Full text of DOI:10.1111/j.1751-0813.2000.tb11895.x

Clinical
In vitro antimicrobial susceptibilities of three
Porphyromonas spp and in vivo responses in the oral
cavity of cats to selected antimicrobial agents
JM NORRIS and DN LOVE
The Department of Veterinary Anatomy and Pathology, The University of Sydney, New South Wales 2006
Objectives
To determine in vitro susceptibility of
lthough the pathogenesis of periodontal disease in
humans and other animals is not understood completely,
Porphyromonas gingivalis, P salivosa and P circumdentaria to
seven antimicrobial agents by agar dilution and Epsilometer
test methods and to assess the effectiveness of these antimi-
crobial agents in reducing the numbers of each
Porphyromonas spp in the oral cavity of 16 domestic cats.
A
certain bacteria and their products in the dental plaque
are amongst several important determinants of the onset and
progression of periodontal disease. The feline oral
Porphyromonas spp have been established as numerically promi-
nent and clinically prevalent in feline periodontal disease1 and
subcutaneous abscesses2 and the presence of putative virulence
factors has been demonstrated.3-4 The susceptibility of these
organisms to antimicrobial drugs is essential information for the
effective management of oral and oral-associated diseases in vet-
erinary practice and in human medicine, where these organisms
are encountered in cat-bite wounds.5
Design
A two-part prospective study involving in vitro
antimicro-bial studies using Porphyromonas spp obtained
from naturally occurring feline infections and in vivo antimicro-
bial response studies using client-owned cats with naturally
occurring periodontal disease.
Procedure Isolates (n = 25) of three feline Porphyromonas
spp from the oral cavity and oral-associated disease were
tested for their in vitro susceptibility to amoxycillin, amoxy-
cillin-clavulanate, benzylpenicillin, clindamycin, doxycycline,
erythromycin and metronidazole, using agar dilution and
Epsilometer test methods. Digoxigenin-labelled whole chro-
mosomal DNA probes directed against P gingivalis VPB 3492,
P circumdentaria NCTC 12469T and P salivosa VPB 3313
were used to quantify organisms taken from two sample sites
at the gingival margins of these cats prior to, and 5 days after,
treatment with one of four commonly used antimicrobial prod-
ucts (amoxycillin-clavulanate, clindamycin, doxycycline or
spiramycin-metronidazole). The response to treatment was
assessed clinically for each cat.
Optimal periodontal therapy in humans has been described
as the total elimination of periodontal pathogens with complete
regeneration of periodontal tissue and return to normal func-
tion.6 The importance of antimicrobial therapy as part of a ther-
apeutic plan has long been recognized in the treatment of
destructive periodontal diseases in humans7 and several antimi-
crobial agents have been tested extensively for their usefulness as
adjunctive treatment for destructive periodontal disease. These
have included tetracyclines, clindamycin, penicillins, metron-
idazole, cephalosporins and erythromycin. Antimicrobial
therapy may be used to decrease bacterial counts before or after
mechanical debridement and to reduce bacterial reservoirs in
periodontal pockets and other intra-oral sites.
Dental scaling, gingival crevicular lavage and periodontal
surgery have been the mainstay of periodontal therapy in veteri-
nary dentistry.8 In veterinary practice over the past 10 years,
increased recognition of the importance of periodontal disease
in domestic cats and greater emphasis on maintaining good
dental hygiene have been seen. In veterinary dentistry, the
necessity for antimicrobial agents as an adjunct to mechanical
debridement has been debated.9 It was commonly stated in
older texts that antimicrobial agents were not required prior to
mechanical debridement, with the justification that normal
dogs clear oral bacteria from the blood within 20 min of bacter-
Results
All isolates were susceptible in vitro to all seven
antimicrobial agents using both methods. The numbers of P
gingivalis were not reduced at the gingival sample sites by
administration of amoxycillin-clavulanate for 5 days, although
this treatment reduced the numbers of P salivosa and P
circumdentaria to below detection levels in six of eight and two
of three of sample sites, respectively; clinical improvement
was not observed in cats treated with amoxycillin-clavulanate.
Treatment with clindamycin, doxycycline or spiramycin-
metronidazole resulted in clinical improvement and a marked
reduction of all Porphyromonas isolates at the sample sites.
