofloxacin after oral and intramuscular dosing of enrofloxacin in rhesus monkeys (Macaca mulatta). J Med Primatol; 37: 177–183.
Kordick DL, Papich MG, Breitschwerdt EB (1997). Efficacy
of enrofloxacin or doxycycline for treatment of Bartonella
henselae or Bartonella clarridgeiae infection in cats. Antimicrob
Agents Chemother; 41: 2448–2455.
Kumar N, Singh SD, Jayachandran C (2003). Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin
and its interaction with diclofenac after intravenous administration in buffalo calves.Vet J; 165: 302–306.
Kung K, Riond JL,Wanner M (1993). Pharmacokinetics of
enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs. J
Vet Pharmacol Ther; 16: 462–468.
Kyriakis SC, Tsiloyiannis VK, Lekkas S et al. (1997). The
efficacy of enrofloxacin in-feed medication, by applying
different programmes for the control of post weaning
diarrhoea syndrome of piglets. Zentralbl Veterinarmed B;
44: 513–521.
Lautzenhiser SJ, Fialkowski JP, Bjorling D et al. (2001). In
vitro antibacterial activity of enrofloxacin and ciprofloxacin
in combination against Escherichia coli and staphylococcal
clinical isolates from dogs. Res Vet Sci; 70: 239–241.
14
Martinez M, McDermott P, Walker R (2006). Pharmacology of the fluoroquinolones: a perspective for the use in domestic animals.Vet J; 172: 10–28.
Meinen JB, McClure JT, Rosin E (1995). Pharmacokinetics
of enrofloxacin in clinically normal dogs and mice and drug
pharmacodynamics in neutropenic mice with Escherichia coli
and staphylococcal infections. Am J Vet Res; 56: 1219–1224.
Mengozzi G, Intorre L, Bertini S et al. (1996). Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin
after intravenous and intramuscular administrations in sheep.
Am J Vet Res; 57: 1040–1043.
Novotny MJ, Shaw DH (1991). Effect of enrofloxacin on
digoxin clearance and steady-state serum concentrations in
dogs. Can J Vet Res; 55: 113–116.
Oluoch AO, Kim CH, Weisiger RM et al. (2001). Nonenteric Escherichia coli isolates from dogs: 674 cases (1990–
1998). J Am Vet Med Assoc; 218: 381–384.
Payot S, Cloeckaert A, Chaslus-Dancla E (2002). Selection
and characterization of fluoroquinolone-resistant mutants
of Campylobacter jejuni using enrofloxacin. Microb Drug Resist; 8: 335–343.
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Randall LP, Cooles SW, Piddock LJ et al. (2004). Mutant
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Wallmann J (2006). Monitoring of antimicrobial resistance
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Schoevers EJ, van Leengoed LA,Verheijden JH et al. (1999).
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ciprofloxacin in the Chinese mitten-handed crab, Eriocheir
sinensis. Anal Biochem; 358: 25–30.
Seguin MA, Papich MG, Sigle KJ et al. (2004). Pharmacokinetics of enrofloxacin in neonatal kittens. Am J Vet Res;
65: 350–356.
Studdert VP, Hughes KL (1992). Treatment of opportunistic mycobacterial infections with enrofloxacin in cats. J Am
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Young LA, Schumacher J, Papich MG et al. (1997). Disposition of enrofloxacin and its metabolite ciprofloxacin after
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Diagnosis and Management of Bacterial Urinary
Tract Infections in Dogs and Cats
Bacterial urinary tract infections
in dogs & cats
The clinical signs of a bacterial cystitis can include
stranguria, pollakiuria, inappropriate urination,
dysuria, and hematuria. Bacterial urinary tract infections (UTIs) occur in approximately 14 % of
dogs during their lifetime, with a variable age of
onset.1 Spayed female dogs have been reported to
be at increased risk for a UTI, which is likely due
to anatomic differences as well as possible protective secretions from the prostate.2 While UTIs are
uncommon in younger cats, the prevalence increases with increasing age. Most young cats that
present with lower urinary tract signs (LUTS) do
not have a positive urine culture. However, in older
cats, a positive urine culture can occur as much as
15–20 % of the time; concurrent illnesses such
as diabetes mellitus, chronic kidney disease, and
hyperthyroidism are often present.3 A slight male
predisposition has been reported in cats, which
likely occurs because male cats can present with
urethral obstruction and are more likely to be
catheterized. In both species, other factors such as
perineal or scrotal urethrostomies,4 a recessed vulva
and perivulvar pyoderma,5 indwelling urinary
catheter,6 or tube cystostomies7 can predispose to
colonization of bacteria in the lower urinary tract.
The entire urinary tract itself has several built-in
“defense mechanisms” to prevent external pathogens from adhering to the urinary mucosa. Normal micturition itself and frequent and complete
voiding can help remove bacteria. Furthermore,
the proximal urethra is sterile and contains
microplicae that expand as urine is voided and
aid in the removal of the bacteria. Although the
distal urethra does contain normal flora, some of
these bacteria can help prevent access of the pathogenic bacteria to the urinary tract by producing
bacteriocin, which can interfere with the metabolism of other pathogenic bacteria.8
16
In addition to anatomic structures and urine voiding, the mucosal surface of the urinary tract has
intrinsic mucosal antimicrobial properties and the
glycosaminoglycan layer can also act as a protective mechanism as well. High urine osmolality
and high concentrations of urea can also inhibit
bacterial growth.While some have stated that dilute (< 1.018) urine may predispose an animal to
bacterial infection, it is likely an underlying disease that causes the animal to produce the dilute
urine that allows the infections to occur. In cats,
we did not find any correlation between decreasing urine specific gravity and positive urine
culture.3 Also, it has been shown in preliminary
data from our laboratory that there does not appear to be a correlation between decreasing specific gravity and positive urine cultures in the
samples that we have obtained from dogs.
Diagnosis of a UTI
As mentioned previously, LUTS can be seen in
many (but not all) dogs and cats that present with
a bacterial cystitis. Many dogs and cats with concurrent diseases do not present with characteristic LUTS, however, a urine culture should be
performed periodically in order to better manage the patient. A thorough physical examination
is important and special attention should be given
to the lower urinary tract. All dogs should have a
rectal examination to evaluate the prostate for size,
pain, and symmetry. The urethra should be palpated for irregularities, mass lesions, or calculi. In
female dogs, the vulva should be examined to
evaluate if it is recessed (“hooded”) and examined for the presence of urine leakage.
In all patients suspected of having a UTI, a urinalysis and bacterial culture provide the most diagnostic information. If a UTI is present, pyruria,
bacteruria, and hematuria can be identified on
the sediment examination. Although not specific
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Jodi L. Westropp, DVM, PhD, DACVIM
University of California
Davis, California, USA
for a UTI, these factors were all associated with
positive urine culture outcome in cats3 and presumably dogs.The sediment is more reliable than
the dipstrips for evaluating these factors; the nitrate and leukocyte assays are unreliable in dogs
and cats.9 The clinician should be aware that bacteria can often be difficult to see on sedimentation unless there are more than 10,000 bacteria/
ml for rods or 100,000 bacteria/ml for cocci.10
A quantitative urine culture obtained by cystocentesis is the gold standard for documenting a
UTI. Ideally, the urine should be cultured within
30 minutes of collection; if this is not possible, it
should be refrigerated and then plated for culture
within 6–8 hours of collection.11 If one cannot
obtain a urine sample by cystocentesis, urine collected by catheterization can be cultured. The
quantitative urine culture allows the clinician to
interpret if the numbers of bacteria present are
considered significant or more likely represent an
artifact. As a general rule, ≥ 1,000 cfu/ml of urine
obtained by cystocentesis is considered significant
in both cats and dogs. If urine is obtained by
catheterization in male dogs and cats, ≥ 10,000
cfu/ml is considered significant.This number increases to ≥ 100,000 cfu/ml for catheterization of
female dogs due to the possible contamination
with bacteria when the catheter passes through
the vestibule. Free catch samples are generally of
no diagnostic value unless the culture is negative.
In most uncomplicated UTIs, a complete blood
count (CBC) and biochemical profile is not
warranted, as the results of these tests are usually
normal. However, if one suspects pyelonephritis
or prostatitis, blood work should be evaluated because significant elevations in the white blood cell
count and renal values can be seen in both of
these conditions. Imaging studies such as radiographs and abdominal ultrasound would also be
indicated if systemic organ involvement were
suspected. Furthermore, if prostatitis is suspected,
aspirates of the prostate can be obtained for
cytology and culture. Advanced diagnostics are
usually warranted in dogs and cats with recurrent
infections (see below).
Sensitivity testing
There are two primary methods for obtaining this
information, the minimal inhibitory concentration (MIC) determination and the Kirby-Bauer
method (disk diffusion test).The MIC is the most
preferred and widely used methodology and determines the least amount of an antimicrobial
agent that causes the complete inhibition of
growth of the infecting species or strain of bacteria. Discriminatory antimicrobial concentrations are used in the interpretation of results of
susceptibility testing to define isolates as Sensitive
(S), Intermediate (I), or Resistant (R). Clinical,
pharmacological, and microbiological considerations are used to set these levels. MIC values are
expressed in μg/ml. The average urine concentration of an antibiotic must exceed the growthinhibiting concentration (MIC value) for the
infecting bacteria by at least fourfold. If the average urine concentration is greater than or equal
to that of the MIC value x 4, the drug will be at
least 90 % effective. Some commercial laboratories
use Kirby-Bauer plates or run only trays set up
with attainable serum levels of antibiotics. Attainable urine concentrations can be 100 times
the attainable serum concentration. For example,
enrofloxacin (and its active metabolite, ciprofloxacin) can achieve concentrations of 200 μg/ml
in the urine, which is 100 times the attainable
serum concentration after a standard oral dose of
5 mg/kg in healthy dogs. The clinician should
consult the laboratory used to determine if serum
concentrations or urine concentrations of the antibiotics were used in the susceptibility testing for
urinary bacterial isolates.With regard to organisms
isolated from urine cultures, if only serum level
17
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Diagnosis and Management of Bacterial Urinary Tract Infections in Dogs and Cats
Jodi L. Westropp
and some Gram-positive uropathogens. Enrofloxacin (Baytril®) is absorbed readily from the
gastrointestinal tract, is poorly bound to plasma
proteins, and has good penetration in tissues to
achieve the necessary high concentrations needed
to eradicate urinary infections.The dose of enrofloxacin approved for dogs in the United States is
5–20 mg/kg once daily or divided12, but one
should not exceed 5 mg/kg/day in the cat due to
the possibility of ocular toxicity characterized by
retinal degeneration and blindness. Once-daily
dosing is preferred and the author recommends
giving the antibiotic at night after the last void.
This will allow the concentration of the antibiotic
to remain in the bladder for an extended interval
to achieve maximal effect against the pathogen.
The fluoroquinolones currently available should
not be used in young growing dogs, in which degeneration of articular cartilage could occur.
trays for determining the MIC value or the KirbyBauer method is used, any antibiotic that is listed
as sensitive will be effective. It is important to
note, however, that the kidneys must be functioning properly for this previous statement to be
true. If the infection is thought to be present in
the kidneys or prostate, or kidney disease is diagnosed, serum MIC concentrations should be obtained to treat the animal appropriately.
Treatment and management of
uncomplicated and recurrent UTIs
In order to select an appropriate antibiotic for a
UTI, susceptibility testing should be performed,
although empiric use of antibiotics for uncomplicated, first occurrence UTIs in dogs can be
tried based on the bacteria isolated. Antibiotics
for common pathogens are shown in Table 1. For
any systemic or recurrent infections, for those animals that have received prior antibiotic therapy,
or for infections that occur in cats, susceptibility
testing should be performed. As shown in Table 1,
the fluoroquinolones (e.g., enrofloxacin) offer
good activity against many Gram-negative species
Escherichia
coli
Appropriate antibiotic therapy and periodic urine
cultures are ideal when treating dogs for bacterial UTIs. Several outcomes can arise which are
depicted in Figure 1. Many animals will have a
simple UTI, whereby the urine is sterilized during treatment, and it remains sterile after the ces-
Proteus
mirabilis
Agent
Coagulasepositive
staphylococci
Amoxicillin
+a
+
+
+
+
+
+
+
+
+
+
+
+
+
Amoxicillin-clavulanate
+
Ampicillin
Cephalexin
+
Chloramphenicol
Klebsiella
pneumoniae
Pseudomonas
aeruginosa
Streptococcus Streptococcus
viridans
canis
+
+
+
+
Enrofloxacin
+
+
+
+
+
Gentamicin
+
+
+
+
+b
Trimethoprimsulfamethoxazole
+
+
+
Table 1 Data obtained from the G. V. Ling Urinary Stone Analysis Laboratory.
+a = > 90 % of strains susceptible based on MIC tests; +b Only 89 % of strains susceptible based on MIC tests
18
+
+
+
+
+
+
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Intact male or female dog/cat?
Recent (past 3mo) UTI?
Predisposing urogenital/Systemic disorder?
(yes)
(no)
Uncomplicated UTI
Complicated UTI
Perform further diagnostics
to look for underlying cause
Tx UTI with appropriate
antibiotic for 10–14 days
Tx UTI with appropriate
antibiotic for 4–6 weeks
Culture 5–7 days after
starting antibiotic
(-)
Culture 5–7 days
post antibiotics
(-)
Cure
(+)
Relapsing/
Reinfection
Culture 5–7 days after
starting antibiotic
(+)
Evaluate owner
compliance &
antiobiotic choice
(+)
(-)
Culture 5–7 days post
antibiotics
Good compliance
Lack of compliance/wrong
antibiotic
Reinstitute
therapy
Persistent
infection
Figure 1 Algorithm for managing bacterial UTIs.
sation of therapy. Although there are no studies
in dogs and cats to determine duration of antibiotic therapy for simple, uncomplicated UTIs, by
convention, these are usually treated for 10–14
days. Proper dosing and administration are essential to prevent the misuse of antibiotics which
could promote antibiotic resistance.
If the dog’s or cat’s urine is sterile during therapy,
but the infection recurs weeks or months later, a
reinfection or relapsing infection has occurred.
Reinfections imply that a new organism or strain
of bacteria has invaded the host, while a relapsing
infection implies that the previous pathogen is
still present. If the urine yields a positive culture
while on antibiotics, the infection is said to be
persistent. Antibiograms were thought to help determine different strains of bacteria, however,
molecular probes using pulse gel electrophoresis
appears to be a superior methodology to determine whether recurrent infections are due to the
acquisition of new isolates or failure to eradicate
previous isolates.13 In both reinfections and relapsing or persistent infections, a search for predisposing causes for the infection should be initiated.
Before pursuing an extensive diagnostic workup,
the clinician should question the client to be certain that the correct medication for the previous
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Jodi L. Westropp
a
b
Figure 2 Cytoscopic views of a bladder mucosal biopsy technique in a female dog with recurrent urinary tract infections. Tissue
obtained should be submitted for aerobic culture; cultures for Mycoplasma spp. and anaerobes can also be considered for specific cases.
UTI was given and that no doses were missed.
Improper dosing can lead to bacterial resistance.
For recurrent (> 3/year) or persistent infections,
other diagnostics such as contrast radiography or
ultrasound should be performed to evaluate for
mass lesions or non-radiopaque stones. Cystoscopy with mucosal biopsy should be considered to evaluate the patient for deep-seated
infections (Fig. 2). It has been reported for dogs
that although urine cultures can be negative, the
bladder mucosa or uroliths (if present) can yield
positive growth.14 Furthermore, in mouse models, Escherichia coli have been noted to develop
within the superficial epithelial cells of the mouse
bladder, forming intracellular bacterial communities.15 Culture of the mucosal biopsies can help
ascertain if this occurs in dogs and cats. If
pyelonephritis or a deep-seated bacterial infection is suspected, antibiotics that achieve good tissue concentrations are warranted.
Recurrent infections can also be due to other
predisposing factors such as metabolic diseases
(e.g., hyperadrenocorticism, diabetes mellitus),
therefore a CBC and biochemical profile should
be evaluated in all dogs and cats that have recurrent or persistent infections. Other differentials
for recurrent infections include a multitude of ab20
normalities that can occur within the urinary system. In dogs, a recessed vulva can predispose the
animals to UTIs; performing an episioplasty can
prevent perivulvar pyoderma and improve
anatomic defenses against uropathogens (Fig. 3).16
Antibiotics should be continued for at least 2–3
weeks after surgery before cessation. Micturition
disorders such as urinary incontinence or urine
retention should be addressed if present. If polypoid cystitis (Fig. 4) or a urachal diverticulum is
noted with imaging studies, removal of these
structures can help remove the nidus for infections. In older dogs that present with recurrent
UTIs, a search for urinary tract neoplasms should
be performed.
Uroliths can also predispose an animal to UTIs
by acting as a nidus for infection.The most common uroliths in cats and dogs are calcium oxalate
and struvite. If an infection is found in an animal
with a calcium-oxalate stone, the infection likely
occurred secondary to the uroliths presence.
However, struvite stones in dogs are usually
formed secondary to a UTI with a urease-producing bacteria such as Staphylococcus intermedius
or Proteus spp. Dissolution of these stones can be
attempted with diet and antibiotic therapy. Penicillins and the fluoroquinolones can be good
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Seite 21
choices for these pathogens, and enrofloxacin is
usually quite effective against these bacteria. The
antibiotics must be given throughout the dissolution protocol, which can take as little as a few
weeks or last as long as 10–12 weeks.
Many dogs will present with chronic UTIs,
weight loss, or prepucial discharge. Prostatic abscesses can also occur after acute or chronic prostatitis, and can cause life-threatening peritonitis
if the abscess were to rupture.
The key to successfully managing both uncomplicated and complicated UTIs in dogs and cats is
by evaluating urine cultures throughout therapy.
Ideally, the urine should be collected by cystocentesis and cultured 5–7 days after therapy has started
and 5–7 days after cessation of the antibiotic. This
will allow the clinician to ascertain the difference
between persistent infections and reinfections and
guide further workup that may be necessary.
Most dogs with bacterial prostatitis have a bacterial cystitis as well. The commonly isolated
pathogens are very similar to isolates obtained
from the bladder. Although in most dogs, a urine
culture will suffice, cultures of the prostate can be
necessary when there is a negative urine culture
or the animal has clinical signs despite appropriate treatment. Diagnostic imaging such as an abdominal ultrasound (Fig. 5a) or retrograde contrast
study (Fig. 5b) should be performed to evaluate
the prostate for size, cysts, abscesses, as well to evaluate for findings compatible with neoplasia. Prostatic fluid can be obtained by ejaculation,
prostatic massage, and most commonly by ultrasound-guided fine-needle aspirate of the prostate.
The fluid should be analyzed for cytological abnormalities as well as cultured for pathogens.
Bacterial prostatitis
Bacterial prostatitis is a chronic or acute condition
in sexually intact male dogs. Acute prostatitis can
have serious systemic ramifications including depression, dehydration, and leukocytosis.Vomiting
and diarrhea and septic shock may also occur.
Chronic prostatitis can also occur and clinical signs
can be vague. The prostate is usually symmetrical
and non-painful upon palpation in chronic cases.
a
Treatment of prostatitis involves appropriate antibiotics and castration. If castration is not an option for a breeding animal, the 5-alpha-reductase
b
Figure 3 A severely recessed vulva with moderate perivulva pyoderma (a) in a 3.5-year-old FS Golden Retriever with recurrent UTIs.
The same dog immediately post operatively after an episioplasty was performed (b).
21
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Jodi L. Westropp
inhibitor, finasteride, can be used to help decrease
the size and secretions from the prostate.17 Surgical
removal of the prostate is rarely performed due to
the high morbidity associated with that procedure.18
However, prostatic abscesses often need to be surgically addressed and omentalization of the prostatic abscess is often performed to prevent fluid and
purulent material from accumulating in the area.
Figure 4 Cystoscopic view of a polypoid mass diagnosed in a
dog with recurrent UTIs.
a
b
Figure 5 An ultrasonographic image of an infected canine
prostate and multiple intraparenchymal cysts (a) and a contrast
urethrogram which illustrates the extravasation of contrast which
can occur in prostatitis (b).
22
Antibiotic treatment for acute prostatitis should
be continued for at least 4 weeks; longer treatment regimens are often warranted for chronic
prostatitis. Due to the blood-prostate barrier, it
can be difficult to achieve levels of antibiotics
above the desired MIC for the bacterial pathogen.
Although the blood-prostate barrier is often broken in acute prostatitis, antibiotics should still be
chosen that will penetrate the blood-prostate barrier, which is important as the infection resolves.
Due to this barrier, an antibiotic with high lipid
solubility, low protein binding, and an appropriate pKa should be used. Non-ionized forms of
antibiotics pass through lipid membranes, whereas
the ionized forms do not. For Gram-negative infections in the prostate, trimethoprim/sulfa, chloramphenicol and the fluoroquinolones are the
most appropriate choices. Enrofloxacin is considered the drug of choice for canine bacterial prostatitis due to its high lipid solubility, low protein
binding, low MIC profile, and broad spectrum of
activity against many uropathogens.19 Furthermore, unlike the other two antibiotics, side effects with enrofloxacin are rare. Oral enrofloxacin
is readily absorbed from the GI tract and approximately 20–40 % is metabolized to its active
metabolite, ciprofloxacin; the fractions of metabolized enrofloxacin were reported to be similar
after intravenous and oral administrations of the
drug.20 Oral ciprofloxacin should not be used as
a substitute for enrofloxacin because the bioavailability of ciprofloxacin is only approximately 40 %
in dogs and is widely variable. The routine dose
of enrofloxacin for prostatitis is usually 10 mg/kg
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once daily. Higher doses may be needed for certain strains of Pseudomonas spp. Once-daily dosing
is preferred, because higher maximum concentrations of the antibiotic are achieved compared
to dividing the dose over the day.
Summary
owner compliance, the appropriate and judicious
use of antibiotics, and periodic urine cultures to
monitor for urine sterility. In complicated UTIs,
a search for and eradication of an underlying cause
will often prevent the occurrence of future infections. When infections are present in the kidney or prostate, a longer course of the appropriate
antibiotic should be implemented and cultures are
warranted to prove the efficacy of the treatment.
Diagnosing and treating UTIs in dogs and cats
can be very rewarding, but does require good
References
1. Ling GV (1984).Therapeutic strategies involving antimicrobial treatment of the canine urinary tract. J Am Vet Med
Assoc; 185: 1162–1164.
2. Seguin MA, Vaden SL, Altier C et al. (2003). Persistent
urinary tract infections and reinfections in 100 dogs
(1989–1999). J Vet Intern Med; 17: 622–631.
3. Bailiff NL, Westropp JL, Nelson RW et al. (2008). Evaluation of urine specific gravity and urine sediment as risk
factors for urinary tract infections in cats. Vet Clin Pathol;
37: 317–322.
4. Griffin DW, Gregory CR (1992). Prevalence of bacterial
urinary tract infection after perineal urethrostomy in cats.
J Am Vet Med Assoc; 200: 681–684.
5. Crawford JT, Adams WM (2002). Influence of vestibulovaginal stenosis, pelvic bladder, and recessed vulva on response to treatment for clinical signs of lower urinary tract
disease in dogs: 38 cases (1990–1999). J Am Vet Med Assoc;
221: 995–999.
6. Smarick SD, Haskins SC, Aldrich J et al. (2004). Incidence
of catheter-associated urinary tract infection among dogs
in a small animal intensive care unit. J Am Vet Med Assoc;
224: 1936–1940.
7. Stiffler KS, McCrackin Stevenson MA, Cornell KK et al.
(2003). Clinical use of low-profile cystostomy tubes in four
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8. Mooney JK, Hinman F (1974). Surface differences in cells
of proximal and distal canine urethra. J Urol; 111: 495–501.
9. Klausner JS, Osborne CA, Stevens JB (1976). Clinical
evaluation of commercial reagent strips for detection of
significant bacteriuria in dogs and cats. Am J Vet Res;
37:719–722.
10. Ling GV, Biberstein EL, Hirsh DC (1980). Bacterial
pathogens associated with urinary tract infections.Vet Clin
North Am Small Anim Pract; 9: 617–630.
11. Lees GE (1996). Bacterial urinary tract infections. Vet
Clin North Am Small Anim Pract; 26: 297–304.
12. Plumb DC (2005).Veterinary Drug Handbook. 5th edn.,
Pharm Vet Inc, Stockholm, WI; pp. 295–298.
13. Drazenovich N, Ling GV, Foley J (2004). Molecular investigation of Escherichia coli strains associated with apparently persistent urinary tract infection in dogs. J Vet Intern
Med; 18: 301–306.
14. Gatoria IS, Saini NS, Rai TS et al. (2006). Comparison
of three techniques for the diagnosis of urinary tract infections in dogs with urolithiasis. J Small Anim Pract;
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15. Anderson GG, Dodson KW, Hooton TM et al. (2004).
Intracellular bacterial communities of uropathogenic
Escherichia coli in urinary tract pathogenesis. Trends Microbiol; 12: 424–430.
16. Lightner BA, McLoughlin MA, Chew DJ et al. (2001).
Episioplasty for the treatment of perivulvar dermatitis or
recurrent urinary tract infections in dogs with excessive
perivulvar skin folds: 31 cases (1983–2000). J Am Vet Med
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17. Sirinarumitr K, Johnston SD, Kustritz MV et al. (2001).
Effects of finasteride on size of the prostate gland and semen
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Bacterial Pathogens of the Respiratory Tract in Dogs
and Antimicrobial Therapy
Introduction
Infectious airway diseases in dogs and cats are associated with symptoms of cough and/or dyspnea,
as well as general signs of disease such as fever,
loss of appetite and apathy. The following article
specifically addresses the interpretation of bacteriological findings in the respiratory tract of the
dog, and their therapeutic relevance.
