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Veterinary Microbiology 131 (2008) 164172

www.elsevier.com/locate/vetmic

Antimicrobial resistance and genetic characterization of

fluoroquinolone resistance of Pseudomonas
aeruginosa isolated from canine infections
J. Rubin a, R.D. Walker b,c, K. Blickenstaff d, S. Bodeis-Jones d, S. Zhao d,*
a

Department of Veterinary Microbiology, Western College of Veterinary Medicine,

University of Saskatchewan, 52 Campus Drive, Saskatoon S7N 5B4, Canada
b
Anti-infectives Research Consultants, Glade Park, CO 81523, United States
c
Department of Biological Sciences, Mesa State College, Grand Junction, CO 81501, United States
d
Office of Research, Center for Veterinary Medicine, U.S. Food & Drug Administration,
8401 Muirkirk Road, Laurel, MD 20708, United States
Received 21 December 2007; received in revised form 26 February 2008; accepted 28 February 2008

Abstract
Infections with antimicrobial-resistant bacteria are a great challenge in both human and veterinary medicine. The purpose of
this study was to determine antimicrobial susceptibility of 106 strains of Pseudomonas aeruginosa isolated from dogs with otitis
and pyoderma from 2003 to 2006 in the United States. Three antimicrobial panels, including 6 classes and 32 antimicrobial
agents, were used. A wide range of susceptibility patterns were noted with some isolates being resistant to between 8 and 28
(mean 16) of the antimicrobials tested. Among the b-lactams, all isolates were resistant to ampicillin, cefoxitin, cefpodoxime,
cephalothin and cefazolin followed by amoxicillin/clavulanic acid (99%), ceftiofur (97%), ceftriaxone (39%), cefotaxime
(26%), and cefotaxime/clavulanic acid (20%), whereas less than 7% of isolates were resistant to ceftazidime/clavulanic acid,
ceftazidime, piperacillin/tazobactam or cefepime. Two isolates were resistant to the carbapenems. Among the quinolones and
fluoroquinolones, the most isolates were resistant to naladixic acid (96%), followed by orbifloxacin (52%), difloxacin (43%),
enrofloxacin (31%), marbofloxacin (27%), gatifloxacin (23%), levofloxacin (21%), and ciprofloxacin (16%). Among the
aminoglycosides, the most resistance was seen to kanamycin (90%), followed by streptomycin (69%), gentamicin (7%), and
amikacin (3%). Of the remaining antimicrobials 100% of the isolates were resistant to chloramphenicol followed by tetracycline
(98%), trimethoprim/sulfamethoxazole (57%), and sulfisoxazole (51%). Point mutations were present in gyrA, gyrB, parC, and/
or parE genes among 34 of the 102 naladixic acid-resistant isolates. Two isolates contained class 1 integrons carrying aadA gene
conferring streptomycin and spectinomycin resistance. The findings suggest that many antimicrobial agents commonly used in
companion animals may not constitute appropriate therapy for canine pseudomonas infections.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Pseudomonas aeruginosa; Antimicrobial resistance; Fluoroquinolones; Canine; Class 1 integron; QRDR

* Corresponding author. Tel.: +1 301 210 4472.

E-mail address: shaohua.zhao@FDA.HHS.GOV (S. Zhao).
0378-1135/$ see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.02.018

J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

1. Introduction

2. Materials and methods

Pseudomonas aeruginosa, a Gram-negative rod, is

an important pathogen to both humans and animals
(Hillier et al., 2006; Gales et al., 2001). The bacterium
can be resistant to all classes of antimicrobial
agents making it especially difficult to successfully
treat patients with compromised immune defenses
(Jalal et al., 2000; Pirnay et al., 2003). In dogs,
P. aeruginosa is a common cause of pyoderma, otitis
media/external and urinary tract infections (Cole
et al., 2006; Gatoria et al., 2006; Hariharan et al.,
2006; Hillier et al., 2006).
Due to the presence of several drug efflux
systems and porins, P. aeruginosa is intrinsically
resistant to a wide range of antimicrobials including
benzylpenicillins, aminobenzylpenicillins, carboxypenicillins, first and second generation cephalosporins, chloramphenicol and tetracycline (Li et al.,
1994; Nikaido, 1994). It also forms biofilms
which are impervious to antimicrobials, further
complicating therapy (Hall-Stoodley et al., 2004).
The major classes of antimicrobials used for the
systemic treatment of infections include the antipseudomonal penicillins, third and fourth generation
cephalosporins, carbapenems, aminoglycosides,
and fluoroquinolones. Unfortunately, resistance to
these drugs is frequently encountered in clinical
practice. Due to highly variable resistance patterns,
empiric therapy may result in inappropriate treatment. Thus, antimicrobial susceptibility testing
should be a crucial step in the selection of
appropriate therapy.
With increasing utilization of fluoroquinolones in
both human and veterinary medicine, emerging
resistance is a concern (Hariharan et al., 2006; Linder
et al., 2005). Resistance to fluoroquinolones is
frequently due to point mutations in the DNA gyrase
(gyrA and gyrB) and topoisomerase IV ( parC and
parE) genes. Plasmid-mediated resistance and efflux
systems have been reported as alternate mechanisms
of resistance. Class 1 integrons are important in the
dissemination of resistance in Gram-negative bacteria
(Hall, 1997). The objectives of this study were to
define susceptibility patterns, characterize quinolone
resistance and screen for class 1 integrons in 106
clinical dog isolates of P. aeruginosa from the United
States.

