Vet Dermatol 2012; 23: 376–e69 DOI: 10.1111/j.1365-3164.2012.01038.

Comparative mutant prevention concentration and

mechanism of resistance to veterinary fluoroquinolones
in Staphylococcus pseudintermedius
Elias Gebru Awji, Dereje Damte Tassew, Joong-Su Lee, Seung-Jin Lee, Myung-Jin Choi, Mohammad
Ahsanur Reza, Man-Hee Rhee, Tae-Hwan Kim and Seung-Chun Park
College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, South Korea
Correspondence: Seung Chun Park, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, South Korea.
E-mail: parksch@knu.ac.kr

Background – The problem of antibacterial drug resistance is increasing worldwide, in part due to the therapeu-
tic concentrations currently used based on the minimal inhibitory concentration (MIC) as a measure of potency
are often the very concentrations required to selectively enrich the resistant mutant portion of the population. A
mutant prevention concentration (MPC)-based dosing strategy is suggested to improve the therapeutic outcome
based on the MIC.
Objective – Our aim was to investigate the MPC and mechanism of resistance to various fluoroquinolones using
recent Staphylococcus pseudintermedius isolates from canine pyoderma.
Methods – The broth microdilution method for MIC and a series of agar plates containing different concentra-
tions of fluoroquinolones were inoculated with 1010 colony-forming units of the bacterial culture for MPC were
used. PCR was used to identify mutation in the resistant isolates.
Results – The rank order of potency based on MIC and MPC was ciprofloxacin = enrofloxacin ‡ marbofloxacin >
difloxacin ‡ orbifloxacin. Integrating our data with reported pharmacokinetic data at the recommended dose
ranges revealed that only high doses of ciprofloxacin, enrofloxacin and marbofloxacin could achieve a maximal
plasma concentration (Cmax) greater than the MPC of 90% of isolates (Cmax ⁄ MPC90). The overall rank of potency
against S. pseudintermedius, based on Cmax ⁄ MIC, Cmax ⁄ MPC, the area under concentration–time curve (AUC) ⁄
MIC and AUC ⁄ MPC values, was in decreasing order: enrofloxacin > ciprofloxacin ‡ marbofloxacin ‡ orbifloxa-
cin = difloxacin. Sequencing of the quinolone resistant determining region of gyrA, gyrB, grlA and grlB of resistant
strains showed a base-pair substitution in both gyrA and gyrB that resulted in Ser-84 to Leu and Ser-80 to Arg
amino acid changes, respectively.
Conclusions and clinical importance – High doses of ciprofloxacin, enrofloxacin and marbofloxacin could
minimize the selection of resistant mutants, whereas the possibility of selecting mutants with the conventional
doses of difloxacin and orbifloxacin, and low clinical doses of all fluoroquinolones, seems high.

S. pseudintermedius between dogs and humans have

Introduction
Staphylococcus pseudintermedius is one of the most
commonly isolated organisms from canine pyoderma,
and fluoroquinolones are among the antibacterial agents
reported to be efficacious for the treatment of deep bac-
terial pyoderma in dogs.1 However, recent reports from
various countries have indicated an increasing resistance
of this organism and failures to treat canine pyoderma or
otitis with a number of fluoroquinolones.2 Furthermore,
cross-resistance between fluoroquinolones and other
antibacterial agents as well as zoonotic transmission
of both meticillin-susceptible and meticillin-resistant
Accepted 8 January 2012
Sources of Funding: This study was supported in part by
Technology Development Program for Agriculture and Forestry,
Ministry for Agriculture, Forestry and Fisheries and in part by
Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (no. 2011-0021670).
Conflict of Interest: No conflicts of interest have been declared.
376
been recent concerns.3,4
Although various fluoroquinolones, including difloxacin,
enrofloxacin, marbofloxacin and orbifloxacin, are approved
for use in companion animals, clinically relevant differ-
ences have not been adequately described for these
agents and they are often used interchangeably. The indis-
criminate use of these agents in veterinary medicine is a
potential factor of utmost significance in the development
of drug resistance.5
The problem of antibacterial drug resistance is increas-
ing worldwide, in part because the therapeutic concentra-
tions currently used, which are based on the minimal
inhibitory concentration (MIC) as a measure of potency,
are often the very concentrations required to selectively
enrich the resistant mutant portion of the population.6,7
For example, as seen with Staphylococcus aureus coloniz-
ing the noses of people who have tuberculosis, acquired
resistance and eradication of susceptible bacteria can
occur concurrently, and restricting acquired resistance
may require direct suppression of mutant growth and
viability in addition to elimination of susceptible bacteria.8
ª 2012 The Authors. Veterinary Dermatology
ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 376–e69.

