Journal of Antimicrobial Chemotherapy (2007) 60, 999– 1003
doi:10.1093/jac/dkm346
Advance Access publication 14 September 2007
Comparative activity of pradofloxacin against anaerobic bacteria
isolated from dogs and cats
Peter Silley1,2*, Bernd Stephan3, Heinrich A. Greife3 and Andrew Pridmore4
1
MB Consult Limited, Lymington, SO41 3TQ, UK; 2Department of Biomedical Sciences, University of Bradford,
Bradford, UK; 3Bayer HealthCare AG, Leverkusen, D-51368, Germany; 4Don Whitley Scientific Ltd, Shipley,
West Yorkshire, BD17 7SE, UK
Received 10 July 2007; returned 25 July 2007; revised 9 August 2007; accepted 10 August 2007
Objectives: To compare the intrinsic activity of pradofloxacin, a new fluoroquinolone developed for
use in veterinary medicine, with other fluoroquinolones, against anaerobic bacteria isolated from dogs
and cats.
Methods: One hundred and forty-one anaerobes were isolated from dogs and cats and comparative
MICs of pradofloxacin, marbofloxacin, enrofloxacin, difloxacin and ibafloxacin were determined according to standardized agar dilution methodology.
Results: Pradofloxacin exerted the greatest antibacterial activity followed by marbofloxacin, enrofloxacin, difloxacin and ibafloxacin. Based on the distinctly lower MIC50, MIC90 and mode MIC values, pradofloxacin exhibited a higher in vitro activity than any of the comparator fluoroquinolones.
Conclusions: Pradofloxacin, a novel third-generation fluoroquinolone, has broad-spectrum antianaerobe activity and offers utility as single-drug therapy for mixed aerobic/anaerobic infections.
Keywords: fluoroquinolones, anaerobes, companion animals
Introduction
Quinolones were first described in the early 1960s,1 with limited
in vitro activity against Gram-negative species. Modifications to
nalidixic acid in the 1970s gave rise to compounds that were
generally used for oral treatment of urinary tract infections.
Improvements in activity against Gram-negative and -positive
pathogens followed the introduction of piperazine substitution at
position 7 of the naphthyridine core and fluorination at position
6 of the molecule, this group being commonly referred to as the
fluoroquinolones. Norfloxacin was the first member of this class.
Substitution of a carbon atom for nitrogen resulted in ciprofloxacin, a 1-cyclopropanyl, and ofloxacin and levofloxacin, both
1,8-cyclo compounds; all three of these latter mentioned compounds have been classified as second-generation fluoroquinolones.2 The next development included compounds such as
moxifloxacin, resulting from a 7-azabicyclo modification that
enhanced antibacterial activity and pharmacokinetic properties,
consequently referred to as a third-generation fluoroquinolone.2
While the spectrum of activity of the first-generation fluoroquinolones was essentially against Enterobacteriaceae, the secondgeneration fluoroquinolones have a wider spectrum including
activity against many Gram-negative species (bacilli and cocci),
some Gram-positive species, intracellular organisms (Rickettsia
spp. and Mycobacterium spp.) and Mycoplasma spp.3 – 6 Thirdgeneration fluoroquinolones such as moxifloxacin have enhanced
activity against Gram-positive bacteria relative to first- and
second-generation compounds and good activity against anaerobes.7 Fluoroquinolones substituted at position C-8 by a
methoxy group such as moxifloxacin have been shown to have
greatly improved bactericidal activity.8,9 Pradofloxacin is being
exclusively developed for use in veterinary medicine. It is a
third-generation fluoroquinolone structurally similar to moxifloxacin2,7 and can therefore be expected to show enhanced activity
against Gram-positive organisms and anaerobes; this will differentiate pradofloxacin from earlier generation fluoroquinolone
compounds used in veterinary medicine. It is structurally distinguished from enrofloxacin, the first veterinary fluoroquinolone,10
by two elements: a bicyclic amine, S,S-pyrrolidino-piperidine,
replacing the ethyl-piperazine moiety located at position C-7 of
enrofloxacin, and a cyano group that is attached to the C atom
at position 8 (Figure 1). The increased potency of pradofloxacin
is mainly attributed to the S,S-pyrrolidino-piperidine moiety at
C-7, but the cyano group at C-8 extends activity to first- and
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*Corresponding author. Tel: þ44-1590-678700; Fax: þ44-1590-678751; E-mail: [email protected]
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999
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Silley et al.
