Detection of Fusobacterium Nucleatum and fadA Adhesin Gene in Patients with Orthodontic Gingivitis and Non-Orthodontic Periodontal Inflammation
et al. (2014) Detection of Fusobacterium Nucleatum and fadA Adhesin Gene in Patients with Orthodontic Gingivitis
and Non-Orthodontic Periodontal Inflammation. PLoS ONE 9(1): e85280. doi:10.1371/journal.pone.0085280
Detection of Fusobacterium Nucleatum and fadA Adhesin Gene in Patients with Orthodontic Gingivitis and Non-Orthodontic Periodontal Inflammation
Ping Liu 0
Yi Liu 0
Jianning Wang 0
Yang Guo 0
Yujie Zhang 0
Shuiqing Xiao 0
Anil Kumar, University of Missouri-Kansas City, United States of America
0 1 Department of Orthodontics, Jinan Stomatological Hospital , Jinan , China , 2 Pediatric Research Institute, Qilu Children's Hospital of Shandong University , Ji'nan, China , 3 Department of Periodontics, Jinan Stomatological Hospital , Jinan , China
Fusobacterium nucleatum is one of the most abundant gram-negative bacilli colonizing the subgingival plaque and closely associated with periodontal disease. However it is unclear whether F. nucleatum is involved in gingival inflammation under orthodontic appliance. A novel adhesin, FadA, which is unique to oral Fusobacteria, is required for F. nucleatum binding and invasion to epithelial cells and thus may play an important role in colonization of Fusobacterium in the host. In this study, we evaluated the prevalence of F. nucleatum and its virulence factor FadA adhesion gene (fadA) in 169 subgingival biofilm samples from 55 cases of gingivitis patients with orthodontic appliances, 49 cases of gingivitis patients without orthodontic treatment, 35 cases of periodontitis patients and 30 cases of periodontally healthy people via PCR. The correlations between the F. nucleatum/fadA and gingivitis index(GI)was also analyzed. The detection rate of F. nucleatum/fadA in periodontitis group and non-orthodontic gingivitis group was higher than the other two groups (p,0.01) while it was higher in orthodontic gingivitis group than in health people (p,0.05). An obviously positive correlation was observed between the prevalence of F. nucleatum/fadA and GI. F. nucleatum carrying fadA may be more closely related to the development of gingivitis and periodontal disease compared with orthodontic gingivitis.
. These authors contributed equally to this work.
Fixed orthodontic treatment is currently the preferred and most
common method for malocclusion which is a frequently-occurring
disease affecting facial appearance and chewing function. During
orthodontic therapy, orthodontists are frequently confronted with
gingivitis . Studies have reported that orthodontic attachments
can accelerate the accumulation of bacterial plaque for the
difficulties in maintaining oral hygiene . Also the placement of
orthodontic appliances affects the subgingival microbial
composition even during the early period of orthodontic treatment,
increasing the prevalence of periodontopathogens .
F. nucleatum is a gram-negative anaerobes ubiquitous in the oral
cavity, presenting in both healthy and diseased periodontal sites
and associated with various forms of periodontal diseases . The
bacterium has been reported to induce apoptosis in gingival
epithelial cells and polymorphonuclear blood cells; in addition, it
suppresses immunological defense mechanisms [4,5] and induces
innate immune responses [6,7].A novel adhesin, FadA, was
identified to be involved in F. nucleatum attachment and invasion
to host cells and highly conservative among oral Fusobacteria species
. Previous studies have shown that F. nucleatum is closely related
to adult and juvenile periodontitis [9-11], but little researches on
gingival response to F. nucleatum and its virulence factor FadA
adhesin during orthodontics.
Our previous research has showed that Porphyromonas gingivalis,
the gram-negative oral anaerobe, is one of the risk factors that are
responsible for orthodontic gingivitis and periodontitis.
However, another periodontitis-associated bacterium, F. nucleatum
with little available information has not been detected. The
purpose of this study was to evaluate the prevalence of F. nucleatum
and FadA adhesin in subgingival biofilm samples from the gingivitis
lesions of orthodontic patients and compared them with that of
non-orthodontic gingivitis and periodontitis patients as well as
periodontal healthy people who showed healthy periodontal tissues
before wearing orthodontic appliances. Also, the correlation
between detection rate of F. nucleatum/fadA and GI was analyzed.