With respect to fluoroquinolone resistance, the ‘mutant
selection window (MSW)’ hypothesis has been developed
to describe how drug exposures below the mutant pre-
vention concentration (MPC) may create the selection of
resistant bacterial strains.7 This hypothesis maintains that
drug-resistant mutant subpopulations present prior to
initiation of antimicrobial treatment are enriched and
amplified during therapy when antimicrobial concentra-
tions fall within a specific range (the MSW). The upper
boundary of the MSW is the MIC of the least drug-
susceptible mutant subpopulation, a value called the
MPC, and the lower boundary of the MSW is the lowest
concentration that blocks the growth of the majority of
drug-susceptible cells, often approximated by the minimal
concentration that inhibits colony formation by 99%
(MIC99).7,9 Hence, several recent studies have empha-
sized the importance of MPC-based dosing strategies to
improve therapeutic outcome and restrict the selection of
resistant mutants.
The aim of this study was to evaluate, for the first time,
the comparative activity, in terms of both MIC and MPC,
of various fluoroquinolones used in small animals against
recent S. pseudintermedius isolates from dogs with pyo-
derma. It was also our objective to determine the genetic
basis of fluoroquinolone resistance in S. pseudintermedi-
us, as well as to provide information on the achievable
ratios of the pharmacokinetic–pharmacodynamic (PK–PD)
indices at the recommended dose ranges for each agent
using reported PK data and PD data obtained in this
study.
Materials and methods
Drugs and bacterial strains
Pure standards of ciprofloxacin, difloxacin, enrofloxacin, marbofloxa-
cin (Sigma Aldrich, St Louis, MO, USA) and orbifloxacin (Samyang
Anipharm, Seoul, Korea) were used. Stock solutions (1 mg ⁄ mL) were
prepared weekly according to the manufacturers’ instructions, and
working solutions were prepared daily by appropriate dilutions. A
total of 52 S. pseudintermedius strains were included in this study.
The isolates were obtained from diagnostic specimens of diseased
dogs that visited the veterinary teaching hospital of Kyungpook
National University and from sample collections obtained from a pro-
vincial veterinary service laboratory. All samples were collected in
2006 (n = 14) and 2008 (n = 38) from adult and juvenile dogs, of both
sexes, showing clinical signs of pyoderma. Inclusion of bacterial
strains was based on one sample per animal. All samples from dogs
with a history of antibiotic medication in the previous 2 weeks were
excluded.
Cultures were grown on 5% sheep blood agar (Difco, Detroit, MI,
USA). Initial identification was based on standard microbiological
procedures, including colony pigment, coagulase reaction, haemo-
lysin, hyaluronidase, and growth characteristics in mannitol–salt agar.
Isolates identified by the above methods were further tested using
the API Staph system (BioMerieux, Marcy l’Etoile, France), and
species differentiation, especially from S. aureus, was made by PCR
tests based on the nuc gene of Staphylococcus intermedius and
S. aureus and a conserved region of 16S rDNA (as a positive control)
using previously reported methods.10 Further species confirmation
was based on a PCR-restriction fragment length polymorphism
method described earlier.11
Determination of MIC and MPC
The MIC of all fluoroquinolones against all clinical isolates and two
quality control strains was determined in triplicate using the Clinical
ª 2012 The Authors. Veterinary Dermatology
ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 376–e69.
Fluoroquinolone resistance in S. pseudintermedius
and Laboratory Standards Institute (CLSI) broth microdilution
method.12 Staphylococcus aureus ATCC 29213 was used as a quality
control strain with each MIC determination. The MIC interpretative
criteria of CLSI, i.e. ‡4 (difloxacin, enrofloxacin and marbofloxacin)