Conclusion The Epsilometer test is a simple and accurate
method for determining the minimum inhibitory concentration
for P gingivalis, P salivosa and P circumdentaria. All strains
were susceptible in vitro to all the antimicrobial agents tested
,although clinical improvement of gingival disease was not
noted with amoxycillin-clavulanate when given for 5 days at
usual doses. This appears to be the first report of the disparity
between the in vivo and in vitro susceptibility of oral bacterial
strains to amoxycillin-clavulanate in the veterinary dental litera-
ture. This also appears to be the first report in which clinical and
microbiological responses to commonly used antimicrobial
agents for periodontal disease in cats has been documented
and quantified. It was shown that treatment with clindamycin,
spiramycin-metronidazole or doxycycline not only produced a
substantial reduction in the number of Porphyromonas spp (in
the majority of cases to below detection levels), but also
resulted in substantial clinical improvement. This would indi-
cate that these antimicrobial agents are useful adjunctive
therapy to mechanical debridement in domestic cats.
ATCC American-type culture collection
CFU Colony forming units
E-test Epsilometer test
MIC Minimum inhibitory concentration
NCCLS National committee for clinical laboratory standards
NCTC National collection of type cultures
Pc Porphyromonas circumdentaria
PgF feline strain Porphyromonas gingivalis
PO Per os
PRBHIB Prereduced brain heart infusion broth
Ps Porphyromonas salivosa
PT Total Porphyromonas spp
SBA Sheep blood agar
SBLA Supplemented Brucella laked blood agar
Aust Vet J. 2000, 78:533-537
VPB Veterinary pathology and bacteriology
Keywords: Porphyromonas spp, antimicrobial susceptibility, periodontal diseas
antimicrobial agents, clinical responses.
e,
Aust Vet J Vol 78, No 8, August 2000
533

Clinical
aemia.10 However, the need for antimicrobial agents in animals
with moderate to severe destructive periodontal disease and
with other systemic illness (such as renal, hepatic or cardiovas-
cular disease) has since been recognized.11
pretation of the antimicrobial susceptibility for each microor-
ganism.
One E-test strip (Epsilometer AB Biodisk, Solna, Sweden)
containing amoxycillin, amoxycillin-clavulanate, benzylpeni-
cillin, erythromycin, clindamycin, doxycycline or metronida-
zole was applied to each 90 mm plate containing SBLA, which
had been inoculated in accordance with manufacturers’ instruc-
tions. Plates were incubated as described above and the MIC
value was read at 4 days, as recommended by the manufacturer,
as the point of intersection between the inhibition ellipse edge
and the E-test strip.
In vitro testing has been the mainstay for determining the
susceptibility of organisms to antimicrobial agents. The agar
dilution method and more recently the E-test have proven effec-
tive and reliable methods to evaluate antimicrobial susceptibility
of human isolates of Porphyromonas.11-13 There have been rela-
tively few quantitative studies on drug susceptibility of anaer-
obic bacteria of animal origin because of the labour-intensive
and time-consuming nature of the agar dilution method.
Because the agar dilution method is seldom practicable for
testing individual clinical isolates, interest has been aroused in
the reliability of the E-test. Antimicrobial susceptibility testing
of Porphyromonas isolates of animal origin has shown these
organisms to be highly susceptible in vitro to many antimicro-
bial agents, with the obvious exception of the aminoglyco-
sides.14-17
The current study aimed to provide veterinarians with
antimicrobial susceptibility data for Porphyromonas spp in order
to assist with the management of periodontal disease and oral-
associated diseases and was presented in part at the Anaerobe
Society of the Americas Conference, Buenos Aires, May 1998.
The study used the in vitro methods of agar dilution and the E-
test and compared those results with the response in vivo to four
commonly prescribed antimicrobial preparations.
In vivo antimicrobial response studies
Cats that had not been treated with any antimicrobial agent
in the previous 12 months and with grade 2 or 3 periodontal
disease24 were chosen for the study from cats presented to a
busy suburban veterinary practice for a variety of procedures.
Owner consent was obtained for involvement in the trial.
Within these limits, only cats with oral disease clearly unrelated
to gingival/periodontal disease were excluded from the study.
The oral cavity was examined as part of a full physical examina-
tion and an evaluation made of the general status of the peri-
odontal tissue. A grade was assigned to the periodontium over
the upper right canine and third upper right premolar teeth.