2.1. Bacterial strains

165

One hundred and six strains of P. aeruginosa

isolated from dogs with soft tissue infections, e.g.,
otitis externa, otitis medius and pyoderma were used
in this study. All strains were isolated from veterinary
diagnostic laboratories from nine states in the U.S.
between 2003 and 2006. History on prior antimicrobial chemotherapy was not available. Following the
initial isolation and identification, the isolates were
sent to the Center for Veterinary Medicine (CVM), the
U.S. Food and Drug Administration (FDA). Upon
receipt, the bacterial isolates were subcultured onto
trypticase soy agar (TSA) plates supplemented with
5% defibrinated sheep blood (Becton Dickinson
Microbiology Systems, Cockeysville, MD). Isolates
were confirmed as P. aeruginosa using VITEK
Gram-negative identification cards (BioMerieux
Inc., Hazelwood, MO) following the manufacturers
instructions and were then suspended in trypticase soy
broth (TSB; Difco) containing 15% glycerol and
stored at 80 8C until used.
2.2. Antimicrobial susceptibility testing
Antimicrobial minimum inhibitory concentrations
(MICs) were determined using the sensititre automated antimicrobial susceptibility system in accordance with the manufacturers instructions (Trek
Diagnostic Systems, Cleveland, OH). Initially, all
isolates were tested using a panel designed by the
National Antimicrobial Resistance Monitoring System (NARMS). This panel included ceftriaxone,
ceftiofur, amoxicillin/clavulanic acid, ampicillin,
cefoxitin, ciprofloxacin, naladixic acid, amikacin,
gentamicin, streptomycin, kanamycin, sulfamethoxazole, trimethoprim/sulfamethoxazole, tetracycline,
and chloramphenicol. A panel containing only
fluoroquinolones was also tested, including ciprofloxacin, levofloxacin, gatifloxacin, marbofloxacin, enrofloxacin, difloxacin, and orbifloxacin. In addition, all
isolates were tested with a panel of b-lactam
antimicrobials which included imipenem, meropenem, cefepime, piperacillin/tazobactam, ceftazidime,
ceftazidime/clavulanic acid, cefotaxime/clavulanic
acid, cefotaxime, ceftriaxone, cefoxitin, cefpodoxime,

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J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

ampicillin, cephalothin, and cefazolin. Results were

interpreted in accordance with CLSI interpretive
criteria with the exception of streptomycin (resistant
breakpoint 64 mg/ml). However, among these 32
antimicrobials tested, not all had CLSI interpretive
criteria for P. aeruginosa. For those drugs that lacked
specific P. aeruginosa interpretive criteria, CLSI
breakpoints for the Enterobacteriaceae were used
(CLSI, 2004, 2006). Escherichia coli ATCC 25922,
Enterococcus faecalis ATCC 29212, Staphylococcus
aureus ATCC 29213 and P. aeruginosa ATCC 27853
were used as quality control organisms in the
antimicrobial MIC determinations.
2.3. Preparation of DNA template and
amplification of the quinolone resistance
determining regions (QRDRs) in gyrA, gyrB,
parC, and parE
Chromosomal DNA from 102 nalidixic acidresistant P. aeruginosa isolates was prepared by
suspending one loop full of bacteria in 500-ml distilled
water and boiling for 10 min, followed by centrifugation
for 30 s. PCR amplification of QRDR of the gyrA, gyrB,
parC, and parE genes was achieved using previously
described PCR primers and amplification conditions.
The primer sequences were listed in Table 1. The PCR
was performed in 50-ml volumes consisting of a
200 mM of dNTP, 1.5 mM MgCl2, 1 U of gold Taq DNA
polymerase, and 50 pmol of each primer. The PCR was
carried out using a PerkinElmer 9700 thermal cycler
(PerkinElmer, Foster City, CA) with an initial denaturing cycle at 95 8C for 3 min; followed by 35 cycles at
95 8C for 30 s, 55 8C (for gyrA and parC) or 60.0 8C (for
gyrB and parE) for 30 s and 72 8C for 1 min; a final