Materials and Methods
The study subjects consisted of four groups who visited Jinan
Stomatological Hospital for orthodontics or periodontitis
treatment from 2011 to 2013. Of four groups, orthodontic group (OG)
included 55 patients, 21 females and 34 males, aged between 11
and 27 years (mean 16.25) who got gingival inflammation during
orthodontic treatment; control group (CG) contained 30
periodontal healthy people, 18 females and 12 males, aged between 12
and 26 years (mean 19.40) before orthodontic treatment;
nonorthodontic gingivitis group (NOG) was made up of 49 gingivitis
patients without orthodontic treatment, 26 females and 23 males,
aged between 12 and 25 years(mean 16.62); periodontitis group
(PG) was composed of 35 periodontitis patients, 16 females and 19
males, aged from 22 to 68 years (mean 46.46). These patients with
any systemic diseases, antibiotics therapy within the last 3 months
and pregnant or lactating females were excluded.
This work was approved by the Medical Ethics Committee of
the Jinan Stomatological Hospital. We obtained written informed
consents from the patients or parents on the behalf of all children
participants involved in the study before the examination was
performed. The relevant regulations and institutional polices were
The reference strains of F.nucleatum ATCC25586 and
Aggregatibacter actinomycetemcomitans ATCC29522 were from the
WestChina Dental School of Si Chuan University. Porphyromonas
gingivalis W83 and Streptococcus mutans ATCC25175 were from
Beijing Oral Research Institute of Capital Medical University.
Evaluation of gingival status
According to the standard revised by Loe , gingival status
was checked and recorded in four gums areas: buccal gingival
papilla, mesial buccal marginal gingiva, buccal and distal gingival
papilla, lingual marginal gingiva. Gingival inflammation was
divided into three levels, 0, 1, 2 and gingival index (GI) was
assessed. All clinical examinations were carried out by the same
Sample collection and DNA extraction
Subgingival biofilm was obtained from the deepest periodontal
pockets as described before [14,15] In brief, before collecting,
saline solution was used to rinse out food debris and then each site
was cleaned by cotton rolls. Visible supragingival plaque was
removed. A sterile paper point was inserted into the pocket for 30
seconds until a minimum of resistance was felt. The paper point
was immediately transferred into a sterile microcentrifuge tube
containing 0.5 ml of 16PBS. The tubes were mixed thoroughly
and stored at 220uC until analyzed. The bacterial DNA was
extracted by the boiling method [12,16]. In short, a 10 ml aliquot
of each stored sample was added to 10 ml of 2 6lysis buffer (2 mM
EDTA, 1% X-100). The mixture was boiled for 10 minutes and
then placed on ice. The supernatant was used as the template for
Specificity of the 16S rRNA-based PCR
Specificity of the 16S rRNA-based PCR was evaluated by using
specific primers of 16SrRNA gene and the reference strains,
including F.nucleatum ATCC25586, A. actinomycetemcomitans
ATCC29522, P. gingivlis ATCC33277 and S. mutans ATCC25175l.
The amplified products from clinical samples were randomly
chosen for sequencing.
The 16S rRNA-based PCR and FadA specific PCR
The 16S rRNA-based PCR was used to determine the
prevalence of F. nucleatum in subgingival biofilm. The PCR was
performed on DNA extracts from subgingival biofilm samples
using F. nucleatum primers of 16S rRNA-F (59-AGA GTT TGA
TCC TGG CTC AG -39) and 16S rRNA-R (59-GTC ATC GTG
CAC ACA GAA TTG CTG-39) to amplify a 360-bp region of the
16S rRNA gene, while using fadA primers of fadA-F (59-CAC
AAG CTG ACG CTG CTA GA -39) and fadA-R (59-TTA CCA
GCT CTT AAA GCT TG -39) to amplify a 232-bp region of the
FadA gene (designed for this study) from positive samples of
F.nucleatum. Amplification reaction was run in a Tetrad Thermal
Cycler (MJ Research, South San Francisco, USA) in a 25 ml
reaction mixture containing 4.5 ml 106PCR buffer (100 mM
TrisHCl, 500 mM KCl, and 15 mM MgCl2), 0.25 mM of each
deoxynucleoside triphosphate (dNTP), 10 mM of each primers,
5 ml of DNA extracts from subgingival biofilm samples, and 1.5
units of Taq DNA polymerase (Transgen Biotech, Beijing). The
16S rRNA PCR of F. nucleatum was carried out for 5 min at 94uC
and 30 cycles, with each cycle consisting of denaturation at 94uC
for 30 sec, annealing at 58uC for 30 sec, extension at 72uC for
1 min, and final extension for 10 min. The PCR of fadA was
carried out for 4 min at 94uC and 30 cycles, with each cycle
consisting of denaturation at 94uC for 30 sec, annealing at 55.8uC
for 30 sec, extension at 72uC for 40 sec, and final extension for
The amplified products were then electrophoresed on 1.5%
agarose gel in Tris-acetate buffer (40 mM Tris acetate, 1 mM
EDTA, pH8.0). The products were visualized with ethidium
bromide by UV transillumination.