Following antimicrobial therapy, the periodontal tissue was
assessed again for the degree of gingival erythema and oedema
and degree of halitosis.
Cats in the four treatment groups were allocated by sequen-
tial rotation and received the following antimicrobial prepara-
tions for 5 days at dose rates recommended by the manufac-
turers: amoxycillin-clavulanate (4:1;Clavulox, Pfizer Animal
Health) at 12.5 mg/kg PO twice daily; doxycycline (Vibravet,
Pfizer Animal Health) at 5 mg/kg loading dose then 2.5 mg/kg
every 12 hours for two doses then once daily PO; clindamycin
(Antirobe, Pharmacia Upjohn) at 5.5 mg/kg twice daily PO,
and spiramycin 75 000 units/kg and metronidazole 12.5 mg/kg
(Stomorgyl, Rhone Merrieux) at 12.5 mg/kg PO once daily.
Some animals were lost to the study between the first sample
and the postantimicrobial-administration sample leaving the
four unequal groups as indicated in Table 2. The cats that
comprised the study ranged in age from 3 to 18 years (median 6
years) and consisted of eight males and eight females.
Immediately before antimicrobial therapy was instituted, and
again 24 h after treatment ceased, samples for bacteriological
investigation were taken from each cat from the gingival margin
of the buccal surface of the right upper canine tooth (canine
site) and the right upper third premolar tooth (premolar site).
Sampling method and bacteriological procedures were routine
and have been described elsewhere.1-3 Samples collected onto
swabs from each site were placed into PRBHIB and 5 mL of
well-mixed aliquots were grown anaerobically for 7 days on SBA
supplemented with haemin and formate-fumarate; digoxigenin-
labelled whole chromosome DNA probes of PgF VPB 3492, Ps
NCTC 11632 and Pc NCTC 12469T were used to enumerate
each species from colony lifts taken from replicate plates 25 and
these counts were used also to derive total CFU of PT per
sample. Successful treatment was defined as a reduction of
greater than one standard deviation of the mean CFU present
prior to treatment.
Materials and methods
In vitro antimicrobial susceptibility studies
For the agar dilution method, 25 feline-origin Porphyromonas
spp were used, comprising 11 isolates of PgF, 10 of Ps and 4 of
Pc. For the E-test, 19 feline-origin Porphyromonas spp were used
comprising 9 PgF, 7 Ps and 3 Pc. All organisms had been
isolated from naturally occurring subcutaneous abscesses,
pyothorax or the oral cavity of cats18-20 and had been identified
by phenotypic characteristics and DNA-DNA hybridization by
in solution methods21 or whole chromosomal probes labelled
with digoxigenin (Boehringer-Mannheim).22 Bacteroides fragilis
ATCC 25285 and B thetaiotaomicron ATCC 29741 were used
as quality control strains.
Inocula for each sensitivity test were prepared by subculturing
to 5 mL PRBHIB (Oxoid) organisms taken from a 5 day
culture grown on SBA supplemented with haemin and formate-
fumarate23 and growing them anaerobically overnight at 37°C.
The following morning, each culture was diluted with PRBHIB
to the turbidity of a 0.5 McFarland density tube standard.
SBLA plates prepared (containing a range of concentrations of
each antimicrobial agent) were inoculated with 1 mL of each
diluted culture before incubation for 4 days at 37°C. All antimi-
crobial agents used in the agar dilution method were free-acid
standards of known potency and supplied as indicated:
benzylpenicillin (Sigma), amoxycillin (Pfizer), clavulanic acid
(Pfizer), erythromycin (Sigma), doxycycline (Pfizer), clin-
damycin (Sigma) and metronidazole (Sigma).
The MIC values were determined using the NCCLS refer-
ence agar dilution method13 and Brucella agar (Difco
Laboratories) supplemented with haemin-menadione, formate-
fumarate23 and 5% laked sheep blood. The MIC was deter-
mined after 4 days’ incubation at 37°C as the concentration at
which there was a marked change in growth compared with the
control. MIC readings were compared with the NCCLS-
approved breakpoints13 for antimicrobial agents to enable inter-
Clinical Assessment
The oral cavity was assessed for gingival erythema (marginal,
+, ++, +++), gingival oedema (+, ++, +++), bleeding on probing
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Clinical
Table 1. Agar dilution and E-test determinations of antimicrobial susceptibility of isolates Porphyromonas gingivalis, P salivosa and P circumdentaria from
oral cavity and oral-associated diseases of cats.
