Anaerobe 43 (2017) 94e98

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Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe

Antimicrobial susceptibility of anaerobic bacteria

Low antibiotic resistance among anaerobic Gram-negative bacteria in

periodontitis 5 years following metronidazole therapy
G. Dahlen a, *, H.R. Preus b
a
Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Sweden
b
Department of Periodontology, Institute of Clinical Odontology, Faculty of Dentistry, University of Oslo, Norway

a r t i c l e i n f o a b s t r a c t

Article history: The objective of this study was to assess antibiotic susceptibility among predominant Gram-negative
Received 26 September 2016 anaerobic bacteria isolated from periodontitis patients who 5 years prior had been subject to mechan-
Received in revised form ical therapy with or without adjunctive metronidazole. One pooled sample was taken from the 5 deepest
9 December 2016
sites of each of 161 patients that completed the 5 year follow-up after therapy. The samples were
Accepted 10 December 2016
Available online 14 December 2016
analyzed by culture. A total number of 85 anaerobic strains were isolated from the predominant sub-
gingival ora of 65/161 patient samples, identied, and tested for antibiotic susceptibility by MIC
Handling editor: Lyudmila Boyanova determination. E-tests against metronidazole, penicillin, amoxicillin, amoxicillin clavulanic acid and
clindamycin were employed. The 73/85 strains were Gram-negative rods (21 Porphyromonas spp., 22
Keywords: Prevotella/Bacteroides spp., 23 Fusobacterium/Filifactor spp., 3 Campylobacter spp. and 4 Tannerella
Antibiotics forsythia). These were all isolated from the treated patients irrespective of therapy procedures
Metronidazole (/metronidazole) 5 years prior. Three strains (Bidobacterium spp., Propionibacterium propionicum,
Resistance Parvimonas micra) showed MIC values for metronidazole over the European Committee on Antimicrobial
Periodontitis
Susceptibility Testing break point of >4 mg/mL. All Porphyromonas and Tannerella strains were highly
Subgingival microbiota
susceptible. Metronidazole resistant Gram-negative strains were not found, while a few showed resis-
Gram-negative anaerobes
tance against beta-lactam antibiotics. In this population of 161 patients who had been subject to me-
chanical periodontal therapy with or without adjunct metronidazole 5 years prior, no cultivable
antibiotic resistant anaerobes were found in the predominant subgingival microbiota.
2016 Elsevier Ltd. All rights reserved.

1. Introduction anaerobic and Gram-negative microbiota, including spirochetes

Antibiotics have frequently been used in periodontal therapy,
especially aggressive forms. Penicillin (PEN), amoxicillin (AMX),
tetracycline (TET), metronidazole (MTZ) or combinations have
been commonly used [1,2]. Clindamycin (CLI) has also been oc-
casionally used as a second choice in case of penicillin allergies
[2]. Although periodontal diseases have been regarded as poly-
microbial infections, Loesche and coworkers [3] early proposed
MTZ to be the drug of choice adjunctive to mechanical debride-
ment. Their rationale was that since microbiological diagnoses of
the subgingival bacterial ora showed a predominantly strict
* Corresponding author. Department of Oral Microbiology and Immunology,
Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Box 450,
40530 Gothenburg, Sweden.
E-mail address: dahlen@odontologi.gu.se (G. Dahlen).
(Treponema spp.), these would serve as the main target for the full
antimicrobial therapy.
Most anaerobic infections are considered to be polymicrobial by
nature. However, they also involve facultative bacteria, not sus-
ceptible to MTZ, such as streptococci, Actinomyces spp., Aggregati-
bacter spp. and Haemophilus spp. and others. Therefore, a rapidly
growing trend to use broad-spectrum antibiotics alone or in com-
binations to cover all putative periodontal pathogens has evolved
in periodontal therapy, and combination of MTZ and AMX (Com-
bination therapy, CT) has been widely used in clinical trials and
recommended in clinical practice [4e6]. However, recently serious
concerns have been raised against the scientic background for the
wide use of this regimen [7].
Numerous studies of the periodontitis associated microbiota
have supported the importance of proteolytic Gram-negative
anaerobic bacteria in the pathogenesis [8]. Socransky et al. [9]
formulated the red complex (Porphyromonas gingivalis, Tannerella

