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Journal of Clinical Microbiology, October 1999, p. 3141-3145, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Incidence of Prevotella intermedia and
Prevotella nigrescens Carriage among Family Members with
Subclinical Periodontal Disease
Katsuhito
Fukui,1,2
Naoki
Kato,1,*
Haru
Kato,3
Kunitomo
Watanabe,1 and
Norichika
Tatematsu2
Institute of Anaerobic
Bacteriology1 and Department of Oral and
Maxillofacial Surgery,2 Gifu University School
of Medicine, Gifu 500-8705, and Department of Bacteriology,
School of Medicine, Kanazawa University, Kanazawa
920-8640,3 Japan
Received 17 December 1998/Returned for modification 9 March
1999/Accepted 14 June 1999
 |
ABSTRACT |
We established a typing system for Prevotella
intermedia and Prevotella nigrescens using the
combination of PCR ribotyping and arbitrarily primed PCR (AP-PCR)
fingerprinting and applied this system to the study of intrafamilial
incidence of these species in the oral cavity. PCR ribotyping followed
by subtyping by AP-PCR fingerprinting was applied to each type strain
of P. intermedia and P. nigrescens and 54 isolates (32 isolates of P. intermedia and 24 isolates of
P. nigrescens) from extraoral infections, resulting in an
excellent discriminatory power (discrimination index, 0.99) for both
species. A total of 18 subjects from six families, with the subjects
from each family comprising the mother, the father, and a child who had
subclinical early-stage to moderate adult periodontitis or simple
gingivitis and who carried P. intermedia or P. nigrescens, or both, were enrolled in the study of intrafamilial carriage. When 20 colonies per specimen of subgingival plaque, if
available, were picked from primary culture, 115 P. intermedia and 178 P. nigrescens isolates were
recovered from the 18 subjects. Among the subjects studied, family
members shared the same subtype strain(s) but non-family members did
not. Multiple subtypes were found in 8 (57%) of the 14 P. nigrescens-positive subjects but in only 3 (27%) of the 11 P. intermedia-positive subjects; the difference was,
however, not statistically significant (P = 0.14). These results suggest that the combination of PCR ribotyping and AP-PCR
fingerprinting is well suited for the epidemiological study of P. intermedia and P. nigrescens and that each family
seems to carry a distinct subtype(s) of these species.
| |
INTRODUCTION |
Prevotella intermedia
sensu lato is an obligatory anaerobic, black-pigmented,
gram-negative rod that is frequently associated with periodontal
disease: adult periodontitis, acute necrotizing ulcerative
gingivitis, and pregnancy gingivitis. This organism is also involved in
extraoral infections such as nasopharyngeal infection and
intra-abdominal infection (10).
It was recognized that there is heterogeneity within P. intermedia strains in terms of serology and DNA homology. In 1992, a comprehensive DNA-DNA hybridization study proposed that P. intermedia be classified into two genospecies, P. intermedia and Prevotella nigrescens (18).
Previous studies suggest that P. intermedia is likely to be
more associated with periodontal sites, whereas P. nigrescens seems to be more frequently recovered from healthy gingivae, although the site specificities of these two species remain
controversial (7, 11, 20).
It is of interest to investigate how humans get P. intermedia and P. nigrescens in the oral cavity and
what the consequence of acquisition of these organisms is. So far there
are few reports in the literature on the incidence or transmission of
P. intermedia and P. nigrescens within families
(11, 20). Establishment of efficient typing systems for
P. intermedia and P. nigrescens is fundamental
for epidemiological study of infecting or colonizing clones and for
tracing the transmission of microorganisms from person to person. The
techniques which have been used to type P. intermedia and
P. nigrescens include sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2, 19), restriction endonuclease
analysis (2, 15), ribotyping (15), and
arbitrarily primed PCR (AP-PCR) (10, 12). These techniques
are time-consuming or have low levels of reproducibility. PCR
ribotyping, which is expected to amplify the 16S and 23S spacer regions
of bacterial rRNA genes by PCR, has been demonstrated to be a simple,
rapid, and reliable typing technique (1, 8, 13). PCR
ribotyping generates simple banding patterns and, thus, allows
comparison of results obtained in separate PCR amplifications, whereas
AP-PCR does not work in this manner. Recent studies have suggested that
the use of multiple typing systems would be more reliable and would
give a higher discriminatory power than any one typing system for the typing of microorganisms (3, 6, 17).