A number of viral and bacterial pathogens have
been detected in animals with kennel cough, but
their etiological relevance remained unclear for
many years, as they also occur frequently in healthy
dogs.Viruses encountered in this connection include canine parainfluenza virus (CPIV), canine
adenovirus 2 (CAV-2), canine herpesvirus 1
(CHV-1), reoviruses and influenza-A viruses, as
well as canine distemper virus (according to some
literature data). The viral pathogens of kennel
cough usually cause only local infections of the
respiratory tract mucosa. However, damage to
the respiratory epithelium often paves the way for
secondary bacterial infection. The bacterial
pathogens especially involved include Bordetella
bronchiseptica (even without prior viral infection
it is itself an obligate pathogen in the lower respiratory tract), as well as Pasteurella, Streptococcus,
Staphylococcus, Klebsiella, and Mycoplasma. It should
be noted that canine infectious tracheobronchitis
is a disease of underlying factors, i.e., environmental and host factors make a decisive contribution to the expression of the symptoms of
disease. Singular infections of well-maintained
dogs with the aforementioned pathogens usually
lead to only mild or inapparent disease. Multiple
infections and environmental factors such as temperature of the surroundings, relative humidity,
stress (cramped quarters, crowding, etc.), and poor
hygiene promote active disease. The number of
cases increases in the summer (family dogs sent
to kennels) and autumn. However, bacterial infections also arise as a result of other underlying
24
causes such as tracheal collapse, foreign body aspiration, tumors, congestion, etc.
Bacteriological examination of the
respiratory tract
Bacteriological examination with susceptibility
testing of tracheal swabs or bronchoalveolar
lavage (BAL) is an important diagnostic measure,
especially in dogs with chronic or recurrent respiratory tract diseases or in dogs with acute cough/
choking cough with suspected Bordetella bronchiseptica infection. However, when interpreting the
findings, it must be taken into consideration that
the respiratory tract is not sterile even in healthy
animals. In a prospective study, we investigated
the microbial flora of the upper and lower respiratory tract in healthy dogs, as knowledge of the
microfloral composition is particularly relevant
for the evaluation of bacteriological findings in
patients with respiratory disease.The results of the
bacteriological examination of tracheal swabs
were hereby compared with those obtained by
bronchoalveolar lavage in 43 healthy adult dogs
(median age six years) (see Fig. 1).
Bacteria isolated from the respiratory tract
in 43 healthy adult dogs
α-hemolytic Streptococcus
Acinetobacter
E. coli
Neisseria
Staphylococcus intermedius
Erwinia
β-hemolytic Streptococcus
Klebsiella
γ-hemolytic Streptococcus
Pasteurella spp.
aerobic bacillus
Flavobacterium
Staphylococcus epidermidis
Enterobacter
hemolytic E. coli
Proteus
Pseudomonas spp.
Micrococcus
S. aureus/intermedius
Braunella
Corynebacteria
Enterobacteriaceae
Figure 1 Bacterial species isolated from the respiratory tract of
healthy dogs (Bauer et al. 2003).
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Andreas Moritz, Dr med vet, PD, Dipl ECVIM-CA, Assoc. Member ECVCP
Small Animal Clinic, Clinical Pathophysiology and Clinical Laboratory Diagnosis
Justus-Liebig-Universität Giessen, Germany
Bacteria isolated (%) from 151 dogs with respiratory diseases
Bacteria
Tracheobronchitis
Pneumonia
Foreign bodies
Miscellaneous
α-hemolytic Streptococcus
54.9
35.4
28.6
44.4
43.6
31.2
28.6
33.4
γ-hemolytic Streptococcus
15.4
16.7
7.1
5.6
β-hemolytic Streptococcus
7.0
0
7.1
0
E. coli
25.3
35.4
50.0
22.2
Pseudomonas spp.
18.3
16.7
14.2
16.7
Pasteurella spp.
8.4
16.7
21.4
5.6
Klebsiella
9.9
12.5
14.3
11.1
Bordetella bronchiseptica
4.2
4.2
0
0
Proteus
1.4
6.3
14.3
0
hemolytic E. coli
2.8
14.5
0
5.6
Neisseria
15.4
4.2
7.1
16.7
0
6.3
14.2
5.5
No bacteria cultured
Figure 2 Bacterial species isolated from the respiratory tract of sick dogs.
While bacteria could be detected in 95.3 % of
tracheal swabs (tracheal wash), only 44.2 % of
BAL specimens showed bacterial growth. When
bacteria were detected by BAL, only one bacterial species was detected in 61.1 % of the dogs,
compared to 90.2 % of bacteriologically positive
tracheal swabs with mixed flora. In all, the 17 different bacterial species obtained from the trachea
and the eight obtained from BAL could be cultivated in only tiny amounts, with Streptococcus,
bacteria of the Enterobacteriaceae family, and
Staphylococcus dominating. The tracheal and BAL
findings differed completely in 69.8 % of dogs,
with the same results in only 4.7 % of cases and
partially overlapping results in 25.6 % of cases.
The results of our study confirmed that dogs
often have different bacterial flora in the upper
versus the lower respiratory tract. Therefore, the
site from which specimens are obtained for bacteriological examination should absolutely be taken
into account in the evaluation of the findings.
In a retrospective study of 151 dogs (from years
1999–2000) with acute and chronic airway disease (Fig. 2), the patients were divided into the
following groups on the basis of the clinical, laboratory diagnostic and radiographic findings: tracheobronchitis (47 %), pneumonia (12 %), foreign
bodies (9 %), and miscellaneous (12 %, e.g., tracheal collapse, bleeding, tumors). It was shown
that several bacterial species could be isolated in
76.5 % of cases and only one species in 18.5 % of
cases (e.g., E. coli, Pseudomonas spp., Pasteurella
spp., Staphylococcus aureus/intermedius, α-hemolytic
Streptococcus, Klebsiella, as well as Bordetella bronchiseptica).
25
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Bacterial Pathogens of the Respiratory Tract in Dogs and Antimicrobial Therapy
Andreas Moritz
ratory tract. Bordetella bronchiseptica is a short,
Gram-negative rod-shaped bacterium. It is peritrichously flagellated and forms numerous fimbriae as well as other adhesions for attachment of
the ciliated respiratory epithelial cells (see Fig. 4).
The bacterial load varied from slight to severe
(see Fig. 3).
5.3 % of the specimens showed no bacterial
growth. Patients with tracheobronchitis presented
predominantly with Gram-positive bacterial
species, whereas those with pneumonia had predominantly Gram-negative species. Fifty percent
of dogs with tracheobronchial foreign bodies harbored E. coli. In simplified terms, it can be said
that the deeper organisms are found in the respiratory tract, the higher the bacterial content
(colony count) and the more relevant the finding is for the affected animal and for its treatment.
This especially applies to animals in which infection with Bordetella bronchiseptica (with predominately high bacterial levels) could be detected.
Resistance to external influences, especially drying, is limited. Nevertheless, the sources of infection include not only infected animals excreting
the pathogen, but also the contaminated surroundings. Bordetella bronchiseptica exhibits a broad
host spectrum: apart from dogs, species such as
cats, rabbits, guinea pigs, and pigs as well as horses,
seals, and humans (HIV-infected!) have been reported as frequently or occasionally infected.
Pathogenesis
The contagion usually passes from animal to animal via aerosols, and transmission from other animal species must also be taken into account.The
spread of the pathogen is particularly favored by
keeping animals in groups (e.g., kennels, dog-
This very common infection in dogs manifests
itself almost exclusively in the lower respiratory
tract, but to some extent also in the upper respi-
Bacteria isolated from 151 dogs with respiratory diseases
E. coli
Pseudomonas spp.
Pasteurella spp.
S. aureus/interm.
α-hem. Streptococcus
Klebsiella
Proteus
Acinetobacter
hemolytic E. coli.
Aeromonas
Neisseria
Enterobacteriacea
γ-hem. Streptococcus
0
Colony count:
20
40
+, 5–50
60
++, 50–200
Figure 3 Bacterial species isolated from the respiratory tract of sick dogs and respective colony counts.
26
80 % Isolates
+++, > 200
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Seite 27
Figure 4 BAL, three ciliated epithelium cells with cilia (left), two alveolar macrophages (round cells on the right in the picture).
breeding units) or during dog shows.The pathogen adheres to the ciliated epithelium of the respiratory tract primarily via the hair-like fimbriae.
Two other adhesion factors have also been described – a filamentous hemagglutinin and pertactin, acting as mediators via specific receptors.
After successfully colonizing the mucosa, the
pathogen then, by means of various exotoxins
and endotoxins, damages the cilia of the epithelial cells (membrane proteins exhibit adenylate
cyclase activity and thereby lead to lowering of
cilial energy supply, with cilial arrest) so that elimination of the pathogen is no longer possible.The
available phagocytes, responsiveness of the immune system, and mucociliary clearance are also
affected. The bacteria also often penetrate into
the host cells. Ultimately, it has not been definitively determined which factors are responsible
for latent or clinically manifested Bordetella bron-
chiseptica infection. Apart from pathogen-dependent and host-dependent factors, prior damage
due to other microorganisms – viruses, mycoplasma (?), pyogenic bacteria – plays a significant role. This is especially clear in the case of
kennel cough, when dogs are additionally infected with canine parainfluenza virus or canine
adenovirus type 2.
Case history and clinical symptoms
The disease is highly contagious and often arises
in larger dog populations. It is expressed as tracheitis, bronchitis, and tracheobronchitis, advancing as far as purulent-necrotic bronchopneumonia which can be fatal in young dogs. Sick
animals are affected by a sudden onset of a highgrade cough (choking cough), which often sug27
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Andreas Moritz
Figure 5 Oropharyngeal contamination of a tracheal lavage specimen is detectable by the presence of pavement epithelial cells
with Simonsiella bacteria.
gests an inhaled foreign body as a differential diagnosis, especially when initial antibiotic therapy
usually does not lead to the desired response.
Tachypnea and mixed dyspnea, as well as fever
may also arise as signs of bronchopneumonia.
If only the upper respiratory tract is affected, apart
from rhinitis, there may be latent colonization
with the pathogen.
pharyngeal, or nasal swabs are possible in principle
but are less conclusive and difficult to evaluate
due to the normal resident flora. If the cytological examination shows pavement epithelial cells
with Simonsiella bacteria (Fig. 5), the specimen has
certainly suffered oropharyngeal contamination.
Neutrophilic granulocytes (with toxic changes)
in the BAL, which have phagocytized bacteria,
provide important diagnostic evidence for bacterially induced tracheobronchitis (Fig. 6).
Diagnosis
A definite diagnosis is possible only by culturing
the pathogen, as other bacterial infectious agents
(Streptococcus, Staphylococcus, Pasteurella spp., etc.)
may also be involved in this type of localized disease process. The specimen should be obtained
from the lower respiratory tract, for example, by
bronchoalveolar lavage, endoscopically guided
tracheal swab, or transtracheal probe. Laryngeal,
28
The BAL sample must be transported to the test
laboratory without delay and with the use of a
transport medium. Cultivation of Bordetella bronchiseptica can be difficult in mixed cultures and requires incubation for at least 48 hours and the use
of selective media. In cases where Bordetella bronchiseptica is detected in nasal swabs and with low
microbial counts, the possibility of latent infection should be considered.
04.06.2009
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Seite 29
Figure 6 BAL from a dog with cough, showing neutrophilic granulocytes with signs of toxicity and phagocytized bacteria.
Therapy of bacterial tracheobronchitis
While uncomplicated cases of tracheobronchitis/(choking) cough – without detection of Bordetella bronchiseptica – need not be treated with
antibiotics, if there are dyspnoea and signs of systemic disease (apathy, fever), either fluoroquinolones, chloramphenicol, or cephalosporins (see
Tab. 1 for dosage) should be used as the therapy
of first choice. On antibiogram, Bordetella bronchiseptica is usually sensitive to tetracycline, doxycycline, chloramphenicol, enrofloxacin, and gentamicin. However, tetracycline-resistant isolates
have been reported recently. Symptomatic treatments include theophylline to prevent bronchospasms, mucolytics, or inhalation (physiological
NaCl solution, perhaps combined with antibiotic) and, in the case of severe dry cough, the
controlled, and well-timed administration of antitussives, avoiding accumulation of secretions.
The prognosis for tracheal and bronchial infection
is good, but it must be borne in mind that Bordetella may be detectable in the respiratory tract for
up to 140 days and the cough may persist for a
long time (e.g., weeks) despite treatment. If there
is pneumonia, the prognosis is guarded.
Antimicrobial susceptibility testing
Culturing of bacteria from BAL, with susceptibility testing, is very important for targeted
antibiotic therapy. In the retrospective study mentioned above with 151 dogs presenting with various respiratory diseases, the bacteria isolated with
high microbial counts from the lower respiratory
tract were Bordetella bronchiseptica, Pseudomonas,
E. coli, Pasteurella spp., Klebsiella, Staphylococcus
aureus/intermedius, and hemolytic E. coli. Susceptibility testing was performed according to
29
04.06.2009
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Andreas Moritz
DIN 58940. The results are presented in Fig. 7.
From Figure 7 it can be seen that enrofloxacin is
particularly effective against Bordetella bronchiseptica, Pasteurella spp., Klebsiella, and hemolytic E. coli,
and is effective against Staphylocuccus aureus/intermedius infections; amoxicillin/clavulanic acid is
effective against E. coli, hemolytic E. coli, Klebsiella,
Staphylococcus aureus/intermedius, and Pasteurella
spp. In addition, injectable tetracyclines are very
effective against Bordetella bronchiseptica and effec-
tive against Pasteurella spp. and hemolytic E. coli. To
check whether the sensitivity of the bacteria to
the various antimicrobials has changed in recent
years, we again performed a retrospective analysis of the BALs from 77 dogs from the years
2004–2009 with various respiratory diseases. Antimicrobial susceptibility testing (Fig. 8) were performed according to DIN 58940 on bacteria
isolated in larger numbers from the lower respiratory tract: Bordetella bronchiseptica (Fig. 9),
Medicinal
product
Species
Dose
(mg/kg BW)
Route of
administration
Interval
(h)
Amikacin
D, C
10
IV, SC
8
Amoxicillin
D, C
15–20
PO, SC, IV
8
Amoxicillin-clavulanic acid
D, C
15
PO, IV
8
D, C
20
PO, IV
12
Ampicillin
D, C
22–30
PO, SC, IV
8
Cefazolin (1st Gen.)
D, C
20–25
IV
6–8
Cefotaxime (3rd Gen.)
D, C
25–50
IV
6–8
Cefoxitin (2nd Gen.)
D, C
15–30
IV
6–8
Cephalexin (1st Gen.)
D, C
25
PO
12
D
30–50
PO, IV
8
C
30–50
PO, IV
12
D
10
PO, SC, IV
12
C
10–15
PO, SC, IV
12
Doxycycline
D, C
5
PO
12
Enrofloxacin
D, C
5–10
PO, SC
24
Gentamicin
D
3–4
IV, SC
12
C
3
IV, SC
12
Ticarcillin-clavulanic acid
D, C
30–50
IV
6–8
Trimethoprim-sulfonamide
D, C
15
PO, SC, IV
12
Chloramphenicol
Clindamycin
D = dog, C = cat; taken from Reitemeier et. al. 2001, modified according to Green 1998
Table 1 Recommended doses, routes of administration and dosing intervals for antimicrobials used for respiratory diseases in dogs and cats.
30
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Seite 31
Antimicrobial susceptibility (%)
Bacteria
Antimicrobial
n
Enro.
Am/Clv.
Chlora.
Genta.
Sulf/Tr.
Tetra.
Ampi.
Pen.
8
100
63
13
88
25
100
63
0
Pseudomonas spp.
25
72
20
4
96
12
44
8
8
E. coli
12
42
100
33
58
50
0
8
0
Pasteurella spp.
10
100
80
90
80
30
90
70
80
Klebsiella
11
100
91
89
73
36
27
27
0
23
87
91
59
70
70
30
22
30
hemolytic E. coli
6
100
100
100
100
67
83
100
0
Note: for treatment recommendation verify tissue concentrations!
Figure 7 Antimicrobial susceptibility of bacteria isolated from 151 dogs with respiratory diseases. Shown are the number of bacteria
tested and the % of bacteria which are susceptible to the particular antimicrobial agent.
Figure 8 Antimicrobial susceptibility test of Bordetella bronchiseptica
Figure 9 Bordetella bronchiseptica cultured
(cultured on Mueller-Hinton agar plate) by agar diffusion method by using disks
for 24 h (37 °C) on a blood agar plate,
containing antimicrobials.
small gray colonies without hemolysis.
Pseudomonas spp., Pasteurella spp., Klebsiella, E. coli,
and Staphylococcus aureus/intermedius. One therapeutically relevant bacterial species was isolated
in 67/77 dogs, two in 9/77, and three in 1/77.
The antibiogram (antimicrobial susceptibility test)
results are presented in Figures 10–15.
polymyxin B exhibit good efficacy against Bordetella bronchiseptica. Compared to the antibiogram
of Bordetella bronchiseptica isolated 5–10 years previously, the bacteria were much more sensitive to
amoxicillin/clavulanic acid and especially chloramphenicol.
The fluoroquinolones enrofloxacin and marbofloxacin as well as tetracycline, doxycycline and
With regard to Pseudomonas, it can be said that all
isolates were 100 % sensitive to the fluoroquino31
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Andreas Moritz
lones enrofloxacin and marbofloxacin as well as
polymyxin B, whereas sensitivity to sulfonamide/
trimethoprim, amoxicillin, chloramphenicol, and
tetracycline had deteriorated. The susceptibility
pattern for enrofloxacin had improved in comparison to the previous retrospective study.
Whereas all previous Klebsiella isolated were 100 %
sensitive to enrofloxacin, the current succeptibility was 62.5 %. Polymyxin B exhibited 100 %
efficacy by itself.
All Pasteurella spp. isolates were fully sensitive to
enrofloxacin and marbofloxacin as well as amoxicillin, cephalexin, doxycycline, and polymyxin B.
Sensitivity to sulfonamide/trimethoprim and
amoxicillin/clavulanic acid had improved.
The state of E. coli susceptibility pattern is problematic. None of the tested antibiotics/antimicrobials was 100 % effective. Sensitivity to enrofloxacin, sulfonamide/trimethoprim and especially amoxicillin/clavulanic acid had decreased,
but that against tetracyclines has improved (previously 0 %, now 50 %).
Bordetella bronchiseptica (n = 20, *n = 16)
Pseudomonas spp. (n = 7, *n = 4)
Enrofloxacin
Enrofloxacin
Marbofloxacin*
Marbofloxacin*
Polymyxin B*
Polymyxin B*
Sulf./Trim.
Sulf./Trim.
Clindamycin
Clindamycin
Lincomycin
Lincomycin
Gentamicin
Gentamicin
Cephalexin
Cephalexin
Amoxicillin*
Amoxicillin*
Amoxi./Clav.
Amoxi./Clav.
Penicillin*
Penicillin*
Oxacillin*
Oxacillin*
Chloramphenicol
Chloramphenicol
Tetracycline
Tetracycline
Doxycycline
Doxycycline
0%
susceptible, ++
20%
40%
60%
intermediate, +
80%
resistant, –
Figure 10 Antimicrobial susceptibility test of Bordetella
bronchiseptica.
32
100%
0%
susceptible, ++
20%
40%
60%
intermediate, +
80%
100%
resistant, –
Figure 11 Antimicrobial susceptibility test of Pseudomonas spp.
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Seite 33
At present we can only speculate as to the reasons
for the changes in sensitivities to the stated antimicrobials of the bacterial species studied here.
It may be considered that the more frequent or
rarer (e.g., chloramphenicol) use of some medicinal products has contributed. One should keep
in mind, that the total number of isolates tested
for each bacterial species was limited. However,
it can be established that, as before, the fluoroquinolones such as enrofloxacin have very good
efficacy against respiratory diseases of dogs. Additionally, enrofloxacin and its active metabolite
ciprofloxacin have been demonstrated to have
extensive distribution in the respiratory tract,
achieving concentrations in the epithelial lining
Pasteurella spp. (n = 27, *n = 23)
Klebsiella (n = 8, *n = 4)
The tested Staphylococcus aureus/intermedius isolates were fully sensitive to enrofloxacin, marbofloxacin, amoxicillin, cephalexin, doxycycline,
and polymyxin B. Sensitivity to enrofloxacin,
chloramphenicol and tetracyclines had improved.
Conclusion
Enrofloxacin
Enrofloxacin
Marbofloxacin*
Marbofloxacin*
Polymyxin B*
Polymyxin B*
Sulf./Trim.
Sulf./Trim.
Clindamycin
Clindamycin
Lincomycin
Lincomycin
Gentamicin
Gentamicin
Cephalexin
Cephalexin
Amoxicillin*
Amoxicillin*
Amoxi./Clav.
Amoxi./Clav.
Penicillin*
Penicillin*
Oxacillin*
Oxacillin*
Chloramphenicol
Chloramphenicol
Tetracycline
Tetracycline
Doxycycline
Doxycycline
0%
susceptible, ++
20%
40%
60%
intermediate, +
80%
100%
resistant, –
Figure 12 Antimicrobial susceptibility test of Pasteurella spp.
0%
susceptible, ++
20%
40%
60%
intermediate, +
80%
100%
resistant, –
Figure 13 Antimicrobial susceptibility test of Klebsiella.
33
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Andreas Moritz
fluid and alveolar macrophages that exceed
plasma levels. As a supplement to systemically
administered medications, gentamicin and, based
on the results presented here, also polymyxin E
(comparable sensitivity to polymyxin B can be
assumed) can be recommended as inhalation
products, especially for infections with Bordetella
bronchiseptica.
E. coli (n = 14, *n = 12)
Staphylococcus aureus/intermedius (n = 18, *n = 15)
Enrofloxacin
Enrofloxacin
Marbofloxacin*
Marbofloxacin*
Polymyxin B*
Polymyxin B*
Sulf./Trim.
Sulf./Trim.
Clindamycin
Clindamycin
Lincomycin
Lincomycin
Gentamicin
Gentamicin
Cephalexin
Cephalexin
Amoxicillin*
Amoxicillin*
Amoxi./Clav.
Amoxi./Clav.
Penicillin*
Penicillin*
Oxacillin*
Oxacillin*
Chloramphenicol
Chloramphenicol
Tetracycline
Tetracycline
Doxycycline
Doxycycline
0%
susceptible, ++
20%
40%
60%
80%
intermediate, +
100%
resistant, –
Figure 14 Antimicrobial susceptibility test of E. coli.
0%
susceptible, ++
20%
40%
60%
intermediate, +
80%
100%
resistant, –
Figure 15 Antimicrobial susceptibility test of Staphylococcus
aureus/intermedius.
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treatment and prevention. Comp Cont Educ Pract Vet; 25:
902–914.
Weiss R, Moritz A (2007). Bakterielle Infektionskrankheiten und Systemmykosen. In: Klinik der Hundekrankheiten (Eds: Grünbaum EG, Schimke E), 3rd ed., Enke Verlag,
Stuttgart; pp. 1082–1111.
DIN 58940-7:1994-09: Medical microbiology – Susceptibility testing of microbial pathogens to antimicrobial agents
– Determination of the minimum bactericidal concentration (MBC) with the method of microbouillon dilution,
NA 063 Normenausschuss Medizin (NAMed) (New Document: Draft 2008-02).
Wettstein K, Frey J (2004). Comparison of antimicrobial
resistance pattern of selected respiratory tract pathogens isolated from different animal species. Schweiz Arch Tierheilkd;
146: 417–422.
Hawkins EC, Boothe DM, Guinn A, Aaucoin DP, Ngyuen
J (1998). Concentration of enrofloxacin and its active
metabolite in alveolar macrophages and pulmonary epithelial lining fluid of dogs. J Vet Pharmacol Ther; 21: 18–23.
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Update on Clinical Management
of Pyoderma
Introduction
Dogs are considerably susceptible to bacterial skin
infections and many studies have speculated about
the causes related to host factors that enhance the
probability of pyoderma. Current hypotheses for
predisposing factors are: the pH of canine skin
(relatively high), the small amount of intercellular lipids in the canine stratum corneum, the thin
stratum corneum, and the scarce defenses in the
canine follicular ostium.
• Endocrine diseases (hypothyroidism, hyperglucocorticism – primary or iatrogenic)
• Diseases of cornification (“primary seborrhoea”)
• Genodermatoses (follicular dysplasia, color dilution alopecia, sebaceous adenitis)
• Infectious skin diseases (dermatophytosis, Malassezia dermatitis)
• Occult neoplasia (solar-induced squamous cell
carcinoma, epitheliotropic lymphoma)
• Autoimmune diseases (pemphigus complex)
• Immunodeficiency (congenital, acquired).
Underlying diseases, which predispose to disruption of the skin barrier, are considered fundamental to the development and recurrence of
pyoderma.
The role of staphylococci in
pyoderma: carriage, colonization,
infection
Commonly recognized underlying diseases which
play a consistent role in pyogenic infection of the
skin are:
• Allergic skin diseases (canine atopic dermatitis,
food allergy, flea allergy dermatitis)
• Parasitic skin diseases (sarcoptic mange, cheyletiellosis, demodicosis, trombiculosis)
• Systemic parasitic diseases (leishmaniosis)
To this point in time, Staphylococcus intermedius
(recently renamed S. pseudintermedius), the most
frequent isolate in cases of canine pyoderma, is
not considered a potent pathogen but its virulence can be enhanced by many factors leading to
infection. As a member of the residential microflora, S. intermedius may be regularly cultured
from the oral and anal mucosae and perineal region of normal dogs, but this population is considerably higher in dogs with superficial bacterial
folliculitis compared to healthy animals. For this
reason, the paradigm of carriage of staphylococci,
based on the human scheme, is still considered
pertinent for both the pathogenesis and treatment
of recurrent pyoderma.
Recurrent staphylococcal skin infections are frequently seen in dogs. Some studies suggest that
hypersensitivity to bacterial antigens may be involved in the pathogenesis of this clinical syndrome.
Figure 1 Cytological sample of pustule from a case of recurrent
superficial pyoderma. A large number of cocci are evident inside
and outside the neutrophils, which are partially degenerated
(Diff Quick stain 40 X).
36
Atopic dogs with pyoderma have detectable
serum IgE against staphylococcal antigens, and it
has also been demonstrated that staphylococcal
antigens can penetrate the skin.
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Antonella Vercelli, DVM, CES derm., CES opht.
Ambulatorio Veterinario Associato
Turin, Italy
Even if uncommonly, other species of staphylococci have been isolated from dogs with pyoderma and there is growing concern about the
role of more aggressive pathogens such as S. aureus, Staphylococcus schleiferi, and the appearance of
multidrug-resistant cocci. In most text books,
Gram-negative organisms such as Proteus sp.,
Pseudomonas sp., and Escherichia coli are discussed
as secondary invaders in cases of deep or chronic
pyoderma. However, in practice, Pseudomonas
aeruginosa and other Gram-negative organisms can
be the sole pathogen in some clinical cases. Unusual staphylococcal infections or Gram-negative
organisms are more likely to be resistant to multiple antibiotics.