http://dx.doi.org/10.1016/j.anaerobe.2016.12.009
1075-9964/ 2016 Elsevier Ltd. All rights reserved.
 G. Dahlen, H.R. Preus / Anaerobe 43 (2017) 94e98
forsythia and Treponema denticola) and the orange complex con-
sisting of yet another nine Gram-negative anaerobic species to
include the putative periodontal pathogens statistically. The strong
association between the red and orange complex bacteria and
periodontitis (chronic as well as aggressive) has been conrmed in
numerous studies [10e12], although new species have been added
to the list of putative pathogens of which the majority is Gram-
negative anaerobes [13]. Bacteria intrinsically resistant to MTZ,
such as streptococci, Actinomyces and others have been shown
associated with periodontal health [9], and consequently have been
regarded as innocent bystanders in the subgingival microbiota, and
should not be targeted by antibiotic therapy. MTZ is still regarded as
a drug of choice in anaerobic infections [14e16] and therefore
suggested as adjunct antibiotic periodontal therapy when needed.
Several clinical trials [6,17] have been performed with statistical
signicant effects on clinical parameters like probing pocket depth
(PPD), clinical attachment level (CAL) or bleeding on probing (BOP),
but with limited effects on the target bacteria.
Concerns may therefore be raised against the use of antibiotics
in periodontal therapy and whether disadvantages might be
greater than the benets. One such concern is that all use of anti-
biotics has been shown to increase the prevalence of antibiotic
resistant bacteria as well as cause selection of intrinsically resis-
tant/tolerant bacterial strains. While MTZ resistance is found
among anaerobic Gram-positive cocci (peptostreptococci and Par-
vimonas micra) and rods (Clostridium spp., Propionibacterium spp.,
Bidobacterium spp.), the prevalence of Gram-negative anaerobic
isolates resistant to MTZ has been reported low or absent [18]. An
increase over time has been noticed preferentially among non-oral
Bacteroides isolates such as Bacteroides fragilis and other gastroin-
testinal isolates [19]. However, the prevalence of MTZ resistant
isolates among oral Gram-negative anaerobes in odontogenic in-
fections has been infrequently reported.
The objective of this study was to investigate antibiotic sus-
ceptibility (MTZ in particular), among predominant Gram-negative
anaerobic bacteria isolated from periodontitis patients who 5 years
previously had been subject to mechanical therapy with or without
adjunctive MTZ.
2. Materials and methods
2.1. Patients
The data from the present study originate in microbiological
samples taken from 161 chronic periodontitis patients who
participated in a randomized clinical trial on the effect of different
periodontal intervention strategies, and who had completed a 5-
year post-therapy follow-up. The study rationale, design as well
as the one-year clinical results have previously been described in
detail [17,20]. Briey, following a 3-months pre-baseline hygiene
phase, which brought the patients to a very high level of oral hy-
giene, 184 patients suffering from severe periodontitis were ran-
domized to one of four intervention groups that comprised scaling
and root planing (SRP) over weeks or one-day full-mouth disin-
fection (FDIS), with or without adjunctive MTZ therapy (i.e. 400 mg
MTZ (Flagyl, Sano-Aventis, Oslo, Norway) t.i.d. for 10 days). After
all mechanical treatment sessions, in all groups, the patients rinsed
for 1 min with 10 mL of 0.2% chlorhexidine (CHX), and after me-
chanical instrumentation, all sulci and pockets were lled with CHX
gel [17]. 161 of these 184 randomized patients completed the 5-year
follow up, whereas 23 patients left the study for various reasons
during these 5 years [21]. The Privacy Ombudsman for the Nor-