extension step at 72 8C for 7 min. The amplification

products were resolved by electrophoresis in a 1.0%
agarose gel and visualized under UV light.
2.4. DNA sequencing and analysis QRDR
PCR amplification products were purified using a
PCR purification kit (Boehringer Mannheim, Indianapolis, IN), and sequenced using an ABI automatic
DNA sequencer (Model 3700, Applied Biosystems),
at FDA/CVM laboratories, using the above described
forward and reverse primers. DNA sequences were
analyzed by comparison with published GenBank
DNA sequences (Accession numbers L29417,
AB00581, AB003428, and AB003429) using the
National Center for Biotechnology Information via the
BLAST network service. Alignment and comparison
of QRDR sequences were done using ClustalW,
Multiple Sequence Alignment Program (http://
www.ebi.ac.uk/clustalw/, European Bioinformatics
Institute (EBI), Cambridge, UK).
2.5. Screen and DNA sequence analysis of class 1
integrons
The presence of class 1 integrons was determined
using previously described PCR primers (Table 1). The
PCR was performed in the same way as described above,
except the primer annealing temperature at 54 8C. The
amplified products were resolved by electrophoresis in a
1.0% agarose gel, and visualized under UV light. For
each set of PCR reactions, S. typhimurium CVM4499
and E. coli CVM 996 were included as positive and
negative controls, respectively. The DNA sequences of
amplified integrons were then analyzed by comparison

Table 1
Primer sequences used to amplify the QRDRs of gyrA, gyrB, parC, parE, and class 1 integron
Name

Sequence

Reference

gyrAF
gyrAR
gyrBF
gyrBR
parCF
parCR
parEF
parER
50 -CS
30 -CS

50 -AGTCCTATCTCGACTACGCGA T-30
50 -AGTCGACGGTTTCCTTTTCCAG -30
50 - GCGCGTGAGATGACCCGC CGT-30
50 - CTGGCGGTAGAAGAAGGTCAG-30
50 - CTGGAGCC GATTCCAAGCAC-30
5- GAAGGACTTGGGATCGTCCGG-30
50 CGGCGTTCGTCTCGGGCGTGGTGAAGGA-30
50 - TCGAGGGCGTAGTAGATGTCC TTGCCGA-30
50 -GGCATCCAAGCACAAGC-30
50 -AAGCAGACTTGACTGAT-30

Akasaka et al., 2001

Akasaka et al., 2001
Mouneimne et al., 1999
Mouneimne et al., 1999
Mouneimne et al., 1999
Mouneimne et al., 1999
Akasaka et al., 2001
Akasaka et al., 2001
Zhao et al., 2003
Zhao et al., 2003

J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

to sequences in the GenBank database (Accession

number AJ620334) as described above.

3. Results
3.1. Antimicrobial susceptibility phenotypes
A total of 106 clinical isolates of P. aeruginosa were
tested for their susceptibility to 32 antimicrobial agents

167

that are used in human and veterinary medicine. The

results of the susceptibility testing are shown in Table 2.
Among the b-lactam antimicrobials 100% resistance
was seen for ampicillin, cefazolin, cefoxitin, cephalothin, and cefpodoxime, followed by 99% resistance to
amoxicillin/clavulanic acid, and 97% resistance to
ceftiofur. Moderate levels of resistance were seen to the
third generation cephalosporins: ceftriaxone, cefotaxime, cefotaxime/clavulanic acid with 39%, 26%, and
20% resistance, respectively. Cefepime, piperacillin/

Table 2
Antimicrobial resistance phenotypes of Pseudomonas aeruginosa isolated from dogs (n = 106)
Class and/or antimicrobial

MIC range (mg/ml)

Resistant breakpoint (mg/ml)a

Resistant strains (%)

MIC50

MIC90

b-Lactams
Amoxicillin/clavulanic acid
Ampicillin
Cefazolin
Cefepime
Cefotaxime
Cefotaxime/clavulanic acid
Cefoxitin
Cefpodoxime
Ceftazidime
Ceftazidime/clavulanic acid
Ceftiofur
Ceftriaxone
Cephalothin
Imipenem
Meropenem
Piperacillin/tazobactam

1/0.532/16
132
816
116
0.2564
0.12/464/4
0.564
0.2532
0.25128
0.12/4128/4
0.128
0.25128
816
0.516
18
4/464/4