In this study, we established a typing system for P. intermedia and P. nigrescens using the combination of
PCR ribotyping and AP-PCR fingerprinting and applied the established
typing system to the study of intrafamilial carriage of these species
in the oral cavity.
| |
MATERIALS AND METHODS |
Stock strains.
The type strains P. intermedia
ATCC 25611 and P. nigrescens JCM 6322 and 54 clinical
strains of P. intermedia sensu lato isolated from patients
with oral, otopharyngeal, and intra-abdominal infections at the
Institute of Anaerobic Bacteriology, Gifu University School of
Medicine, Gifu, Japan, were used to determine the discriminatory power
of a typing system that combined PCR ribotyping with AP-PCR fingerprinting. P. intermedia sensu lato was identified on
the basis of Gram staining, black-pigmented colony formation on
anaerobic blood agar, and the results obtained with the Rapid ID 32A
system (bioMerieux, Marcy-l'Etoile, France). Identification of
P. intermedia and P. nigrescens was done by the
PCR technique described below.
Subjects.
We first studied an individual (a mother, a
father, or a child) who visited a dental clinic due to dental diseases
other than periodontal diseases and who was found to have early-stage
to moderate adult periodontitis or simple gingivitis with a
pathologically deepened periodontal pocket of 
6 mm. Then, we asked
the remaining family members to be enrolled in this study in order to
cover all family members (the mother, the father, and the child).
Eventually, this study enrolled 18 families; the periodontal diseases
were subclinical for all subjects studied, and no particular treatment for the periodontal disease other than tooth brushing was required. None of the subjects enrolled in the study had systemic diseases.
Bacterial sampling, culture, and isolation.
The
supragingival dental plaque was removed with sterile cotton, and the
tooth surface was dried with compressed air to prevent contamination
with saliva. With exclusion of moisture in the mouth with sterile
cotton rolls, subgingival plaque was collected from the most inflamed
sites by inserting a sterile no. 60 paperpoint into the periodontal
pocket for 30 s (14). A plaque sample was inoculated
onto each of Brucella HK blood agar (Kyokuto Seiyaku, Tokyo, Japan) and
paromomycin-vancomycin Brucella HK blood agar (Kyokuto). The inoculated
media were immediately incubated in an anaerobic environment generated
with the AnaeroPack system (Mitsubishi Gas Chemical, Tokyo, Japan) for
3 or 4 days. Twenty black-pigmented colonies per specimen, if
available, were subcultured onto Brucella HK blood agar and were
subjected to the PCR technique (4) described below for the
identification of the isolates as P. intermedia or P. nigrescens.
DNA extraction.
Three- to 4-day-old cultures on Brucella HK
blood agar were suspended in 500 µl of lysis buffer (50 mM
Tris-hydrochloride [pH 8.0], 5 mM EDTA, 50 mM sodium chloride, 1 mg
of proteinase K per ml, 10% sodium dodecyl sulfate), and the mixture
was incubated for 45 min at 56°C. After centrifugation at 14,000 rpm
(15,000 × g) for 5 min, DNA was extracted twice with
70% phenol-water-chloroform (Perkin-Elmer Applied Biosystems, Foster
City, Calif.), adjusted to pH 7.6 with 1 M Tris-hydrochloride (pH 8.3),
and precipitated with an equal volume of isopropyl alcohol at 
20°C.
Following centrifugation at 14,000 rpm (15,000 × g)
for 10 min, a DNA pellet was rinsed with ice-cold 70% ethanol and was
resuspended in distilled water. The amount of extracted DNA was
determined with a spectrophotometer (Gene Quant II; Pharmacia Biotech,
Uppsala, Sweden), and the DNA sample was prepared to a concentration of
approximately 1,300 ng/ml for PCR amplification.
PCR amplification for identification of P. intermedia
and P. nigrescens.