wegian Universities (#15768) and the Regional Committee for
Medical Research Ethics, (Oslo, Norway) (REC South East
2.2006.3573/S-06458b) approved the protocol, and the trial was
95
registered with the U.S. National Institutes of Health Clinical Trials
Registry (http://www.clinicaltrials.gov) number NCT01318928.
2.2. Sampling and laboratory processing
Microbiological samples from the 161 patients completing the
5-year follow-up were obtained by curette/paper points from the 5
deepest periodontal sites in each patient and pooled to a bottle of
transport medium VMGA III [22]. This transport medium is espe-
cially prepared for preserving oral facultative and anaerobic bac-
teria during transport for 2 days in room temperature [22]. The
bottles were transferred to the laboratory of Oral Microbiology,
Institute of Odontology, University of Gothenburg, Sweden for
analysis. After making a 10-fold dilution series the samples were
inoculated on one reduced Brucella Blood Agar plate (BBL Micro-
biological Systems, Cockeysville, MD) supplemented with hemin
(5 mg/L) and menadione (1 mg/L) for anaerobic incubation and one
NAM-plate in which 10 mg/mL N-acetyl-muraminic acid (Sigma, St.
Louis, Mo.) was incorporated into the Brucella agar for supple-
mental growth of T. forsythia [23].
The total number of colony forming units (CFU) was calculated
and bacterial species tentatively identied by their colony
morphology and Gram-staining. Special attention was paid to the
presence of black-pigmented or corroding colonies. Colonies with a
nacreous appearance were tentatively diagnosed as Fusobacterium
spp. All Gram-negative bacteria/colonies that were found pre-
dominantly on the agar plate (>0.5% of CFU) were pure cultured
and stored until further identication.
2.3. Identication
As a second step, all isolates were further identied using the
DNA-DNA hybridization method (Checkerboard) against 12 bac-
terial DNA-probes used in the panel for periodontitis associated
bacteria routinely in the department [24]. The panel included
probes of the following species: Aggregatibacter actino-
mycetemcomitans, Campylobacter rectus, Filifactor alocis, Fusobacte-
rium nucleatum, Parvimonas micra, Porphyromonas endodontalis,
Porphyromonas gingivalis, Prevotella intermedia, Alloprevotella tan-
nerae, Tannerella forsythia, Treponema denticola.
As a third step in the identication of the isolates were by using
API Anae and API zym (Analytab Products, Plainview, N.Y.). Strain
identication was conrmed or strains with doubtful identication
were also analyzed according to tests recommended by the
Anaerobic Systems (www.anerobesystem.com, Morgan Hill, CA) or
by the CCUG (Culture Collection University of Gothenburg, Goth-
enburg, Sweden). Distinction between Porphyromonas spp. and
black-pigmented Prevotella species was also made by hemaggluti-
nation test and autouorescence test according to Slots and Rey-
nolds [25].
2.4. Susceptibility testing
The MIC values for isolated strains were determined using the E-
test (bioMerieux, Marcy l'Etoile, France) and for PEN, AMX, amox-
icillin and clavulanic acid (AMC), CLI, TET and MTZ. Bacterial sus-
pensions of ca 109 cells/mL (as measured by McFarland optical
density at 660 nm) were prepared in PBS and spread uniformly
onto reduced Brucella Blood Agar plates and incubated in anaerobic
jars for 3e5 days. For Tannerella strains NAM-plates were used.
The range and MIC90 were determined for each antibiotic.
Percentage resistance was determined using the breakpoints
advised by the European Committee on Antimicrobial Susceptibil-
ity Testing (EUCAST) for all antibiotics tested [26]. Chi-square test
was used for calculating statistical differences among the 4
96 G. Dahlen, H.R. Preus / Anaerobe 43 (2017) 94e98