32/16
32
32
32
64
64
32
8
32
32
8
64
32
16
16
128/4

99
100
100
4
26
20
100
100
6
7
97
39
100
1
1
5

>32/16
>32
>16
4
32
>64/4
>64
>32
2
4/4
>8
32
>16
1
1
4/4

>32/16
>32
>16
16
>64
>64/4
>64
>32
8
16/4
>8
>128
>16
4
2
32/4

Quinolones and fluoroquinolones

Ciprofloxacin
Difloxacin
Enrofloxacin
Gatifloxacin
Levofloxacin
Marbofloxacin
Nalidixic Acid
Orbifloxacin

0.01532
0.01532
0.01532
0.01516
0.01532
0.01532
0.532
0.01532

4
4
4
8
8
4
32
8

16
43
31
23
16
27
96
52

0.25
2
1
2
0.25
1
>32
8

8
>32
32
16
8
16
>32
>32

Aminoglycosides
Amikacin
Gentamicin
Kanamycin
Streptomycin b

0.564
0.2516
864
3264

64
16
64
64

3
7
90
69

4
4
>64
32

16
8
>64
>64

51
57
98
100

>256
4/76
32
>32

>256
>4/76
3
>32

Sulfonamides and potentiated sulfonamides

Sulfisoxazole
16256
Trimethoprim/sulfamethoxazole
0.12/2.384/76
Tetracycline
432
Chloramphenicol
232
a

512
4/76
16
32

MIC (mg/ml) determined via microdilution broth methods in accordance with CLSI standards (Clinical and Laboratory Standards Institute,
2004; Clinical and Laboratory Standards Institute, 2006).
b
No CLSI breakpoint.

168

J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

Table 3
Antimicrobial susceptibility profiles of fluoroquinolones and QRDR mutations
CVM #

Antimicrobials (MIC mg/ml)

QRDR genes

DIF

ORB

MAR

ENR

CIP

LEV

GAT

GyrA

GyrB

ParC

ParE

35819
35820
35825
35826
35828

>32
4
8
32
16

>32
16
32
>32
32

>32
1
4
8
4

>32
2
8
16
8

>32
0.5
1
4
2

>32
2
4
8
8

>16
4
8
8
8

Thr83Ile

Asp87Tyr

Thr83Ile

Val471Phe
Ser468Phe

Glu459Lys

35832
35833
35840
35841
35850
35851
35852
35856
35857
35858
35861
35878
35883
35885

16
>32
16
>32
>32
>32
32
16
>32
8
16
2
2
2

32
>32
32
>32
>32
>32
>32
>32
>32
16
>32
4
4
2

8
16
8
>16
>16
8
8
8
16
4
8
1
1
1

Ala53Thr
Asp87Asn

Thr83Ile
Thr83Ile

Thr83Ile
Asp87Asn
Asp87Asn

Ser468Phe

Ser468Tyr
Ser468Phe

Ser468Phe

His509Asp

35887
35888
35889
35895
35896

8
16
4
32
>32

16
32
16
32
>32

4
4
2
4
>32

4
8
2
8
>32

2
2
0.5
2
>32

4
4
2
8
>32

4
8
4
4
>16

Asp490Tyr

35897

>32

>32

>32

>32

>32

>32

>16

Ser87Leu

Ala473Val

35903
35905
35906

>32
>32
>32

>32
>32
>32

32
16
>32

>32
32
>32

16
8
32

32
16
>32

>16
>16
>16

Ser87Leu

Ala473Val

35912

>32

>32

>32

>32

>32

>32

>16

Ser87Leu

Ala473Val

35913

>32

>32

>32

>32

>32

>32

>16

Ser87Leu

Ala473Val

35916
35918
35921
35926

1
2
32
2

2
8
>32
4

Asp87His
Thr83Ile

Thr83Ile
Thr83Ile,
Asp87Asn
Thr83Ile,
Asp87Gly
Thr83Ile
Asp87Asn
Thr83Ile,
Asp87Gly
Thr83Ile,
Asp87Gly
Thr83Ile,
Asp87Gly

Lys64Thr
Thr83Ile

Ser87Leu
Val94Glu

Glu91Asp,
Ala92Ser

Tyr89Ser

Met93Arg,
Val94Asp
Met93Arg

Ser87Leu

Arg442Ser

Ala473Val

4
8
8
16
32
16
8
4
16
4
8
0.5
0.5
0.5

0.5
1
8
0.5

8
32
8
32
>32
32
16
8
>32
8
8
1
1
1

0.5
2
8
1

2
4
2
8
16
8
4
2
16
2
2
0.25
0.25
0.12

0.06
0.25
4
0.12

4
16
8
16
32
16
8
4
16
4
8
1
0.5
0.5

0.25
1
8
0.5

0.5
2
8
0.5

Ala503Val

Ala473Val
Ala473Thr
Ala473Val

Note: DIF, difloxacin; ORB, orbifloxacin; MAR, marbofloxacin; ENR, enrofloxacin; CIP, ciprofloxacin; LEV, levofloxacin; GAT, gatifloxacin;
Ala, alanine; Thr, threonine; Lys, lysine; Ile, isoleucine; Asp, aspartic acid; Asn, asparagine; Ser, serine; Leu, leucine; Gly, glycine; Val, valine;
Glu, glutamic acid; Lys, lysine; Tyr, tyrosine; Phe, phenylalanine; His, histidine; Met, methionine; Arg, arginine.