Chi-squared test was used to compare detection rates of F.
nucleatum and fadA among four groups. The Spearmans rank
correlation analysis was utilized to determine the correlation
between prevalence of F. nucleatum/fadA genes and GI in four
research groups. All statistical analyses were done by using a
statistical software package (SPSS for Windows 17.0). p,0.05
were considered to be statistically significant.
Detection and confirmation of 16S rRNA-based PCR for F.
The reference strains were first amplified by the 16S
rRNAbased PCR to evaluate the specificity of it. Agarose gel
electrophoresis showed that a 360bp specific amplification was
obtained only from F.nucleatum ATCC25586, not from P.gingivalis
W83, A.actinomycetemcomitans ATCC29522, S.mutans ATCC25175
and double distilled water.
F. nucleatum was detected in 122 (72.19%) cases of subgingival
biofilm samples from 169 cases of four groups, 38 (69.09%) from
OG, 14 (46.67%) from CG, 41(83.67%) from NOG, and 29
(82.86%) from PG (Fig 1.a, Table1).
Ten out of 122 F. nucleatum positive samples were randomly
selected for sequencing in Invitrogen Company (Invitrogen,
Shanghai) to confirm the validity of the 16S rRNA-based PCR
in clinical subgingival biofilm samples (Fig. 2).
PCR amplification of FadA gene
fadA specific PCR was used to amplify FadA gene firstly from the
reference strains of F.nucleatum ATCC25586 and then from 122
positive samples of F. nucleatum to generate a 232-bp product.
There were 101 fadA positive samples when fadA primers were
used to amplify FadA gene from 122 F.nucleatum positive samples.
The detection rate of fadA in all cases of subgingival samples from
four groups was 59.79%, 58.18% from OG, 33.33% from CG
69.39% from NOG, and 71.43% from PG, individually (Fig 1.b,
Figure 1. Detection and distribution of F. nucleatum/fadA. a. Detection of F. nucleatum in clinical subgingival biofilm samples. M:Marker;lane
1:positive control of F. nucleatum ATCC25586;lane 2:blank; lane 3,10:positive clinical samples;lane 11 and12:negative clinical samples; b. Detection of
fadA in clinical subgingival biofilm samples. M:Marker;lane 1:positive control of F. nucleatum ATCC25586;lane 2:blank;lane 3,4,6,10:positive clinical
samples;lane 5: negative clinical sample; c. Distribution of F. nucleatum in four groups. d. Distribution of fadA in four groups. ** P,0.01 between GI 2
and GI 0 in PG and NOG (c, d) (Chi-squared test).
For both of F. nucleatum and fadA, the detection rate was higher
in group OG than that in group CG(P,0.05); while the detection
rates in group PG and NOG were significantly higher than that in
group CG(P,0.01)(Table 1).
Correlation of F. nucleatum/fadA and GI
We found that the prevalence of F nucleatum and fadA increased
with GI value. For detection of F. nucleatum, 20 out of 40 (50%)
cases in level 0 of GI were positive; in level 1 of GI, 48 (75%) were
positive and 54 (80.85%) were positive in level 2 of GI; For fadA, in
GI 0, 14 out of 40(35%) were positive; in GI 1, 39 (60.94%) were
positive and in GI 2, 48 (73.85%) were positive. From 122 positive
cases for F. nucleatum, 101(82.79%) were also positive for fadA. The
detection rates of F. nucleatum and fadA rose with GI in clinical
samples. An obvious positive correlation(P,0.05)was observed
between GI and the prevalence of F. nucleatum/fadA by using
Spearmans rank correlation analysis. However, there was no
statistical difference between positive rate of F.nucleatum/fadA and
GI in OG. The prevalence of F.nucleatum/fadA was observed
significantly higher only in GI 2 from PG and NOG than from
CG (Fig.1c, 1d).