treatment groups. Table 2

Anaerobic bacterial species isolated from the subgingival plaque of periodontitis
patients treated with or without metronidazole, regardless of mechanical treatment
3. Results strategy (FDIS or SRP).

Species Number of strains

3.1. Bacterial strains
MTZ treated group Control group Total

Table 1 shows the number of patients and the mean CFU for the Gram-negative rods (Total) 40 33 73
samples included in each treatment group as well as the number of Campylobacter rectus 3 0 3
Filifactor alocis 0 1 1
patients with samples containing predominant Gram-negative
Fusobacterium nucleatum 12 7 19
anaerobic species (>0.5% of CFU). No signicant differences were Fusobacterium varium 0 2 2
observed among the 4 therapy groups, although the FDIS MET Fusobacterium mortiferum 1 0 1
group seemed to have a higher CFU than the other 3 groups (i.e. Parabacteroides distasonis 2 0 2
Porphyromonas gingivalis 7 5 12
FDIS, SRP MET, SRP). The variation in CFU was substantial ranging
Porphyromonas endodontalis 2 4 6
2e3 10log units in each treatment group. Porphyromonas asaccharolytica 0 3 3
Ninety-four tentative anaerobic isolates were pure cultured and Prevotella intermedia 8 7 15
identied. Six strains were misclassied as anaerobes and deleted Prevotella bivia 2 1 3
from further identication. Another 3 Gram-negative rods, sus- Prevotella disiens 1 0 1
Prevotella melaninogenica/oralis 0 1 1
pected to be A. actinomycetemcomitans, were MTZ resistant,
Tannerella forsythia 2 2 4
showed weak or catalase-negative reaction, did not need growth Gram-positive cocci (Total) 6 2 8
factor X or V and were classied as Aggregatibacter aphrophilus Parvimonas micra 3a 2 5
strains. Thus 85 anaerobic strains remained of which 12 were Peptostreptococcus anaerobius 1 0 1
Other peptostreptococci 2 0 2
Gram-positive rods and 73 were Gram-negative rods (Table 2). The
Gram-positive rods (Total) 2 2 4
latter group was dominated by Porphyromonas spp. (21 strains), Actinomyces meyeri/odontolyticus 0 1 1
Prevotella/Bacteroides spp. (22 strains) and Fusobacterium/Filifactor Bidobacterium spp. 1a 0 1
spp. (23 strains) as well as 3 strains of Campylobacter rectus and 4 Propionibacterium propionicum 0 1a 1
strains of T. forsythia. There was no statistically signicant differ- Propionibacterium acnes 1 0 1
Total 48 37 85
ence between the number of Gram-negative anaerobes that were
a
recovered from the two groups treated with MTZ and those of the Including one metronidazole resistent strain.
two groups treated with SRP or FIDS alone. The type of mechanical
strategy (scaling and root planing in one day (FDIS) or over weeks
by adjunct, MTZ medication (400 mg MTZ t.i.d. for 10 days)
(SRP)) did not inuence on these results (Table 2).
together with mechanical therapy 5 years prior. The control pop-
ulation was treated with mechanical therapy only. The clinical re-
3.2. Susceptibility sults have been published recently [17,20] and showed a successful
5-year outcome for the majority of the patients, irrespective of
Table 3 shows the antibiotic susceptibility (MIC90 and range) of treatment strategy. Regarding bacterial sampling 5 years following
all anaerobic strains recovered from the predominant subgingival therapy in this population that kept a very high standard of oral
microbiota. Three (one strain of Bidobacterium spp., Propioni- hygiene [17], most samples did not produce predominant anaer-
bacterium propionicum and P. micra respectively) of these anaerobic obes and only 64 (39.8%) patients harbored anaerobes in their
isolates were resistant >4 mg/mL (EUCAST breakpoint), whereas all predominant ora. All of which showed a low degree of resistance
Porphyromonas spp. and Tannerella spp. isolates were highly sus- to MTZ, beta-lactam antibiotics, tetracycline and clindamycin.
ceptible to MTZ (MIC90 < 0.032 and 0.25 mg/mL respectively). All The present study dealt with the predominant anaerobic bac-
these strains were highly susceptible to PEN, AMX and CLI, while terial species using culture on non-selective media. It is possible
one strain of T. forsythia was TET resistant (MIC 16 mg/mL). Resis- that more resistant strains would have been disclosed using agar
tance against beta-lactam antibiotics (but susceptible for AMC) was plates containing breakpoint concentrations of the antibiotics, in
noted for 2 strains of F. nucleatum, and one strain each of Para- this case 4 mg/mL of MTZ. The disadvantage with such a method is
bacteroides distasonis and C. rectus. that most facultative oral bacterial species (e.g. streptococci, lac-
tobacilli, Actinomyces, Propionibacterium, Haemophilus and Neisseria
4. Discussion spp.) which are intrinsically resistant to metronidazole, and which
are commonly associated with periodontal health [9], would have
This study was conducted in order to investigate whether the overgrown a low number of resistant Gram-negative anaerobes.
resistance pattern among predominating anaerobes from the sub- Therefore, we cannot exclude the possibility that undetected MTZ
gingival microbiota of periodontitis patients had been inuenced

Table 1
Number of patients with Gram-negative anaerobes less or more than 0.5% of CFU in the 4 different periodontal therapy groups comprising scaling and root planing in 1 day
(FDIS) or over 3 weeks (SRP) with or without adjunctive MT.