a
Antimicrobial agent
Minimum inhibitory concentration ranges for isolates tested (mg/mL)
Break point
(mg/mL)
P gingivalis
Agar dilution
P salivosa
P circumdentaria
Agar dilution E-test
E-test
(n = 9)
Agar dilution
(n = 10)
E-test
(n = 7)
(n = 11)
(n = 4)
(n = 3)
Amoxycillin
< 0.008 -0.064
< 0.00 - 0.064
0.004 - 0.512
0.002 - 0.004
0.032 - 0.256
0.128 - 1.024
0.001 - 0.064
< 0.016 - 0.032
< 0.016
1.024 - 2.048
0.064 - 0.256
0.512 - 1.024
0.004 - 0.016
0.128 - 0.256
0.512 - 1.024
0.002 - 0.064
2.0
0.008 - 1.024
0.008 - 1.024
0.004 - 1.024
0.002 - 0.008
0.008 - 0.512
0.016 - 1.024
0.032 - 0.064
< 0.016 - 1.0
< 0.016 - 1.0
0.002 - 0.75
< 0.016
4
4
4
4
Amoxycillin/clavulanic acid
Benzylpenicillin
Clindamycin
Doxycycline
0.050 - 0.075
0.50 - 0.75
< 0.016
0.002 - 0.125
< 0.016
0.064 - 0.125
0.25 - 0.50
< 0.002 - 0.023
0.094 - 0.125
0.25 - 0.50
< 0.002 - 0.032
< 0.016 - 0.25
0.023 - 1.0
8
Erythromycin
Metronidazole
a
NA
16
0.023 - 0.032
Break points approved by the NCCLS13
NA Not available
Table 2. Mean colony forming units of total Porphyromonas spp and P gingivalis at canine and
premolar sample sites pre and postantimicrobial treatment.
(±, +, ++, +++) and halitosis (nil, mild,
moderate, foul) as outlined by West-Hyde
and Floyd.24
Treatment group (n)
Colony forming units at canine site (mean ± SD)
Total Porphyromonas P gingivalis
Pretreatment Posttreatment
Results
Pretreatment
Posttreatment
In vitro antimicrobial susceptibility studies
The MIC values obtained by both
methods for PgF, Ps and Pc are shown in
Table 1. Agreement between E-test and agar
dilution MIC values within ± log2 dilution
was 100% for all antimicrobial agents tested
against all three species. PgF, Ps and Pc were
susceptible in vitro to all antimicrobial
agents tested using both methods.
Amoxycillin-
2.59 x 105
(± 1.11 x 105)
1.83 x 105
(± 8.97 x 104)
2.09 x 105
(± 7.13 x 104)
1.77 x 105
(± 8.39 x 104)
clavulanate (6)
Doxycycline (4)
1.50 x 105
1.13 x 104
9.35 x 104
1.05 x 104
(± 7.21 x 104)
(± 1.40 x 104)
(± 3.51 x 104)
(± 1.28 x 104)
Spiramycin-
metronidazole (3)
2.54 x 105
(± 1.72 x 105)
0
0
1.57 x 105
(± 7.34 x 104)
0
0
Clindamycin (3)
1.75 x 105
(± 2.26 x 105)
1.37 x 105
(± 1.59 x 105)
In vivo antimicrobial response studies
Colony forming units at premolar site (mean ± SD)
As can be seen from Table 2, there was a
successful response to treatment with doxy-
cycline, clindamycin and spiramycin-
metronidazole, as indicated by reduction in
PT. There was similarly a reduction in total
numbers of PgF from treatment with these
three antimicrobial products but not with
amoxycillin-clavulanate. Similarly, as indi-
cated for individual cats in Table 3, there
was a successful response as indicated by
reduction in numbers of Ps and Pc after
treatment with doxycycline, clindamycin
and spiramycin-metronidazole but not with
amoxycillin-clavulanate.