tazobactam, ceftazidime, and ceftazidime/clavulanic

acid had under 7% resistance. The carbapenems
(imipenem and meropenem) exhibited the greatest
anti-pseudomonas activity with a single isolate being
resistant to imipenem and another to meropenem.

For the quinolones and fluoroquinolones tested the

most resistance was seen with nalidixic acid (96%)
followed by orbifloxacin (52%), difloxacin (43%),
enrofloxacin (31%), marbofloxacin (27%), gatifloxacin (23%), and levofloxacin (21%). Ciprofloxacin

J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

had the greatest activity against Pseudomonas isolates

with 16% of isolates being resistant.
Among the aminoglycosides, most isolates were
resistant to kanamycin (90%) and streptomycin
(69%). Low levels of resistance were seen to
gentamicin (7%) and amikacin (3%). Of the remaining
antimicrobials tested 100% of the isolates were
resistant to chloramphenicol, 98% to tetracycline,
57% to trimethoprim/sulfamethoxazole, and 51% to
sulfisoxazole.
Susceptibility patterns varied greatly. On average
the isolates were resistant to 16 of 32 antimicrobials
with a range of 8 (n = 1) to 28 (n = 2). This included
those antimicrobial agents to which P. aeruginosa is
intrinsically resistant to, e.g. benzylpenicillins, aminobenzylpenicillins, carboxypenicillins, first and
second generation cephalosporins, chloramphenicol,
and tetracycline. Of particular interest was an isolate
resistant to all antimicrobials with the exception of
sulfisoxazole, trimethoprim/sulfamethoxazole and the
carbapenems. Three isolates that were resistant to all
b-lactams with the exception of carbapenems and two
isolates resistant to carbapenems (one resistant to
imipenem, one to meropenem) were also identified.
This study demonstrated that canine isolates of P.
aeruginosa were frequently resistant to the antimicrobial agents most commonly used in veterinary
medicine including the fluoroquinolones.

169

which had MICs above the highest tested concentration

for all fluoroquinolones. These 6 isolates also had gyrA
hot spot mutations. The remaining five isolates had
parC mutations at Tyr 89, Glu 91 and Ala 92, Met 93,
Met 93 and Val 94, and Val 94. Ten isolates had
mutations on the parE gene occurring at Ala 473, Glu
459, and Ala 503. With the exception of one, all isolates
resistant to ciprofloxacin contained QRDR mutations.
There was a high correlation between QRDR mutations
and increased fluoroquinolone MICs (Table 3). High
level fluoroquinolone resistance was seen, particularly
in isolates with both gyrA and parC mutations. There
were, however, a number of isolates with decreased
fluoroquinolone susceptibility without QRDR mutations.
3.3. Class 1 integrons
Class 1 integrons were amplified from two isolates,
and subsequently sequenced. Sequence analysis
revealed that both 1 kb fragments encoded aadA, a
gene conferring resistance to streptomycin and
spectinomycin. Phenotypically, these isolates had
MICs of >64 mg/ml to streptomycin. Additionally,
as class 1 integrons are flanked by sulfa resistance
genes, MICs to sulfamethoxazole of >256 mg/ml
were noted (Hall, 1997).

3.2. QRDR mutations

4. Discussion

Of 102 naladixic acid resistant isolates, 34 had

QRDR mutations in one or more of the genes analyzed
(Table 3.). Of the 22 altered gyrA sequences, 20 isolates
had mutations at the Thr 83 and/or Asp 87 positions.
These isolates were all resistant to difloxacin,
orbifloxacin, marbofloxacin, and enrofloxacin. The
MICs for ciprofloxacin, levofloxacin, and gatifloxacin
for these isolates were above the MIC50. Two isolates
had gyrA mutations outside the hotspots, at Ala 53
and Lys 64. The isolate with the Ala 53 mutation
showed resistance to all fluoroquinolones, the strain
with Lys 64 mutation was only resistant to orbifloxacin
with decreased susceptibility to the other fluoroquinolones. Nine isolates had gyrB mutations; five occurring
at Ser 468 with the remaining four located at Val 471,
Arg 442, Asp 490 and His 509, respectively. Of the 11
isolates with parC mutations, 6 occurred at Ser 87; all of