and ‡8 lg ⁄ mL (orbifloxacin) as resistance breakpoint were used.
The human drug ciprofloxacin has also been widely used in
small animals owing to its activity against various bacteria and the
availability of several less expensive generic products;2,5 therefore,
we included ciprofloxacin in our investigation. A CLSI-approved
breakpoint for ciprofloxacin does not exist for canine bacteria.
However, taking into account the lower oral bioavailability of cipro-
floxacin in dogs compared with humans, and based on reported
pharmacokinetic data in dogs, a resistant breakpoint of ‡4 lg ⁄ mL
in dogs has been suggested.5 We adopted this breakpoint in the
present study.
The MPC of all fluoroquinolones was determined as described
elsewhere.13 Briefly, the tested micro-organisms were cultured in
Mueller–Hinton broth (MHB) and incubated for 24 h. Then the sus-
pension was centrifuged (at 4000g for 10 min) and resuspended in
MHB to yield a concentration of 1010 colony-forming units (CFU) ⁄ mL.
The inocula were further confirmed by serial dilution and plating of
100 lL samples on drug-free medium. A series of agar plates con-
taining known fluoroquinolone concentrations were then inoculated
with 1010 CFU of the bacterial culture. In addition to twofold
increases of MICs (2·, 4·, 8·, 16· etc.), we tested some intermedi-
ate concentrations (narrow concentration increments) to obtain more
precise values of MPCs. The inoculated plates were incubated for
48–72 h at 37C and screened visually for growth. To estimate the
MPC, logarithms of bacterial numbers were plotted against fluoroqui-
nolone concentrations. The MPC was taken as the point where the
plot intersected the x-axis, i.e. the lowest fluoroquinolone con-
centration that completely inhibited growth. All experiments were
performed in triplicate.
Pharmacokinetic–pharmacodynamic integration
To obtain further insight into the clinical utility of our in vitro data, we
integrated the PD data obtained in this study with published PK data
at the recommended clinical doses of the five fluoroquinolones in
dogs, and computed various PK–PD indices, including the maximal
serum concentration (Cmax) ⁄ MIC (or Cmax ⁄ MPC), area under the
concentration–time curve (AUC) ⁄ MIC (or AUC ⁄ MPC), and the per-
centage of a dosage interval in which serum level exceeds the MIC
(T > MIC) or MPC (T > MPC).
Mechanism of resistance
To amplify the quinolone resistant determining region (QRDR) of
gyrA and grlA, we used DNA samples of independent isolates and
previously reported forward and reverse primers for each.14 The PCR
conditions consisted of 35 cycles of denaturation at 94C for 45 s
(90 s for the first cycle), annealing at 50C for 30 s, and elongation at
72C for 90 s (300 s for the last cycle).
To amplify the QRDRs of gyrB and grlB, forward and reverse
primers (Macrogen, Seoul, Korea), synthesized based on reported
sequence data for S. pseudintermedius, were used.15 The primers
used were as follows: gyrB, 5¢-AAGTTCGGCGGCGGTGGATAC-3¢
(forward) and 5¢-AAAGCCGTCTTCGTGCGTCC-3¢ (reverse); and grlB,
5¢-TCGCTCACGGTGGCGTACCT-3¢ (forward) and 5¢-TCTTGCTTCTG-
GCGTGCCAAGT-3¢ (reverse).
The PCR conditions consisted of 35 cycles of denaturation at 94C
for 60 s, annealing at 56C for 30 s, and elongation at 72C for 45 s
(300 s for the last cycle). After confirming the amplicon sizes by aga-
rose gel electrophoresis and using size markers, duplicates of all PCR
products were purified and sequenced in both directions using the
same sets of forward and reverse primers by a sequencing service
(Macrogen). The sequences were finally aligned with previously
reported sequences and deposited in the Genbank database.
Furthermore, to determine reserpine-sensitive efflux, all resistant
isolates were screened for any change in fluoroquinolone MICs in
the presence of 25 lg ⁄ mL reserpine (Sigma Chemical Co., St Louis,
MO, USA), an efflux pump inhibitor.
377
Awji et al.