Amoxycillin
2.40 x 105
(± 2.05 x 105)
2.48 x 105
(± 1.65 x 105)
1.53 x 105
(± 8.69 x 104)
2.09 x 105
(± 1.01 x 105)
-clavulanate (6)
Doxycycline (4)
2.08 x 105
(± 1.27 x 105)
3.15 x 104
(± 3.12 x 104)
1.47 x 105
(± 1.04 x 105)
3.0 x 104
(± 3.04 x 104)
Spiramycin-
3.35 x 105
(± 1.29 x 105)
0
1.91 x 105
(± 9.42 x 104)
0
metronidazole (3)
Clindamycin (3)
1.59 x 105
6.67 x 102
1.47 x 105
6.67 x 102
(± 1.23 x 105)
(± 1.15 x 103)
(± 1.17 x 105)
(± 1.15 x 103)
Amoxycillin-clavulanate (Clavulox), doxycycline (Vibravet), spiramycin and metronidazole (Stomorgyl),
clindamycin (Antirobe)
SD standard deviation
Clinical assessment after antimicrobial therapy
showed similar susceptibility patterns of feline oral
On examination 24 h after cessation of antimicrobial therapy,
cats in the doxycycline, clindamycin and spiramycin-metronida-
zole, but not in the amoxycillin-clavulanate, treatment groups
showed a marked decrease in halitosis, gingival oedema and
erythema (at least one grade reduction in each parameter).
Porphyromonas spp.15 The additional information provided here
is the susceptibility of these organisms to antimicrobial prepara-
tions currently used in veterinary practice. Other recent studies
have found unspeciated members of the genus Porphyromonas
from dogs and cats17 or P gingivalis from cats16 to be susceptible
to antimicrobial agents including metronidazole, chloram-
phenicol, amoxycillin-clavulanate, ampicillin, cefadroxil and
clindamycin. The results of in vitro antimicrobial susceptibility
testing were also comparable to those found in previous studies
with human strains of P gingivalis and other species14, 26-27 that
showed that the strains tested were very susceptible to
benzylpenicillin, ampicillin, cefaclor, cefoxitin, cefuroxime,
Discussion
The in vitro antimicrobial susceptibility studies reported here
have shown that recent isolates of PgF, Ps and Pc were suscep-
tible to benzylpenicillin, amoxycillin, amoxycillin-clavulanic
acid, erythromycin, doxycycline, clindamycin and metronida-
zole. These results were comparable to previous studies that
Aust Vet J Vol 78, No 8, August 2000
535

Clinical
Table 3. Colony-forming units (x103) of Porphyromonas spp isolated from the canine and premolar sample sites pre and postantimicrobial treatment.
a
Cat
Number
Antimicrobial agent
Grade
at C/P
Canine sample site
Premolar sample site
P gingivalis
P salivosa
Pre Post
P circumdentaria
P gingivalis
P salivosa
Pre Post
P circumdentaria
Pre
Post
Pre
Post
Pre
Post
Pre
Post
1
Amoxycillin-clavulanate
Amoxycillin-clavulanate
Amoxycillin-clavulanate
Amoxycillin-clavulanate
Amoxycillin-clavulanate
Amoxycillin-clavulanate
Doxycycline
3/3
3/3
3/3
3/3
2/2
3/3
3/3
2/2
3/3
2/2
3/3
3/3
3/3
2/2
2/2
3/3
232
120
264
280
120
240
124
80
64
128
232
300
140
200
26
16
0
0
0
0
0
84
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
148
148
70
300
100
136
360
156
200
40
0
0
0
0
0
0
0
0
8
0
0
0
0
0
0
0
0
0
0
48
28
320
40
0
0
36
0
2
80
8
3
68
0
36
0
20
104
20
0
4
320
120
110
176
46
0
200
0
5
0
0
0
6
0
0
0
0
7
80
0
3
30
0
64
0
92
0
6
8
Doxycycline
0
0
9
Doxycycline
120
50
0
0
64
20
0
280
86
68
12
0
0
0
0
10
11
12
13
14
15
16
a
Doxycycline
0
30
200
90
0
0
46
180
136
90
0
40
0
0
Spiramycin-metronidazole
Spiramycin-metronidazole
Spiramycin-metronidazole
Clindamycin
240
132
100
42
0
0
300
142
132
60
0
0
0
0
0
12
12
14
0
0
0
0
0
0
0
0
0
0
0
0
0
Clindamycin
48
0
0
0
0
102
280
2
0
0
Clindamycin
320
0
116
0
0
0
132
20
0
24
Grade of periodontal disease
Amoxycillin-clavulanate (Clavulox), doxycycline (Vibravet), spiramycin and metronidazole (Stomorgyl), clindamycin (Antirobe)
C/P canine/premolar sample site
Pre pretreatment
Post posttreatment
in vitro tests showed all strains were susceptible to amoxycillin
and amoxycillin-clavulanic acid. This appears to be the first
report of a disparity between the in vivo and in vitro suscepti-
bility of oral bacterial strains to amoxycillin-clavulanate in the
veterinary dental literature. There does not appear to be a
simple explanation for these findings but several factors may
have played a role. These include the possibility that the total
bacterial load in the periodontal pocket may have been too large
in relation to the maximal achievable antibiotic concentration.