Currently, most dermatologists agree on the fact
that although the treatment of pyoderma was easily performed based on empirical choice of antibiotics in the past, now, especially in referral
practice, managing the infection is a more complicated process, requiring bacterial culture and
susceptibility testing and selection of appropriate
treatment based on the individual patient
requirements.
methoprim/sulfamethoxazole is frequently observed.
Methicillin-resistant Staphylococcus aureus (MRSA)
is a well-recognized pathogen in human medicine and is mainly associated with human hospital-acquired infections worldwide; it can cause
life-threatening infection after surgical procedures
and the multidrug resistance increases health care
costs due to prolonged hospitalization. In Staphylococcus aureus, methicillin resistance is conferred
by a protein, a penicillin-binding protein, known
as PBP2A. This protein is coded by the mecA
gene which confers an intrinsic resistance to all
beta-lactam antibiotics and their derivatives. Multidrug resistance to aminoglycosides, fluoroquinolones, fusidic acid, and mupirocin may be observed. The presence of individuals carrying these
bacteria, without clinical disease on mucosae or
the skin is now a growing public health concern.
Resistance to cephalosporins and/or fluoroquinolones by Staphylococcus intermedius has remained low in Europe for many years, with
effective drugs for systemic therapy in pets generally available.
Facing MRSI, MRSA …
zoonosis or anthropozoonosis
At present, antimicrobial chemotherapy is the
most practical way to treat staphylococcal infections and there have been many studies on the
“in vitro effects” of antimicrobial agents against
strains of S. intermedius isolated from the dog.The
percentage of strains resistant to various classes of
antibiotics varies in different continents, even
countries, and changes with time. Irrespective of
the place of isolation, most S. intermedius strains are
susceptible to amoxicillin/clavulanic acid, gentamicin, oxacillin, cephalosporins, and enrofloxacin; however, resistance to penicillin, ampicillin,
tetracycline, erythromycin, lincomycin, and tri-
Figure 2 German Shepherd pyoderma: a multiresistant
Staphylococcus intermedius was isolated from this 8-year-old
male dog.
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Antonella Vercelli
improve identification of multiresistant isolates.
In clinical practice, zoonosis must be a significant
concern in any veterinary patient bearing large
numbers of pathogenic bacteria that display worrisome antibiotic resistance, but, conversely, the
possibility of anthropozoonosis should also be
considered due to the presence of carriers among
the human population.
Figure 3 A macroscopic view of the ulcerative lesion of the
same case.
However, multiresistant, mecA-positive S. intermedius isolated from dogs and cats is now emerging in Europe.
Prior antibiotic use is a known risk factor for
selection of resistant strains of bacteria. A first report of multiresistant, mecA-positive Staphylococcus intermedius (MRSI) in Europe in a veterinary
dermatology referral clinic in Germany indicated
an incidence of 23 % among the isolates, and included resistance to cefalexin, methicillin, and
enrofloxacin. More recently, the author has observed similar data in Italy, with an incidence of
18.5 % of MRSI/MRSA from selected cases of
pyoderma in dogs previously treated with several
antibiotics.Twenty-one of 113 cases were phenotypically identified as multidrug- and methicillinresistant staphylococci.They were represented by
9 cases involving S. aureus, 10 Staphylococcus intermedius, and 2 with Staphylococcus xylosus. Only one
S. aureus was resistant to the entire panel of antibiotics tested, while the other 20 were resistant
to more than four antimicrobial classes and sensitive at least to rifampicin. Based on these observations, treatment with antibiotics must be
based on culture and sensitivity of swabs, and inclusion of oxacillin (methicillin) in antimicrobial
susceptibility testing panels is advisable and may
38
In summary;
• MRSA is primarily a human pathogen and infections in animals remain infrequent.
• Some cases of MRSI/MRSA have been identified in referral dermatological practice, in dogs
previously treated with antibiotics.
• MRSA is a zoonotic pathogen that can have
possible consequences for human health, but
may also be transfered by humans to pets and
vice versa.
• MRSA transfer is considered a possible indicator of inadequate practice hygiene.
• Human beings, dogs, and cats can carry MRSA
or MRSI on skin and mucosae, without signs of
clinical disease.
Empirical treatment of staphylococcal infection
has been the norm in veterinary dermatology.
Historically, usually only refractory cases were cultured, leading to an underestimation of the occurrence of MRSA/MRSI in pets. A more
rational approach would be the use of the right
antibiotic based on bacterial culture and sensitivity testing. For the majority of cases, it is possible
to identify the same pathogen in all the skin lesions, but in a small number of dogs there are bacteria with different susceptibility to antibiotics,
which can lead to a clinical failure despite a presumably “good antibiotic choice”.
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Common trends in treating skin
infections
The aim of the treatment is resolving the infection and the skin lesions, providing relief to the
patient (controlling pruritus and pain) and preventing recurrence (with an extensive diagnostic
workup). This goal is better achieved combining
topical and systemic antimicrobial therapy. However, surface pyoderma, in mild or localized cases,
can be managed with topical therapy, thus avoiding antimicrobial selective pressure in the intestinal tract. Weekly antibacterial shampoos, daily
sprays or foams containing antibacterial ingredients are usually employed such as chlorhexidine,
ethyl lactate, triclosan, and benzoyl peroxide, because they aid in decreasing the surface bacterial
counts and limiting decolonization. Adverse reactions to topical agents can occur, but they are
infrequent.
Clipping enhances the probability of efficacy of
the topical treatment even if it is not always well
accepted by the owner.
The use of antibiotic creams such as neomycin,
fusidic acid, or mupirocin was suggested for lo-
Figure 4 Chronic pododermatitis (furuncolosis) in a 3-year-old
male Corso dog.
calized lesions in the past, however, concerns
about the use of mupirocin has emerged because
this molecule is considered the “gold standard” in
human medicine to treat nasal carriers of MRSA.
Successful management of diffuse superficial pyoderma or extensive bacterial overgrowth and of
all deep pyoderma requires systemic antibiotic
therapy. The first step is the selection of an appropriate antibiotic and establishment of an appropriate dose for a sufficient duration to ensure
cure, rather than transient remission.Time frames
of one to three weeks of antimicrobial therapy
beyond clinical cure are empirical concepts, although currently widely accepted. For this reason,
it is very important to re-evaluate every case of
pyoderma within two to three weeks after starting the treatment; this enhances the probability
of discovering compliance problems due to owner
or to the pet, and identifying factors that may
contribute to the occurrence of clinical failure.
Antimicrobial dosage recommendations for different infections may underestimate dosing
needed in deep pyoderma. A concern about the
“mutant selection window hypothesis” was first
proposed in human medicine and is also beginning to be an important concept in the treatment
of infections in pets.We know now that there may
be a “dangerous drug concentration zone”, which
enhances the survival probability of organisms
having reduced susceptibility to the drug. The
lower boundary of that selection window is represented by the MIC (minimum inhibitory concentration) and the upper boundary is the MPC
(mutant prevention concentration). If we use a
drug dosing that achieves concentrations above
the MIC level but below the MPC, the majority
of bacteria will be eliminated, but a small amount
of them will constitute a subpopulation of resistant mutants. So using antibiotics within the mutant selection window will favor the increase of
resistant mutant subpopulation. However, if drug
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Antonella Vercelli
concentrations above MPC are achieved, bacteria
must acquire two different mutation genes at the
same time, which is a very rare event. Therefore,
newer drugs and pharmacological studies are oriented towards employing safe dosing above the
MPC for the optimal management of infections.
Further complication of the treatment of skin infections may be due to the “Darwinian evolution” of microorganisms – this concept states that
mutations are continuously randomly occurring
to improve their survival capability. Microbes are
also able to elaborate biofilms that help in producing aggregates of organisms protected from
the toxicity of antimicrobials and from host defenses, and develop signals that influence the settling behavior of other organisms, activating
transcription of genes (the mechanism of “quorum sensing”).
So, from the clinician’s point of view, we must also
evolve our therapeutic model and start to use the
antibiotic drug that has the highest probability of
efficacy for a particular infection, with regard to
antibacterial activity, as well as one with favorable
ability to rapidly penetrate and diffuse into tissues,
and that is easily administered to the patient (hit
quickly, hit rapidly, hit for an adequate time).
The choice of drug
An antibiotic chosen empirically should have a
known spectrum of activity against S. intermedius
and should not be inactivated by beta-lactamases,
and must have bactericidal activity.
Presently, antibiotics considered useless for pyoderma include erythromycin, penicillin, amoxicillin, and tetracycline.
Sulfonamides are usually not prescribed due to
the potential for drug reactions, but could be used
40
Figure 5 Acral lick granuloma secondary to neuropathy in a
mixed breed dog, 11 years old, female.
in cases of MRSA/MRSI, if indicated by susceptibility tests. Drugs such as clindamycin and
lincomycin can still be used, but a risk of
10–25 % of resistant strains of cocci should be expected in practice.
Amoxicillin and clavulanic acid is currently employed, but some dermatologists advise using at a
higher dose than on the label, three times a day.
Cephalosporins have been the favorite antibiotic
class in dermatology for a number of years, with
different options for administration and posology.
They are still very effective as an empirical choice,
however, some resistant bacterial populations are
identified both in superficial and deep pyoderma.
Fluoroquinolones are still extremely active for the
treatment of skin infections, including Gram-negative isolates; resistance when employed as firstline therapy is very rare, but some cases of resistance in recurrent superficial and deep chronic
pyoderma have been observed in referral dermatological practice.
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Suggested use of enrofloxacin for
the treatment of pyoderma (more
than twenty years later)
Enrofloxacin is a fluorinated quinolone-carboxylic acid derivative developed exclusively for
use in animals. It is licensed for use in many countries in Europe and the rest of the world at a
dosage of 5 mg/kg once daily, and in the USA at
a dose range of 5 to 20 mg/kg once daily for the
treatment of skin and soft-tissue infections with
a spectrum of activity including efficacy against
S. intermedius, selected Pseudomonas spp. and other
Gram-negative invaders.
Enrofloxacin has been on the market for many
years and is still a first-line choice antibiotic for
the treatment of deep pyoderma and granulomatous lesions of the skin due to its ability to achieve
high accumulation in tissues and inside macrophages and neutrophils in the dog, with relatively
low incidence of clinical bacterial resistance.
Fluoroquinolones are rapidly bactericidal, acting
on the bacterial nuclear material (disruption of
DNA replication) and, contrary to other antibiotic classes, they do not select for plasmid-mediated resistance. One of the metabolic products of
enrofloxacin is ciprofloxacin, which has been
shown to increase the production of interferon
and that of interleukin-2 enhancing its killing effect on bacteria including Pseudomonas aeruginosa.
A recent study determined that the concentration of enrofloxacin in the extracellular fluid in
healthy dogs after oral and intravenous administration was equivalent to the plasma concentrations.
to predict the efficacy of fluoroquinolones in
tissues.
Statistically significant higher skin levels of enrofloxacin were seen in dogs with deep pyoderma
versus superficial pyoderma, and in contrast to
levels in normal skin, thus indicating that the drug
is transported into the tissue by inflammatory cells.
Synergistic effects have been noted between fluoroquinolones and beta-lactam penicillins. The
effects of penicillins on bacterial cell wall permeability may allow better penetration of fluoroquinolones. Fluoroquinolones should not be
given in conjunction with drugs that inhibit protein or RNA synthesis (e.g., chloramphenicol and
rifampin); these agents may diminish the activity
of the fluoroquinolone.
As a personal choice, I usually recommend enrofloxacin as a first-line antibiotic for the treatment
of mucocutaneous pyoderma, for German Shepherd pyoderma, pododermatitis complex and in
cases of acral lick granuloma, and confirmed by
susceptibility testing.
Precautions in use are related to possible damage
to the articular cartilage in rapidly growing large
breed dogs, so avoid use in giant breed dogs before 18 months of age and in other breeds prior
to 12 months of age. In cats, it is advised not to
use dosages exceeding 5 mg/kg due to the occurrence of retinal damage at elevated doses.
Treatment with enrofloxacin would not be recommended for a bacterial organism intermediate or resistant in susceptibility to enrofloxacin as
appropriate levels of enrofloxacin would not be
expected to be attained.
Enrofloxacin and ciprofloxacin binding to plasma
proteins is low in dogs and does not impair diffusion to the interstitial space. Therefore, plasma
concentrations may be used as a surrogate marker
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Conclusion
The therapeutic choice of an antimicrobial in
clinical practice is often empirical. In dermatology, the common opinion in the past was
oriented towards the use of molecules with increasing potency in cases with therapeutic failure
of the previous drugs. The use of the most effective and powerful antibiotic was reserved for the
more serious clinical case, or the ones characterized by recurrences. In reality, over the past few
decades, the empirical selection of the antibiotic,
and the use of inappropriate dosing or duration of
the treatment (sometimes secondary to lack of
compliance), has potentially influenced the adaptability of bacteria in many cases, with appearance
of resistant pathogens.
Currently, this approach is being replaced by a
more rational therapeutic attitude that immediately employs the use of the antibiotic with the
greatest probability of potency and effectiveness
against the involved bacteria, in order to avoid
the selection of resistant strains and the appearance
of chronic skin lesions.
References
DeBoer DJ (1995). Management of chronic and recurrent
pyoderma in the dog. In: Kirk’s Current Veterinary Therapy XII (Ed: Bonagura JD), W. B. Saunders, Philadelphia;
pp. 611–617.
Miller WH Jr (1992).The use of enrofloxacin in canine and
feline pyodermas and otitis in dogs. Proc 1st Int Baytril Symposium, Bonn; pp. 41–48.
Ehrlich P (1913). Chemotherapeutics: scientific principles,
methods and results. Lancet; ii: 445–451.
Rantala M, Holso K, Lillas A et al. (2004). Survey of condition-based prescribing of antimicrobial drugs for dogs at a
veterinary teaching hospital.Vet Rec; 155: 259–262.
Ganiere JP, Medaille C et al. (2001). Antimicrobial activity
of enrofloxacin against Staphylococcus intermedius strains
isolated from bacterial pyodermas. Vet Dermatol; 12(3):
171–175.
Riond JL, Wanner M (1993). Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous
and oral administration of enrofloxacin in dogs. J Vet Pharmacol Ther; 16: 462–468.
Ihrke PJ (1996). Experiences with enrofloxacin in small animal dermatology. Suppl Comp Cont Educ Pract Vet; 18(2):
35–39.
Tillotson GS (2003). Deja-vu: a ‘new’ approach to managing bacterial infections. Pulmon Perspec; 20: 7–9.
Koch HJ, Peters S (1996). Antimicrobial therapy in German
Shepherd pyoderma (GSP): an open clinical study.Vet Dermatol; 7: 177–181.
Loeffler A, Linek M, Guardabassi L et al. (2007). First report
of multiresistant, mecA-positive Staphylococcus intermedius in
Europe: 12 cases from a veterinary dermatology referral
clinic in Germany.Vet Dermatol; 18(6): 412–421.
42
Vercelli A, Carnevale M, Cornegliani L (2008). Multidrug
and meticillin – resistance in Staphylococcus sp. Canine recurrent superficial and deep pyoderma in Italy. Scientific
abstract of the 6th World Congress of Veterinary Dermatology, November 19–22, 2008.Vet Dermatol; 19 (Suppl 1): 37.
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Bacterial Diseases and Antimicrobial Therapy in
Exotic Species – Overview on the Use of Enrofloxacin
Introduction
The importance of bacterial diseases in exotics
and wild animals is well documented. Writing
about “exotic animal medicine” means dealing
with thousands of species. Even within the same
animal class, the anatomy and physiology of
the digestive, renal or respiratory systems differ
widely among species. However, after taking into
account these several specific considerations, medicating exotics is accomplished by the same methods of administration used in domestic mammals.
Following their arrival in clinical medicine in the
later 1980s, the fluoroquinolones have become a
widely used group of synthetic antimicrobials in
veterinary medicine. They have provided small
animal clinicians with a truly exciting new class
of antimicrobials. Never before had veterinarians
a drug with such a broad spectrum of activity,
combined with the pharmacokinetic properties
that allow for oral administration on a once-aday basis. This has allowed clinicians to treat not
only a larger number of patients, but also other
species with more assurance.
Enrofloxacin is a member of the family of 6-fluoro-7-piperazinyl-4-quinolones and was designed specifically as a veterinary drug, available for
companion animals in a tablet form and an injectable preparation. Modification of the 4quinolone ring has enhanced the antimicrobial
activity of this compound. It is widely used to
treat bacterial infections in exotics and wildlife
and has become the drug of choice for exotics
and wild animals clinicians. Enrofloxacin is a
highly effective bactericidal compound with relatively low minimum inhibitory concentrations
(MIC). By altering the action of bacterial DNA
gyrase, a type II topoisomerase (an enzyme involved in unwinding, cutting, and resealing
DNA), it has excellent activity against Gram-negative and also Gram-positive pathogens. Enrofloxacin does not readily complex with plasma
44
proteins, which enables metabolites to readily
cross cell membranes. This antibiotic has been
used to control certain intracellular pathogens.
Following some general considerations, this paper
provides an overview on the main bacterial diseases encountered in exotic species. Special emphasis has been made to point out basic principles
and to underscore some of the traps to be
avoided. The main class of vertebrates will be
approached and in each class, an overview on the
use of enrofloxacin is discussed.
General overview
The skills, medications, and protocols used in
medicine and surgery of domestic carnivores are
applicable to exotics. Therefore, thorough physical examination should be followed by several
clinical tests. Because most of the exotic species
are highly dependent on their environmental
conditions, husbandry records are extremely important. Reviewing information on both the sick
individual and apparently healthy animals is part
of the diagnostic process. Before any therapy is
instituted, the clinician must carefully consider
questions on the husbandry situation of the patient, especially its nutritional status. There is no
sense in instituting antimicrobial therapy in exotic
practice without correcting zoo-technical deficits.
One should always have in mind that antimicrobial therapy is only a part of a more general therapeutic plan.
The practitioner must also be careful to avoid the
unnecessary prophylactic use of antimicrobials
that can result in antimicrobial resistance and in
the emergence of infections that were previously
subclinical, or that might interfere with experimental studies if the patients are experimental
models.
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Norin Chai, DVM, MSc, MScVet, PhD
Ménagerie du Jardin des Plantes,
Museum of Natural History Paris, France
Looking more specifically at antimicrobial therapy, the principles of antibiotic used in dogs and
cats apply similarly to exotics. Ideally, before instituting antibiotics, the clinician should qualify
the nature of the infection, predict the most likely
pathogen(s), obtain a culture and a minimum
database, and choose the antibiotic based on those
results. Establishing these basic parameters allows
the clinician to form a reasonable prognosis and
a clear rationale for the therapeutic approach and
length of treatment required. However, in the
author’s experience, many animals are often
presented late in the disease process. Immunosuppression in clinically ill subjects, the rapid progression to life-threatening diseases, and the
suspected presence of mixed infections are indications for empirical use of combination antibiotics while waiting for culture and susceptibility
results. The clinician should select not only the
most efficient drug but also the safest and will
use the most efficacious route of administration,
knowing that restraint is often difficult and parenteral therapy can have its own limitations. Many
dosage regimens have been designed largely on
an empirical basis. In most of the cases, supportive care is as important as antimicrobial therapy, as
well as correcting nutritional deficits.
As a rule, optimal treatment of infectious diseases
depends on accurate diagnosis, susceptibility to the
selected drug, husbandry practices, and anatomical
and physiological differences among the species.
where bacterial infections are a common problem.
They often result in walled-off abscesses with
caseated pus in subcutaneous tissues and/or other
sites, nervous symptoms for infection of the inner
ear, and septicemia. Both Gram-positive and
Gram-negative bacteria may be isolated. Aerobic
bacteria are the most common.
Ferrets
General considerations
Primary bacterial infections in pet ferrets are not
common and should be lower on a list of differential diagnoses. If present, these infections are
rather secondary to another primary disease
process (traumatic, viral, or parasitic). A ferret with
a bacterial disease will often present significant
alterations of its hematologic profile, with white
blood cell counts higher than 8,000, with primarily neutrophilia. Chronic affections tend to be
characterized by monocytosis.
Dental tartar, gingivitis, and periodontal disease
are common in middle-aged and older ferrets.
Tooth root abscesses are not common but can occur at any age.
In ferrets, gastrointestinal disease is common.
However, in practical terms, diarrheas are more
Mammals
Medication and diagnostic process used in domestic carnivores can be applied in ferret medicine. There are no specific intolerances to any
drugs described. In ferrets, primary bacterial infections seem uncommon and are rather secondary to another primary disease process. In rabbits
and rodents, there are numerous organ systems
Figure 1 Primary bacterial infections in pet ferrets are not
common and, if present, are rather secondary to another primary
disease process (traumatic, viral, or parasitic). Here is a ferret
presented with an advanced bronchopneumonia.
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Bacterial Diseases and Antimicrobial Therapy in Exotic Species – Overview on the Use of Enrofloxacin
Norin Chai
likely to result from non-infectious causes: foreign bodies (very common), trichobezoars, neoplasia. A fecal culture can be difficult to interpret.
The prevalence of primary bacterial gastroenteritis is unknown. Helicobacter mustelae infection,
the most commonly described, causes ulceration
of the gastrointestinal mucosa, clinically characterized by lethargy, vomiting, anorexia, melena, and
weight loss. Definitive diagnosis of Helicobacter disease is best made based on clinical signs, gastric
Figure 2 Bacterial infection of the inner ear in a chinchilla.
(Photo courtesy of Dr. Gersende Doumerc)
biopsy, and response to treatment. Other gastrointestinal bacterial infections appear to be uncommon in ferrets.
Urolithiasis may induce bacterial cystitis. Gramnegatives are commonly isolated in bacterial
prostatitis, an infection closely linked with hypertrophy of the adrenal glands. The treatment is
based on surgery (removing the affected adrenal
glands, draining the cysts, and in some case, marsupialisation of the prostate to the body wall) and
administration of appropriate antibiotics. The
disease can endure even when there is no longer
adrenal gland androgen influence on the prostate.
Like the other organ systems, the respiratory tract
is an uncommon area for bacterial infection in
ferrets. Both canine distemper (CDV) and influenza virus can be complicated by secondary
bacterial respiratory disease. In practical terms,
bacterial respiratory disease secondary to influenza virus infection is rare in immune competent
ferrets and bacterial pneumonia secondary to
CDV infection is rarely treated, as CDV itself is
fatal in ferrets.
Primary bacterial dermatitis is uncommon compared to skin tumors, which are more frequently
seen in ferrets. In all cases, ferret dermatology
follows a classical diagnostic process: cytology or
biopsy and culture of the affected area.
Figure 3 Advanced pneumonia in a rat. Infectious respiratory
diseases are the most common health problem in rats and have
multifactorial causes, with Mycoplasma pulmonis often the
major component.
46
(Photo courtesy of Dr. Gersende Doumerc)
Antimicrobial therapy in ferrets
Usually, the antibiotics that are safe to use in dogs
and cats are safe to use in ferrets and the most appropriate antibiotic based on culture results can be
used without regard to serious gastrointestinal upset. Antibiotic administration in ferrets includes
the parenteral (intramuscular, subcutaneous, intraperitoneal, intravenous, or intraosseous), enteral, and topical routes. Because of their small
body size, the intramuscular route is rarely used
for an extended period of time in ferrets. The
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intravenous or intraosseous routes are reserved
primarily for the most severe cases of bacterial
disease. The subcutaneous route of antibiotic administration is favored. In ferrets, the enteral route
is used extensively, especially with antibiotics administered at home, given by syringe or in the food.
The ideal antibiotic for use in ferrets is easily administered and bactericidal. Because it can be
given orally with once-a-day administration and
is effective against serious Gram-negative infections, the most commonly used drug is enrofloxacin.
Enrofloxacin is used at 10–30 mg/kg q24h IM,
SC, PO. Enrofloxacin may be used with metronidazole if anaerobic infections are suspected, at
a dose of 20 mg/kg q12–24h PO. The incidence
of anaerobic infections in ferrets is unknown.
Rabbits and rodents
Rabbits
Unlike ferrets, bacterial infections are common in
rabbits. However, the clinician should keep in mind
before starting an antibacterial therapy that nearly
all important diseases in rabbits are directly or indirectly related to diet and feeding practices or environmental conditions. The basic diagnostic
approach first includes thorough investigation of
the husbandry conditions. Bacterial infections induce an inflammatory reaction characterized by
caseated pus and walled-off abscesses that are not
necessarily associated with an increase in total
white blood cell count. Many of them are
attributed to Pasteurella multocida, even if numerous
species of bacteria are cultured routinely from rabbits. Some additional selected bacterial species
known to infect rabbits include pathogenic species
of Staphylococcus and Streptococcus, Klebsiella, Proteus,
Pseudomonas, Listeria, Actinomycoses, and Actinobacillus. A study demonstrated that anaerobic bac-
teria, not Pasteurella, were common causes of bacterial disease in rabbit abscesses. As a rule, for abscess treatment, antibiotic administration alone will
not cure the problem and surgical debridement of
the area is always necessary.
Respiratory tract infections are very common
in rabbits. While the clinical diagnosis may be
obvious, characterization of the disease is not
(is this a recently developed upper respiratory bacterial disease or a chronic infection that becomes
apparent only now?), and neither is the treatment.
Furthermore, bacterial infections of the respiratory tract may act as the nidus for which bacteria spread to other areas of the body either by
local invasion (for example, in the ear canal causing otitis) or septicemia.
Bacterial otitis is common in rabbits.There is also
a potential complication with infection of the
central nervous system by invasion of the cranial
nerves. Aural discharge, a vestibular syndrome,
and hyperthermia are often observed. The treatment is based on antibiotic therapy, antihistaminic
and antiinflammatory drugs administration, and
careful nursing.
Conjunctivitis, keratitis, naso-lacrimal duct and
retrobulbar infections are also common in rabbits, as well as infection of the urinary tract. Bacterial cystitis might be an important pre-disposing
cause for urolithiasis, and so in cases of urolithiasis in rabbits, the clinician should make a culture.
Subcutaneous abscess are walled off with caseated
pus. It appears rather spontaneously and the clinician should always search for a bacterial nidus
of infection somewhere else, which might be
challenging. Bacterial dermatitis is generally superficial and secondary to another infectious or
non-infectious disease. Dermatology of rabbits
follows a classical diagnostic process: cytology or
biopsy and culture of the affected area.