Statistical analysis recommended dose ranges of all fluoroquinolones,

Statistical analysis was performed using GraphPad Instat (GraphPad
Software Inc., San Diego, CA, USA). Potency statistics, including
geometric means, ranges, and the 50th and 90th percentiles of MIC
and MPC, were generated. One-way analysis of variance was used
to compare the log-transformed values of Cmax ⁄ MIC, Cmax ⁄ MPC,
AUC ⁄ MIC and AUC ⁄ MPC among the five fluoroquinolones. Bonfer-
roni’s test was used to determine differences among geometric
means, and a value of P < 0.05 was considered significant.
Results
The in vitro activities, in terms of MIC, MPC and
MPC ⁄ MIC ratios, and the proportion of resistant isolates
are presented in Table 1. The rank order of potency based
on MIC was ciprofloxacin = enrofloxacin = marbofloxa-
cin > difloxacin > orbifloxacin, and based on MPC was
ciprofloxacin = enrofloxacin > marbofloxacin > difloxacin =
orbifloxacin.
Pharmacokinetic parameters at clinically recommended
doses in dogs of the five fluoroquinolones, obtained from
package inserts or published sources,16 and calculated
PK–PD parameters are listed in Table 2. The mean PK–PD
indices of activity against S. pseudintermedius are shown
in Figure 1. The overall rank of potency of the five fluor-
oquinolones against S. pseudintermedius, in decreasing
order, based on the AUC ⁄ MIC value, was marbofloxacin
> ciprofloxacin = enrofloxacin > orbifloxacin = difloxacin,
and based on AUC ⁄ MPC value was marbofloxacin > cipro-
floxacin > enrofloxacin > orbifloxacin = difloxacin (Table 2
and Figure 1).
As the therapeutic usefulness of MPC is dependent on
having its value below the attainable serum and tissue
drug concentrations after administration of drug doses
that are safe for patients,6 we compared our MPC data
with the Cmax values of each agent. The MPC90 values of
all fluoroquinolones were higher than the respective Cmax
concentrations achievable at the lowest doses within the
clinically recommended dose ranges (5–20 mg ⁄ kg for
ciprofloxacin and enrofloxacin, 5–10 mg ⁄ kg for difloxacin,
2–5 mg ⁄ kg for marbofloxacin and 2.5–7.5 mg ⁄ kg for
orbifloxacin). However, it was possible to attain Cmax
values higher than the MPC90 of S. pseudintermedius
isolates with the highest doses within the clinically
except difloxacin.
Out of 52 S. pseudintermedius strains tested, three
strains were resistant to all fluoroquinolones, with MIC
values in the range of 4–8 lg ⁄ mL for ciprofloxacin,
8–16 lg ⁄ mL for enrofloxacin, 4–32 lg ⁄ mL for marboflox-
acin and 8–64 lg ⁄ mL for both difloxacin and orbifloxacin.
An additional strain was resistant to difloxacin and orbi-
floxacin (MIC 8 lg ⁄ mL for both), while maintaining inter-
mediate susceptibility to ciprofloxacin, enrofloxacin and
marbofloxacin (MIC 2 lg ⁄ mL for all agents). Amplification
and sequencing of the QRDR of gyrA and grlA of the strains
resistant to all fluoroquinolones showed single base-pair
exchanges in both genes that resulted in Ser-84 to Leu and
Ser-80 to Arg amino acid exchanges, respectively. The
S. pseudintermedius strain resistant to only difloxacin and
orbifloxacin had a single base-pair change in grlA that
resulted in a Ser-80 to Arg amino acid substitution, while
containing no alterations in the gyrA. None of the resistant
strains had mutations in the QRDR of gyrB and grlB.
We also sequenced the QRDR of gyrA and grlA for five
representative mutants selected from MPC plates with
the highest fluoroquinolone concentrations, along with
the parent strains. Three mutants with intermediate
susceptibility to orbifloxacin (MIC 4–6 lg ⁄ mL) and other
fluoroquinolones (MIC 2–3 lg ⁄ mL) had a single base-pair
change in grlA that resulted in a Ser-80 to Arg amino acid
substitution, while the other two mutants with fully sus-
ceptible phenotype for all fluoroquinolones contained no
mutations in all tested genes. None of the mutant strains
had mutations in the QRDR of gyrB and grlB. Sequences
determined in this study have been deposited in the
GenBank database under accession numbers HQ690824–
HQ690830.
All resistant isolates and representative mutants
selected from MPC plates were further tested for any
change in fluoroquinolone MICs in the presence of
25 lg ⁄ mL reserpine. A S. pseudintermedius strain resis-
tant to only difloxacin and orbifloxacin (MIC 8 lg ⁄ mL for
both) had shown a fourfold reduction in the MICs of both
agents when tested in the presence of reserpine. Reser-
pine-sensitive efflux was not observed in all other strains
(MIC changes twofold or less).