The PCR primers used for the
identification of P. intermedia and P. nigrescens
have been described by Guillot and Mouton (4); Pi754-1
(5'-CAGCACCCACAACGATATGA-3') and Pi754-2
(5'-TTCCATCTTCTCTGCCTGTC-3') were used for P. intermedia and Pn1100-1 (5'-TTATGTTACCCGTTATGATGGAAG-3') and Pn1100-2 (5'-ATGGCGAAATAGGAATGAAAGTTA-3') were
used for P. nigrescens. PCR amplification was performed as
described previously (16). In brief, DNA amplification was
performed in a 30-µl reaction mixture consisting of 10 mM
Tris-hydrochloride (pH 9.0), 50 mM potassium chloride, 2.5 mM magnesium
chloride, each deoxynucleoside triphosphate at a concentration of 200 µM, and 0.75 U of Taq DNA polymerase (Pharmacia Biotech).
DNA amplification was done for 35 cycles in a DNA thermal cycler 480 (Perkin-Elmer Applied Biosystems). The PCR cycle included denaturation
for 20 s at 95°C and primer annealing-extension for 2 min at
50°C. As a final step, a 5-min extension was done at 74°C. The
reaction products were subjected to polyacrylamide gel electrophoresis
with a 5% polyacrylamide gel and a 100-bp DNA ladder (GIBCO BRL, Life
Technologies, Rockville, Md.) and were visualized under UV
transillumination following ethidium bromide staining.
PCR ribotyping and AP-PCR for P. intermedia and
P. nigrescens.
The primers used for PCR ribotyping
were Primer-1 (5'-TTGTACACACCGCCCGTCA-3') and Primer-pin2
(5'-GGTACCTTAGATGTTTCAC-3'); Primer-1 is a primer used for
PCR ribotyping of Burkholderia cepacia (9). PCR
was run for 35 cycles as follows: denaturation for 1 min at 95°C,
annealing for 1 min at 53°C, and extension for 2 min at 74°C. A
final extension was done for 5 min at 74°C.
AP-PCR was carried out with the primer set of ERIC1R
(5'-ATGTAAGCTCCTGGGGATTCAC-3') and ERIC2
(5'-AAGTAAGTGACTGGGGTGAGCG-3') (21). DNA
amplification was performed for 35 cycles, with each cycle comprising 1 min at 95°C, 1 min at 45°C, and 2 min at 74°C, with a single
final extension step for 5 min at 74°C.
The PCR amplicons were resolved by a polyacrylamide gel electrophoresis
with a 4% polyacrylamide gel. The banding patterns were analyzed with
Advanced Quantifier 1-D Match version 2.2.0 for Windows software
(Genomic Solutions Inc., Ann Arbor, Mich.) to obtain the percent
similarity of electrophoresis mobilities with the dendrogram of
averages (unweighted pair group method with arithmetic means) algorithm.
Statistical evaluation for discriminatory power.
The index
of discriminatory power (D) was obtained by the following
equation described by Hunter (5):
where
s is the number of the types,
xj is the number of population members falling
into the
jth type, and
N is the size of
the
population.
Data analysis.
For statistical analysis Fisher's exact
probability test was performed with StatView 4.0 (Abacus Concepts,
Berkeley, Calif.).
| |
RESULTS |
PCR ribotyping of stock strains of P. intermedia and
P. nigrescens.
Of 54 stock strains of P. intermedia sensu lato, 31 (57.4%) were identified as P. intermedia and 23 (42.6%) were identified as P. nigrescens by PCR amplification (data not shown).
PCR ribotyping was applied to
P. intermedia ATCC 25611,
P. nigrescens JCM 6322, and 54 stock strains (31
P. intermedia strains
and 23
P. nigrescens strains). Since
amplicons smaller than 600
bp were poorly reproducible, we analyzed a
few reproducible major
bands larger than 600 bp. Two or three DNA
products were generated
by most strains (Fig.
1).
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FIG. 1.
PCR ribotyping patterns of P. intermedia (A)
and P. nigrescens (B). (A) Lanes 1 and 10, 100-bp DNA
ladder; lanes 2 to 8, P. intermedia types A to G,
respectively; lane 9, negative control without DNA. (B) Lanes 1 and 13, 100-bp DNA ladder; lanes 2 to 11, P. nigrescens types A and
E to M, respectively; lane 12, negative control without DNA.