Prevotella [P. buccae (1), P. corporis (2), P. dentalis (2),
P. denticola (3), P. disiens (1), P. heparinolytica (1), P. oralis
(7), P. oris (1), P. zoogleoformans (2)]; Propionibacterium [P. acnes
(3), P. granulosum (1) P. propionicum (1)]; Rhodococcus
fascians (1); Ruminococcus torques (2); Sebaldella termitidis (1);
Selenomonas sputigena (2); Sporomusa [S. acidovorans (3),
S. sphaeroides (3)]. There were a number of isolates for which species
names could not be defined: Clostridum spp. (7), Bacteroides spp.
(1), Bifidobacterium spp. (1), Fusobacterium spp. (2), Megasphaera
Figure 1. Chemical structures of the C-8 substituted third-generation
fluoroquinolones, pradofloxacin and moxifloxacin.
second-step fluoroquinolone-resistant strains. Pradofloxacin has
been shown to be highly active in vitro against aerobic clinical
isolates from dogs and cats. Typical MIC90 values for pradofloxacin against organisms such as Pasteurella multocida,
Escherichia coli, Staphylococcus intermedius and Streptococcus
spp. have been shown to be in the range 0.016 – 0.25 mg/L.11
Low mutant prevention concentrations (MPCs) have been
reported for pradofloxacin against E. coli and Staphylococcus
spp.12 Low MPC has recently been reported also for one strain
of the anaerobic bacterium Porphyromonas gingivalis.13
Therefore, pradofloxacin should possess an exceptional potential
in eliminating not only large populations of wild-type but also
selected first-step resistant clones.12
In order to evaluate its potential for use against anaerobes
from cats and dogs, we studied the comparative activity of pradofloxacin relative to other fluoroquinolones used in companion
animals against 141 anaerobic strains isolated in the period
2000 –2002.
Table 1. MIC data for pradofloxacin and other veterinary
fluoroquinolones against anaerobic bacteria from dogs and cats; only
genera where n 5 are included
MIC parameters (mg/L)
Bacterial genus (n)
Clostridium (32)
Bacteroides (28)
Fusobacterium (22)
Prevotella (20)
Materials and methods
Bacterial strains
Anaerobic bacteria were isolated from oral infections, abscesses and
wound infections and also from faecal flora of dogs and cats. A
total of 141 strains were isolated from dogs (94) and cats (47) in the
period 2000–2002, all of which were from the UK and obtained
from animals that had not received antimicrobial agents for at least
3 months prior to sampling. Individual species were isolated from
separate animals and thus all bacterial species can be considered as
unrelated. Identification was to species level using the BiologTM
system (Hayward, CA, USA) and isolates were stored at 2808C
prior to testing. The strains identified to species level were:
Actinomyces [A. bovis (n ¼ 2), A. israelii (1)]; Bacteroides
[B. capillosus (3), B. cellulosolvens (1), B. eggerthii (1), B. forsythus
(2), B. fragilis (3), B. helcogenes (4), B. putredinis (1), B. stercoris
(1), B. suis (3), B. uniformis (1), B. ureolyticus (1), B. vulgatus (6)];
Clostridium [C. absonum (4), C. carnis (2), C. cellobioparum
(3), C. glycolicum (1), C. hydroxybenzoicum (3), C. irregularis (1),
C. oroticum (1), C. perfringens (4), C. ramosum (2), C. rectum (1),
C. sporosphaeroides (1), C. subterminale (1), C. tetanomorphum (1)];
Desulfomonile tiedjei (1); Eubacterium [E. biforme (1), E. cylindroides (1), E. plautii (1)]; Fusobacterium [F. naviforme (1),
F. necrogenes (5), F. necrophorum (1), F. nucleatum (9), F. russii (1),
F. ulcerans (1), F. varium (2)]; Hallella seregrens (1); Macrococcus
bovicus (1), Megamonas hypermegale (2); Peptostreptococcus
anaerobius (3); Porphyromonas [P. gingivalis (5), P. macacae (1)],
Porphyromonas (6)
Sporomusa (6)
Propionibacterium (5)
All strains (141)
Antimicrobial
agent
PRA
MAR
ENR
DIF
IBA
PRA
MAR
ENR
DIF
IBA
PRA
MAR
ENR
DIF
IBA
PRA
MAR
ENR
DIF
IBA
PRA
MAR
ENR
DIF
IBA
PRA
MAR
ENR
DIF
IBA
PRA
MAR
ENR
DIF
IBA
PRA
MAR
ENR
DIF
IBA
range
MIC50 MIC90
0.062– 2
0.25
0.5
0.25– 8
1
2
0.125– 8
1
8
0.25– 16 2
8
0.125– 8
4
4
0.062– 1
0.25
1
0.062– 8
1
4
0.125– 16 2
8
0.25– 16 2
8
0.25– 32 8
32
0.031– 2
0.5
1
0.25– 64 4
64
0.25– 64 8
32
0.25– 16 2
16
0.5– 64 4
32
0.016– 1
0.25
1
0.25– 8
1
4
0.25– 16 2
8
0.125– 32 1
16
1– 32 8
16
0.062– 0.5 0.062 NC
0.5– 8
0.5
1– 16 8
0.5– 1
1
0.25– 16 16
0.016– 1
0.25 NC
0.062– 4
0.5
0.25– 16 1
0.125– 16 1
0.25– 32 2
0.125– 1
0.25 NC
0.5– 4
1
0.5– 8
2
1– 16 2
2– 8
4
0.016– 2
0.25
1
0.062– 64 1
8
0.031– 64 2
16
0.062– 32 2
16
0.062– 64 4
16
PRA, pradofloxacin; MAR, marbofloxacin; ENR, enrofloxacin; DIF,
difloxacin; IBA, ibafloxacin; NC, not calculated.