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Dental abscesses are common in rabbits and may
involve the mandibular or maxillar teeth. In cases
of odontogenic abscesses, multiple adjacent teeth
may be affected. The most common bacterial
species isolated are a mixture of anaerobic Gramnegative rods, such as Fusobacterium nucleatum,
anaerobic Gram-positive non-spore-forming rods,
predominantly Actinomyces species, and aerobic
Gram-positive cocci of the Streptococcus milleri
group. Other reported pathogens include Pasteu-
rella and Staphylococcus species. Dental abscesses always have an origin in infection of tooth roots,
with or without osteomyelitis of the mandible.
Mandibular abscesses are non-resectable and
therefore carry a poor prognosis. The clinician
must remember that jaw abscesses are frequently
due to organisms that travel from other sites; failing to recognize this may lead to antibiotic treatment failure.
Enteritis complex due to proliferation of pathogenic bacteria is the most common gastrointestinal disease in rabbit medicine. Causes involve
diet, antibiotics, stress, and genetic predisposition
to gut dysfunction. Maintaining optimal husbandry, minimal stress, avoiding sudden changes
in the diet, and offering good quality grass hay
are the most important factors for prevention. Primary bacterial enteritis is less common and mostly
involves pathologic strains of E. coli. Use of enrofloxacin (10 mg/kg, PO q12h) in early treatment, while awaiting culture and sensitivity test
results, has provided positive clinical results. It also
strongly advised to provide supportive care and
to prevent the overgrowth of other pathogenic
bacteria and the production of toxins. For this,
administration of cholestyramine (2 g in 20 ml
water q24 h PO), a strong anionic bile-acid exchange resin capable of binding bacterial toxins,
has shown good results.
Figure 4 Ocular bacterial infections are common in rabbits,
presenting with blepharospasm, tearing, discharge, and swelling
of the ocular tissues. Here is a rabbit with panophthalmia that
required the enucleation of the eye.
48
Small rodents
In rodents, there are numerous organ systems
where bacterial infections are a common problem.
Bacterial infections of the respiratory tract and
gastrointestinal system are the most common in
the majority of rodents. Guinea pigs are very susceptible to respiratory disease caused by Bordetella
bronchiseptica (Gram-negative rod commonly carried by rabbits, dogs, and non-human primates)
and Streptococcus pneumoniae (Gram-positive coccus). Salmonella typhimurium and S. enteritidis are
the most common causes of bacterial enteritis in
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guinea pigs. Stress and nutritional deficiencies increase susceptibility to diseases. Enteropathies are
the most common problem seen in pet hamsters.
Lawsonia intracellularis and Clostridum sp. are the
common pathogens isolated in the young and
adult, respectively. In rats, infectious respiratory
diseases are the most common health problem,
characterized by two major clinical syndromes:
chronic respiratory disease and bacterial pneumonia. In most of the cases, these diseases have
multifactorial causes with Mycoplasma pulmonis as
the major component. In general, bacterial infections of the respiratory tract are secondary to a
viral disease process in rodents. Ectoparasites represent the first primary predisposal cause of bacterial dermatitis in rodents. Following the
example of the rabbit, dental infections are very
common.
do not have the same susceptibility. At low doses,
lincomycin administration is fatal to hamsters and
guinea pigs, but a much higher dose of this antibiotic given to rats for 30 days exhibited no toxic
effects. Tetracyclines may induce dysbiosis in
guinea pigs but apparently not in other species.
Antimicrobial therapy in rabbits and rodents
Antimicrobial therapy in rodents and rabbits entails greater risk than in most other species because inappropriate therapy can result in death of
the patient due to enterotoxemia. Some antibiotics provoke a disruption of the normal enteric flora in rodents and rabbits, which can be
potentially fatal.This dysbiosis is caused by a sudden loss of microbial diversity in the cecum of
affected animals, which subsequently leads to the
overgrowth of opportunists such as Clostridium sp.
and E. coli. Ultimately, the production of toxins
from these pathogens, specifically Clostridium spiroforme (iota toxins) leads to enterotoxemia. Antibiotics that may induce these gastrointestinal
diseases include parenteral penicillin, oral or injectable cephalosporins, tetracycline, and erythromycin. Antibiotics that are highly likely to cause
gastrointestinal dysbiosis include amoxicillin,
ampicillin, clindamycin, and lincomycin. Lincomycin has been shown to consistently provoke
cecal dysbiosis, leading to a fatal diarrhea. Ampicillin has been shown to kill up to 40 % of rabbits
when administered orally. However, all rodents
Medicating the drinking water is advantageous
in that no stress of capture or restraint is involved
and large numbers of animals can be treated easily. However, exact dosing is impossible since water intake, disease status, dehydration, age vary
among individuals. Interspecies differences must
also be considered. For example, guinea pigs consume relatively large amounts of water, whereas
gerbils consume very little. Additionally, if the
drinking water has an undesirable taste from the
antibiotic, the patient may stop drinking and become dehydrated. Direct oral dosing is much preferred to medication of the drinking water, since
the amount administered can be accurately determined based on the needs of the individual.
The drug can be hidden in a small piece of fruit
for instance or directly administered with a syringe after a proper restraint technique.
The ideal antibiotic to use in rabbits and rodents
is one easily administered, bactericidal, and which
not cause gastrointestinal disease. The clinician
should be aware of the high basal metabolism of
small mammals, thus, it is important not to give
too low of a dosage and to institute a twice-daily
(or even three times daily) administration. In our
rodent and lagomorph practice, it is common to
complement the antibiotic therapy with probiotic
administration such as Lactobacillus sp., which is
appropriate in general.
Enrofloxacin is one of the most commonly used
antimicrobials to manage many different bacterial
diseases in lagomorphs, from Pasteurella multocida
to Mycoplasma sp. Oral dosing with enrofloxacin
does not appear to lead to the development of
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antibiotic dysbiosis. Enrofloxacin is well distributed through the tissues, including milk, and
should be used cautiously in lactating rabbits.
Dosing rabbits with 5 mg/kg q12h enrofloxacin
PO or IM will achieve MIC levels appropriate
for most bacterial pathogens. Some doses at 20
mg/kg SC, PO q12h have been given to rabbits
as well.
To date, there have not been any pharmacokinetic
studies for enrofloxacin performed on rodents.
Although it is empirical, doses for most rodents
should be 5 to 20 mg/kg IM once, followed by
oral administration every 12 or 24 hours. Small
specimens would need a dose at 10 mg/kg PO,
IM, q12h. Mice require higher dosing of 25–85
mg/kg q24h PO.
Depending on the indication, it is common to
give enrofloxacin with metronidazole at a dose
of 20 mg/kg q12–24h PO.
Birds
Microbial diseases are common in companion and
aviary birds. Bacterial infections may be primary,
however, secondary infections due to poor husbandry conditions, stress, nutritional deficiencies,
viral processes, or parasitic burden are the most
common. Additionally, many secondary invaders
are able to maintain a disease process independent of other infectious agents or predisposing
conditions. Thus, with suspicion of bacterial infection, the clinician should always try to find the
predisposing causes and should employ all practical diagnostic techniques to direct primary care
before initiating any therapy.
The status of certain patients highly suggests
either an existing infection or the potential for
one – birds presented with wounds, purulent si50
Figure 5 A very advanced pododermatitis in a pheasant; the
main causes of the disease are environmental deficiencies and
penetrations. Note that in birds, purulent material is rather solid.
nus discharge, odiferous feces, increased warmth
of the feet or beak. However, the differentiation
between primary or secondary infection may be
challenging as laboratory examinations (biochemical or serologic) are rarely of any help in
this differentiation. Again, historical data and husbandry records are essential. Blood work may at
least suggest the presence of an infectious process.
Elevated white cell counts accompanied by a heterophilia or monocytosis support this hypothesis.
Further diagnosis will be based on hematology,
cytology, Gram’s strains, and specific stains (such
as Ziehl-Neelsen staining for mycobacteriosis
diagnosis), and culture and sensitivity testing. It is
important to underscore here that there is not an
obvious relation between the symptoms observed
in the patient and the microbiological isolate obtained through standard culture techniques. Obtaining mixed cultures of different bacterial
isolates suggests secondary infections are occurring and that the primary pathogen and/or the
predisposing conditions still needed to be identified. Just as isolating a bacteria that is part of the
autochthonous flora may suggest an opportunistic overgrowth. For this kind of interpretation, it
is highly advantageous for the clinician to have
databases or good literature on specific normal
flora, from which bacterial results may be dis-
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cussed with consistency. Finding viruses or other
known pathogenic agents that play a significant
role in the cause of illness in pet avian species
(such as Chlamydophila or Mycoplasma) also assumes that the bacterium may be a secondary
pathogen.Yet organisms such as Chlamydophila or
Mycoplasma are not easily detected through routine in-house screening. Isolation of an almost
pure culture may indicate that the bacterium is
an important, or even the main, component in
the disease process. Just as having a positive bacterial culture with (only) fungi and protozoa suggests its prominent pathogenic role.With necropsy
or living biopsy, isolating small to moderate numbers of bacteria from the liver or kidney is not
pathognomonic.These organs should be expected
to contain autochthonous flora, due to the avian
hepatic and renal portal circulations and the lack
of lymph nodes that filter blood before it drains
into the liver and kidney.
The most common causes of primary and secondary bacterial infections in psittacine birds are
Gram-negative bacteria (Escherichia coli, Klebsiella,
Pseudomonas) and Chlamydophila psittaci. Gramnegative bacteria are frequently resistant to routine
antibiotics, however, most isolates are susceptible
to enrofloxacin. Enterobacteriaceae (Salmonella,
Citrobacter, Proteus, Serratia), Enterococcus faecalis
(canaries) are common as well. Less common
infectious agents of psittacine birds are Staphylococcus aureus and Streptococcus spp., Mycoplasma, Bordetella, Mycobacterium, and Pasteurella multocida.The
causative organisms of mycobacteriosis are Mycobacterium avium spp. avium, M. intracellulare, and
M. genavense. The disease may be asymptomatic
for long periods and the main clinical symptoms
are chronic wasting, weakness, labored respiration,
diarrhea, skin granulomas, lameness, and death.
There is a zoonotic potential, particularly for immunocompromised individuals. M. genavense is of
greatest zoonotic concern.Thus, therapeutic management must be considered with caution or per-
haps not even recommended except for valuable
and endangered species. M. avium is resistant to
common antituberculosis drugs, however, combination therapy (isoniazid, ethambutol, and rifampicin) for extended periods (up to 18 months)
has resulted in clinical remission in some exotic
birds.
Once the antimicrobial therapy is decided, it is
important to ensure the antibiotic reaches therapeutic levels in all target sites. For example, direct
flushing or nebulization is needed to bring effective concentrations of antibiotics in the upper respiratory tract. Avian abscesses are usually presented
with solid pus and are completely unavailable to
antibiotic penetration. Surgical excision or debridement followed by topical medication are often essential parts of the therapy. In all cases,
antibiotic therapy is one part of the therapeutic
process. Correcting husbandry and nutritional deficiencies, giving supportive care (placing the animal in its optimal thermal zone, fluid therapy and
gavage if needed) are also essential.
Antimicrobial therapy in birds –
the use of enrofloxacin
The ideal antibiotic to use in birds is bactericidal,
readily absorbed and widely distributed with therapeutic concentrations in tissues, easily administered, and does not cause adverse effects. Because
of the high basal metabolism of birds, it is important to ensure the availability of therapeutic
concentrations. Thus, medicated water, a traditionally favored route for poultry, should not be
adapted in companion and aviary birds and would
rather be a last choice (e.g., multiple-bird flocks).
Serious microbial infections and critically ill birds
should be treated with parenteral medications (or
PO by gavage) to establish effective drug concentrations quickly. It’s important not to give too
low of a dosage and to institute a twice-daily, or
even three times daily, administration.
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Enrofloxacin is widely used to treat bacterial infections in companion birds. Enrofloxacin is readily absorbed and widely distributed, and partially
metabolized to an active metabolite, ciprofloxacin,
in many species, including psittacine birds. Enrofloxacin is bactericidal for many Gram-negative
bacteria and species of Staphylococcus and Mycoplasma. Enrofloxacin is highly active against most
Enterobacteriaceae recovered from psittacine birds.
A recent study has compared the efficacy of enrofloxacin, oxytetracycline, and sulfadimethoxine
for the control of morbidity and mortality caused
by Escherichia coli in broiler chickens. Chickens
that received enrofloxacin had significantly less
mortality (p < 0.01), lower average gross pathology (colibacillosis) scores (p < 0.01), and better
feed-conversion ratios (p < 0.05) than chickens
that received either oxytetracycline or no medication. Chickens that received enrofloxacin had
significantly less mortality and lower pathology
scores than those that received sulfadimethoxine,
and numerically lower feed conversion than the
sulfadimethoxine group. Results from the study
showed that enrofloxacin was superior to oxytetracycline and sulfadimethoxine for the control
of morbidity and mortality caused by E. coli in
broiler chickens.
Enrofloxacin has variable activity against Pseudomonas aeruginosa and species of Streptococcus and
Chlamydophila; it has little activity against anaerobes. It may also be used for the treatment of
chlamydophilosis and reduces clinical signs in
birds infected. The duration of treatment should
be 21 days for all species. Practical experience indicates that total eradication with clearing the
carrier state is often difficult and not routinely
achieved.
Route of Administration
Selecting the route of drug administration in birds
requires careful consideration. Available routes
52
Figure 6 Enrofloxacin is most effective if dosed per os or by
intramuscular injection; intramuscular injection achieves greater
peak concentrations than oral administration. IM dosing is
performed in the pectoral muscle.
include medicated water, medicated food, oral,
intramuscular, intravenous, subcutaneous, intraosseous, intratracheal, inhalation, and topically.
Water-based drug administration is easiest and less
stressful route of administration. However, consumption is erratic and therapeutic serum concentrations are rarely achieved, especially during
the night when less water is consumed. Several
studies have measured the plasma concentrations
of enrofloxacin achieved by offering drinking water medicated with an injectable enrofloxacin formulation. However, practical applications of these
results must be taken with caution as the animal
models were healthy animals. A debilitated patient
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would have a different behavior and wouldn’t
have the water consumption.
One study concluded that 0.19 to 0.75 mg/ml
enrofloxacin in drinking water should provide appropriate MIC levels for susceptible infections in
African gray parrots (Psittacus erithacus).The study
also showed that African gray parrots tend to consume less water as the doses get higher (0.09–3.0
mg/ml). Another study measured the plasma concentrations of enrofloxacin with 200 mg/l enrofloxacin in drinking water in 16 psittacine birds
for 10 days. The birds included 6 cockatoos (Cacatua species), 4 conures (Aratinga species), 2 Senegal parrots (Poicephalus senegalus), 2 red-shouldered
macaws (Ara nobilis), and 2 gray parrots (Psittacus
erithacus). The authors concluded that water
medicated with the injectable formulation of enrofloxacin at 200 mg/l maintains plasma concentrations in psittacine birds that are adequate only
for treating systemic infections caused by highly
susceptible bacteria. Administration ad libitum to
8 healthy sandhill cranes (Grus canadensis) of enrofloxacin in the drinking water at a concentration
of 50 ppm would only be effective for treating infections of highly susceptible bacteria. In this
study, enrofloxacin and ciprofloxacin concentrations were both below accepted therapeutic
plasma concentrations for birds.
Enrofloxacin is most effective if dosed per os or
by injection. Given orally, the injectable formulation of enrofloxacin produces therapeutic plasma
concentrations, even more, it induces higher peak
plasma concentrations than the water-soluble formulation. The major disadvantage of parenteral
administration is intramuscular pain and irritation at the site of injection. However, in the Amazon parrot and cockatoo, intramuscular injection
achieves greater peak concentrations compared
to oral administration.
Enrofloxacin can be administered orally but is bitter, and many birds will refuse to accept it. It may
be necessary to dilute the drug in a palatable vehicle such as fruit juice or lactulose syrup, or to
deliver it via a gavage tube. Oral administration
accomplished by feeding red-tailed hawks (Buteo
jamaicensis) and great-horned owls (Bubo virginianus) a small freshly killed mouse that had received an intraperitoneal injection of enrofloxacin
(15 mg/kg) had provided appropriate MIC levels for susceptible bacteria. Peak plasma levels were
achieved between 4 to 8 hours for orally administered enrofloxacin (compared to 0.5 to 2 hours
for IM dosing). Studies on the single-dose kinetics
of enrofloxacin in healthy African gray parrots,
blue-fronted and orange-winged Amazons, and
Goffin’s cockatoos indicate that a dose of 7.5–
15 mg/kg administered IM or PO BID should
maintain effective concentrations in these species.
Again, intramuscular injection achieves greater
peak concentrations (3–5 mg/ml versus 1–1.5
mg/ml with oral administration at 15 mg/kg). Enrofloxacin can be given with metronidazole to
broaden the spectrum as well as to take advantage
of the ability to control anaerobic bacteria.
The dose of enrofloxacin in birds used by the author is 20 mg/kg PO, IM q24h for most of companion birds. For small specimens, a dose of
10–15 mg/kg PO q12h would be recommended.
Larger birds such as ratites would need lower drug
dosages: 1.5–2.5 mg/kg PO, IM q12h.
Reptiles
Infectious diseases are an important cause of illness and mortality in all reptilian species. Again,
the therapeutic plan will begin first by correcting
environmental and nutritional deficiencies, which
are the most important predisposing causes of diseases in reptiles.Without this first step, there is no
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pathogens, infections caused by Gram-negative
bacteria are most common. Aeromonas hydrophila,
Klebsiella oxytoca, Morganella morganii, Providencia
rettgeri, Pseudomonas aeruginosa, and Salmonella arizonae are prominent among the microorganisms
isolated from healthy and ill captive reptiles.These
bacteria can remain dormant and become invasive
when conditions decrease the immune resistance
of the host and/or follow primary viral infection.
Anaerobic infections are more common than
once thought and may be involved in up to 40 %
of all bacterial infections.
Figure 7 Abscesses caused by traumatic injury, bite wounds,
or poor environmental qualities are seen in all orders of reptiles.
The pus is generally solid, as in birds. Mandibular abscess in a
green iguana (top) and a frilled lizard.
sense in starting other treatments. Environmental
temperatures should be maintained near the upper limit preferred by the species to enhance
immune function. Higher metabolic rates of anorectic reptiles may necessitate force feeding. Fluid
therapy should be considered as well. Ideally,
severely affected reptiles should be isolated and
antibiotic therapy initiated. In general, good sanitation is paramount in prevention of all diseases.
The enclosure should be set up to reduce stress,
with addition of hide boxes. Arboreal animals
should be furnished with a secluded branch on
which to lay.
Although a wide variety of bacteria have been
incriminated as either primary or secondary
54
Abscesses caused by traumatic injury, bite wounds,
or poor environmental conditions are seen in all
orders of reptiles. Differential diagnoses include
parasitic nodules, tumors, and hematomas. Isolates
of the anaerobic organism Peptostreptococcus and
of the aerobes Pseudomonas, Aeromonas, Serratia,
Salmonella, Micrococcus, Erysipelothrix, Citrobacter
freundii, Morganella morganii, Proteus, Staphylococcus,
Streptococcus, Escherichia coli, Klebsiella, Arizona, and
Dermatophilus have been recovered from reptilian
abscesses, often in combinations. Antibiotic administration will always be combined with surgery that can be quite invasive in large abscesses,
in order to remove as much material as possible.
Aural abscesses are commonly seen in chelonians,
most frequently in box turtles and aquatic turtles.
Marked swelling is seen at the tympanic membrane, and caseous material is present. Proteus sp.,
Pseudomonas sp., Citrobacter sp., Morganella morganii,
Enterobacter sp., and other bacteria have been isolated. The tympanic membrane must be incised,
followed by an aggressive curettage and flushing
of the area, with diluted povidone-iodine or
chlorhexidine.
All snakes and some lizards posess a transparent
spectacle located over the cornea. Subspectacle
infections are common in snakes. Drainage is
achieved by surgically removing a small wedge
from the spectacle and flushing with an antibi-
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otic solution directly onto the globe and within
the space. In the other orders, conjunctivitis is
sometimes seen, ranging from mild inflammation
to panophthalmitis, and may occur as a result of
ascending infectious stomatitis.Topical antibiotic
ointments are used in turtles, lizards without
spectacles, and crocodilians.
Trauma, local abscessation, parasitism, or environmental stress may induce septicemia, a common cause of death. Aeromonas and Pseudomonas
sp. are frequently isolated in such cases. Petechiae
may be found on the ventral abdomen, and
chelonians develop erythema of the plastron.
Infectious stomatitis is frequently seen in snakes,
and less so in lizards and turtles.The disease course
begins with petechiae in the oral cavity, followed
by caseous material appearing along the dental
arcade, and the infection can extend into the bony
structures of the mouth. Aeromonas and Pseudomonas spp. are most frequently isolated. Debridement, irrigation with antiseptics, systemic
antibiotics, and supportive therapy are indicated.
Stomatitis is a secondary infection. The animal’s
environment should be modified as necessary to
aid in recovery.
Respiratory infections are common in reptiles.
The incidence can be influenced by respiratory or
systemic parasitism, unfavorable environmental
temperatures, unsanitary conditions, concurrent
disease, malnutrition, and hypovitaminosis A. In
snakes with neurologic symptoms, one should
always consider involvement of a viral disease
process.Turtles often have an underlying vitamin
A deficiency. Increased temperatures are important not only to stimulate the immune system but
also to help mobilize respiratory secretions. If the
reptile does not respond to environmental correction and the antibiotic therapy, a culture and
sensitivity along with histology should be performed.
Figure 8 Advanced stomatitis in a boa constrictor (left) and a
red-eared turtle. Stomatitis is most commonly seen in snakes
and lizards, less frequently in chelonians. Pockets of caseous pus
may occur in the soft tissues. Left untreated, the condition may
progress to osteomyelitis of the mandibular and cranial structures, and teeth may be found loose within the necrotic tissue.
Antimicrobial therapy
Culture and sensitivity are essential in determining
appropriate therapy. Because most infected reptiles
have some level of immunosuppression, bactericidal drugs are preferable to bacteriostatic ones. Enrofloxacin has a wide spectrum of antimicrobial
activity which includes the common reptile
pathogens and a large volume of distribution and,
thus, has become a commonly used antimicrobial
in reptile medicine. Enrofloxacin is effective in the
treatment of Mycoplasma infections such as conjunctivitis and soft tissue infections. A recent study
showed that against Mycoplasma iguanae, a proposed
species nova isolated from vertebral abscesses of
two feral iguanas (Iguana iguana), clindamycin,
doxycycline, oxytetracycline, and tylosin were bacteriostatic from 0.1 to 0.5 µg/ml, whereas enrofloxacin was bactericidal at 20 ng/ml.The MIC
for common reptile pathogens is lower for enrofloxacin than other commonly used antimicrobials. Enrofloxacin, and its active metabolite
ciprofloxacin, are capable of inhibiting growth of
pathogenic bacteria at serum levels of approximately 0.1 µg/ml. Mixed or resistant infections
may require combinations of antibiotics. Enrofloxacin can be given with metronidazole to
broaden the spectrum as well as to take advantage
of the ability to control anaerobic bacteria.
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In most cases, antimicrobials will be given by injection, either SC or IM. Oral administration is
reserved for primary infection of the gastrointestinal tract, for species that do not tolerate injections or extremely small specimens (some
chameleons and geckos) that lack adequate muscle mass for a painless injection. Since most species
of reptiles have a renal portal system, with blood
from the caudal half of the body going to the kidneys before reaching systemic circulation, it has
been recommended that SC and IM injections
should be given in the cranial half of the body.
However, there are few studies that have looked
at this potential problem scientifically.
For the dosing, if metabolic scaling may work in
mammals and birds, it does not in reptiles. Differences in body temperature, season, reproductive
status, nutritional, and overall specific physiology
are just a few of the variables that may ultimately
influence metabolic rates and thus make any
equation of metabolic scaling invalid.
Because IM injection is often painful and may
induce adverse local effects, the pharmacokinetics of enrofloxacin disposition following oral
administration have been investigated in green
iguanas (Iguana iguana), savannah monitors (Varanus
exanthematicus), red-eared sliders (Trachemys scripta
elegans), American alligators (Alligator mississippiensis), and in loggerhead sea turtles (Caretta caretta).
There is a markedly varied disposition among
species, indicating that extrapolation between reptile species is likely to result in inaccurate dosing
of enrofloxacin. Green iguanas given injectable
enrofloxacin PO at 5 mg/kg had therapeutic plasma concentrations of enrofloxacin (> 0.2 µg/ml).
However, enrofloxacin does not appear to be metabolized to ciprofloxacin in significant amounts
in green iguanas. This result suggested that the
parenteral route would be more suitable than oral
administration for the treatment of critical infections in green iguanas. Savanna monitors fed mice
56
containing 10 mg/kg of injectable enrofloxacin
showed similar but delayed therapeutic plasma
concentrations as compared to IM injection.
Thus, in Savanna monitors, an initial dose of enrofloxacin (10 mg/kg IM) followed by oral administration for continued therapy would be
beneficial for acute infections. In American alligators, 5 mg/kg PO is not expected to achieve
minimum inhibitory values for susceptible organisms based on a pharmacokinetic study in this
species, however, IV administration every 36
hours would. In loggerhead sea turtles, a dosage
rate of 20 mg/kg PO no more often than once
per week may be recommended to treat susceptible pathogens.
The use of enrofloxacin in reptiles depends then
upon the species. The primary doses for most
species are 5–10 mg/kg q24h PO, SC, IM.
The following doses are adapted from Carpenter
JW, Mashima TY, Rupiper D (2001), and Prescott
JF, Baggot JD, Walker RD (2000):
Green iguanas: 5 mg/kg PO, IM q24 h
Monitors: 10 mg/kg IM q5d
Pythons: 6.6 mg/kg IM q24h or 11 mg/kg IM
q48h or 10 mg/kg IM, then 5 mg/kg q48h
Herman’s tortoises: 10 mg/kg IM q24h
Gopher tortoises: 5 mg/kg IM q24-48h
Star tortoises: 5 mg/kg IM q12-24h
Sea turtles: 5 mg/kg IM q48h or 20 mg/kg PO
q1week (loggerhead sea turtles)
Alligators: 5 mg/kg IV q36h
Amphibians
Amphibian medicine is an emerging field – an
emerging field close to a “emergency” field. The
amphibian patient is often presented late in the
disease process and the most frequently apparent
clinical signs are non-pathognomonic. Because
amphibians are – more than any terrestrial verte-
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Figure 9 Antibiotic therapy is always indicated for traumatic wounds. Here a young monitor (top and bottom left) that recovered
uneventfully after a bite wound, and traumatic wounds on a Madagascarian boa (upper right) and a tortoise.
brate – very dependent on their environmental
conditions, husbandry records are critical for the
clinician.