This has also been described as the ‘inoculum effect’. The deter-
mination of MIC uses a standard inoculum of approximately
105 CFU/mL and it has been shown that the periodontal
pocket and supragingival tissue harbour many more bacteria
than this. Also, the subgingival microbiota can be considered an
adherent layer of bacterial microcolonies (biofilms). The
production of glycocalyx by plaque organisms may induce a
biofilm phenomenon whereby the microbes are more resistant
to the bactericidal actions of antimicrobial agents.
While it may be argued that these factors should have affected
all the antimicrobial agents used, the concentration of amoxy-
cillin-clavulanate reached in the gingival crevicular fluid and the
rest of the oral cavity may not have been sufficient to cope with
a large bacterial inoculum existing in the biofilm of plaque. In
the human dental literature it is reported that a single oral dose
of 500 mg amoxycillin produces a peak serum concentration of
8 mg/mL and a peak gingival crevicular fluid concentration of 3
to 4 mg/ml.28 The NCCLS-approved breakpoint MIC for
amoxycillin against anaerobes is 2 mg/mL. In a complex envi-
ronment, such as the oral cavity, in which the inoculum exceeds
105 CFU/ml, this concentration of amoxycillin may be insuffi-
cient to reduce the numbers of all bacteria present. Further
investigations are required to examine the concentration of
amoxycillin achieved in the gingival crevicular fluid of animals
with various grades of periodontal disease to determine whether
cefotaxime, imipenem, erythromycin, tetracycline, doxycycline,
metronidazole and clindamycin by either the agar dilution or E-
test methods. We have also shown good agreement between the
results obtained by agar dilution method and E-test methods for
these feline oral Porphyromonas spp. While the agar dilution
method remains the reference method for anaerobes, the E-test
has considerable advantages by being simpler and less labour-
intensive. The agreement between these methods has made the
E-test a feasible option for use in commercial laboratories to aid
medical and veterinary practitioners in the correct choice of
antimicrobial agent to treat oral-associated infections involving
these organisms. While antimicrobial susceptibility testing of
clinical isolates is not always required due to the frequently
favourable clinical response to empirical therapy and the
inevitable delay in growth of fastidious obligate anaerobes such
as Porphyromonas, it is valuable to know that the E-test may be
used if required.
This appears to be the first report in which clinical and
microbiological responses to commonly used antimicrobial
agents for periodontal disease in cats has been documented and
quantified. It was shown that treatment with clindamycin,
spiramycin-metronidazole or doxycycline not only produced a
substantial reduction in the number of Porphyromonas spp (in
the majority of cases to below detection levels), but also resulted
in substantial clinical improvement. This would indicate that
these antimicrobial agents are useful adjunctive therapy to
mechanical debridement in domestic cats.
In vivo findings raise some interesting questions about the
correlation between in vitro antimicrobial susceptibility testing
and in vivo responses of microbes in complex polymicrobial
infections such as periodontal disease. Treatment with amoxy-
cillin-clavulanate did not reduce PgF numbers at the canine and
premolar sample sites by greater than one standard deviation
from the mean in any of the cats tested, while the results of the
536
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Clinical
their characterization by biochemical tests and arbitrarily primed polymerase
chain reaction fingerprinting. Clin Infect Dis 1996;23 (1 Suppl):78S-82S.
6. Collins JG, Offenbacher S, Arnold RR. Effects of a combination therapy to
eliminate Porphyromonas gingivalis in refractory periodontitis. J Periodontol
1993;64:998-1007.
7. Genco RJ. Antibiotics in the treatment of human periodontal disease. J
Periodontol 1981;52:545-558.