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|
The dendrograms for PCR ribotyping of
P. intermedia and
P. nigrescens are constructed separately (Fig.
2). Although the type
strain and 31 stock
strains of
P. intermedia were grouped into
seven types (Fig.
2A), the discrimination index was as low as
0.44 since type A is the
most predominant type, with 24 strains
(75%) being this type.
Twenty-four
P. nigrescens strains including
the type strain
were differentiated into 10 types (Fig.
2B), resulting
in a
discrimination index of 0.82. Type E was the most prevalent;
42% of
the 24 strains tested were this type. Types A, E, F, and
G were found
in two species (Fig.
2).
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FIG. 2.
Dendrograms of PCR ribotyping results for P. intermedia ATCC 25611 and 31 clinical strains of P. intermedia (A) and P. nigrescens JCM 6322 and 23 clinical strains of P. nigrescens (B). Each type was
designated by a letter from A to M. Types A, E, F, and G were found in
two species.
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|
Subtyping of stock strains by AP-PCR.
To generate a higher
discriminatory capability, the strains were further subtyped by AP-PCR
fingerprinting. All strains of the same PCR ribotyping type were
analyzed by one run of AP-PCR to evade the run-to-run variations in PCR
amplification efficiencies. Strains were subtyped by duplicate AP-PCR.
A representative result of the AP-PCR patterns of P. intermedia type A strains is shown in Fig.
3. Of the P. intermedia
strains, 24 of type A and 3 of type B were separated into 20 and 3 subtypes, respectively (data not shown). Of the P. nigrescens strains, 10 type E strains were grouped into 8 subtypes
and 3 type A, 2 type F, 2 type H, and 2 type I strains were all
discriminated into separate subtypes (data not shown). As a result, the
discrimination index of PCR ribotyping followed by AP-PCR was up to
0.99 for both P. intermedia and P. nigrescens.
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FIG. 3.
AP-PCR patterns of P. intermedia type A
strains. Lane 1, 100-bp DNA ladder; lanes 2 to 8, subtypes A1 to A7,
respectively; lane 9, negative control without DNA.
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|
Typing and subtyping of P. intermedia and P. nigrescens strains isolated in family study.
Mothers,
fathers, and children of 18 families were tested for carriage of
P. intermedia sensu lato. In 6 of the 18 families tested,
all three family members were found to carry P. intermedia sensu lato. Thus, the 18 subjects in the six families were analyzed further (Table 1). The ages of the
mothers and fathers studied ranged between 36 and 49 years (mean, 42.6 years), while the ages of the children were between 4 and 15 years
(mean, 11.0 years).
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TABLE 1.
Occurrence of P. intermedia and P. nigrescens in family members with adult periodontitis and simple
childhood gingivitisa
|
|
A total of 293 strains of
P. intermedia sensu lato were
isolated from 18 subjects and were identified by the PCR method; 115
were
P. intermedia and 178 were
P. nigrescens.
The number of isolates
per subject varied between 5 and 20 (mean,
16.3). Of the 18 subjects,
7 (39%) harbored both
species.
These isolates were first typed by the PCR ribotyping method (Table
1).
Of the 115
P. intermedia strains isolated, 93 were
type A, 1 was type E, 1 was type L, and 20 were type O, while
of the 178
P. nigrescens strains isolated, 2 were type A, 161
were type E, 1 was
type H, 1 was type L, 3 were type N, 8 were
type P, 1 was type Q, and 1 was type R. Of 18 subjects, 12 (67%)
carried more than one type of the
same species.
P. intermedia type A was recovered from 10 subjects (56%) and types E, L, and
O were recovered from only 1 subject each, while
P. nigrescens type E was found in 13 subjects (72%) and types A, H, L, N, P,
Q, and R were recovered from 1 subject
each.