Bacterial diseases have a high prevalence in amphibian facilities. Most of environmental bacterial
agents become pathogens in stressed amphibians:
transportation, bad husbandry, changes in the environment. Red-leg syndrome in amphibians is so
named due to the hyperemia of the ventral skin
of the thighs and abdomen of septicemia anurans,
and is now synonymous with any generalized
bacterial infection in amphibians. It is better to
talk about bacterial dermosepticemia. If historically this syndrome is associated with Aeromonas
hydrophila, many other infectious agents produce
similar integumentary signs. For the diagnostic
process, body fluids are taken when possible.
Smears and fast staining are done in the first approach. Biopsy for histology, bacteriology, and
sensitivity are conducted as rapidly as possible.
Isolating ill animals, optimizing the husbandry
conditions, combating dehydration, and putting
the animal in the upper limit of its preferred thermal zone are the first steps of treatment.
Antimicrobial therapy
Safe, efficacious treatment for common anuran
bacterial infections requires knowledge of specificity, pharmacokinetics, and toxicity of antibacterial agents in frogs. Three readily available
antibiotic agents – tetracycline, enrofloxacin,
amikacin – which have specificity for common
anuran bacterial pathogens were selected for investigation. Tetracycline was the first and most
commonly recommended antibiotic for treatment
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Norin Chai
of bacterial disease in frogs. However, tetracyclineresistant organisms from clinically ill amphibians
have been isolated and widespread bacterial resistance to tetracycline has also been reported in
mammals and reptiles. On the other hand, bacterial resistance to enrofloxacin has only rarely been
reported and amphibian pathogens have been
uniformly susceptible.
Enrofloxacin and its active metabolite ciprofloxacin are frequently effective in inhibiting growth of
pathogenic bacteria at serum levels of approximately 0.1 µg/ml. In bullfrogs (Rana catesbeiana),
a study has shown that dosages of 5 and 10 mg/kg
once daily maintained the plasma concentration
above this level throughout the dosing interval.
A single 10 mg/kg intramuscular dose did not induce any significant hematological or biochemical abnormalities. Any route of administration is
possible: PO, IM, percutaneous. All antibiotic
therapy must last at least 7 days. In most amphibians, enrofloxacin is reported to be used at
5–10 mg/kg PO, SC, IM q24h.The weight is very
variable depending on the state of hydration and
one should not hesitate to reweigh the animal.
and dose-related, and the drug has not been studied in all species. As a rule, with many animal
species, this class of antimicrobial agents should
not be administered to animals less than 8 months
of age or to large-breed dogs less than 12 months
of age.
In reptiles, the injectable form can be extremely
irritating, and this always needs to be considered
when this route of administration is selected.
Focal to diffuse areas of necrosis at intramuscular
injection sites in snakes and chelonians, and excessive salivation in juvenile Galapagos tortoises
(Geochelone elephantopus) that were administered
a single IM injection have been reported.
Conclusion
Adverse effects
Due to its bactericidal, wide distribution to tissues
and the extracellular space, and because it can penetrate nearly every tissue in the body, enrofloxacin
is among the most effective drugs for treating most
bacterial infections in exotics. Enrofloxacin also
offers the advantages of oral administration. Oral
bioavailability of enrofloxacin is excellent in
monogastric mammals and pre-ruminant calves,
with up to 80 % of the ingested dose being absorbed into systemic circulation. However, the metabolism and elimination half-life of enrofloxacin
varies greatly between species. More pharmacokinetic studies are required in veterinary medicine
for non-empiric use in more species.
The main adverse effects associated with fluoroquinolones are primarily associated with abnormal development of immature cartilage. Arthropathies have been reported in immature rats,
beagles, guinea pigs, and foals. However, in birds,
enrofloxacin has been widely used in psittacine
nurseries without reports of side effects. Despite
this fact, the drug should be used with caution in
growing birds as toxic effects are species-specific
Enrofloxacin is generally well tolerated. At therapeutic doses, it has proven to be relatively safe
in all species, with few reported side effects. In
addition, effective treatment with twice-daily, or
once-daily in some species, administration is a
clear advantage over some other antibiotics.The
major disadvantage of parenteral administration
is intramuscular pain and irritation at the site of
injection.
The author finds that the percutaneous route by
bath is the less stressful. A dosage of 0.3 mg/ml
water bath for 15 days has demonstrated routinely
favorable results.
58
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References
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Brown SA (1996). Fluoroquinolones in animal health. J Vet
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Westfall ME, Demcovitz DL, Plourdé DR, Rotstein DS,
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37(2): 206–208.
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(2000). Application of fluoroquinolone pharmacodynamics.
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Use of Enrofloxacin in Cats
with Resistant Mycoplasma spp. Infections
Hemotropic Mycoplasma spp.
The new names for Haemobartonella felis are Mycoplasma haemofelis (Mhf), ‘Candidatus M. haemominutum’ (Mhm), and ‘Candidatus M. turicensis’
(Mtc).1-3 The organisms are now classified as hemotropic mycoplasmas and clinical syndromes associated with the agents are collectively known as
hemoplasmosis. In the studies of experimentally
infected cats performed to date, Mhf is apparently
more pathogenic than Mhm; however, some cats
with Mhm alone develop clinical illness.4 Cats
with chronic Mhm infection had more severe
anemia and longer duration of anemia when experimentally infected with Mhf when compared
to cats infected with Mhf alone.5 Cats infected
with Mtc can be clinically ill or develop a chronic
carrier state like Mhm and Mhf.
In a recent study, we collected fleas from cats in
the United States and attempted to amplify hemoplasma DNA from flea extracts as well as the
blood of the cat using polymerase chain reaction
assay (PCR).6 The prevalence rates for Mhf in
cats and their fleas were 7.6 % and 2.2 %, respectively. The prevalence rates for Mhm in cats and
their fleas were 20.7 % and 23.9 %, respectively.
In another study of cats in the United States, the
overall prevalences of Mhm, Mhf, and Mtc infection were 23.2 % (72/310), 4.8 % (15/310),
and 6.5 % (20/310), respectively.7 In addition,
fleas ingest Mhm and Mhf from infected cats
when feeding. In one cat, we documented flea
feeding to transfer Mhf.8 However, when we fed
Mhf- or Mhm-infected fleas to cats, infection was
not documented.9 In other studies, hemoplasmas
have been transmitted experimentally by IV, IP,
and oral inoculation of blood. Clinically ill
queens can infect kittens; whether transmission
occurs in utero, during parturition, or from nursing has not been determined. Transmission by
biting has been hypothesized and the organisms
are in the mouths of cats.2,10 Red blood cell de62
struction is due primarily to immune-mediated
events; direct injury to red blood cells induced by
the organism is minimal.
Clinical signs of disease depend on the degree of
anemia, the stage of infection, and the immune
status of infected cats. Coinfection with FeLV can
potentiate disease associated with Mhm.11 Clinical signs and physical examination abnormalities
associated with anemia are most common and include pale mucous membranes, depression, inappetence, weakness, and, occasionally, icterus and
splenomegaly. Fever occurs in some acutely infected cats and may be intermittent in chronically
infected cats. Evidence of coexisting disease may
be present.Weight loss is common in chronically
infected cats. Cats in the chronic phase can be
subclinically infected only to have recurrence of
clinical disease following periods of stress.
The anemia associated with hemoplasmosis is
generally macrocytic, normochromic. Chronic
non-regenerative anemia is unusual in cats with
hemoplasmosis. Neutrophilia and monocytosis
have been reported in some hemoplasma-infected cats. Diagnosis is based on demonstration
Figure 1 Cytology of a blood smear of a cat with anemia after
experimental infection with Mycoplasma haemofelis.
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Michael R. Lappin, DVM, PhD, Professor, Diplomate ACVIM (Internal Medicine)
Department of Clinical Sciences,
Colorado State University, Fort Collins, Colorado, USA
Enrofloxacin 10 mg/kg/d
9
8
Log copy no
of the organism on the surface of erythrocytes on
examination of a thin blood film (Fig. 1) or PCR
assay.12,13 Organism numbers fluctuate and so
blood film examination can be falsely negative up
to 50 % of the time. The organism may be difficult to find cytologically, particularly in the
chronic phase. Thus, PCR assays are the tests of
choice due to sensitivity. Primers are available that
amplify a segment of the 16S rRNA gene common to both hemoplasmas. Real-time PCR to
quantify hemoplasma DNA has now been
titrated and can be used to monitor response to
treatment.14 Many healthy cats are positive for
hemoplasma DNA in blood and so not all PCRpositive cats are clinically affected.
7
6
5
4
3
2
1
0
7
14
21
28
35
42
Figure 2 Real-time PCR assay results showing the rapid decrease
in Mycoplasma haemofelis copy numbers in the blood of a cat
Doxycycline has fewer side effects than other
tetracyclines in cats and so is the preferred drug
in this class. Doxycycline is often administered at
10 mg/kg, PO, every 24 hours for 7 days. If there
is a positive response to treatment at the day 3 or
day 7 recheck evaluation and the cat is tolerating
the drug, treatment is continued for 28 days. In
the United States, doxycycline is administered as
a flavored suspension or water is given after
pilling to avoid esophageal strictures.15 Tetracyclines utilized to date appear to lessen parasitemia
and clinical signs of disease but probably do not
always clear the organism from the body and so
recurrence is possible.16
Some cats with clinical hemoplasmosis appear to
be resistant to administration of tetracyclines. In
these cats, administration of antibiotics in the fluoroquinolone class appears to be frequently effective; enrofloxacin was the first drug studied.17-19
The drug was well tolerated in experimentally
infected cats when administered 5 mg/kg or 10
mg/kg, PO, every 24 hours for 14 days.17 The
drug was equally effective or more effective than
doxycycline for the treatment of the clinical
manifestations of hemoplasmosis in this study.
administered enrofloxacin at 10 mg/kg, PO, daily.
Administration of enrofloxacin rapidly decreased
DNA copy numbers in blood, but some cats
are still PCR-positive after a course of therapy
(Fig. 2).14 Cats administered marbofloxacin also
are PCR-positive after treatment. Pradofloxacin is
a fluoroquinolone with potent effect against hemoplasmas that is currently under investigation.
This drugs shows promise for the elimination of
the hemoplasma carrier phase.20
Other antimicrobial agents that have been attempted for the treatment of hemoplasmosis in
cats include azithromycin and imidocarb.5,21,22
Azithromycin was not effective for the treatment
of hemoplasmosis in one study.5 Imidocarb has
been administered safely to research cats harboring Mhf or Mhm and when administered at 5
mg/kg, IM, every 2 weeks for at least 2 injections
was used successfully in the management of five
naturally-infected cats that had failed treatment
with other drugs.21,22
Hemoplasmosis and primary immune hemolytic
anemia are difficult to differentiate based on clinical and laboratory findings.23 Thus, cats with se63
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Michael R. Lappin
vere, regenerative hemolytic anemia, particularly
those with rapidly dropping PCV or autoagglutination, are often treated with glucocorticoids
and antibiotics. In these cats, prednisolone is often
prescribed at 1 mg/kg, PO, every 12 hours for
a
the first 7 days or until autoagglutination is no
longer evident while waiting for hemoplasma
PCR assay results to return.
To attempt to prevent hemoplasma infections, it
might be prudent to control fleas. Cats should be
housed indoors to avoid other potential vectors
and fighting. Blood donor cats should be
screened by PCR assay prior to use.24
Mycoplasma spp.-associated
bronchitis and rhinitis
b
Figure 3 Lateral (a) and VD (b) radiographs from a cat with
bronchitis consistent with that seen with Mycoplasma spp.
infection.
64
Almost all cats with mucopurulent or purulent
nasal discharge have a bacterial component to
their disease. In addition, bacterial bronchitis can
occur in cats (Fig. 3). Primary bacterial disease is
associated with Bordetella bronchiseptica, Mycoplasma
spp., and Chlamydophila felis.25-29 Recently it was
shown that Bartonella spp. are not causes of rhinitis in cats.30 Mycoplasma spp., are normal commensal organisms of the nose and mouth.
However, some strains may be primary pathogens
(i.e., M. felis), and other strains may be associated
with clinical illness if another primary disease
process has occurred. Primary infection with feline herpesvirus 1 or feline calicivirus with secondary bacterial infections are also very common
causes of rhinitis. In these situations, Pasteurella
spp., Streptococcus spp., and Staphylococcus spp. infections are likely isolates.
Veterinarians in the United States frequently
administer amoxicillin-clavulanate to cats with
mucopurulent rhinitis or suspected bacterial
bronchitis and this therapy is often effective.
However, this class of antibiotic is ineffective for
Mycoplasma spp. infections as these organisms
do not have a cell wall. It is often difficult for
commercial veterinary laboratories to culture
Mycoplasma spp. successfully and antimicrobial
susceptibility testing for Mycoplasma spp. is not
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Seite 65
routinely available.Thus, cats with suspected bacterial rhinitis or bronchitis that fail to respond to
penicillins should be treated with an alternate
drug class effective for Mycoplasma spp. The author has frequently prescribed enrofloxacin at
5 mg/kg, PO, daily to cats with mucopurulent
rhinitis or bronchitis suspected to be from Mycoplasma spp. infection with clinical success. In addition, most B. bronchiseptica isolates in the United
States are susceptible to enrofloxacin. Cats with
acute mucopurulent rhinitis or bacterial bronchitis only need to be treated for 7 to 10 days.
Cats with chronic rhinitis can have osteomyelitis
and may need to be treated for several weeks.
Doxycycline administered at 10 mg/kg, PO, once
daily can also be effective for treatment of the
primary bacterial pathogens. Most cases of bacterial rhinitis are secondary to other diseases including trauma, neoplasia, inflammation induced
by viral infection, foreign bodies, inflammatory
polyps, and tooth root abscessation. Cats with suspected bacteria bronchitis often have an underlying cause. Thus, if routine antibiotic therapy fails,
a diagnostic workup should be performed.
Mycoplasma spp.-associated
polyarthritis
Polyarthritis in cats from Mycoplasma spp. infections are rare.31-33 Most cats evaluated at Colorado
State University have had fever and a stiff gait.
Some cats have had a history of rhinitis. Multiple
cats can be involved in the same household. Joint
pain can be very severe.
Cytology of synovial fluid reveals septic suppurative inflammation and both M. felis and other
Mycoplasma spp. have been grown or amplified
from affected joints by PCR assay. Doxycycline at
10 mg/kg, PO, daily has been clinically effective
for the treatment of some cats. In others, administration of doxycycline failed to sterilize the
joints. Enrofloxacin administered at 5 mg/kg, PO,
daily has been used to successfully as a primary
treatment as well as to eliminate Mycoplasma spp.
from the joints of affected cats that failed treatment with doxycycline.
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bartonellosis in cats with chronic rhinosinusitis. J Am Vet
Med Assoc; 233: 1084–1089.
31. Zeugswetter F, Hittmair KM, de Arespacochaga AG, Shibly S, Spergser J (2007). Erosive polyarthritis associated with
Mycoplasma gateae in a cat. J Feline Med Surg; 9(3): 226–231.
32. Liehmann L, Degasperi B, Spergser J et al. (2006). Mycoplasma felis arthritis in two cats. J Small Anim Pract; 47:
476–479.
33. Ernst S, Goggin JM (1999).What is your diagnosis? Mycoplasma arthritis in a cat. J Am Vet Med Assoc; 215:
19–20.
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Unique Pharmacologic Characteristics of Baytril®
Introduction
As the first fluoroquinolone developed exclusively for veterinary use, Baytril® (enrofloxacin)
offered high potency, broad-spectrum activity
with the advantages of both oral and parenteral
administration, extensive tissue distribution, and
a favorable safety profile, for the treatment of
bacterial infections in companion animals.With
numerous pharmacokinetic, microbiologic, and
clinical investigations since its availability, and well
over 1,100 publications in the scientific literature,
it has become one of the most thoroughly investigated compounds in veterinary medicine. A few
of the unique features of Baytril that continue to
distinguish its utility will be highlighted here.
Structure-activity relationships of
enrofloxacin and fluoroquinolones
The precursors to Baytril and other compounds in
the fluoroquinolone class, the ‘early’ quinolones such
as nalidixic and oxolinic acid, were used almost exclusively for urinary tract infections in humans and
had limitations with respect to spectrum (soley
Gram-negative bacteria, except Pseudomonas aeurginosa), potency, oral bioavailability, and tissue distribution.1 Advances in the understanding of chemical
structure-function relationships for the quinolones
led to synthesis of numerous derivatives and analogues of the original core quinolone structure,
with markedly improved pharmacologic characteristics. The pivotal addition of a fluorine atom at position 6 of the basic 4-quinolone ring structure
significantly broadened the antibacterial spectrum
and gave rise to the modern ‘fluro’quinolones.2
This structural modification increased spectrum of
activity to include Gram-positive bacteria and increased potency against Gram-negative organisms,
as well as enhancing oral bioavailability and tissue
penetration.The substitution of a piperazinyl ring at
position 7 resulted in increased activity against bacteria such as Pseudomonas.3 Figure 1 highlights these
structure-activity relationships for enrofloxacin as
an example.
Enrofloxacin
Fluorine atom at C6
– enhances efficacy against
Gram-negative bacteria
– broadens spectrum against
Gram-positive organisms
Basic Quinolone
Parent Structure
co-planar carbonyl groups
– hydrogen bond with
DNA gyrase complex
O
O
R6
R7
R4
COOH
R3
piperazine ring at C7
– enhances antimicrobial activity,
especially against
Pseudomonas spp.
R1
F
OH
N
N
O
N
N
H5C2
N-ethyl group
– enhances tissue penetration
– decreases CNS toxicity
amine (basic) and carboxylic acid (acidic) functional groups
– endow amphoteric and zwitterionic properties
– lipid solubility enhanced between pKa's of acidic
and basic functional groups
Figure 1 Chemical structure of enrofloxacin with key modifications to quinolone core molecule.
68
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Joy Olsen, DVM
Global Veterinary Services
Bayer Animal Health GmbH, Germany
Enrofloxacin is structurally similar to ciprofloxacin, differing in the addition of an ethyl group
on the piperazinyl ring, which enhances the absorption of enrofloxacin as compared to ciprofloxacin.4 Ciprofloxacin has been reported to
have enhanced potency against organisms such as
Pseudomonas aeruginosa. However, as will be later
highlighted, enrofloxacin is partially metabolized
to ciprofloxacin in many species, notably dogs.5
Low bioavailability following oral administration
of ciprofloxacin has also been demonstrated in
cats; a pharmacokinetic study in 2004 reported
oral bioavailability of only 33 % in cats, with considerable differences between the drug exposure
(AUC) observed with IV administration and that
of oral administration (of pure drug substance).13
A highly active metabolite
Bioavailability
While the structural enhancements of the fluoroquinolones dramatically improved bioavailability
in general, oral bioavailability may vary significantly both among different species and also between different fluoroquinolone drugs. Many
compounds are characterized by favorable bioavailability with oral administration in monogastric animals, however, ciprofloxacin has been
shown to have poor and variable oral bioavailability in dogs6–8 in contrast to that of enrofloxacin, which approaches 100 %.9 Underscoring the
fact that dogs are not “small furry humans”, ciprofloxacin’s oral bioavailability is approximately 70 %
in humans,10 whereas that reported in dogs is
roughly 40 %.7,8 Mean peak serum concentrations
(Cmax) of enrofloxacin (Baytril tablets) in dogs
were four-fold higher than those of ciprofloxacin
(Ciprobay tablets) following administration of
each drug at a dosing rate of 5 mg/kg.11 Additionally, differences in rates of absorption may be
seen as the dosage of ciprofloxacin is increased.7
Laboratory studies involving administration of
radio-labeled ciprofloxacin [14C] to rats, dogs, and
monkeys observed that following oral administration, ciprofloxacin was partially absorbed and
achieved only 30– 40 % of the area under the
serum concentration versus time curves (AUCs)
of that obtained with intravenous dosing, indicating low overall exposure.12
A prominent unique pharmacologic feature of
enrofloxacin is that it is partially metabolized in
vivo by hepatic N-dealkylation to ciprofloxacin,
a highly potent metabolite which contributes to
overall antibacterial activity.9,14–16 This transformation has been documented in a variety of domestic animal species including dogs,9,16 cats,17,18
adult horses,19 sheep20, and cattle21. Pharmacokinetic studies in a diverse array of exotic animals
have also identified additional species in which
metabolism to ciprofloxacin occurs to a considerable extent, such as Burmese pythons,22 tortoises,22 rhesus monkeys,23 rabbits24, and psittacine
birds,25 to name just a few examples.
The resulting amount of ciprofloxacin conversion
varies among species; therapeutic levels exceeding
minimum inhibitory concentrations of relevant
pathogens are produced in many animals following administration of enrofloxacin.8 Cester and
Toutain conducted a pharmacokinetic study using
a comprehensive model to quantitatively determine enrofloxacin to ciprofloxacin transformation
in dogs following oral and IV administration.16
Their results indicated that approximately 40 % of
enrofloxacin was metabolized to ciprofloxacin following both routes of administration. This conversion contributes to both the magnitude and
duration of antimicrobial drug exposure.26
Additional pharmacokinetic investigations have
demonstrated significant concentrations of cipro69
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Joy Olsen
fore, each square (representing one well) in the
checkerboard configuration contains a unique
combination of the two compounds.35 Drug
concentrations encompassing a range of multiples (and fractions) of the minimum inhibitory
concentration(s) (MIC) in serial two-fold dilutions are used. Each checkerboard plate also contains a row and a column in which a serial dilution of each respective agent alone is present.
Bacteria are added to the test wells and, following incubation, observation for visible growth
is done to determine the lowest combined inhibitory concentrations.
floxacin in numerous tissues and body fluids following administration of enrofloxacin, including
urine,27 alveolar macrophages and respiratory epithelial lining fluid,28 prostate gland,29 peripheral
leukocytes,30 dermal tissues31, and bone32.
Additive activity
Enrofloxacin and its microbiologically active
metabolite ciprofloxacin share the same mechanism of action, rapidly killing bacteria in a concentration-dependent manner. For this reason, in
vitro studies have investigated what the interaction of enrofloxacin and ciprofloxacin is against
various small animal pathogens.33,34 In these particular studies, a checkerboard assay method was
used to ascertain the interaction and antibacterial activity of the combination of the two compounds. The “checkerboard” consists of a pattern
of microtiter wells with multiple dilutions of the
two drugs tested, such that each row (and column) contains a fixed amount of one drug and
increasing amounts of the second drug. There-
Pirro et al. investigated enrofloxacin and ciprofloxacin interactions on clinical isolates and
reference strains of Staphylococcus intermedius, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Pasteurella multocida; a total of 56
Gram-negative and Gram-positive strains were
included.33 Figure 2 illustrates an example of a
checkerboard assay from this study for a strain of
P. aeruginosa. For each combination experiment,
the fractional inhibitory concentration (FIC) of
ciprofloxacin
enrofloxacin
FIC
4
2
1 (MIC)
1/2
1/4
1/8
1/16
1/32
1/64
1/128
µg/ml
2.0
1.0
0.5
0.25
0.125
0.063
0.031
0.015
0.008
0.004
0
2
4.0
-
-
-
-
-
-
-
-
-
-
-
++
1 (MIC)
2.0
-
-
-
-
-
-
-
-
-
-
-
++
1/2
1.0
-
-
-
-
+
+
++
++
++
++
++
++
1/4
0.5
-
-
-
-
++
++
++
++
++
++
++
++
1/8
0.25
-
-
-
-
++
++
++
++
++
++
++
-
1/16
0.125
-
-
-
-
++
++
++
++
++
++
++
-
1/32
0.063
-
-
-
++
++
++
++
++
++
++
++
-
0
-
-
-
++
++
++
++
++
++
++
++
-
growth
control
Figure 2 Checkerboard combination of enrofloxacin and ciprofloxacin with P. aeruginosa ATCC 27853.
–: no visuable growth +: reduced growth ++: complete growth
70
(Adapted from Pirro et al. 1997, 14th Congress ESVD-ECVD)
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each drug was determined by dividing the inhibitory concentration when used in combination by the MIC of the agent alone, for example:
FICenr = Cenr/MICenr. The FIC index (FICI) is
the sum of FICs for the two compounds and defines the nature of the interaction. Criteria used
for the assessment of the FIC indices in this particular study were as follows: synergistic interactions FICI of ≤ 0.5; additive interactions 0.5 <
FICI ≤ 1; antagonism FICI > 2; and indifference
1 < FICI ≤ 2. Figure 3 is a plot of median FICs
for some reference strains that graphically depicts
the classification of the interactions. More recent
investigations have used criteria of FICI > 4 to
indicate antagonism,35 and FICI between 0.5 and
4 classification as additive.34 The FIC indices for
all reference strains and field strains in this study
were in the range of 0.5 to 1, clearly demonstrating additive inhibitory effects of the combination
on all bacterial species tested.
molytic, coagulase-positive Staphylococcus isolates
cultured from dogs treated for dermal infectious,
otitis externa, cystitis, pyometra, and respiratory
tract infections; a total of 50 isolates of E. coli and
50 staphylococci were included.34 The results of
MIC testing indicated that ciprofloxacin and enrofloxacin had equivalent potency against E. coli
and staphylococci, and the results of the checkerboard assay again revealed that the compounds
acted in an additive fashion against these bacteria.
Both of the above studies demonstrated that the
combination of enrofloxacin and ciprofloxacin
in vitro has additive inhibitory effects on major
Gram-negative and Gram-positive pathogens.