1000
Comparative anti-anaerobe activity of pradofloxacin
spp. (1). Isolates were sub-cultured on Fastidious Anaerobe Agar
(LabM, LAB103, Bury, UK) prior to susceptibility testing.
Susceptibility testing
The test compounds, pradofloxacin, marbofloxacin, enrofloxacin,
ibafloxacin and difloxacin were all supplied with a certificate of
analysis detailing purity. MICs of each of the test compounds
against 141 anaerobic bacteria were determined using agar dilution
methodology as described by the CLSI (formerly NCCLS) in complete accordance with the procedures detailed in M11-A5, Methods
for Antimicrobial Susceptibility Testing of Anaerobic Bacteria,
using Brucella blood agar (Difco, D0964-17) supplemented with
haemin (5 mg/L) and vitamin K1 (1 mg/L) and incubating for up to
48 h.14 B. fragilis ATCC 25285 and Eubacterium lentum ATCC
43055 were used as quality control organisms. All susceptibility
testing was carried out under strict anaerobic conditions using an
anaerobic workstation (Don Whitley Scientific Limited, Shipley, UK).
Results and discussion
The MIC data for pradofloxacin and the other veterinary
fluoroquinolones are summarized in Table 1 for all genera
where n was 5, i.e. Clostridium, Bacteroides, Fusobacterium,
Prevotella, Porphyromonas, Sporomusa and Propionibacterium.
Comparative MIC data were also generated for species where
n was between 2 and 4, namely Actinomyces where n ¼ 3
( pradofloxacin, 0.125 – 0.25; marbofloxacin, 0.5 –1; enrofloxacin,
0.25 – 1; difloxacin, 1; ibafloxacin, 0.5 –2), Eubacterium, n ¼ 3
( pradofloxacin, 0.25– 0.5; marbofloxacin, 0.5 –2; enrofloxacin,
0.25 – 4; difloxacin, 1 –2; ibafloxacin, 4), P. anaerobius, n ¼ 3
( pradofloxacin, 0.25; marbofloxacin, 1; enrofloxacin, 1 – 2;
difloxacin, 2; ibafloxacin, 4), M. hypermegale, n ¼ 2 ( pradofloxacin, 1– 2; marbofloxacin, 2 – 16; enrofloxacin, 16 – 32; difloxacin,
4 – 16; ibafloxacin, 2 –8), R. torques, n ¼ 2 ( pradofloxacin,
0.062 –0.5; marbofloxacin, 1– 4; enrofloxacin, 0.5– 8; difloxacin,
0.25 – 16; ibafloxacin, 0.5 –2) and S. sputigena, n ¼ 2 ( pradofloxacin, 0.25 –1; marbofloxacin, 1– 8; enrofloxacin, 2– 16; difloxacin, 2– 16; ibafloxacin, 4 –32). Additionally, MICs for single
species were Bifidobacterium spp. ( pradofloxacin, 0.125; marbofloxacin, 0.5; enrofloxacin, 0.5; difloxacin, 1; ibafloxacin, 2),
D. tiedjei (pradofloxacin, 0.25; marbofloxacin, 0.125; enrofloxacin,
0.5; difloxacin, 0.5; ibafloxacin, 0.5), H. seregens ( pradofloxacin,
0.25; marbofloxacin, 0.5; enrofloxacin, 2; difloxacin, 8; ibafloxacin, 4), M. bovicus ( pradofloxacin, 0.5; marbofloxacin, 1;
enrofloxacin, 0.5; difloxacin, 0.5; ibafloxacin, 4), Megasphaera
spp. ( pradofloxacin, 0.031; marbofloxacin, 0.062; enrofloxacin,
Figure 2. MIC distribution of anaerobic bacteria from dogs and cats (n ¼ 141) for (a) pradofloxacin, (b) marbofloxacin, (c) enrofloxacin, (d) difloxacin and
(e) ibafloxacin.