Treatment No of Mean CFU x 106 No of samples with Gram-neg strains present in >0.5% Mean CFU x 106 in samples with Gram-neg present in >0.5%
Group patients (range) of CFU (range)

FDIS MTZ 36 72.4 (0.3e300) 16 71.6 (0.3e300)

FDIS 43 48.3 (0.67e304) 15 28.6 (0.67e304)
SRP MTZ 41 47.6 (0.36e304) 19 48.4 (0.21e304)
SRP 41 47.1 (2.8e208) 14 41.7 (2.8e208)
Total 161 64a
a
Totally 73 Gram-negative anaerobic strains were isolated. In 9 samples 2 strains were isolated.
 G. Dahlen, H.R. Preus / Anaerobe 43 (2017) 94e98 97

Table 3
Antibiotic susceptibility (MIC90 and range) for the anaerobic strains isolated from the predominant subgingival ora of all patients treated 5 years prior regardless of treatment
strategy (FDIS or SRP MTZ).

Bacteria Number of strains MTZ MIC90 (range) PEN MIC90 AMX MIC90 AMC MIC90 (range) TET MIC90 CLI MIC90 (range)
(range) (range) (range)

Fusobacterium/Filifactor 23 0.047 (0.016e1.0) 12 (0.016e24)b 12 (0.016e24)b 1.5 (0.016e2.0) 2.0 (0.016e2.0) 0.016 (0.016)
spp.
a b b
Prevotella/Bacteroides spp. 22 0.047 (0.016e0.5) 4.0 (0.016e128) 1.0 (0.016e64) 0.5 (0.016e1.0) 1.0 (0.016e0.38) 0.016 (0.016)
Porphyromonas spp. 21 0.25 (0.016e0.38) 0.064 (0.016e1.5) 0.016 (0.016e4.0) 0.032 (0.016 0.019 (0.016e4.0) 0.016 (0.016)
e0.032)
Tannerella forsythia 4 0.032 (0.016 0.38 (0.016e0.38) 0.19 (0.016e0.19) 0.125 (0.016 16 (0.016e16)b 0.016 (0.016)
e0.032) e0.125)
Campylobacter rectus 3 0.047 (0.032 16 (0.016e16)b 16 (0.016e16)b 0.016 (0.016) 0.016 (0.016) 0.016 (0.016
e0.047) e0.032)
Peptostreptococcus group 8 0.38 (0.016e0.38) 0.125 (0.016e1.0) 0.125 (0.16e0.25) 0.064 (0.016e0.35) 1.5 (0.016e2.0) 0.125 (0.016
e0.125)
b
Gram-positive rods 4 12 (0.016e12) 0.25 (0.016e0.25) 0.25 (0.016e0.38) 0.038 (0.016e0.38) 0.5 (0.016e0.5) 0.5 (0.016e0.5)
a
Including one strain of Parabacteroides distasonis.
b
Including 1e3 resistant strains. (Parvimonas micra, Prpionibacterium propionicum, Bidobacterium spp. Campylobacter rectus, parabacteroides distasonis and 2 strains of
Fusobacterium nucleatum).