P. aeruginosa is an opportunistic bacterial pathogen that is well known for its intrinsic and acquired
resistance and ability to cause serious infections in
animals. Consistent with its reputation of being
resistant to many antimicrobial agents the isolates
tested in this study were resistant to between 8 and 28
drugs with a mean of 16. These isolates were all
resistant to multiple compounds, including intrinsic
resistant antimicrobial agents as well as acquired
resistance to newer synthetic antimicrobial agents that
are commonly used in canine therapies. However,
while some highly resistant isolates were identified,
none met the criteria of multidrug-resistant P.
aeruginosa (MDRPA) (Gales et al., 2001; Paramythiotou et al., 2004) which is defined as resistance
to piperacillin, ceftazidime, imipenem, and gentamicin. Highly variable susceptibility profiles were noted

170

J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

among these isolates, for example, some isolates were

resistant to the aminoglycosides (amikacin, gentamicin, kanamycin, and streptomycin) while susceptible
to the fluoroquinolones and vice versa. As would be
expected, the first generation cephalosporins, aminobenzylpenicillins, and potentiated amoxycillin, which
are commonly used in veterinary medicine (Prescott
et al., 2002), had no activity against the isolates tested
in this study.
The results presented in this study are consistent
with those presented elsewhere on the P. aeruginosas
highly variable resistance patterns (Gales et al., 2001).
These studies underlined the importance of performing antimicrobial susceptibility tests on this bacterial
pathogen. However, perhaps due to less selective
pressure, resistances to anti-pseudomonal drugs are
lower in veterinary isolates than in human clinical
isolates (Paramythiotou et al., 2004; Prescott et al.,
2002). For example, in one canine study an MIC90 of
2 mg/ml was reported for ciprofloxacin (Tejedor et al.,
2003), while a study on human MDRPA revealed
30.2% resistance (MIC  4 mg/ml) to the same drug
(Paramythiotou et al., 2004). In the current study, 16%
of isolates showed resistance to ciprofloxacin
(MIC  4 mg/ml), although the MIC90 is 8 mg/ml.
Paramythiotou et al. (2004) also found 21.9%, 23.5%,
and 55.9% resistance to ceftazidime, imipenam and
piperacillin, respectively, compared to 6%, 1%, and
5%, respectively in this study. It has been postulated
that MDRPA is most likely selected for by the use of
antimicrobials with specific anti-pseudomonal activity; therefore, these drugs should be reserved for cases
where other treatment options are not available
(Paramythiotou et al., 2004). In our study, imipenem/meropenem, amikacin, cefepime and piperacillin/tazobactam had the most conserved activity with
1%, 3%, 4%, and 5% resistance, respectively. The blactams tested here with the most anti-pseudomonal
activity, are not labeled for use in veterinary medicine
(CLSI, 2004; Health Canada, 2007, www.hc-sc.ga.ca/
dhp-mps/prodpharma/databasdon/index_e.html.)
highlighting the lack of registered effective veterinary
anti-pseudomonal drugs. Of the fluoroquinolones, the
least isolates were resistant to ciprofloxacin 16%,
while 27% were resistant to marbofloxacin, the most
active of the veterinary labeled fluoroquinolones. It is
important for veterinarians to know that many of the
available anti-pseudomonal drugs are also key drugs

in the treatment of human infections, so that restricted

use of these drugs would help to prolong the
effectiveness of these drugs in treating human
infections.
With detection in only two isolates, the relative lack
of class 1 integrons was surprising. These genetic
elements have been found to be common in
Enterobacteriaceae from dogs and other domestic
species (Goldstein et al., 2001; van Duijkeren et al.,
2005). In one study 82% of Enterobacteriaceae from
horses and dogs contained class 1 integrons, suggesting an important role in the dissemination of
antimicrobial resistance (van Duijkeren et al., 2005)
and another study documented class 1 integrons in the
flora of wild animals (Goldstein et al., 2001). To the
best of our knowledge, class 1 integrons have never
been reported in P. aeruginosa isolated from dogs.
Class 1 integrons are commonly found in human
isolates of P. aeruginosa with a prevalence of 40.8
63.5% (Fonseca et al., 2005; Gu et al., 2007). Both
integrons characterized here contained the aadA gene,
which is commonly identified in P. aeruginosa
isolated from human infections (Gu et al., 2007).
In the present study 102 of 106 isolates were
resistant to naladixic acid, but only 34 of the isolates
had QRDR mutations. Since first step mutations
commonly occur in the gyrA or gyrB genes (Hooper,
1999), we expected a higher portion of mutants in the
naladixic acid resistant sub-population. Additionally,
in one study 100% of Shigella dysenteriae that were
naladixic acid resistant had gyrA mutations (Talukder
et al., 2006). Furthermore, one isolate resistant to
ciprofloxacin contained no QRDR mutations. Despite
this, a strong association appeared to exist between the
gyrA mutation Thr 83-Ile, the parC mutation Ser 87Leu and high levels of resistance to all fluoroquinolones tested. However, due to the lower than expected
prevalence of QRDR mutations, other mechanisms of
resistance are likely involved. Such mechanisms
include: porin deficiencies (decreased drug entry to
cell), increased efflux pumps (removal of drug prior to
target contact) and plasmid mediated (protection of
drug targets) (Martinez et al., 2006; Li, 2005).
The emergence of antimicrobial resistance in
Gram-negative pathogens will continue to pose
therapeutic challenges in both human and veterinary
medicine. Further research defining the relationship
between specific therapies and the development of