Table 1. Comparative activity of five fluoroquinolones against clinical isolates of Staphylococcus pseudintermedius from dogs
Potency Isolates Ciprofloxacin Difloxacin Enrofloxacin Marbofloxacin Orbifloxacin
MIC (lg ⁄ mL) n* 52 52 52 52 52
Range 0.03–8 0.13–64 0.03–16 0.06–32 0.25–64
MIC50 0.13 0.5 0.13 0.25 0.5
MIC90 0.5 1 0.5 0.5 2
R† (%) 3 (5.77) 4 (7.69) 3 (5.77) 3 (5.77) 4 (7.69)
MPC (lg ⁄ mL) Range 0.2–3.5 0.5–9.5 0.1–3.0 0.1–4.2 0.4–16.5
MPC50 0.25 1.71 0.27 0.5 1.65
MPC90 2.7 8.5 2.7 3.3 13.6
MPC ⁄ MIC Range 1.15–7 1.78–18.5 1.05–6.0 1.20–15.1 1.56–12.7
(MPC ⁄ MIC)50 2.0 3.4 2.2 2.0 3.3
(MPC ⁄ MIC)90 5.33 8.5 5.3 6.6 6.8
Abbreviations: MIC, minimal inhibitory concentration; MPC, mutant prevention concentration; and MIC50, MIC90, MPC50 and MPC90, the 50th and
90th percentiles of MIC and MPC, respectively.
*n, number of tested strains.
†Resistant strains (R) were not included in the MPC determination.

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378 ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 376–e69.
 Fluoroquinolone resistance in S. pseudintermedius

Table 2. Pharmacokinetic–pharmacodynamic relationship of five fluoroquinolones towards S. pseudintermedius*†

Low dose High dose

Cip Dif Enr Mar Orb Cip Dif Enr Mar Orb
Dose (mg ⁄ kg) 5 5 5 2 2.5 20 10 20 5 7.5
AUC (lg h ⁄ mL) 12.7 9.3 8.7 13.1 12.7 28.4 29.0 57.0 57.3 42.9
Cmax (lg ⁄ mL) 0.8 1.1 1.4 1.5 1.4 3.1 3.6 5.2 3.7 6.9
Tmax (h) 2.3 2.8 1.8 1.8 2.4 3.0 2.8 3.6 1.8 0.8
T½ (h) 3.8 6.9 4.1 9.1 7.1 5.3 9.3 5.2 10.9 5.6
AUC ⁄ MIC50 101.6 18.7 69.9 52.3 25.4 227.2 58.0 456.0 229.2 85.8
AUC ⁄ MIC90 25.4 9.3 17.5 26.1 6.4 56.8 29.0 114.0 114.6 21.5
AUC ⁄ MPC50 50.8 5.5 32.4 26.1 7.7 113.6 17.0 211.1 114.6 26.0
AUC ⁄ MPC90 4.8 1.1 3.3 4.0 0.9 10.7 3.4 21.5 17.4 3.2
Cmax ⁄ MIC50 6.0 2.2 11.3 5.9 2.7 24.6 7.2 41.6 14.9 13.8
Cmax ⁄ MIC90 1.5 1.1 2.8 2.9 2.7 6.2 3.6 10.4 7.5 3.5
Cmax ⁄ MPC50 3.0 0.6 5.2 2.9 0.8 12.3 2.1 19.3 7.5 26.0
Cmax ⁄ MPC90 0.3 0.1 0.5 0.4 0.1 1.2 0.4 2.0 1.1 0.5
T > MIC50 (h) 10 8 14 23 10 >24 >24 >24 >24 21
T > MIC90 (h) 2 1 6 14 0 14 17 18 >24 10
T > MPC50 (h) 6 0 10 14 0 19 10 22 >24 12
T > MPC90 (h) 0 0 0 0 0 1 4 5 2 0
Abbreviations: Cip, ciprofloxacin; Dif, difloxacin; Enr, enrofloxacin; Mar, marbofloxacin; and Orb, orbifloxacin.
*Area under concentration–time curve (AUC), maximal observed plasma concentration (Cmax), Tmax (time for maximal observed serum concentra-
tion) and T½ (elimination half-life) values were based on prescription information and published sources given in the text.
†Time above minimal inhibitory concentration or mutant prevention concentration (T > MIC or T > MPC) computations were based on a 24 h dos-
ing interval for all fluoroquinolones.