Therefore, the in vivo transformation of enrofloxacin to ciprofloxacin may enhance the overall
antibacterial activity. This activity is not reflected
with traditional culture and susceptibility testing
with enrofloxacin as it cannot account for presence of the highly active metabolite.26
A subsequent study reported in 2001 evaluated
the in vitro interaction between enrofloxacin and
ciprofloxacin against Escherichia coli and beta-he1
C enr
C cip
FICI = —–—— + —–——
MIC enr MIC cip
FIC enrofloxacin
0.75
E. coli:
P. multocida:
P. aeruginosa:
S. intermedius:
E. coli
ATCC 10536
FICI = 0.56
0.5
P. multocida
DSM 5281
FICI = 0.625
0.25
P. aeruginosa
ATCC 27853
FICI = 0.56
S. intermedius
ATCC 29663
FICI = 0.53
0
0
0.25
0.5
0.75
0.5
0.5
0.06
0.03
+ 0.06
+ 0.125
+ 0.5
+ 0.5
=
=
=
=
0.56
0.625
0.56
0.53
synergism
addition
indifference
1
FIC ciprofloxacin
Figure 3 Median FICs of different reference strains.
(Adapted from Pirro et al. 1997, 14th Congress ESVD-ECVD)
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Joy Olsen
Summary
Over the past two decades, a substantial body of
scientific and clinical research has been compiled
on enrofloxacin, making it one of the most ex-
tensively evaluated drugs in veterinary medicine.
Despite the advent of additional compounds in
the veterinary field, Baytril remains unique in
both its pharmacology and breadth of scientific
support and clinical applications.
References
1. Emmerson AM, Jones AM (2003). The quinolones:
decades of development and use. J Antimicrob Chemother;
51(Suppl S1): 13–20.
2. Tillotson G (1996). Quinolones: structure-activity relationships and future predictions. J Med Microbiol; 44:
320–324.
3. Martinez M, McDermott P, Walker R (2006). Pharmacology of the fluoroquinolones: A perspective for the use in
domestic animals.Vet J; 172: 10–28.
4. Walker, RD (2000). Fluoroquinolones. In: Antimicrobial
therapy in veterinary medicine (Eds: Prescott JF, Baggot JD,
Walker RD), 3rd edn., Iowa State University Press, Ames,
Iowa; pp. 315–338.
5. Tychowska K, Hedeen KM, Aucoin DP, Aronson AL
(1989). High performance liquid chromatographic method
for the simultaneous determination of enrofloxacin and its
primary metabolite ciprofloxacin in canine serum and prostatic tissue. J Chromatogr; 493: 337–346.
6. McKellar QA (1996). Clinical relevance of the pharmacologic properties of fluoroquinolones. Suppl Comp Cont
Educ Pract Vet; 18(2): 14–21.
7. Abadia AR, Aramayona JJ, Munoz MJ, Pla Delfina JM,
Bregante MA (1995). Ciprofloxacin pharmacokinetics
in dogs following oral administration. J Vet Med A; 42:
505–511.
8. Boothe DM, Boeckh A, Simpson RB, Dubose K (2006).
Comparison of pharmacodynamic and pharmacokinetic indices of efficacy for 5 fluoroquinolones toward pathogens of
dogs and cats. J Vet Intern Med; 20: 1297–1306.
9. Küng K, Riond JL, Wanner M (1993). Pharmacokinetics
of enrofloxacin and its metabolite ciprofloxacin after intravenous and oral administration of enrofloxacin in dogs.
J Vet Pharmacol Ther; 16: 462–468.
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10. Drusano GL, Standiford HC, Plaisance K, Forrest A,
Leslie J, Caldwell J (1986). Absolute oral bioavailability of
ciprofloxacin. Antimicrob Agents Chemother; 30(3): 444–
446.
11. Heinen E (1999). Comparative pharmacokinetics of enrofloxacin and difloxacin as well as their main metabolites
in dogs. Suppl Comp Cont Educ Pract Vet; 21(12): 12–18.
12. Dalhoff A, Bergan T (1998). Pharmacokinetics of fluoroquinolones in experimental animalsanimals. In: Quinolone
antibacterials (Eds: Kuhlmann J, Dalhoff A, Zeiler H-J),
Springer, Berlin; pp. 179–206.
13. Albarellos GA, Kreil VE, Landoni MF (2004). Pharmacokinetics of ciprofloxacin after single intravenous and repeat oral administration to cats. J Vet Pharmacol Ther; 27:
155–162.
14. Tychowska K, Hedeen KM, Aucoin DP, Aronson AL
(1989). High performance liquid chromatographic method
for the simultaneous determination of enrofloxacin and its
primary metabolite ciprofloxacin in canine serum and prostatic tissue. J Chromatogr; 493: 337–346.
15. Küng K, Riond JL,Wolffram S,Wanner M (1993). Comparison of an HPLC and bioassay method to determine
antimicrobial concentrations after intravenous and oral administration of enrofloxacin in four dogs. Res Vet Sci; 54:
247–248.
16. Cester CC,Toutain PL (1997). A comprehensive model
for enrofloxacin to ciprofloxacin transformation and disposition in dogs. J Pharm Sci; 86(10): 1148–1155.
17. Richez P, Monlouis JD, Dellac B, Daube G (1997).Validation of a therapeutic regimen for enrofloxacin in cats of
the basis of pharmacokinetic data. J Vet Pharmacol Ther;
20(Suppl 1): 152–153.
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18. Seguin MA, Papich MG, Sigle KJ, Gibson NM, Levy JK
(2004). Pharmacokinetics of enrofloxacin in neonatal kittens. Am J Vet Res; 65(3): 350–355.
19. Papich MG, Van Camp SD, Cole JA, Whitacre MD
(2002). Pharmacokinetics and endometrial tissue concentrations of enrofloxacin and the metabolite ciprofloxacin
after i.v. administration of enrofloxacin to mares. J Vet Pharmacol Ther; 25: 343–350.
20. Mengozzi G; Intorre L; Bertini S; Soldani G (1997).
Pharmacokinetics of Enrofloxacin and Its Metabolite Ciprofloxacin after Intravenous and Intramuscular Administrations in Sheep. Am J Vet Res; 57(7): 1040–1043.
21. De Lucas JJ, San Andrés MI, González F, Froyman R,
Rodríguez C (2008). Pharmacokinetic behaviour of enrofloxacin and its metabolite ciprofloxacin after subcutaneous
administration in cattle.Vet Res Commun; 32(4): 275–279.
22. Papich MG (1999). Pharmacokinetics of enrofloxacin in
reptiles. Suppl Comp Cont Educ Pract Vet; 21(12): 110–114.
23. Klein H, Hasselschwert D, Handt L, Kastello M (2008).
A pharmacokinetic study of enrofloxacin and its active
metabolite ciprofloxacin after oral and intramuscular dosing
of enrofloxacin in rhesus monkeys (Macaca mulatta). J Med
Primatol; 1–7.
24. Elmas M, Yazar E, Bas AL, Tras B, Bayezit M, Yapar K
(2002). Comparative pharmacokinetics of enrofloxacin and
tissue concentrations of parent drug and ciprofloxacin after
intramuscular administrations of free and liposome-encapsulated enrofloxacin in rabbits. J Vet Med; 49: 507–512.
25. Flammer K, Aucoin DP, Whitt DA (1991). Intramuscular and oral disposition of enrofloxacin in African grey parrots following single and multiple doses. J Vet Pharmacol
Ther; 14: 359–356.
26. Boothe DM, Boeckh A, Boothe HW, Wilkie S, Jones S
(2002). Plasma concentrations of enrofloxacin and its activie metabolite ciprofloxacin in dogs following single oral
administration of enrofloxacin at 7.5, 10 or 20 mg/kg.Vet
Ther; 34(4): 419.
27. Monlous JD, De Jong A, Limet A, Richez P (1997).
Plasma pharmacokinetics and urine concentrations of enrofloxacin after oral administration of enrofloxacin in dogs.
J Vet Pharmacol Ther; 20(Suppl 1): 61–63.
28. Hawkins EC, Boothe DM, Guinn A, Aucoin DP,
Ngyuen J (1998). Concentration of enrofloxacin and its
active metabolite in alveolar macrophages and pulmonary
epithelial lining fluid of dogs. J Vet Pharmacol Ther; 21:
18–23.
29. Dorfman M, Barsanti J, Budsberg SC (1995). Enrofloxacin concentrations in dogs with normal prostate and
dogs with chronic bacterial prostatitis. Am J Vet Res; 56(3):
386–390.
30. Boeckh A, Booth D, Wilkie S, Jones S (2001).Time
course of enrofloxacin and its active metabolite in peripheral leukocytes of dogs.Vet Ther; 2(4): 334–344.
31. Cole LK, Papich MG, Kwochka KW, Hillier A, Smeak
DD, Lehman AM (2009). Plasma and ear tissue concentrations of enrofloxacin and its metabolite ciprofloxacin in dogs
with chronic end-stage otitis externa after intravenous administration. Vet Dermatol; 20(1):51–59.
32. Ehinger AM, Meyer-Lindenberg A, Vick M, Nolte I,
Kietzmann M (2002). Concentration of enrofloxacin and
its metabolite ciprofloxacin in canine matrices of the locomotor system. J Vet Med; 49: 99–104.
33. Pirro F, Scheer M, De Jong A (1997). Additive in vitro
activity of enrofloxacin and its main metabolite ciprofloxacin. Proceedings 14th Annual Congress of the ESVDECVD; p. 199.
34. Lautzenhiser SJ, Fialkowski JP, Bjorling D, Rosin E
(2001). In vitro antibacterial activity of enrofloxacin and
ciprofloxacin in combination against Escherichia coli and
staphylococcal clinical isolates in dogs. Res Vet Sci; 70:
239–241.
35. Eliopoulos GM, Moellering RC (1996). Antimicrobial
combinations. In: Antibiotics in laboratory medicine (Ed:
Lorian V), Williams & Wilkens, Baltimore, MD; pp. 330–
396.
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Pharmacokinetic variables describe absorption,
distribution, metabolism, and elimination (ADME)
of a drug. This short overview will focus on the
most important topics veterinary practitioners
need to know about the pharmacokinetic behavior of antimicrobial drugs with special focus
on fluoroquinolones, such as enrofloxacin. Antimicrobials used in companion animal medicine
generally are administered via the oral route, although parenteral formulations are also available.
Absorption
After oral administration, most drugs are absorbed
in the proximal gastrointestinal tract. Systemic
availability, i.e., the rate and extent to which a
drug appears in the circulation, is usually determined by measurement of the concentration of
the active ingredient in plasma or serum as a
function of time. Ideally, a comparison of plasma
concentrations after oral application and injection of an equivalent intravenous dose should be
made. This will allow the determination of the
absolute systemic exposure (bioavailability). Uptake into the circulation is dependent on the disintegration of the oral formulation, liberation of
the active substance, dissolution in the gastrointestinal fluid, intestinal blood flow, molecular size,
lipophilic properties, ionization of the drug, and,
finally, the pH value at the site of absorption.1
Enrofloxacin possesses a carboxylic acid in position 3 and a basic amine functional group in position 1 of the molecule, making the compound
amphoteric and zwitterionic. At a pH range of the
medium between 6 and 8, the drug is sufficiently
lipophilic to be well absorbed and to penetrate
cell membranes, resulting in high intracellular
concentrations.2 Following oral administration,
the intestinal absorption of the fluoroquinolones
74
is rapid and nearly complete. The oral bioavailability of enrofloxacin is high with almost 100 %
in dogs and 95 % in cats,2 and plasma concentrations after oral and subcutaneous administration
are nearly identical.3 Absorption of the drug is
best after administration on an empty stomach.4
Distribution
Drug concentration-time curves describe the behavior of a drug in serum or plasma and represent
the basis of subsequent pharmacokinetic analysis.
The highest concentration achieved in the reference compartment is denominated Cmax, whereas
Tmax is the time interval between application and
peak concentration.These two parameters can be
directly read from the curve. AUC, the area under
the concentration-time curve, is calculated by
means of an integral function. It is directly proportional to the amount of drug in the organism
and thus is a measure of the total exposure to the
drug. AUClast is based on the time interval between application and time of the last measurable
concentration, whereas AUCinf represents the extrapolated area from time zero to infinity5 (Fig. 1).
Cmax
AUC
Concentration
PK Parameters
0
24 time (h)
Tmax
Figure 1 Concentration-time curve with Cmax, Tmax, AUC.
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Gert Daube, Dr med vet · Sandra Mensinger · Bernd Stephan, Dr med vet
Clinical Research and Development Antiinfectives
Parameter
Doga
Cat
Maximum serum concentration Cmax (µg/l)b
1,711
2,454
1
0.83
6,291
29,376
Volume of distribution Vdssb
3.7
4.0
Half-life T1/2 (h)b
3.24
8.86
Protein binding (%)
< 30
–
95
100
Time of maximum serum concentration Tmax (h)b
Area under the curve AUClast (µg h/l)b
Bioavailability, oral (%)
Table 1 Pharmacokinetic parameters of Baytril after oral administration of tablets at 5 mg/kg BW.
a
Sum of concomitant enrofloxacin and ciprofloxacin concentrations;
For optimum clinical efficacy, aminoglycosides
and fluoroquinolones should achieve high Cmax
values. For these drugs, the rate and extent of bacterial killing and the clinical efficacy increases, as
the concentration of the drug increases. Members of this group are therefore denominated
concentration- or dose-dependent antibiotics.6
After administration of Baytril tablets, the mean
serum peak concentration in cats and dogs was
2,454 µg/l and 1,711 µg/l, respectively (Tab. 1).
For other classes of antimicrobials, e.g., beta-lactams, sufficiently high drug concentrations have
to be maintained above the minimum inhibitory
concentration of the pathogen for almost the entire dose-to-dose interval.This group is therefore
denominated as time-dependent antibiotics.6
b
Daube et al. 2007, unpublished data
thetical volume of body fluid that would be necessary to accommodate the whole amount of
drug based on the concentration actually measured in serum or plasma7 (Fig. 2).
A Vd of above 0.6 l/kg is generally regarded as indicator of intracellular penetration.1 In pharmacokinetic studies with enrofloxacin in cats and
dogs, a Vd of 4.0 and 3.7 l/kg was calculated, suggesting an excellent penetration of the drug into
the target tissues.8 This has been confirmed by
numerous publications on high tissue concentrations after administration of Baytril, not only in
readily accessible target organs, but also in skin,
bones, inflamed tissues, and phagocytic cells.
Vd =
Distribution of a drug into the tissues is dependent on its lipophilic properties, molecular size,
ionization, the permeability of membranes, blood
perfusion, pH values at the infection site, and
plasma protein binding of the drug.1 An indicator of the distribution within the organism is the
volume of distribution (Vd).Vd does not represent
any physical or anatomical structure, but a hypo-
Total amount of drug administered
Concentration in plasma (l/kg)
Vd = 0.05 l/kg – Distribution in plasma
Vd = 0.2 l/kg – Distribution in extracellular space
Vd = 0.6 l/kg – Distribution in extra- and
intracellular space
Vd > 0.6 l/kg – Accumulation in tissues
Figure 2 Volume of Distribution Vd .
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Gert Daube · Sandra Mensinger · Bernd Stephan
Distribution of a substance also is influenced by
its protein binding. Drugs bound to plasma or tissue proteins do not have antimicrobial activity,
cannot cross barriers, nor can they be metabolized or eliminated. Protein binding is strongly
influenced by animal species, age, disease, and nutritional status (e.g., cachectic animals).1 However,
binding in general is reversible following a concentration gradient. In dogs, less than 30 % of enrofloxacin is bound to plasma proteins.Therefore,
it is considered to have low protein binding.8
Metabolism
In general, substances undergoing metabolism are
inactivated, detoxified, and prepared for excretion. Enrofloxacin is dealkylated to ciprofloxacin,
a compound with similar antimicrobial activity.8
In dogs, around 40 % of enrofloxacin is transformed to ciprofloxacin,9 whereas in cats the percentage of the metabolite in serum is much lower.
Elimination and excretion
Further pharmacokinetic parameters routinely
provided include the elimination half-life (T1/2),
clearance (Cl), and Mean Residence Time (MRT).
Half-life is the time period during which the
concentration of a drug is reduced to half of its
initial value. T1/2 has some influence on possible
drug accumulation and is therefore important for
estimation of the dosing interval.10 The term
clearance describes the rate of excretion of a
drug. In the majority of cases, the clearance is determined in plasma, as this matrix can easily be
obtained. It represents the volume of body fluid
which is completely cleared of a substance per
time interval by an elimination process. Clearance can also be defined for each elimination
organ, for example renal or hepatic clearance.
Total body clearance is the sum of all organ clear76
ances. Half-life strongly depends upon the Cl
and Vd parameters.11 Mean Residence Time represents the average time a drug molecule remains
in the circulation, between (intravenous) administration and elimination from the central compartment.1
Fluoroquinolones are excreted via the bile after
conjugation with glucoronic acid, and via the
urine mainly by glomerular filtration. In dogs,
70 % of enrofloxacin is excreted in the bile and
around 30 % in urine.2 Due to the renal concentration process, however, a sufficiently high amount
of active drug is present in the urine. In the intestines, segregation of glucuronic acid from the
fluoroquinolone molecule by bacterial enzymes
subsequently leads to re-absorption thereby enabling enterohepatic re-circulation of the drug.1
PK/PD relations – what do they mean?
The aim of each antimicrobial therapy should not
only be the clinical cure of the patient, but also
elimination of the target pathogens in order to
minimize the selection of resistant bacteria. In the
case of incomplete eradication, less susceptible
bacterial subpopulations may develop and lead
to therapeutic failure. Therefore PK/PD calculations are conducted, whereby pharmacokinetic
parameters are linked to pharmacodynamic processes.12 They enable a rough estimation of the
in vivo antimicrobial effect to be expected and
offer the possibility of a prospective dose setting
for patients in clinical studies (Fig. 3).
For drugs with concentration-dependent antimicrobial activity, e.g., aminoglycosides and fluoroquinolones, the parameters used for PK/PD
calculations (Cmax and AUC0-24) are derived from
dose linearity studies in healthy animals.The data
are subsequently linked to the in vitro minimum
inhibitory concentration (MIC90) of each target
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Pharmacokinetics
Pharmacodynamics
Dose
Concentration
Concentration
Effect
PK / PD
Dose
Effect
Figure 3 PK/PD relationships.
pathogen by simple division. In 1993, Forrest
et al.13 proposed that the ratios of Cmax/MIC ≥ 10
or AUC0-24/MIC ≥ 125 should be considered as
predictors of clinical efficacy, based on observations in critically ill human patients in intensive
care units. Most of these patients suffered from
Gram-negative infections, predominantly caused
by Pseudomonas aeruginosa. Meanwhile it has become evident that these values first proposed
more than 15 years ago are highly conservative
and generally relate to Gram-negative organisms
only. Critical review of several experimental studies resulted in the finding that the magnitude of
the PK/PD ratios necessary for successful therapy is reduced in animal models due to the presence of neutrophils, as antimicrobial drugs and
immune mechanisms act in concert.14–17 Today,
most data from controlled animal studies suggest
that an AUC0-24/MIC ratio of ≥ 40 is sufficient to
predict clinical efficacy when Gram-positive infection is present.14 In case of Gram-negative infections, an AUC0-24/MIC ratio above 125 is now
regarded as conservative, as ex vivo experiments
in bovines and studies in cats have also shown
good results with ratios lower than initially proposed.18,19
Pharmacokinetics and bioequivalence
Registration of generic products in general is
based on the proof of bioequivalence between
the pioneer approved product and the generic
copy. Bioequivalence studies are statistically based
comparisons of two drug formulations to demonstrate that both are interchangeable.The basic assumption underlying the bioequivalence concept
is that essentially similar drug concentration-time
courses in the circulation lead to essentially similar pharmacological and therapeutic effects. Most
bioequivalence studies are conducted in vivo under
good laboratory practice (GLP) requirements on
a homogenous group (age, breed, gender) of
healthy animals of the target species. The reference product usually is the pioneer approved
product with the most extensive dossier. In bioequivalence studies in general, each product is administered as a single dose within a complete
cross-over design, so that each animal serves as its
own control.The blood sampling schedule has to
be adapted to the pharmacokinetic characteristics
of the products, so that absorption, distribution,
and elimination phases are properly represented.
The serum- or plasma-analytic procedure needs to
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Gert Daube · Sandra Mensinger · Bernd Stephan
follow recognized standards. Bioequivalence calculation today is done with computer programs,
e.g., WinNonlin®, Pharsight Corp., Mountain
View, CA, USA. In general, the maximum serum
concentration Cmax and the area under the concentration-time curve (AUClast) of the originally
approved product and the generic are compared
to each other after log-transformation. The 90 %
confidence interval of the ratios of Cmax reference
product/Cmax test product and AUClast reference
product/AUClast test product must then be entirely contained within the reference limits of
80–125 %. Due to the high variability of Cmax values, a wider range of limits may be acceptable, if
sufficiently justified in advance20 (Fig. 4).
Thus, by means of a bioequivalence study only,
generic copies of an original product can officially be licensed without generating much more
than basic pharmacokinetic data. Additionally,
this bioequivalence is granted across a range of
45 % variability compared to the original substance. In contrast, the entire body of information on pharmacokinetics, pharmacodynamics,
safety, toxicology, and clinical efficacy laid down
in dossiers or reported in the literature was generated using the original product.
+ 25 % Deviation* generic
100 % Original product
Plasma concentration
Cmax
– 20 %
Deviation* generic
Original product Generic copy
Figure 4 Bioequivalence between original and generic copy.
78
* Deviation calculated
from AUC generic/
AUC original
Time
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Seite 79
References
1. Kietzmann M (2008). Richtlinien für die Durchführung
pharmakokinetischer Studien in der Entwicklung von Tierarzneimitteln. Proceedings „Seminar Pharmakokinetische
Studien bei Tierarzneimitteln“, Hannover; pp. 1–29.
12. Bäumer W (2008). Vergleich von Formulierungen,
Bioverfügbarkeit, Bioäquivalenz. Proceedings „Seminar
Pharmakokinetische Studien bei Tierarzneimitteln“, Hannover; pp. 70–84.
2. Brown SA (1996). Fluoroquinolones in animal health.
J Vet Pharmacol Ther; 19:1–14.
13. Forrest A, Nix DE, Ballow CH, Goss TF, Birmingham
MC, Schentag JJ (1993). Pharmacodynamics of intravenous
ciprofloxacin in seriously ill patients. Antimicrob Agents
Chemother; 37: 1073–1081.
3. Kroker R (2006). Pharmaka zur Behandlung und Verhütung bakterieller Infektionen. In: Pharmakotherapie bei
Haus- und Nutztieren (Eds: Löscher W, Ungemach FR),
Parey Verlag, Stuttgart; pp. 234–258.
4. Küng K,Wanner M (1993). Einfluss zweier verschiedener
Futter auf die Pharmakokinetik von oral appliziertem
Baytril® (Enrofloxacin) beim Hund. Kleintierpraxis 38:
95–102.
5. Pabst G (2003). The area under the concentration-time
curve. In: Parameters for compartement-free pharmacokinetics (Ed: Cawello), Shaker Verlag, Aachen; pp. 65–80.
6. Meinen JB, McClure JT, Rosin E (1995). Pharmacokinetics of enrofloxacin in clinically normal dogs and mice
and drug pharmacodynamics in neutropenic mice with
Escherichia coli and staphylococcal infections. Am J Vet Res
56: 1219–1224.
7. Cawello W (2003).The physiological basis of pharmacokinetics. In: Parameters for compartement-free pharmacokinetics (Ed: Cawello W), Shaker Verlag, Aachen; pp. 7–22.
8. Kietzmann M (1999). Overview of the pharmacokinetic
properties of fluoroquinolones in companion animals. Proceedings of the Third International Veterinary Symposium
on Fluoroquinolones. Suppl Comp Cont Educ Pract Vet;
21(12): 7–11.
9. Cester CC, Toutain PL (1997). A comprehensive model
for enrofloxacin to ciprofloxacin transformation and disposition in dog. J Pharm Sci; 86(10): 1148–1155.
14. Wright DH, Brown GH, Peterson ML, Rotschafer JC
(2000). Application of fluoroquinolone pharmacodynamics. J Antimicrob Chemother; 46: 669–683.
15. Nicolau DP (1999). Using pharmacodynamic and pharmacokinetic surrogate markers in clinical practice: Optimizing antimicrobial therapy in respiratory-tract infections.
Am J Health Syst Pharm; 56(3): S16–S20.
16. Dudley MN, Ambrose PG (2000). Pharmacodynamics
in the study of drug resistance and establishing in vitro susceptibility breakpoints: ready for prime time. Curr Opin
Microbiol; 3: 515–521.
17. Ambrose PG, Grasela DM, Grasela T, Passarell J, Mayer
HB, Pierce PF (2001). Pharmacodynamics of fluoroquinolones against Streptococcus pneumoniae in patients with
community-acquired respiratory tract infections. Antimicrob Agents Chemother; 45: 2793–2797.
18. Lees P, Aliabadi FS (2002). Rational dosing of antimicrobial drugs: animals versus humans. Int J Antimicrob
Agents; 19: 269–284.
19. Coulet M, Cox P, Lohuis J (2005). Pharmacodynamics
of ibafloxacin in microorganisms isolated from cats. J Vet
20. Guideline for the conduct of Bioequivalence Studies for
Veterinary Medicinal Products, EMEA/CVMP/016/00corr-Final, 11. Juli 2001.
10. Gieschke R (2003). Half-life. In: Parameters for compartement-free pharmacokinetics (Ed: Cawello W), Shaker
Verlag, Aachen; pp. 39–64.
11. Toutain PL, Bousquet-Melou A (2004). Plasma clearance. J Vet Pharmacol Ther; 27: 415–425.
79
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Baytril® Resistance Monitoring –
Susceptibility Status after More Than 20 Years
Introduction
Baytril® has been successfully marketed for more
than 20 years in most European countries. Several
oral and parenteral formulations are available for
the treatment of infectious diseases in both companion animals and livestock. Excellent pharmacokinetic properties and high bactericidal activity
against a broad spectrum of aerobic Gram-negative and Gram-positive bacteria as well as
mycoplasmas make it highly suitable for the treatment of serious bacterial infections.To ensure the
long-term efficacy of enrofloxacin, the active ingredient of Baytril, knowledge on the ongoing
susceptibility status of bacterial pathogens is important.