8. Penman S, Harvey CE. Periodontal disease. In : Harvey CE, Orr HS, editors.
Manual of small animal dentistry. British Small Animal Veterinary Association,
Cheltenham, 1990:37-48.
9. Harvey CE. Oral medicine. In: Harvey CE, editor. Veterinary Dentistry.
Saunders, Philadelphia ,1985:159-178.
10. Silver JG, Martin L, McBride BC. Recovery and clearance of oral microor-
ganisms following experimental bacteraemia in dogs. Arch Oral Biol
1975;20:675-679.
11. Sarkiala E, Harvey C. Systemic antimicrobials in the treatment of periodon-
titis in dogs. Semin Vet Med Surg (Small Animal) 1993;8:197-203.
12. Harvey CE. In: Veterinary dentistry. University of Sydney Post Graduate
Committee in Veterinary Science, Proceedings 1993;(212):170-173.
13. National Committee for Clinical Laboratory Standards. Approved standard
M11-A3. Methods for antimicrobial susceptibility testing for anaerobic bacteria.
Villanova, Philadelphia, 1993.
14. Pajukanta R, Asikainen S, Forsblom B, Piekkola M, Jousimies-Somer H.
Evaluation of the E-test for antimicrobial susceptibility testing of Porphyromonas
gingivalis. Oral Microbiol Immunol 1994;9:123-125.
larger doses achieve favourable results in the treatment of these
and other similar disorders.
There have been a number of cases in the human dental liter-
ature in which bacterial isolates from clinical cases have been
sensitive to antimicrobial agents in vitro but the condition
failed to improve and the organisms were still isolated after
these agents had been administered parenterally.29-31 Most
recently, a randomised double-blind placebo-controlled study of
the clinical and microbiolgical effects of initial periodontal
therapy in conjunction with systemic amoxycillin-clavulanate,
found no advantages in using these agents.32 The in vivo effect
of numerous periodontal pathogens were analysed, amongst
them human strains of P gingivalis; the four patients positive for
this organism remained positive after treatment with amoxy-
cillin-clavulanate. It is clear that bacterial in vitro susceptibility
to antimicrobial agents should be regarded as a guide to poten-
tial activities in vivo but the clinical utility of each antimicrobial
agent should be based on its pharmacological properties and the
results of clinical trials.
Reports on the efficacy of amoxycillin-clavulanate in peri-
odontal disease in small animals have focused on the in vitro
response of commonly isolated oral organisms rather than an
assessment of changes in clinical and microbiological variables
in vivo.17 In one study,33 92% of cats (11 of 12) and 77% of
dogs (17 of 22) treated with amoxycillin-clavulanate were clini-
cally ‘cured’ of gingivitis. In that study, oral isolates were culti-
vated prior to treatment and antimicrobial susceptibility was
assessed in vitro, but isolation of organisms after treatment was
not performed to verify the clinical response to treatment. In
addition, an undisclosed proportion of these 34 animals were
given additional undisclosed medical or surgical treatments but
were still included in the calculations of those responding to
amoxycillin-clavulanate.
Our findings have established the E-test as an acceptable
method of in vitro antimicrobial testing for the feline oral
species of Porphyromonas and confirmed the in vitro suscepti-
bility of these organisms to commonly used antimicrobial
agents. However, important questions remain about the correla-
tion between the clinical response to treatment of a disease with
complex aetiology and the antimicrobial susceptibility of indi-
vidual organisms within the complex. Further studies are
required to investigate the reasons for such clinical failure.
15. Love DN, Bailey M, Johnson RS. Antimicrobial susceptibility patterns of obli-
gatory anaerobic bacteria from subcutaneous abscesses and pyothorax in cats.
Aust Vet Pract 1980;10:168-172.
16. Harvey CE, Thornsberry C, Miller BR. Antimicrobial susceptibility of sub-
gingival flora in cats with gingivitis. J Vet Dent 1995;12:157-160.
17. Jang SS, Breher JE, Dabaco LA, Hirsh DC. Organisms isolated from dogs
and cats with anaerobic infections and susceptibility to selected antimicrobial
agents. J Am Vet Med Assoc 1997;210:1610-1614.
18. Love DN, Johnson JL, Moore LVH. Bacteroides species from the oral cavity
and oral-associated diseases of cats. Vet Microbiol 1989;19:275-281.
19. Love DN, Jones RF, Bailey M, Johnson RS. Isolation and characterisation
of bacteria from abscesses in the subcutis of cats.