Figure 1. Pharmacokinetic–pharmacodynamic indices towards Sta-
phylococcus pseudintermedius at low (left panels) and high clinical
doses (right panels) in dogs for ciprofloxacin (Cip), difloxacin (Dif),
enrofloxacin (Enr), marbofloxacin (Mar) and orbifloxacin (Orb) based
on mean minimal inhibitory concentration (MIC) and mutant preven-
tion concentration (MPC) values from the present study and reported
pharmacokinetic data (listed in Table 2). Abbreviations: AUC, area
under the concentration–time curve; and Cmax, maximal plasma con-
centration. A significant difference is present when the superscript
letters differ between two numerical values for the same variable
(P < 0.05). Values with the same superscript letter do not differ sig-
nificantly from one another.
Discussion
The MPC-based antimicrobial dosing is currently attract-
ing attention as a way to minimize the emergence of
resistance. Studies using various in vitro and in vivo
models of human infection, including streptococcal
pneumonia and staphylococcal tissue-cage models, have
demonstrated the importance of the MPC-based PK–PD
approach for restricting the enrichment and amplification
of resistant subpopulations of bacteria.6,8,17
The MPC ⁄ MIC ratio defines the concentration range in
which resistant mutant subpopulations are selectively
amplified, with lower values of the MPC ⁄ MIC ratio indi-
cating a better ability to prevent the emergence of
mutants.6,7 In the present study, difloxacin had the
highest MPC ⁄ MIC ratio. Ciprofloxacin and enrofloxacin
had a comparable MPC ⁄ MIC value which was the lowest
compared with the others, suggesting their better ability
to restrict the selection of resistant mutants of S. pseud-
intermedius.
For the fluoroquinolone antimicrobial agents, either the
AUC ⁄ MIC or Cmax ⁄ MIC ratio may predict antibacterial
success, with distinct PK–PD end-points required for
different levels of antibacterial activity according to the
host or pathogen. It is generally accepted that an AUC ⁄
MIC ratio of ‡100–125 or a Cmax ⁄ MIC ratio of ‡8–10 is the
surrogate used as an activity indicator against Gram-nega-
tive bacteria; however, lower values of these indices have
been associated with prompt eradication of Gram-positive
bacteria.18 For instance, a previous PK–PD integration
study using orbifloxacin and S. pseudintermedius isolates
from dogs indicated an AUC ⁄ MIC ratio as low as 40 to be
effective in eliminating bacteria.19
When it comes to the restriction of resistant mutants,
the AUC ⁄ MPC value is expected to be more accurate than
the AUC ⁄ MIC value, because MIC determinations gener-

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ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 376–e69. 379
Awji et al.
ally ignore mutant subpopulations.6 Although no
AUC ⁄ MPC threshold is currently available for the fluoroqu-
inolone–S. pseudintermedius combination, mutant-
restrictive values for other species of bacteria have been
reported. For example, the AUC ⁄ MPC for fluoroquinol-
ones of 60–70 h with S. aureus and about one-third of that
value with S. pneumoniae and E. coli were considered to
restrict selection of mutants.7 Therefore, it is logical to
assume that it could be possible to restrict the selection of
resistant S. pseudintermedius mutants with high doses of
ciprofloxacin, enrofloxacin and marbofloxacin, which had
AUC ⁄ MPC50 values of >100, while difloxacin and orbiflox-
acin are expected to enrich mutants easily. In support of
this notion, the possibility of maintaining drug concentra-
tions above the MPC values (T > MPC50) for an extended
duration (85–100% of the dosing interval of 24 h) with high
doses of ciprofloxacin, enrofloxacin and marbofloxacin
(Table 2) may result in a large reduction in viable cells and
less selection of resistant mutants.
Our study confirmed the importance of two muta-
tions, one in gyrA-84 and one in grlA-80, for the devel-
opment of clinical resistance in S. pseudintermedius.
The gyrA and grlA mutations described in this study are
similar to those reported in S. intermedius and Staphy-
lococcus schleiferi.14 The MPC mutants in the present
study contained only a single grlA-80 mutation. This
may suggest that the grlA subunit of topoisomerase IV
is the primary target of fluoroquinolone resistance in
S. pseudintermedius.
In addition, we observed resistance to only two fluor-
oquinolones in one strain, whereas in other strains there
was resistance to all fluoroquinolones tested. A similar
pattern of dichotomous resistance has been reported
with other fluoroquinolones.14 Structural differences may
partly account for this difference. The presence of reser-
pine-sensitive efflux in the isolate resistant to only difloxa-
cin and orbifloxacin, but not in other fluoroquinolones,
may also have contributed to the observed differences.
However, in light of the small number of isolates used in
the present study, these assumptions need to be vali-
dated in further studies using a large number of strains.
In conclusion, this is the first report on MPC of vari-
ous fluoroquinolones against S. pseudintermedius iso-
lates from dogs. Based on these results, the highest
doses within the clinically recommended dose ranges
of ciprofloxacin, enrofloxacin and marbofloxacin could
minimize the selection of resistant mutants in vitro,
whereas the possibility of selecting mutants with the
currently used clinical doses of difloxacin and orbifloxa-
cin, and the lowest doses within the clinically recom-
mended dose ranges of all fluoroquinolones, seems
high. Target mutations in gyrA-84 and grlA-80 contribute
to clinical resistance to fluoroquinolones in S. pseud-
intermedius. Although we attempted to integrate our
in vitro data with published PK data, the in vitro
approach employed here may have some potential
limitations in addressing the problem of fluoroquinolone
resistance with respect to other issues, such as the con-
centration of drugs at sites of infection and the immune
⁄ health status of the host. Therefore, additional in vivo
studies would be required to establish the importance of
our findings in the clinical setting.
380
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ª 2012 The Authors. Veterinary Dermatology
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 Fluoroquinolone resistance in S. pseudintermedius