Bayer has established several susceptibility monitoring programs, starting in 1992, involving
veterinary pathogens as well as zoonotic and
commensal bacteria. In all, more than 40,000 individual bacterial strains have been collected up to
this time, comprising target pathogens (> 20,000)
and food-borne bacteria (22,000) such as Salmonella spp. Regarding pathogens of dogs and cats,
more than 12,000 isolates have been collected,
the majority from dogs. Additionally, several external monitoring surveys have been organized
by Bayer Animal Health. In the last decade, various
national resistance monitoring programs have been
initiated (e.g., CIPARS, DANMAP, MARAN,
NARMS, SVARM) which include a wide variety of bacterial pathogens from food-producing
and companion animals. However, the focus of
these programs is on resistance monitoring of
zoonotic/commensal organisms, and relatively
few pathogens from companion animals have
been collected. One exception is the German
GERM-Vet program which was initiated in 2001
by the German Federal Office of Consumer Protection and Food Safety (BVL), supplemented by
an industry association (BfT) sponsored project
(BfT-GermVet), which focused on pathogens
80
from both livestock and companion animals
(Schwarz et al. 2007a).
The current susceptibility status for enrofloxacin
with respect to major pathogens relevant for dogs
and cats is presented here. Results of the Bayer
monitoring programs are compared to recent literature data, particularly that of the BfT-GermVet
monitoring program.
Materials and methods
Two different European programs have been conducted by Bayer Animal Health. In one program
starting in 1992, samples were taken from four
major clinical indications in three large regions of
Germany. These regions (South, Central, North)
are considered representative of Germany. In this
survey it was not possible to collect historic data
and it is assumed that in many cases the patients
had received antimicrobial treatment prior to sampling. In the second program, samples of the same
four indications have been collected from 2003
onwards in various veterinary practices from animals not recently exposed to antimicrobial treatment. Four countries (Hungary, Poland, Sweden,
United Kingdom) were involved.
Bacterial isolates were recovered by local diagnostic laboratories and transferred to Bayer Animal Health laboratories in Monheim am Rhein,
Germany. The isolates were identified by colony
morphology, Gram stain, and biochemical tests
(e.g., API). The minimum inhibitory concentration (MIC) to enrofloxacin was determined by
agar dilution according to the principles of the
Clinical and Laboratory Standards Institute (CLSI;
M31-A3, 2008). MIC50, MIC90, and MIC90s
values as well as the percentage of resistance were
calculated for each bacterial species, divided into
different indications if data from a sufficient number of strains was collected.
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Hans-Robert Hehnen, Dr med vet · Sonja M. Friederichs, Dr rer nat
Julia C. Heimbach · Anno de Jong, Dr · Bernd Stephan, Dr med vet
Clinical Research and Development Antiinfectives
According to Guideline EMEA/CVMP/627/
01-FINAL, the MIC90 should be determined
against the susceptible population reporting the
percentage of resistant strains separately, where
field isolates show a bimodal or multimodal distribution distinguishing susceptible and resistant
isolates.The MIC90s is thus defined as the MIC90
of the susceptible bacterial population. To differentiate between enrofloxacin resistant and susceptible strains, the CLSI breakpoint of 4 µg/ml was
used throughout all calculations although CLSI
has not confirmed this value for each bacterial
species and indication yet. Escherichia coli strain
ATCC 25922 was used as quality control strain.
n = 12,032
E. coli
Staphylococci
Pseudomonas
Proteus
Pasteurella
Salmonella
Bordetella
Klebsiella
Others
Streptococci
Enterococci
Figure 1 Distribution of canine and feline bacterial pathogens
sampled from 1992 onwards in the German Bayer monitoring
program.
Clinical indication
Bacterial species
Number
of isolates
MIC50
(µg/ml)
MIC90
(µg/ml)
MIC90s
(µg/ml)
Resistance
(%)
Respiratory tract
357
0.5
1
1
2.5
E. coli
342
0.03
32
0.125
15
P. multocida
289
0.015
0.03
0.03
0
Staphylococcus spp.
251
0.125
0.25
0.125
4
E. coli
1,607
0.03
16
0.06
15
Klebsiella spp.
127
0.125
64
0.25
39
Proteus spp.
230
0.25
8
0.25
21
P. aeruginosa
238
1
16
2
21
P. multocida
126
0.015
0.03
0.03
0
Proteus spp.
226
0.25
8
0.5
17
P. aeruginosa
611
1
16
2
28
Staphylococcus spp.
1,638
0.125
0.5
0.125
4
E. coli
821
0.03
16
0.06
16
Urinary/genital tract
Skin/ear/mouth
Gastrointestinal tract
Table 1 Antimicrobial susceptibility to enrofloxacin of canine and feline pathogens from the German Bayer monitoring program
(2000–2008 subset; n = 6,863).
Results and Discussion
In total, 12,000 isolates were investigated encompasing the most important bacterial diseases
of dogs and cats in the German Bayer program.
The frequency of recovery of the pathogens is
depicted in Figure 1. The antimicrobial suscepti-
bility to enrofloxacin for a subset of the collection
(2000–2008) is summarized in Table 1 and includes MIC50 and MIC90 values as well as the
prevalence of resistance. The MIC50 and MIC90s
values underline the high susceptibility of the
major key pathogens to enrofloxacin. Prevalence
of resistance varied from 0 to 39 %; the resistance
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Baytril® Resistance Monitoring – Susceptibility Status after More Than 20 Years
Hans-Robert Hehnen · Sonja M. Friederichs · Julia C. Heimbach · Anno de Jong · Bernd Stephan
level was notable for strains from infections of the
urinary and genital tract. It should be noted,
however, that collecting samples in diagnostic laboratories without knowledge of the treatment
history can be considered as a worst-case scenario. We analyzed the data set for possible trends,
but low annual numbers per bacterial species precluded conclusions on any trends. However, a
combination of several years showed that the rate
of resistance is constant for the major pathogens.
It is of interest to compare our findings with
the results of BfT-GermVet survey, 2004–2006
(Tab. 2), the isolates of which also reflect the sus-
ceptibility status in Germany. It can be seen that
MIC50 and MIC90s values are very similar to our
data and underline the high susceptibility of
pathogens from all German regions. Prevalence
of resistance varied from 0 to 29 % and is predominantly seen for isolates from urinary/genital tract and skin/ ear/mouth infections. For all
pathogens but one (Proteus spp.), resistance rates of
our collection exceeded those of the BfT-GermVet study. The fact that pathogens recovered from
untreated animals – as usually applies to the BfTGermVet study – have a higher susceptibility than
frequently treated animals is reassuring.
Clinical
indication
Bacterial species
Number
of isolates
MIC50
(µg/ml)
MIC90
(µg/ml)
MIC90s
(µg/ml)
Resistance
(%)
Respiratory tract
42
0.25
0.5
0.5
0
Schwarz et al.
2007b
E. coli
28
0.03
0.06
0.03
7
Grobbel et al.
2007a
P. multocida
72
0.015
0.03
0.03
0
Schwarz et al.
2007b
Staphylococcus spp.
coagulase-positive
57
0.125
0.5
0.25
4
Schwarz et al.
2007c
E. coli
100
0.03
0.5
0.06
7
Grobbel et al.
2007a
Klebsiella spp.
17
0.03
≥ 32
0.25
29
Grobbel et al.
2007b
Proteus spp.
37
0.125
8
0.25
22
Grobbel et al.
2007b
P. aeruginosa
28
1
2
1
11
Werckenthin
et al. 2007
P. multocida
20
0.015
0.06
0.06
0
Schwarz et al.
2007b
Proteus spp.
30
0.125
4
0.125
27
Grobbel et al.
2007b
P. aeruginosa
71
1
≥ 32
2
24
Werckenthin
et al. 2007
Staphylococcus spp.
coagulase-positive
101
0.125
0.25
0.25
2
Schwarz et al.
2007c
E. coli
100
0.03
0.06
0.06
2
Grobbel et al.
2007b
Urinary/genital tract
Skin/ear/mouth
Gastrointestinal
tract
Table 2 Summary of findings of BfT-GermVet: Antimicrobial susceptibility to enrofloxacin of canine and feline pathogens.
82
Reference
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Seite 83
n = 2,378
E. coli
Staphylococci
Pseudomonas
Proteus
Pasteurella
Klebsiella
Enterococci
Figure 2 Distribution of canine and feline bacterial pathogens
during 2003–2004 of the European Bayer monitoring program.
The results obtained from the European study are
summarized in Table 3 for isolates recovered in
2003–2004; the distribution frequency is shown
in Figure 2. Isolates of different indications are
combined.The spectrum of species differs from the
German program due to the inclusion of a relatively high number of samples from skin infections.
It is shown that the overall susceptibility is very
high. MIC50 values of seven species ranged from
0.016 to 0.125 µg/ml; only for P. aeruginosa and
Enterococcus spp. 0.5 and 1 µg/ml were determined,
respectively. Resistance levels of major pathogens
barely exceeded 5 % and usually did not exceed 2 %.
The resistance rate for E. coli amounted to 5.1 %.
Bacterial species
Number
of isolates
MIC50
(µg/ml)
MIC90
(µg/ml)
MIC90s
(µg/ml)
Resistance
(%)
E. coli
472
0.03
0.25
0.06
5.1
P. multocida
177
0.016
0.016
0.016
0
Staphylococcus spp.
290
0.125
0.5
0.5
1.7
Staphylococcus aureus
49
0.125
0.25
0.25
0
Staphylococcus intermedius
1150
0.125
0.125
0.125
1.0
Klebsiella spp.
27
0.03
0.5
0.5
0
Proteus mirabilis
74
0.125
0.25
0.25
1.4
P. aeruginosa
88
0.5
2
2
5.7
Enterococcus spp.
51
1
1
1
3.9
Table 3 Antimicrobial susceptibility to enrofloxacin of canine and feline pathogens of the European Bayer monitoring program.
Conclusions
The data demonstrate that the susceptibility of
major canine and feline pathogens to enrofloxacin is usually excellent even after two decades of
therapeutic use of fluoroquinolones in veterinary
medicine. This is consistent with the findings of
other national monitoring surveys. In spite of this
favorable status, prudent and rational use of key
antibiotics such as fluoroquinolones, and all antibiotics in general, is crucial for maintaining high
susceptibility in the future. Ongoing monitoring
will allow detection of any emerging resistance
or shifts in susceptibility.
Acknowledgement
The excellent technical assistance of Nora Schröter, Ilona Fietz, and Stefan Buschmann is gratefully acknowledged.
83
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Baytril® Resistance Monitoring – Susceptibility Status after More Than 20 Years
Hans-Robert Hehnen · Sonja M. Friederichs · Julia C. Heimbach · Anno de Jong · Bernd Stephan
References
Grobbel M, Lübke-Becker A, Alesik E, Schwarz S, Wallmann J, Werckenthin C, Wieler LH (2007a). Antimicrobial
susceptibility of Escherichia coli from swine, horses, dogs and
cats as determined in the BfT-GermVet monitoring program 2004–2006. Berl Munch Tierarztl Wochenschr; 120:
391–401.
Grobbel M, Lübke-Becker A, Alesik E, Schwarz S, Wallmann J, Werckenthin C, Wieler LH (2007b). Antimicrobial
susceptibility of Klebsiella spp. and Proteus spp. from various
organ systems of horses, dogs and cats as determined in the
BfT-GermVet monitoring program 2004–2006. Berl
Munch Tierarztl Wochenschr; 120: 402–411.
Schwarz S, Alesik E, Grobbel M, Lübke-Becker A, Wallmann J, Werckenthin C, Wieler LH (2007a). The BfTGerm-Vet monitoring program – aims and basics. Berl
Munch Tierarztl Wochenschr; 120: 357–362.
Schwarz S, Alesik E, Grobbel M, Lübke-Becker A,Werckenthin C, Wieler LH, Wallmann J (2007b). Antimicrobial susceptibility of Pasteurella multocida and Bordetella bronchiseptica
from dogs and cats as determined in the BfT-GermVet
monitoring program 2004–2006. Berl Munch Tierarztl
Wochenschr; 120: 423–430.
84
Schwarz S, Alesik E, Werckenthin C, Grobbel M, LübkeBecker A, Wieler LH, Wallmann J (2007c). Antimicrobial
susceptibility of coagulase-positive and coagulase-variable
staphylococci from various indications of swine, dogs and
cats as determined in the BfT-GermVet monitoring program 2004–2006. Berl Munch Tierarztl Wochenschr; 120:
372–379.
Werckenthin C, Alesik E, Grobbel M, Lübke-Becker A,
Schwarz S, Wieler LH, Wallmann J (2007). Antimicrobial
susceptibility of Pseudomonas aeruginosa from dogs and cats
as well as Arcanobacterium pyogenes from cattle and swine as
determined in the BfT-GermVet monitoring program
2004–2006. Berl Munch Tierarztl Wochenschr; 120: 412–422.
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Clinical Efficacy, Rapid Bactericidal Action and
Low Potential for Resistance Selection of Baytril®
Introduction
Enrofloxacin (Baytril®) is a fluoroquinolone antimicrobial agent with broad-spectrum activity
against a wide variety of Gram-positive and
Gram-negative bacteria, and is rapidly bactericidal against key companion animal pathogens. Its
pharmacokinetic/pharacodynamic profile is favorable for optimal dosing against susceptible
bacterial strains. Clinical trial data indicate enrofloxacin is clinically efficacious in dogs and cats
for a range of bacterial infections, including treatment of infections of the respiratory and urinary
tracts and for skin and soft tissue infections. More
recent data suggest enrofloxacin has a low potential to select for resistance. This manuscript is organized to provide an overview of antimicrobial
resistance issues, pharmacology, resistance selection/prevention, and kill studies measuring bactericidal activity. A summary of this data in
reference to achievable and sustainable enrofloxacin drug concentrations will be presented.
Antimicrobial resistance is a worldwide problem
requiring worldwide solutions. Increasing awareness of the issues surrounding resistance has demanded a rethinking about antimicrobial use in
infectious diseases. Many variables complicate our
understanding of the interaction of pathogenic
microorganisms and antimicrobial agents, especially when one considers that susceptibility testing is conducted in vitro, yet the infection is
treated in vivo. In vitro measurements are unable to
replicate the role that immunity plays in combating infection, however, we do believe that these
measurements are useful in guiding appropriate
therapy. It is becoming increasingly clearer that
traditional in vitro measurements of susceptibility
or resistance may not fully predict the heterogenous nature of bacterial populations associated
with infection, as the number of bacterial cells
tested by minimum inhibitory concentration
testing is less than the number of bacterial cells
86
typically associated with infection. We have argued that susceptibility testing utilizing higher
bacterial burdens may more accurately measure
the amount of drug required to inhibit bacterial
pathogens. This is especially true when one considers the amount of drug required to inhibit
high density bacterial growth with the pharmacokinetic and pharmacodynamic characteristics
of the drug or drugs considered for therapy. Optimal versus suboptimal therapy has been debated,
as we continue to evolve our understanding of
infection, antimicrobial therapy and antimicrobial
resistance selection. For optimal therapy, the combined goals of infectious diseases therapy need to
be a successful clinical outcome and reducing the
likelihood of resistance selection to the therapeutic drug(s) used. Are such combined goals
possible in today’s environment and with our
current antimicrobial compounds? The answer
appears to be both yes and no. Clearly, one difficulty is the syndromic empiric approach to antimicrobial therapy. For respiratory tract, urinary
tract and skin and skin structure infections, the
etiology can be one of several different pathogens
that may be either Gram-negative or Gram-positive. It is well known that not all antimicrobial
agents exert the same degree of in vitro microbiological potency (defined by in vitro minimum
inhibitory concentration (MIC) measurements),
nor the same degree of pharmacological potency
as defined by pharmacokinetic (PK) or pharmacodynamic (PD) measurements/principals. Ideally,
the pathogen(s) identification and drug susceptibility/resistance would be known prior to antimicrobial administration and in all instances, the
correct drug(s) would be used at the correct
dosage, dosing intervals, and duration of therapy
to ensure pathogen eradication and minimization
of resistance.
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Joseph M. Blondeau, MSc, PhD, RSM(CCM), SM(AAM), SM(ASCP), FCCP
Departments of Microbiology, Microbiology and Immunology and Pathology,
Royal University Hospital and University of Saskatchewan, Canada
Pharmacokinetics and pharmacodynamics of antimicrobial therapy
Pharmacokinetics (PK) determines the fate of the
drug in the body (i.e., absorption, transformation,
distribution, elimination). Pharmacodynamics is
the effect of the drug on the body and includes
its mechanism of action and efficacy. Much emphasis has been placed on PK/PD parameters and
antimicrobial agents in an attempt to establish optimal antimicrobial therapy. Figure 1 summarizes
some of the key points. For compounds classified
as concentration-dependent agents (e.g., fluoroquinolones, aminoglycosides), two relationships
have defined their antibacterial activity: 1) the
maximum serum drug concentration (Cmax) to
MIC ratio and 2) the area under the drug concentration curve (AUC) to MIC ratio (AUC/
MIC), which is sometimes referred to as the
AUIC or area under the inhibitory curve. From
investigations by Forrest et al. (1993), it was suggested that a Cmax-to-MIC ratio of 8/12 and
an AUC/MIC ratio of > 125 correlated with
favorable clinical outcomes and minimization of
resistance. Considerable controversy has emerged
regarding an AUC/MIC ratio of ≥ 125.1-4 Some
argue that for Gram-positive organisms (particularly Streptococcus pneumoniae), the AUC/MIC
ratio needs only be in the range of 30–50. File
et al. (2009) investigated human patients with
chronic obstructive pulmonary disease.5 Such patients may frequently have acute infectious exacerbations (AECB) of their chronic lung disease.
The study investigated various factors that may
influence the progression from an acute exacerbation to community-acquired pneumonia
(CAP). Factors that were associated with progression to CAP included infection with S. pneu-
Concentration (mg/l)
Cmax =
Peak serum concentration
AUC =
Area under the curve
MPC =
Mutant prevention concentration
MIC =
Minimal inhibitory concentration
T > MIC
Concentration-dependent
– Peak/MIC > 8–12
– AUC/MIC = AUIC
• > 125 Gram –
• ~ 30–50 Gram + (??)*
• > 100 Gram +**
– AUC/MPC = 22***
• AUIC-PMA
Time-dependent
– T > MIC; 40–50 %
* Schentag et al. (2001). CID;
32 (Suppl. 1): S39–46. Drusano
et al. (2001). CID; 32: 2091–2092.
Schentag et al. (2001). CID (Response);
33: 2092–2096.
** File et al. (2009). For human pathogen
S. pneumoniae, reported that patients
with an acute exacerbation of chronic
bronchitis were statistically less likely
to progress to pneumonia If the AUIC
was >100. Int J Antimicrob Agents;
33: 58–64.
*** Oloffson et al. (2006). J Antimicrob
Chemother; 57(6): 1116–1121.
Time (h)
Blondeau, updated 2009
Figure 1 PK/PD relationships: surrogate markers.
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Clinical Efficacy, Rapid Bactericidal Action and Low Potential for Resistance Selection of Baytril®
Joseph M. Blondeau
moniae versus other pathogens (p < 0.001), an
AUIC > 100 was more likely in patients with
AECB, whereas patients with an AUIC of < 100
were associated with CAP (p < 0.001).The authors
concluded that for S. pneumoniae-infected patients,
achieving an AUIC > 100 can attenuate progression to CAP. To date, the absolute value for this
AUC/MIC ratio remains unresolved, however,
this most recent study suggests that for S. pneumoniae-infected patients a higher ratio is preferential to a lower ratio.The antibacterial activity of
time-dependent antimicrobial agents (i.e., betalactams) is linked to the length of time-drug concentrations remain in excess of the MIC over the
dosing interval. It has been suggested that this
value needs to be in the range of 40–60 % of the
dose where the drug concentration exceeds the
MIC. Figure 2 summarizes the PK/PD classification of antimicrobial agents used in veterinary
medicine.
The pathogenesis of infection begins when organisms invade otherwise sterile body sites and
are not initially eliminated by non-specific immune processes. The host is often damaged as a
direct result of invasion by the pathogen, by liberation of exotoxins, by endotoxin, and by stimulating the immune response. While recovery
from infection requires a functioning immune
system, symptoms associated with infection may
be partly or mostly related to the inflammatory
response. As such, antimicrobial agents are adjunct
therapies to the bodies own natural defences for
fighting infection.This in no way lessons the importance of adjunctive antimicrobial therapies but
does stress the importance of correctly using
these agents for a successful clinical outcome and
not inadvertently contributing to antimicrobial
resistance. Unfortunately, most decisions regarding antimicrobial drug selection are based solely
on clinical outcome (as determined in clinical tri-
Concentration (mg/l)
Cmax =
Peak serum
concentration
AUC =
Area under the curve
T > MIC
MIC =
Minimal inhibitory
concentration
Time (h)
Figure 2 PK/PD relationships: surrogate markers.
88
Cmax/MIC: streptomycin,
gentamicin, tobramycin,
omikacin, danofloxacin,
enrofloxacin, marbofloxacin,
difloxacin, sarafloxacin,
metronidazole
AUC/MIC: streptomycin,
gentamicin, amikacin,
tobramycin, danofloxacin,
enrofloxacin, marbofloxacin,
difloxacin, sarafloxacin,
metronidazole, colistin,
oxytetracycline, chlortetracycline, doxycycline,
azithromycin, clarithromycin,
vancomycin
T > MIC: benzylpenicillin,
amoxicillin, cloxacillin,
carbenicillin, cephalixin,
ceftiofur, cephapirin,
florphenicol, chloramphenicol, erythromycin, tilmicosin,
tulathromycin, aivlosin,
clindamycin, sulfadiazinesulfadoxime, trimethoprim
03.06.2009
17:32 Uhr
als) with little or no consideration for microbiological or pharmacological potency. In today’s environment where few new antimicrobial agents
are under clinical development, a renewed interest in existing agents seems necessary and nonclinical outcome parameters require investigation
for optimal drug use. In human medicine, numerous guidelines have been assembled and published by expert working groups, whereby
recommended therapies for various infectious
diseases are based on the best evidence available.6
In the most recent CAP guidelines, Mandel et al.
wrote: “Because overall efficacy remains good for
many classes of agents, the more potent drugs are
given preference because of their benefit in decreasing the risk of selection for antibiotic resistance. Other factors for consideration of specific
antimicrobials include pharmacokinetics/pharmacodynamics, compliance, safety, and cost”.
Such statements suggest that the potential for the
prevention of resistance selection is important
and that PK/PD parameters are also important
considerations: clinical outcome alone should not
be the sole criteria for drug selection for therapy.
Resistance prevention
In vitro susceptibility testing has been the cornerstone of measuring the activity of antimicrobial
agents against various pathogens, and in an ideal
world, the results of such testing impact of appropriate therapy. Current routine susceptibility testing is based on utilizing a bacterial inoculum of
105 colony forming units per mililiter (cfu/ml)
and is standardized for a number of variables.
Such testing may not fully appreciate the true dynamics of higher bacterial populations present
during infection, where bacterial numbers likely
fluctuates. During acute infections, bacterial loads
may exceed 109 cfu7-11 – a value relevant in the
context of antimicrobial resistance, as the frequency with which a spontaneous mutant that
Seite 89
confers drug resistance occurs ranges from 1 x 10-7
to 1 x 10-9 or 1 spontaneous mutant for every 107
to 109 bacterial cells.12 As such, an acute infection
with ≥ 109 cfu is likely to harbor a resistant cell (or
resistant cells) and in the presence of an antimicrobial agent, may allow for the selective amplification of the resistant subpopulation as the susceptible population is being eliminated by the drug.
Resistance prevention is being recognized as a
goal of antimicrobial therapy. Dong et al. published the mutant prevention concentration
(MPC) approach in 1999.13 As stated above, high
bacterial burdens may be present during infection and from these higher bacterial burdens, mutant cells may be present spontaneously within
the population and under antimicrobial selective
pressure may be selectively amplified during therapy. In the landmark experiments by Dong et al.,
increasing bacterial populations of Staphylococus
species and Mycobacterium species were exposed
to increasing fluoroquinolone drug concentrations in an in vitro assay. For these experiments,
approximately 1010 bacterial cells were inoculated
to the surface of agar plates containing increasing drug concentrations of the fluoroquinolones
being investigated. Following incubation under
ideal conditions, the lowest drug concentration
blocking all growth was termed the mutant prevent concentration (MPC). It was readily demonstrated in the experiments by Dong et al. that
mutant bacterial cells could be readily selected
off drug-containing agar plates over drug concentrations ranging between the measured MIC
and MPC drug concentrations. As such, the drug
concentration that blocked all growth (including
those of the mutants) was termed the MPC.
Since the initial description of MPC by Dong
et al., a number of other reports have appeared in
the peer reviewed literature expanding on this
concept and reporting on the results for the testing of numerous different antimicrobial and microorganism combinations.14, 15
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Joseph M. Blondeau
Figure 3 is a schematic representation demonstrating how mutant cells may be selectively amplified in the presence of the MIC drug concentration. This schematic representation assumes
that the MIC drug concentration is insufficient to
block the growth of mutant cells that may be
present in the population. As shown in the figure,
spontaneously resistant cells naturally present in
the population may be amplified in the presence
of the MIC drug concentration as the susceptible
cells are being eliminated.This would occur if the
achievable drug concentration is insufficient to
block the mutant cells. In a patient with a healthy
and functioning immune system, all of these organisms would be cleared. In patients that may be
immunocompromised, had prior infection, had
prior antibiotic exposure or patients that appeared to be failing therapy for acute infection,
continued proliferation of the bacterial population to a point where it breaches the immune
threshold may result in a patient being colonized
or infected with the mutant population. As
shown in the bottom right hand section of the
figure, mutant cells that were selectively ampli-
fied in the presence of the drug may ultimately be
eliminated over time with factors such as competitive inhibition with normal flora and this in
some way may relate to the relative fitness of the
mutant cells. Figure 4 shows a schematic representation of the blocking of mutant cells in the
presence of the MPC drug concentration. This
schematic assumes that the MPC drug concentration is high enough to block the growth of the
mutant cells present in the population. As shown
in the figure, MPC drug concentrations would
essentially eliminate the mutant cells along with
the susceptible bacteria and control resistance to
the spontaneous rate seen in high density bacterial populations or eliminate these mutant cells
from the population. As depicted, some microorganisms tested by MPC may require a centrifugation step in order to concentrate the number of
bacterial cells. As well, the number of starter plates
required to be inoculated per organism varies.