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Silley et al.
0.125; difloxacin, 0.125; ibafloxacin, 0.25), R. fascians
( pradofloxacin, 0.031; marbofloxacin, 0.062; enrofloxacin,
0.031; difloxacin, 0.062; ibafloxacin, 0.062) and S. termitidis
( pradofloxacin, 0.062; marbofloxacin, 1; enrofloxacin, 2; difloxacin, 1; ibafloxacin, 2). All individual strain MIC values are
provided in Table S1, available as Supplementary data at JAC
Online (http://jac.oxfordjournals.org/).
Pradofloxacin demonstrates enhanced activity relative to the
other tested compounds, consistent with what would be expected
from a third-generation fluoroquinolone.15 – 17 This is best
expressed in Figure 2 where the total data set is presented as
susceptibility distributions for the respective antimicrobial
agents from which it can be seen that the mode MIC for pradofloxacin was 0.25 mg/L compared with 1 mg/L for marbofloxacin, 2 mg/L for enrofloxacin and difloxacin and 4 mg/L for
ibafloxacin. Figure 2 also shows that all isolates were susceptible
to pradofloxacin at 2 mg/L, whereas the MIC range extended to
32 mg/L for difloxacin and 64 mg/L for marbofloxacin, enrofloxacin and ibafloxacin.
Goldstein18 emphasized that due to increasing development
of resistance of anaerobic bacteria to all antimicrobial agents
there is a need to find new agents active against anaerobes.
Additionally, the point was made that it would be useful to
have oral antimicrobial agents with broad-spectrum activity
against both aerobes and anaerobes. Currently available fluoroquinolones in veterinary medicine only have modest activity
against anaerobes, as evident from the data in Table 1. It is
clear that pradofloxacin has enhanced anaerobic activity and
further provides broad-spectrum coverage against aerobic
organisms.11 While data supporting the activity of thirdgeneration fluoroquinolones against human isolates are available, such data have not previously been reported for animal
isolates. Indeed, there has been contrasting data reported,
suggesting that animal isolates may not be equally susceptible.
In this context, Wexler et al.19 reported that 96% of 557 anaerobes tested were susceptible to trovafloxacin at 2 mg/L;
Goldstein et al.20 showed trovafloxacin to be active against
anaerobic pathogens isolated from human and animal bitewounds, all were susceptible at 2 mg/L with the exception of
Fusobacterium spp., which were susceptible at 4 mg/L. The
authors commented on this small one dilution difference in
susceptibility of Fusobacterium spp. with the Wexler study19
and pointed out that the same methods were used and the
reason for the disparity was unclear except that they studied
veterinary isolates recovered from human infections, whereas
Wexler et al.19 used human isolates from other sources. The
data from our study suggest that animal isolates exhibit similar
susceptibility as human isolates.
Moxifloxacin is probably the most relevant example of how
third-generation fluoroquinolones demonstrate enhanced antianaerobe activity because it is structurally similar to pradofloxacin.
Indeed, the reported MIC data for pradofloxacin are consistent
with MIC data reported for moxifloxacin.21 – 23 This study provides the first comparative MIC data for veterinary fluoroquinolones against anaerobes isolated from dogs and cats and reveals
a high level of anti-anaerobe activity for pradofloxacin. It is
clear that third-generation fluoroquinolones, such as pradofloxacin, may have important utility in veterinary medicine as singledrug therapy for infections caused by mixed aerobic/anaerobic
infections15,24 and in this context they could also be used to treat
bacteria associated with dental infections as has been
demonstrated in a large pan-European study investigating the
in vitro activity of a range of anti-anaerobe antimicrobials against
Gram-negative bacilli.25
Funding
This study was funded by Bayer HealthCare AG.
Transparency declarations
P. S. has received funds for speaking at symposia organized on
behalf of Bayer HealthCare AG and also acts as a consultant to
Bayer HealthCare AG. B. S. and H. A. G. are Bayer employees.
A. P.: none to declare.
Supplementary data
Table S1 is available as Supplementary data at JAC Online
(http://jac.oxfordjournals.org/).
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