Résumé
Contexte – Une méthode de dosage basée sur la MPC (Mutant prevention concentration) est étudiée afin
d’améliorer les résultats thérapeutiques basés sur la concentration minimale inhibitrice (MIC).
Objectif – Notre but était d’étudier la MPC et le mécanisme de résistance de différentes fluoroquinolones
sur de récentes souches de S. pseudintermedius prélevées à partir de pyodermites canines.
Méthodes – Nous avons utilisé une méthode de microdilution pour la MIC et une série de plaques d’agar
contenant des concentrations de fluoroquinolones inoculées avec 1010 CFU de culture bactérienne pour
MPC. Les mutations dans les isolats résistants ont été déterminées par PCR.
Résultat – Le classement par ordre de puissance basé sur la MIC et la MPC était ciprofloxacine = enroflox-
acine ‡ marbofloxacine > difloxacine ‡ orbifloxacine. L’intégration à nos données des valeurs de PK aux
doses recommandées a révélé que seules les doses élevées de ciprofloxacine, enrofloxacine et marboflox-
acine pourraient atteindre une concentration plasmatique maximale (Cmax) supérieure à la MPC de 90%
des souches (Cmax ⁄ MPC90). Le grade général de puissance contre S. pseudintermedius, basé sur Cmax ⁄
MIC, Cmax ⁄ MPC, l’aire sous la courbe concentration-temps (AUC) ⁄ MIC et les valeurs de AUC ⁄ MPC, était
par ordre décroissant: enrofloxacine > ciprofloxacine ‡ marbofloxacine ‡ orbifloxacine = difloxacine. Le
séquençage du QRDR de gyrA, gyrB, grlA et grlB des souches résistantes a révélé une substitution d’une
paire de base à la fois pour gyrA et gyrB qui a mené au remplacement respectivement de Ser-84 en Leu et
Ser-80 en Arg.
Conclusions et importance clinique – De fortes doses de ciprofloxacine, enrofloxacine et marbofloxacine
pourraient minimiser la sélection de mutants résistants. En revanche, la possibilité de sélectionner des
mutants avec des doses conventionnelles de difloxacine et d’orbifloxacine et de faibles doses cliniques de
toute fluoroquinolone semble élevée.