One limitation with MPC testing relates to the
fact that it is technically more demanding than
performing a MIC test (Fig. 5). In comparison,
TIME
(hours to days)
20 in 1 billion
MIC
2 in 1 billion
200 in 1 billion
Immunocompromised state
Prior infection
Prior antibiotic exposure
Patient colonized or
infected with
mutant population
Acute infections/
failed therapy
HEALTHY
IMMUNE
SYSTEM
Potential
Clearance
Mutant cells may be cleared
over time, i.e., competitive
inhibition from
normal flora (fitness)
IMMUNE THRESHOLD BREACHED
Blondeau et al. (2004). J Chem, updated 2009
Figure 3 Selective amplification of resistant mutants at MIC.
90
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Seite 91
the MPC assay is based on testing of ≥ 109 cfu on
drug containing agar plates, whereas MIC testing
is conducted in a microbroth assay utilizing 105
cfu/ml. More recently, Hesje and Blondeau reported on MPC testing using a modified microbroth dilution assay.16,17 Of the data generated to
date, it appears that the modified microbroth
dilution assay yields consistent results to those
demonstrated by the agar dilution method that initially defined the MPC approach. Further testing
of the modified microbroth dilution method is
necessary in order to confirm this method as suitable assay for generating MPC results.
The mutant selection window (MSW) defines
the antimicrobial drug concentration that falls
between the MIC and MPC drug concentrations
and it has been previously argued that the time
the drug concentration remains within the MSW
or above the MPC has an impact on the selective
amplification of resistant subpopulations that may
be present in high density heterogeneous bacterial populations.14,18–20 Figure 6 is a schematic
representation of the MSW. When drug concen-
trations are below the MIC drug concentration,
neither susceptible nor mutant cells would be inhibited and as such, the mutant fraction is not
selectively amplified. Similarly, for drug concentrations in excess of the MPC, susceptible and
mutant populations are inhibited or killed. However, when drug concentrations remain within
the MSW – i.e., above the MIC but below the
MPC – the susceptible cells are inhibited and the
mutant fraction selectively amplified in the presence of the drug. While clinical evidence for the
selection window hypothesis is lacking, two in
vitro experiments using fluctuating fluoroquinolone concentrations support the idea. In one,
Firsov et al.21 simulated dosing of fluoroquinolones with S. aureus. They found that organisms
with elevated values of MIC were obtained only
when the drug concentration remained inside the
selection window, not when it was above the
MPC or below the MIC. Similar results were
found with quinolones and S. pneumoniae.20
Croisier et al. showed in a rabbit pneumonia
model that when drug concentrations remained
within the MSW for 45 % or greater of the dos-
TIME
MPC
2 in 1 billion
Colorization or
new infection
may occur
Mutant and
susceptible cell
inhibited
Immunocompromised state
Prior infection
Prior antibiotic exposure
Acute infections/
failed therapy
HEALTHY
IMMUNE
SYSTEM
Potential
Clearance
IMMUNE THRESHOLD BREACHED
Hansen & Blondeau (2002); Blondeau et al. (2004). J Chem, updated 2009.
Figure 4 Blocking of resistant mutants at MPC.
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Joseph M. Blondeau
Basic method: (varies by organism)
• Inoculate plates and incubate
• Transfer to fresh media (~ 100 ml)
• Centrifuge and resuspend in fresh media
• Inoculate drug containing plates with 1010 cfu
1)
1) Inoculate 3* plates per organism;
incubate 18–24 h at 35–37 °C in O2
2)
2) Transfer contents of plates to flask with
100 ml fresh media.
Incubate 18–24 h at 35–37 °C in O2
3) Centrifuge** culture media at 5,000 xg
for 30 min at 4 °C
Centrifuge**
3)
4) Resuspend in 3 ml of media
5) Inoculate drug containing plates with
1010 organisms; incubate for 18–24 h in O2,
examine for growth, reincubate for 18–24 h
in O2 and reexamine. The lowest drug
concentration preventing = MPC
4)
5)
0.06
0.12
16
8
0.25
0.5
1
MPC
2
4
Organism
# Starter plates
inoculated*
E. coli
2–3
S. intermedius
2–3
P. multocida
3–4
P. aeruginosa
2–3
M. haemolytica
4–5
A. pleuropneumonia
7–8
Centrifugation
required**
No
No
No
No
Yes
Yes
Figure 5 Schematic method of MPC testing.
Serum or tissue drug concentration
• Above MPC – both susceptible and
1st-step resistant cells inhibited –
no selective amplification of resistance
subpopulation.
• Double mutants may not be inhibited.
MPC
MSW
MIC
Sub-MIC – neither susceptible bacteria
nor 1st-step resistant mutants killed/
inhibited – no selective amplification of
resistant subpopulation.
Time post administration
Figure 6 Mutant selection window (MSW).
92
• Susceptible cells killed/inhibited.
• 1st-step or 2nd-step resistant cells not
inhibited – selective amplification may occur.
• Longer times in MSW = greater risk for
mutant selection/amplification.
03.06.2009
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Seite 93
sponse clearly contributes to organism killing and
eradication is currently unknown!
ing interval, it correlated 100 % with the selection of a mutant subpopulation conferring
quinolone resistance.22
Smith et al. previously argued that while the
MPC approach applied to the testing of fluoroquinolone compounds, it does not apply to betalactam, macrolide, or aminoglycoside compounds
as the principal mechanisms or resistance to these
compounds were not as a result of de novo resistance but rather acquisition of various resistance determinants.28 Zhao argued that when a
mutant was present within a bacterial population,
the issue is really how do you prevent that mutant
from being selectively amplified during drug
therapy, not how the mutant came to be.29 Prevention of mutant amplification and not mutation prevention is the goal of this approach and,
as such, might readily be applied to any scenario
where restricting mutant growth could be
achieved. It may be that for some pathogens and
drugs, prevention of mutant amplification could
only be achieved with combinations of drugs.30
To date, MPC measurements have been made
Based on the MSW approach, drug dosages
should be administered to be in excess of the
MPC (i.e., above the MSW) for as long as necessary to affect a substantial reduction in viable organisms. One unknown is how long do drug
concentrations need to remain in excess of the
MPC and what percentage of viable organisms
needs to be killed – 99 % versus 100 %. Is 1 % or
less of 1–10 billion cells surviving following drug
exposure too many – especially if they are all mutants? From kill experiments carried out with
human and veterinary pathogens and fluoroquinolone and non-fluoroquinolone compounds,
it was shown that at least 6–12 hours above the
MPC was necessary to yield a > 99 % reduction
in viable organisms when 106 to 109 bacteria
were exposed to MPC drug concentrations.23–27
How such observations translate to infection in
animals, where the impact of the immune reAntimicrobial
agent
MIC50
MIC90
MIC Range
MPC50
MPC90
MPC Range
Amikacin
2
4
1–8
32
32
16–32
b
Ampicillin
2
2
1–2
16
32
16–>128
Cefazolinb
2
2
1–8
64
64
16–128
Cefotaxime
0.063
0.125
0.031–0.25
8
16
0.5–32
Ceftriaxone
0.063
0.125
0.031–0.25
2
16
0.5–16
Doxycycline
0.5
1
0.25–8
64
> 64
> 8–>64
Enrofloxacin
0.008
0.016
< 0.008–0.063
0.125
0.25
0.063–0.25
Gentamicin
0.5
1
0.25–1
0.5
16
0.25–>8
Marbofloxacin
0.016
0.016
0.004–0.016
0.125
0.5
0.063–0.5
Nitrofurantoin
8
16
4–32
64
≥ 64
64–≥64
Tobramycin
1
1
0.5–16
8
16
8–≥8
Table 1 Comparative in vitro activity of several antimicrobial agents tested against E. coli a isolates from companion animals
(studies combined).
a
Some isolates complements of Dr. Heinz Wetzstein of Germany.
b
For organisms with MICs ≤ 2 µg/ml;
MPCs for organisms with MICs ≥ 4 µg/ml ranged from 64–≥ 256 µg/ml
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Joseph M. Blondeau
with macrolides 31, aminoglycosides30, tetracyclines, beta-lactams, glycopeptides32, and glycylcycline.33,34 In some instances, the mechanisms
associated with the observations have not been
fully elucidated and further investigations are
necessary (reviewed in Hesje et al.).15,35,36
MPC values were higher against organisms with
MICs ≥ 4 µg/ml.
Comparative MIC and MPC data for enrofloxacin
and comparator antimicrobial agents tested against
companion animal isolates of S. intermedius are
shown in Table 2. For enrofloxacin, the MIC90
value was 0.063 and the MPC90 was 0.5 µg/ml; by
comparison, values for marbofloxacin were 0.5 and
1 µg/ml, respectively. For amikacin, gentamicin, and
tobramycin, MIC90 values ranged from 0.25 to 2
µg/ml being lowest for gentamicin and highest for
amikacin: MPC90 values respectively were 32, 4 and
8 µg/ml. For cefazolin, cefotaxime and ceftriaxone,
MIC90 values were 0.063, 2, and 1 µg/ml and
MPC90 values were 16, 4, and 8 µg/ml, respectively.
Veterinary data
Table 1 is a summary of MIC and MPC data for
enrofloxacin and comparator antimicrobial agents
tested against companion animal isolates of E. coli.
For enrofloxacin, the MIC90 value was 0.016 µg/
ml and the MPC90 was 0.25 µg/ml. By comparison, MIC90 values for amikacin, gentamicin, and
tobramycin were 4, 1, 1 µg/ml, respectively, and
MPC90 values were 32, 16, 16 µg/ml. For cefotaxime and ceftriaxone, MIC90 values were
0.125 µg/ml and MPC90 values were 16 µg/ml.
The MPC90 value for cefazolin was 64 µg/ml.
Enrofloxacin MIC and MPC testing has also been
completed against a more than 100 strains of
Pasteurella multocida. The MIC90 value was 0.008
µg/ml and the MPC90 was 0.125 µg/ml.
Antimicrobial
agent
MIC50
MIC90
MIC Range
MPC50
MPC90
MPC Range
Amikacin
1
2
1–2
32
32
16–>32
Ampicillin
0.12
0.25
0.031–0.25
> 128
> 128
0.125–>128
Cefazolin
0.063
0.063
0.031–0.063
0.25
16
0.125–16
Cefotaxime
0.5
2
0.25–2
1
4
0.5–8
Ceftriaxone
0.5
1
0.5–2
4
8
4–8
< 0.063–0.125
8
16
4–6
Doxycyclineb
≤ 0.063
≤ 0.063
Enrofloxacin
0.063
0.063
0.031–0.063
0.5
0.5
0.5–1
Erythromycin
0.25
0.5
0.125–0.5
>8
>8
0.5–>8
Gentamicin
0.125
0.25
0.063–0.5
2
4
2–8
marbofloxacin
0.25
0.5
0.125–0.5
1
1
0.5–2
Nitrofurantoin
8
8
8
32
32
32–64
Tobramycin
0.25
0.5
< 0.125–0.5
8
8
2–≥8
Table 2 Comparative in vitro activity of several antimicrobial agents tested against S. intermediusa isolates from companion animals
(studies combined).
a
Some isolates complements of Dr. Heinz Wetzstein of Germany.
organisms with MICs ≥ 2 µg/ml had MPCs ≥ 64 µg/ml.
94
b
For organisms with MICs ≤ 2 µg/ml;
03.06.2009
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Seite 95
Figure 7 shows enrofloxacin serum drug concentrations in dogs for 3 dosages.The mutant selection window is bordered by a MIC90 of 0.016
and MPC90 of 0.25 µg/ml for E. coli. For S. intermedius, the mutant selection window is bordered
by a MIC90 of 0.063 µg/ml and a MPC90 of
0.5 µg/ml. As such, serum drug concentrations
for all dosages remain in excess of the mutant
selection window for 7–>12 hours. From kill
studies conducted by our laboratory with enrofloxacin and E. coli, 98–99 % of a 106–107 cfu/ml
bacterial burden were killed in the presence of
enrofloxacin following 1–2 hours of mutant prevention drug concentrations in kill assays. Serum
drug concentrations would remain in excess of
the MPC90 of P. multocida for ~ 12 hours. Figure 8
shows serum drug concentrations for enrofloxacin in cats to be 5 mg/kg dosage. For E. coli, the
mutant selection window was bordered by a MIC90
of 0.016 µg/ml and the MPC90 of 0.25 µg/ml.
For S. intermedius, the mutant selection window is
bordered by a MIC90 of 0.063 µg/ml and a MPC90
of 0.5 µg/ml. As such, serum drug concentrations
would be in excess of the mutant selection window for ~ 9–>12 hours of the dosing interval.
Serum drug concentrations would remain in excess of the MPC90 for P. multocida for >12 hours.
Previously, it was mentioned that PK/PD parameters have been utilized to characterize various
antimicrobial agents and their antibacterial activity. To date, most measurements related to pharmacology have included MIC measurements of
the respective organisms being investigated.
Given the recent swell of data related to MPC
measurements, what impact does such a measurement have on our understanding of resistance
prevention with the ratio derived from Cmax/
MIC or AUC/MIC? To date, limited data is available defining Cmax/MPC or AUC/MPC ratios.
Olofsson et al. working with ciprofloxacin and
E. coli reported that an AUC/MPC ratio of ≥ 22
Concentration µg/ml
Dose-dependent
8
E. coli
MIC90 0.016 µg/ml
MPC90 0.25 µg/ml
AUC/MIC = 656
AUC/MPC = 42
7
6
S. intermedius
MIC90 0.063 µg/ml
MPC90 0.5 µg/ml
AUC/MIC = 166
AUC/MPC = 21
5
4
Elimination half-life = 4 h
20 mg/kg PO OD
10 mg/kg PO OD
5 mg/kg PO OD
Mutant Selection Window
3
MAX SUSCEPTIBILITY
2
MPC90 0.25 µg/ml
1
2 µg/ml
MIC 90 0.016 µg/ml
0
0.5
1
2
3
4
5
6
Hours
7
8
9
10
11
12
Figure 7 Enrofloxacin – E. coli/S. intermedius.
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Joseph M. Blondeau
correlated with the prevention of resistance development (Fig. 1).37 Clearly, additional studies
are required considering MPC rather than MIC
measurements for defining PK/PD relationships
and to help predict or prevent the selection of resistance subpopulations. Recently, the idea of
“best-in-class” has emerged as a potential way to
optimizing therapy: maximize successful patient
outcome while minimizing resistant mutant selections. Once the class of agent to be used for
therapy has been decided, the most potent (microbiologically, pharmacologically) agent in the
class would be used based on the organism(s)
most likely to be the cause of infection. One
major problem with this approach is that patients
are most often treated empirically; consequently,
microbiological investigation may not be performed to identify the pathogen(s) and its/their
susceptibility profile. A more fundamental problem is that two compounds can have very similar
activity with susceptible cells, but very different
activities against resistant mutants.12,38,39 The more
mutant-active agent is more likely to restrict the
enrichment of mutant subpopulations. Ideally,
“best-in-class” should include a strong consideration of anti-mutant activity and not be based
solely on MIC measurement.
In vitro kill studies
Fluoroquinolones such as enrofloxacin may be
characterized as having excellent bactericidal activity and tissue penetration and, in general, have
broad in vitro and in vivo activity against Grampositive and -negative pathogens. In previous sections, in vitro susceptibility testing was discussed
and the MPC concept reviewed. Measurements
such as MIC testing and MPC testing are measurements of inhibition of bacterial growth and
not a measurement of killing. Traditional measurements of in vitro killing are performed by ex-
Concentration µg/ml
Dose-dependent
8
E. coli
MIC90 0.016 µg/ml
MPC90 0.25 µg/ml
AUC/MIC = 656
AUC/MPC = 42
7
6
S. intermedius
MIC90 0.063 µg/ml
MPC90 0.5 µg/ml
AUC/MIC = 166
AUC/MPC = 21
5
4
3
Elimination half-life = 6 h
5 mg/kg PO OD
Mutant Selection Window
MPC90 0.25 µg/ml
1
MIC 90 0.016 µg/ml
0
0.5
Figure 8 Enrofloxacin – E. coli/S. intermedius.
96
2 µg/ml
MAX SUSCEPTIBILITY
2
1
2
3
4
5
6
Hours
7
8
9
10
11
12
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17:32 Uhr
posing ~ 105 cfu/ml of bacteria to varying drug
concentrations, i.e., 2x, 4x, 10x the MIC. Such
studies have been used to categorize antibacterial agent as either bacteriostatic or bactericidal
based on a log10 reduction in viable cells of ≤ 2
log10 versus ≥ 3 log10, respectively.40 We have previously argued that kill studies might be more
meaningful if higher bacterial densities were used,
as such burdens were more indicative of bacterial
infections. Additionally, we also argued that drug
concentrations utilized in kill studies should be
based on the measured MIC, MPC, maximum
Seite 97
serum and maximum tissue drug concentrations.26,40-43
Figure 9 is a schematic representation of the
modified in vitro kill method for determining
killing of bacterial inocula of 106 cfu/ml, 107
cfu/ml, 108 cfu/ml, and 109 cfu/ml. Briefly, bacteria are sub-cultured to 3–7 (varies based on organism) agar plates and incubated under optimal
conditions for 18–24 hours and, following this,
the bacterial cells are transferred to liquid media,
incubated for ~ 2 hours and then diluted to yield
• inoculate bacteria
to three agar plates
to produce confluent
growth
• incubate 18–24 h
at 35–37 °C in O2
• Transfer content of plates
to broth media
• Incubate 2 h at 35–37 °C in O2*
• Cell density approximately 109 cfu/ml
PERFORMED IN TRIPLICATE
• Dilute to give bacterial
inocula ranging from
106–109 cfu/ml
• Add drug and sample
at predetermined times
• Dilute to broth to facilitate
counting and inoculate agar
plates, incubate as described
• Determine colony counts
and calculate log10 and %
reduction (kill) in viable cells
• inoculate to plates • incubate 18–24 h
at 35–37 °C in O2
* 5% CO2 depending on organism being tested
Figure 9 A new approach to in vitro kill measurements.
Traditional kill studies utilize 105 colony forming units per milliliter (cfu/ml) of bacteria exposed to varying drug concentrations –
usually as multiples of the MIC – i.e., MIC, 5 times (x) MIC, 10 x MIC, etc. The new method involves testing of higher bacteria
inocula – 106 (1 million) to 109 (1 billion) cfu/ml against drugs as shown schematically above.
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Joseph M. Blondeau
bacterial densities ranging from 106–109 cfu/ml.
Drug is added to the bacteria and then bacteria
are sampled at predetermined intervals and inoculated to the surface of drug-free agar plates and
incubated for 18–24 hours under optimal conditions. Colony counts are performed and the log10
reduction and percentage kill of viable cells
recorded. Each assay is performed in triplicate
and the results averaged. Where multiple strains
are tested, the results are averaged.
As previously stated, the MIC drug concentration may be ineffective in inhibiting the growth
of high density bacterial populations, as less susceptible cells may be present in this population.
To inhibit these less susceptible or resistant cells,
the MPC drug concentration is necessary. In kill
studies conducted in our laboratory with enrofloxacin and E. coli, the killing was slow and incomplete when high density inocula (106–109
cfu/ml) were exposed to the measured MIC drug
concentrations for the organisms tested (Figs. 10,
11). For example, 6–25 % of bacterial cells were
killed following 1–2 hours of drug exposure.
These kill values did not increase substantially
after 12–24 hours of drug exposure. These observations are consistent with previous reports
from our laboratory for human pathogens exposed to fluoroquinolones in identical kill studies.26,39,43 This observation is also consistent with
observation with Mannheimia haemolytica recovered from cattle with bovine respiratory disease
and enrofloxacin.44–46 Exposure of high density
bacterial populations of E. coli to enrofloxacin at
the measured MPC drug concentration yielded
more rapid and complete killing (Figs. 10, 11). For
example for 106 cfu/ml, a 2.3–3.4 log10 reduction
in viable cells (98–> 99 % kill) was seen after 1–2
hours of drug exposure; for 107 cfu/ml, 2.2–2.9
log10 reduction (98–>99 % kill); for 108 cfu/ml,
1.1–1.3 log10 reduction (90–92 % kill); 109 cfu/ml,
0.1 log10 reduction (10–19 % kill). Killing rates
exceeded 99 % following 12–24 hours of drug
exposure. This data clearly indicates that against
high density bacterial populations – such as those
potentially present during acute infection – enrofloxacin was rapidly bactericidal with 90–>99 %
of viable cells being killed in the presence of the
2
MIC 10^6
1
MIC 10^7
0
MIC 10^8
–1
MIC 10^9
–2
MPC 10^6
–3
MPC 10^7
–4
MPC 10^8
–5
MPC 10^9
–6
–7
0.5
1
2
4
6
Time
Figure 10 The killing of E. coli at the MIC and MPC enrofloxacin concentration.
98
12
24
03.06.2009
17:32 Uhr
drug. At present, kill studies are not yet completed utilizing the maximum serum and timedrug concentrations against bacterial inocula
ranging from 106–109 cfu/ml. However, from
studies completed with M. haemolytica, killing
using maximum serum or tissue drug concentration (1.9–2 µg/ml in cattle; higher than MPC
drug concentrations), killing was more rapid and
complete – i.e., > 99 % kill for 106–108 cfu/ml following 1 hour of drug exposure and ≥ 96–98 %
kill for a 109 cfu/ml inocula following 1 hour of
drug exposure. These observations with enrofloxacin testing against E. coli and M. haemolytica indicate rapid in vitro bactericidal activity.
In summary, antimicrobial susceptibility/resistance is defined by testing a known density of
bacterial cells to varying drug concentrations in
vitro and the MIC results are compared to susceptibility breakpoints of an established base of
microbiological and pharmacological parameters.
An organism with a MIC at or below the susceptibility breakpoint is considered susceptible,
whereas an organism with a MIC above the sus-
Seite 99
ceptibility breakpoint is considered non-susceptible or resistant. More recently, mutant prevention concentration testing has been proposed as
an alternative form of susceptibility testing and
takes into account higher bacterial densities such
as those seen associated with acute infection. In
addition to providing a level of susceptibility or
resistance to a higher density of bacterial cells,
MPC also provides a means to predict the likelihood of the selection of resistant subpopulations
during therapy when antimicrobial agents are
dosed to be above the mutant selection window
or within the mutant selection window. Clearly,
the MPC approach to antimicrobial susceptibility testing is still in its infancy and numerous
other investigations need to be completed with
various bug/drug combinations and in some instances, combinations of antimicrobial agents together. Utilization of strategies such as MPC may
provide guidance, optimal therapy of infectious
diseases and reduce the rate at which resistance
will escalate antimicrobial compounds.
3,500
MIC 10^6
3,000
MIC 10^7
MIC 10^8
2,500
MIC 10^9
2,000
MPC 10^6
1,500
MPC 10^7
1,000
MPC 10^8
500
MPC 10^9
0
– 500
0.5
1
2
4
6
12
24
Time
Figure 11 The percent killing of E. coli at the MIC and MPC enrofloxacin concentration.
99
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Seite 100
Joseph M. Blondeau
Infection
Number
Clinical diagnosis
25 dogs
Dose (mg/kg
BW/day)
Duration of
treatment
Clinical
response rate
Reference
Complicated upper and
lower respiratory infections
6–12
96 %
(48)
30 cats
Feline rhinotracheitis
Bronchopneumonia
6–12
96 %
(48)
178 dogs
Upper and lower
respiratory infections
6–10
89.9 %
(49)
87 cats
Feline rhinotracheitis
Bronchopneumonia
6–10
91.9 %
(49)
Respiratory
20 dogs
5
90 %
(48)
30 cats
5
90 %
(48)
14 dogs
5
100 %
(50)
49 cats
5
89.5 %
(50)
129 dogs
5
89.9 %
(51)
68 cats
5
88.2 %
(51)
48 dogs
5
89.6 %
(52)
50 dogs
5
93.3 %
(53)
30 dogs
5
93.3 %
(54)
100 dogs
10
90 %
(55)
16 dogs
5
94 %
(56)
25 cats
10
88 %
(55)
Urinary
Pyoderma
Table 3 Clinical studies investigating the efficacy of enrofloxacin (Baytril) in companion animals.
The 1, 2, 3 punch of Baytril
Clinical efficacy
Enrofloxacin (Baytril) is approved for the treatment of infectious diseases in companion animals
(dogs and cats) and the clinical efficacy has been
established and reported by numerous investigators (Tab. 3). Specifically, enrofloxacin is indicated
for the treatment of skin, respiratory and urinary
tract infections. For pyoderma, clinical efficacy
rates for enrofloxacin were reported to range
from 89.6–94 % in digs and 88 % in cats. For respiratory tract infections, enrofloxacin clinical efficacy rates ranged from 91.9–96 % in cats and
89.9–96 % in dogs. For urinary tract infections,
100
clinical efficacy rates ranged from 88.2–90 % for
cats and from 89.9–100 % for dogs.
Rapid bactericidal action
As summarized in this report, in vitro measurements
showing killing of high density populations of
E. coli yielded reductions of viable cells of 90–>
99 % by 1–2 hours against 1 million to 100 million cells/ml by 1–2 hours of drug exposure at the
MPC drug concentrations. Dagan et al. previously
indicated that eradication of bacterial cells correlated with clinical outcome in human patients with
respiratory tract infections.47 As shown, enrofloxacin demonstrates rapid bactericidal activity
against the organisms reported in this manuscript.
03.06.2009
17:32 Uhr
Low potential for resistance selection
As summarized, traditional measurements of antimicrobial susceptibility or resistance, while useful, may not fully measure the true dynamics of
the interaction of an antimicrobial agent and bacteria in vivo, where the number of bacteria associated with infection may exceed the number
of cells utilized by MIC testing. As such, MPC
measurements have evolved to determine the
drug concentration required to block the growth
of the least susceptible cells present in high density bacterial populations – such as those seen
during acute infection. Suboptimal dosing may
lead to the selective amplification of the mutant
Seite 101
subpopulations present in high density bacterial
populations. Ideally, antimicrobial therapy would
eliminate both susceptible and resistant cells present in the population. E. coli, S. intermedius, and
P. multocida are amongst the most common pathogens associated with infections in dogs and cats.
Both MIC and MPC values were below the
achievable serum drug concentrations with approved dosing of enrofloxacin in dogs and cats;
enrofloxacin serum drug values remain in excess
of the MSW for no less than 7 hours (lowest
dosage) and for higher dosages, longer. As such,
in the MPC/MSW model, enrofloxacin demonstrates a low potential for resistance selection.
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4 International Baytril Symposium Proceedings of the

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