J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

resistance to anti-pseudomonal antimicrobials would

be useful. The present study indicated that in dog,
infections caused by resistant to anti-pseudomonal
antimicrobials of P. aeruginosa are occurring at
different levels. The findings stress the need for
continued monitoring of antimicrobial resistance
among animal bacterial pathogens and the value of
laboratory antimicrobial susceptibility testing as the
basis for clinical treatment decisions.

Acknowledgments
We would also like to thank Mr. Donald Bade,
Microbial Research, Fort Collins, CO, and Dr. Dave
Bemis from the University of Tennessee for providing
some of the clinical isolates used in this study.

References
Akasaka, T., Tanaka, M., Yamaguchi, A., Sato, K., 2001. Type II
topoisomerase mutations in flouroquinolone-resistamt clinical
strains of Pseudomonas aeruginosa isolated in 1998 and 1999:
role of target enzyme in mechanism of fluoroquinolone resistance. Antimicrob. Agents Chemother. 45, 22632268.
Cole, L.K., Luu, D.H., Rajala-Schultz, P.J., Meadows, C., Torres,
A.H., 2006. In vitro activity of an ear rinse containing tromethamine, EDTA, and benzyl alcohol on bacterial pathogens from
dogs with otitis. Am. J. Vet. Res. 67, 10401044.
CLSI, 2004. Performance standards for antimicrobial disk
and dilution susceptibility tests for bacteria isolated from
animals, informational supplement. CLSI/NCCLS document
M31-S1. Clinical and Laboratory Standards Institute, Wayne,
PA.
CLSI, 2006. Performance standards for antimicrobial susceptibility
testing, sixteenth informational supplement. CLSI/NCCLS
ducument M100-S16. Clinical and Laboratory Standards Institute, Wayne, PA.
Fonseca, E.L., Vieira, V.V., Cipriano, R., Vicente, A.C.P., 2005.
Class 1 integrons in Pseudomonas aeruginosa isolates from
clinical settings in Amazon region, Brazil. FEMS Immunol.
Med. Microbiol. 44, 303309.
Gales, A.C., Jones, R.N., Turnidge, J., Rennie, R., Ramphal, R.,
2001. Characterization of Pseudomonas aeruginosa isolates:
occurrence rates, antimicrobial susceptibility patterns, and
molecular typing in the global SENTRY antimicrobial surveillance program, 19971999. Clin. Infect. Dis. 32 (Suppl. 2), 146
155.
Gatoria, I.S., Saini, N.S., Rai, T.S., Dwivdei, P.N., 2006. Comparison of three techniques for the diagnosis of urinary tract
infections in dogs with urolithiasis. J. Small Anim. Pract. 47,
727732.