resistant anaerobes may have been present in the subgingival ora
of these patients. On the other hand MTZ resistance among an-
aerobes is limited and MTZ is still the drug of choice in anaerobic
infections [14,15], including periodontal diseases [27].
An increasing prevalence of MTZ-resistance has been reported
[19]. In many countries MTZ, in combination with AMX, is used in
the treatment of Helicobacter pylori of peptic ulcer. Probably, as a
consequence, the incidence of antibiotic resistance of H. pylori to
MTZ has been reported as high as 70%, whereas the resistance rates
for AMX and TET were shown to be 0% and 5.8% respectively in the
same population [28]. In a study on 640 Bacteroides strains (mainly
B. fragilis) Soki et al. [29], found only 3 strains to be MTZ resistant,
all harboring chromosomally located nim genes. Four nim genes (A-
D) have been identied, all able to confer moderate to high MTZ
resistance levels [30,31]. The nim genes code for the enzyme 5-
nitroimidazole reductase, and have also been found in plasmids
[32,33], which make them able to spread by horizontal transfer to
other anaerobes resident of the gastrointestinal tract, including the
oral cavity. MTZ resistance in oral bacteria has only been sparsely
reported. Kuriyama et al. [34] did not nd any MTZ resistance (4 mg/
mL) among 800 anaerobic isolates from patients with dentoal-
veolar infection, and similarly Ready et al. [35] did not nd any child
to harbor MTZ resistant anaerobic bacteria in the oral anaerobic
microbiota. Rams et al. [36] found one strain each of P. intermedia
and F. nucleatum to be MTZ-resistant (16 mg/mL), while 33.8% and
6.6% respectively were resistant to AMX (8 mg/mL). A signicantly
higher number of antibiotic (including MTZ) resistant strains of
subgingival anaerobes from adult periodontitis patients were found
in Spain as compared to ditto strains isolated in The Netherlands
[37,38], explained by a more widespread use of antibiotics in Spain
as compared to that of the Netherlands. The present study shows
that antibiotic resistance in general, and to MTZ in particular, is
comparatively low in Norway where the prescription rate of anti-
biotics has been shown to be low. Norwegian dentists are only
responsible for 5% of the total antibiotic prescriptions in Norway
[39]. However, they stand for more than 10% of MTZ prescriptions
alone [40], and although this does not seem to inuence upon the
MTZ resistance development in the periodontal microora, the
resistance pattern of the subgingival ora may be changed and
should be continuously followed up in Norway as well as in other
populations in the future.
Many studies on adjunct MTZ in periodontal therapy have been
conducted with signicant clinical improvement but with unclear
microbiological outcome [6]. It is still a controversy whether the
reduction/elimination of the periodontopathogens justies the use
of adjunct antibiotics in view of the increasing risk for antibiotic
resistance [2]. Preus et al. [17,20] reported the 1-year results of the
here presented 5-year longitudinal project on different periodontal
treatment strategies using clinical (BOP, PPD, CAL) and microbio-
logical outcome variables. The results showed that in the group of
patients treated with MTZ P. gingivalis was reduced/eliminated,
whereas T. forsythia seemed more persistent one year following
therapy. However, although the outcome showed a statistically
signicant positive association between MTZ therapy and clinical
parameters, only modest effects were observed on the microbial
parameters [17,20]. It was argued that the observed discrepancy
between clinical and microbiological results could be explained by
the short follow-up (1 year) or a low susceptibility of the target
bacteria in vivo.
Some of the target bacteria in this study were the red complex
species, P. gingivalis and T. forsythia, both of which are extremely
susceptible to most antibiotics. No strains of Porphyromonas spp.
have to our knowledge been reported MTZ resistant. Oral species
such as P. gingivalis and P. endodontalis have proven highly sus-
ceptible to common oral antibiotics like PEN, AMX, AMC, CLI and
MET [34,37,38,41], while penicillin resistance has been reported in
gastrointestinal associated P. asaccharolytica [42]. One isolate of
P. assacharolytica in the present study had a MIC90 for AMX of 8 mg/
mL, showing that normally non-oral microorganisms may tran-
siently be present in the oral cavity and thereby participate in a
spread of transferrable elements into oral bacteria. Information on
antibiotic susceptibility patterns of T. forsythia is almost non-
existing. Takemoto et al. [23] reported a high susceptibility
among 15 strains for common antibiotics used in periodontal
therapy. Four strains of T. forsythia in the present study showed
high susceptibility to beta-lactam antibiotics, CLI and MTZ while
one strain showed increased resistance against TET (MIC 16 mg/
mL).
5. Conclusions
In this population of 161 patients who had experienced me-
chanical periodontal therapy with or without adjunct metronida-
zole 5 years prior to sampling, no cultivable MTZ resistant
anaerobes were found in the predominant subgingival microbiota.
It was concluded that the use of adjunct MTZ, together with sys-
tematic mechanical therapy and a very high level oral hygiene did
not result in antibiotic resistant oral bacteria 5 years following
therapy.
98 G. Dahlen, H.R. Preus / Anaerobe 43 (2017) 94e98
Acknowledgement
There is no conict of interest associated with this report, and
the work was nanced by the Norwegian Research Council, Oslo,
Norway; grant # 185120 and contd # 229029 and Lab of Oral
Microbiology, Institute of Odontology, University of Gothenburg,
Sweden. We are grateful for technical assistance by Mrs Lisbeth
Bengtsson and Mrs Susanne Blomqvist.
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