Resumen
Introducccion – se investiga una estrategia de dosificación de concentración de prevención de mutantes
(MPC) – para mejorar el resultado terapéutico basado en la concentración inhibitoria mı́nima (MIC).
Objetivo – nuestro objetivo fue investigar MPC y el mecanismo de resistencia a varias fluoroquinolonas
usando aislados recientes de S.pseudintermedius en casos de pioderma canina.
Métodos – se utilizaron el método de micro-dilución de la suspensión para la MIC y una serie de placas de
agar que contenı́an concentraciones de fluoroquinolonas inoculadas con 1010 CFU del cultivo bacteriano
para MPC. Se utilizo la técnica de PCR para identificar la mutación en los aislados resistentes.
Resultado – el orden de eficacia basado en MIC y MPC fue ciprofloxacina = enrofloxacina ‡ marbofloxa-
cina > difloxacina ‡ orbifloxacina. Al integrar nuestros datos con datos conocidos de la farmacocinetica a
las dosis recomendadas indicoo que solamente las dosis elevadas de ciprofloxacina, de enrofloxacina y de
marbofloxacina podrı́an alcanzar una concentración máxima en el plasma (Cmax) mayor que el MPC del
90% de los aislados (Cmax ⁄ MPC90). La orden final de potencia frente a S. pseudintermedius, basado en los
valores de Cmax ⁄ MIC, Cmax ⁄ MPC, el área bajo la curva de concentración-tiempo (AUC) ⁄ MIC, y AUC ⁄ MPC,
estaba en orden decreciente enrofloxacina > ciprofloxacina ‡ marbofloxacina ‡ orbifloxacina = difloxacina.
La secuencia del QRDR del gyrA, gyrB, grlA y grlB de cepas resistentes demostró una sustitución de un
par de bases en gyrA y gyrB que resultaron en el cambio de Ser-84 a Leu y Ser-80 a Arg, respectivamente.
Conclusiones e importancia clı́nica – Altas dosis de ciprofloxacina, de enrofloxacina y de marbofloxacina
podrı́an reducir al mı́nimo la selección de mutantes resistentes, mientras que la posibilidad de seleccionar
mutantes con dosis convencionales de difloxacina y de orbifloxacina, y dosis clı́nicas bajas de todas los
fluoroquinolonas parece alta.

Zusammenfassung
Hintergrund – Eine auf einer mutanten Vorbeugekonzentration (MPC-) – basierende Dosierungsstrategie
wurde untersucht, um die therapeutischen Ergebnisse basierend auf der minimalen inhibitorischen Kon-
zentration (MIC) zu verbessern.
Ziel – Unser Ziel war es, die MPC und die Mechanismen der Resistenz jüngster S. pseudintermedius Iso-
late von Fällen mit caniner Pyodermie gegenüber verschiedenen Fluoroquinolonen zu untersuchen.
Methoden – Für die MIC wurde eine Bouillon-Mikroverdünnungs-Methode und für die MPC wurden eine
Reihe von Agarplatten, welche Fluoroquinolonkonzentrationen, die mit 1010 CFU einer bakteriellen Kultur
beimpft waren, verwendet.
Ergebnis – Die auf MIC oder MPC basierende Reihung nach Wirksamkeit war Ciprofloxacin = Enrofloxacin
‡ Marbofloxacin > Difloxacin ‡ Orbifloxacin. Unsere Daten in Verbindung mit bereits veröffentlichten PK
Daten bei den empfohlenen Dosierungen zeigten, dass nur hohe Dosen von Ciprofloxacin, Enrofloxacin
und Marbofloxacin maximale Plasmakonzentrationen (Cmax) erreichen konnten, die höher waren als die
MPCs von 90% der Isolate (Cmax ⁄ MPC90). Die allgemeine Reihenfolge der Wirksamkeit gegen S. pseudin-
termedius basierend auf Cmax ⁄ MIC, Cmax ⁄ MPC, die Fläche unter der Konzentrations-Zeitkurve (AUC) ⁄
MIC, und AUC ⁄ MPC Werte, war umgekehrt: Enrofloxacin > Ciprofloxacin ‡ Marbofloxacin ‡ Orbifloxacin =
Difloxacin. Die Sequenzierung des QRDR des gyrA, gyrB, grlA und grlB von resistenten Stämmen zeigte
die Substitution eines Basenpaares sowohl in gyrA als auch in gyrB, was zu Aminosäureveränderungen
führte (bei Ser-84 zu Leu beziehungsweise bei Ser-80 zu Arg).

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Awji et al.

Zusammenfassungen und klinische Bedeutung – Hohe Dosen an Ciprofloxacin, Enrofloxacin und Mar-
bofloxacin könnten die Selektion von resistenten Mutanten minimieren, während die Wahrscheinlichkeit
einer Selektion von Mutanten mit konventionellen Dosen von Difloxacin und Orbifloxacin und niedrigen kli-
nischen Dosen von Fluoroquinolonen hoch zu sein scheint.

ª 2012 The Authors. Veterinary Dermatology

e69 ª 2012 ESVD and ACVD, Veterinary Dermatology, 23, 376–e69.