171

Goldstein, C., Lee, M.D., Sanchez, S., Hudson, C., Phillips, B.,
Register, B., Grady, M., Liebert, C., Summers, A.O., White,
D.G., Maurer, J.J., 2001. Incidence of class 1 and 2 integrases in
clinical and commensal bacteria from livestock, companion
animals, and exotics. Antimicrob. Agents Chemother. 45,
723726.
Gu, B., Tong, M., Zhao, W., Liu, G., Ning, M., Pan, S., Zhao, W.,
2007. Prevalence and characterization of class 1 integrons
among Pseudomonas aeruginosa and Acinetobacter baumannii
isolates from patients in Nanjing, China. J. Clin. Microbiol. 45,
241243.
Hall, R.M., 1997. Mobile gene cassettes and integrons: moving
antibiotic resistance genes in Gram-negative bacteria. Ciba
Found. Symp. 207, 192202.
Hall-Stoodley, L., Costerton, J.W., Stoodley, P., 2004. Bacterial
biofilms: from the natural environment to infectious diseases.
Nat. Rev. Microbiol. 2, 95108.
Hariharan, H., Coles, M., Poole, D., Lund, L., Page, R., 2006.
Update on antimicrobial susceptibilities of bacterial isolates
from canine and feline otitis externa. Can. Vet. J. 47, 253255.
Health Canada, 2007. Therapeutic Products Directorate: Drug Product Database (DPD). www.hc-sc.ga.ca/dhp-mps/prodpharma/
databasdon/index_e.html (accessed February 21, 2007).
Hillier, A., Alcorn, J.R., Cole, L.K., Kowalski, J.J., 2006. Pyoderma
caused by Pseudomonas aeruginosa infection in dogs20
cases. Vet. Dermatol. 17, 432439.
Hooper, D.C., 1999. Mode of action of fluoroquinolones. Drugs 58
(Suppl. 2), 610.
Jalal, S., Ciofu, O., Hoiby, N., Gotoh, N., Wretlind, B., 2000.
Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob. Agents Chemother. 44, 710712.
Li, X., 2005. Quinolone resistance in bacteria: emphasis on plasmidmediated mechanisms. Int. J. Antimicrob. Agents 25, 453463.
Li, X., Livermore, D.M., Nikaido, H., 1994. Role of efflux pump(s)
in intrinsic resistance of Pseudomonas aeruginosa: resistance to
tetracycline, chloramphenicol and norfloxacin. Antimicrob.
Agents Chemother. 28, 17321741.
Linder, J.A., Huang, E.S., Steinman, M.A., Gonzalas, R., Stafford,
R.S., 2005. Fluoroquinolone prescribing in the United States:
1995 to 2002. Am. J. Med. 118, 259268.
Martinez, M., McDermott, P., Walker, R., 2006. Pharmacology of
the fluoroquinolones: a perspective for the use in domestic
animals. Vet. J. 172, 1028.
Mouneimne, H., Robert, J., Jarlier, V., Cambau, E., 1999. Type II
topoisomerase mutations in ciprofloxacin-resistant strains of
Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 43,
6266.
Nikaido, H., 1994. Prevention of drug access to bacterial targets:
permeability barriers and active efflux. Science 264, 382
388.
Paramythiotou, E., Lucet, J.C., Timsit, J.F., Vanjak, D., PaugamBurtz, C., Trouillet, J.L., Belloc, S., Kassis, N., Karabinis, A.,
Andremont, A., 2004. Acquisition of multidrug-resistant Pseudomonas aeruginosa in patients in intensive care units: role of
antibiotics with antipseudomonal activity. Clin. Infect. Dis. 38,
670677.

172

J. Rubin et al. / Veterinary Microbiology 131 (2008) 164172

Pirnay, J., De Vos, D., Cochez, C., Bilocq, F., Pirson, J., Struelens,
M., Duinslaeger, L., Cornelis, P., Zizi, M., Vanderkelen, A.,
2003. Molecular epidemiology of Pseudomonas aeruginosa
colonization in a burn unit: persistence of a multidrug-resistant
clone and a silver sulfadiazine-resistant clone. J. Clin. Microbiol. 41, 11921202.
Prescott, J.F., Hanna, W.J.B., Reid-Smith, R., Drost, Kelli., 2002.
Antimicrobial drug use and resistance in dogs. Can. Vet. J. 43,
107116.
Talukder, K.A., Khajanchi, B.K., Islam, M.A., Islam, Z., Dutta,
D.K., Rahman, M., Watanabe, H., Nair, G.B., Sack, D.A., 2006.
Fluoroquinolone resistance linked to both gyrA and parC mutations in the quinolone resistance-determining region of Shigella
dysenteriae Type 1. Curr. Microbiol. 52, 108111.

Tejedor, M.T., Martin, J.L., Navia, M., Freixes, J., Vila, J., 2003.
Mechanisms of fluoroquinolone resistance in Pseudomonas
aeruginosa isolates from canine infections. Vet. Microbiol.
94, 295301.
van Duijkeren, E., Box, A.T.A., Schellen, P., Houwers, D.J., Fluit,
A.C., 2005. Class 1 integrons in Enterobacteriaceae isolated
from clinical infections of horses and dogs in the Netherlands.
Microb. Drug Resist. 11, 383386.
Zhao, S., Qaiyumi, S., Freidman, F., Singh, R., Foley, S.L.,
White, D.G., McDermott, P.F., Donkar, T., Bolin, C., Munro,
S., Baron, E.J., Walker, R.D., 2003. Characterization
of Salmonella enterica serotype Newport isolated from
humans and food animals. J. Clin. Microbiol. 41, 5366
5371.