1979;12:207-212.
J Med Microbiol
20. Love DN, Jones RF, Bailey M, Johnson RS, Gamble N. Isolation and char-
acterisation of bacteria from pyothorax (empyema) in cats. Vet Microbiol
1982;7:455-461.
21. Johnson JL. Genetic characterization. In: Gerhardt P, editor. Manual of
methods for general microbiology. American Society for Microbiology,
Washington, DC, 1981:450-472.
22. Love DN, Bailey, GD, Bastin D. Chromosomal DNA probes for the identifica-
tion of asaccharolytic anaerobic pigmented bacterial rods from the oral cavity.
Vet Microbiol 1992;31:287-295.
23. Holdeman LV, Cato EP, Moore WEC. Anaerobe laboratory manual. 4th edn.
Virginia Institute and State University, Blacksburg, 1977.
24. West-Hyde LW, Floyd M. Dentistry. In: Ettinger SJ, Feldman EC, editors.
Textbook of veterinary internal medicine. 4th edn. Saunders, Philadelphia,
1995:1097-1123.
25. Gunaratnam M, Smith GLF, Socransky SS, Smith CM, Haffajee AD.
Enumeration of subgingival species on primary isolation plates using colony
lifts. Oral Microbiol Immunol 1992;7:14-18.
26. Pajukanta R, Asikainen S, Forsblom B, Saarela M, Jousimies-Somer H. b-
lactamase production and in vitro antimicrobial susceptibility of Porphyromonas
gingivalis. FEMS Immunol Med Microbiol 1993;6:241-244.
27. Schieven BC, Massey VE, Lannigan R, Hussain Z. Evaluation of suscepti-
bility of anaerobic organisms by the E-test and the reference agar dilution
method. Clin Infect Dis 1995;20 (2 Suppl ):337S-338S.
28. van Winkelhoff AJ, Rams TE, Slots J. Systemic antibiotic therapy in peri-
odontics. Periodontol 2000 1996;10:45-78.
29. Renvert S, Dahlen G, Wikstrom M. Treatment of periodontal disease based
on microbiological diagnosis. Relation between microbiological and clinical
parameters during 5 years. J Periodontol 1996;67:562-571.
Acknowledgments
This work was supported in part by a grant from the
Australian Research Council. D Wigney, L Patoka and F Taeker
provided valuable assistance throughout. The staff of Inner
West Veterinary Hospital were helpful with access to clinical
cases.
References
1. Norris JM, Love DN. Associations amongst three feline Porphyromonas
species from the gingival margin of cats during periodontal health and disease.
Vet Microbiol 1999;65:195-207.
30. Slots J, Rams TE. Antibiotics in periodontal therapy: advantages and disad-
vantages. J Clin Periodontol 1990;17:479-493.
31. Mishkin D, Javed T, Wood G, Engler W. Effect of topically applied tetracy-
cline on microflora of juvenile periodontitis [abstract]. J Dent Res 1985;64:305A
32. Winkel EG, van Winkelhoff AJ, Barendregt DS, van der Weijden GA,
Timmerman MF, van der Velden U. Clinical and microbiological effects of initial
periodontal therapy in conjunction with amoxicillin and clavulanic acid in
patients with adult periodontitis. J Clin Periodontol 1999;26:461-468.
33. Jones RL, Godinho KS, Palmer GH. Clinical observations on the use of oral
amoxycillin-clavulanate in the treatment of gingivitis in dogs and cats and anal
sacculitis in dogs. Br Vet J 1994;150:385-388.
2. Norris JM, Love DN. The isolation and enumeration of three feline oral
Porphyromonas species from subcutaneous abscesses of cats. Vet Microbiol
1999;65:115-122.
3. Norris JM. Studies in the Porphyromonas species from the oral cavity and
oral-associated diseases of domestic cats [PhD thesis], The University of
Sydney, New South Wales, 1998.
4. Love DN, Binas M. Use of SDS-polyacrylamide-gelatin gels to detect SDS
stable proteinases of feline strains within the genus Porphyromonas. Aust J
Med Sci 1995;16:102-105.
5. Citron DM, Hunt Gerardo S, Claros MC et al. Frequency of isolation of
Porphyromonas species from infected dog and cat bite wounds in humans and
(Accepted for publication 9 December 1999)
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