To clarify family-to-family variations in
P. intermedia and
P. nigrescens strains, AP-PCR fingerprinting was carried out
to
subtype the strains (Table
1); type A, the most predominant type
of
P. intermedia, was subtyped into 7 subtypes (subtypes A1 to
A7), and type E, the most predominant type of
P. nigrescens,
was
subtyped into 10 subtypes (subtypes E1 to E10). As representative
results, the results of PCR ribotyping and AP-PCR for family 2
(Table
1) are shown in Fig.
4. Eventually,
P. intermedia was
isolated from five families and 11 subjects and
P. nigrescens was recovered from six families
and 15 subjects, but the same
subtypes were never shared by different
families (Table
1). The
same subtypes were shared by two pairs of
spouses for
P. intermedia (Table
1, families 1 and 2) and
two pairs of spouses for
P. nigrescens (Table
1, families 4 and 5). The same subtypes were shared by
a parent and the child of
three families for
P. intermedia (Table
1, families 1, 2, and 3) and three families for
P. nigrescens (Table
1,
families 4, 5, and 6). Multiple subtypes were found
in 8 (57%) of the
14
P. nigrescens-positive subjects and 3 (27%)
of the 11
P. intermedia-positive subjects, but the difference
was not
statistically significant (
P = 0.14).
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FIG. 4.
Results of PCR ribotyping (a) and AP-PCR (b) for
P. intermedia strains isolated from family 2. (a) Lanes 1, 5, and 10, 100-bp DNA ladder; lanes 2 and 3, isolates from the mother;
lane 4, an isolate from the father; lanes 6 to 8, isolates from the
child; lane 9, negative control without DNA. (b) Lanes 1 and 7, 100-bp
DNA ladder; lanes 2 and 3, isolates from the mother; lane 4, an isolate
from the father; lanes 5 and 6, isolates from the child.
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|
| |
DISCUSSION |
PCR ribotyping, which demonstrates variations in the length and
the number of the 16S-23S rRNA gene spacer regions of microorganisms, has been used to type several bacterial species (1, 8, 13) since this technique gives rather stable typing results on repeat testing and simple banding patterns and thus allows the comparison of
the results obtained by separate runs. However, in this study PCR
ribotyping proved not to be a highly discriminatory method for strains
of P. intermedia and P. nigrescens. Meanwhile,
AP-PCR for these organisms gave reliable typing results with
significant discriminatory power in a run but had run-to-run variations
(data not shown). Thus, in this study PCR ribotyping was applied to group strains into types, and then strains were subtyped by a separate
AP-PCR run for each type. As a consequence, PCR ribotyping followed by
subtyping by AP-PCR fingerprinting for isolates from patients with
extraoral infections had an excellent discrimination power of 0.99 for
both P. intermedia and P. nigrescens species. This system seems to provide valuable information on the predominant PCR ribotypes of P. intermedia and P. nigrescens
in a population and to be highly discriminating for strains of these
two species.
On the basis of these results, the combination of PCR ribotyping and
AP-PCR was applied to the study of intrafamilial carriage of P. intermedia and P. nigrescens. Our data indicate that a
P. intermedia or P. nigrescens strain(s) of the
same subtype can colonize spouses or parents and children. The results
are compatible with those of previous studies (11, 20).
Recently, studies have reported that P. intermedia seems to
be associated with advanced periodontitis and that P. nigrescens is likely to be predominant at healthy gingival sites
of children (11, 20). This study demonstrated that both
P. intermedia and P. nigrescens were predominant
in adults and children who had subclinical early-stage to moderate
periodontal disease for which no treatment but tooth brushing was
required. To draw conclusions about the pathogenicities of these
species, a comparative evaluation of the incidence of P. intermedia, P. nigrescens, and another established
periodontal pathogen such as Porphyromonas gingivalis should
be performed on the basis of the stage of periodontal disease.
In conclusion, the combination of PCR ribotyping and AP-PCR
fingerprinting proved to be well suited for the epidemiological study
of P. intermedia and P. nigrescens. The same
genotype(s) of both species can colonize spouses or parents and children.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Anaerobic Bacteriology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. Phone: 81-58-267-2342. Fax:
81-58-265-9001. E-mail: nk19{at}cc.gifu-u.ac.jp.
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Journal of Clinical Microbiology, October 1999, p. 3141-3145, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