*P,0.05 between OG/NOG/PG and CG; **P,0.01 between OG/NOG/PG and CG (Chi-squared test).
Sallum et al.  investigated the clinical and microbiologic
changes after removal of orthodontic appliances and found
periodontal pathogens such as A.actinomycetemcomitans and B.forsythus
were associated with gingival inflammation during orthodontic
treatment. F. nucleatum is reported playing an important role for
periodontal diseases [3,18]. In this study, we detected prevalence
of F. nucleatum and FadA adhesin gene in subgingival biofilm in
local patients of orthodontic gingivitis, non-orthodontic gingivitis,
periodontitis as well as periodontally healthy people to evaluate the
distribution of F. nucleatum and fadA in different periodontal health
status, then further deduced the pathogenicity of F. nucleatum
We randomly collected subgingival biofilm samples with sterile
paper point from 169 patients. The prevalence of F. nucleatum was
detected and the correlation of it with GI was analyzed. There
were significant differences among the four group(P,0.01).
Meanwhile there was a positive correlation between the positive
rate and GI by using Spearmans rank correlation analysis. The
detection rate of F. nucleatum as one of main periodontal
inflammation pathogens increased with the severity of periodontal
lesion. However, there were no statistical differences among
positive rates of F. nucleatum in three GI levels in OG, while
detection rate of F. nucleatum in GI2 from PG and NOG was
significantly higher than that from both groups of CG and OG.
After wearing the fixed appliance, such as brackets, bands and
arch wires, the accumulation of bacterial plaque increases the
difficulty of maintaining oral hygiene, which may result in
increased sulcus bleeding index, gingival inflammation and
hyperplasia . Orthodontic treatment may create a living
environment more conducive to periodontal anaerobe such as F.
nucleatum, which might imply a potential risk for periodontal health
in certain patients after longtime orthodontic treatment.
Some relevant clinical studies confirmed the differences between
orthodontic gingivitis and periodontitis. Polson et al.  found
that orthodontic treatment during adolescence had no distinct
effect upon later periodontal health. Gingival inflammation and
gingival bleeding will increase in teenagers as a result of the
hormone changes that occur during puberty . A systematic
review identified an absence of reliable evidence describing
positive effects of orthodontic treatment on periodontal health,
but many findings indicated that orthodontic therapy resulted in
small detrimental effects to the periodontium . A controlled
clinical study of persons who had completed orthodontic therapy
at least 10 years previously compared to a group of adults with
untreated malocclusion demonstrated that orthodontic treatment
during adolescence had no distinct effect upon later periodontal
health . In this study, we analyzed correlation of patients age
and occurrence of F. nucleatum and found the age of both F.
nucleatum positive and negative was statistically different which
implied the prevalence of F. nucleatum may increase with
patientsage, while the incidence of periodontal disease also increases.
Therefore, longitudinal studies including large amount of samples
are required to find the impact of F. nucleatum colonization on
periodontal conditions during and after orthodontic therapy.
Bacterial adhesion is usually the first step for a periodontal
pathogen to infect and invade the host cells. In 2005, a novel
adhesin, FadA, which is unique to oral Fusobacteria was identified by
Han et al . It was required for F. nucleatum to attach epithelial
cells and thus may play an important role in Fusobacterium
colonization in the host. In this study, we further detected the
distribution of fadA in four groups to investigate whether it is
gingival inflammation under orthodontic appliance. The
detection rate of fadA decreased in turn from NOG, PG, OG to
CG group. Also, it had an upward trend with the increase of
gingival index. A clear positive correlation was indicated between
GI and FadA gene by using Spearmans rank correlation analysis.
However, only the prevalence of fadA in GI 2 from PG and NOG
was significantly higher than that in CG, while there were no
statistical difference among positive rate of fadA in three GI levels
in OG. The F. nucleatum carrying fadA may have a higher
pathogenicity and could lead to a classification of these strains,
which is more closely related to the development of
nonorthodontic periodontal inflammation rather than gum
inflammation during orthodontic treatment. On the contrary, the F.
fadA may represent the avirulent or weak virulence genotype of
In summary, F. nucleatum carrying fadA is one of the potential
risks that are responsible for non-orthodontic periodontal
inflammation. All orthodontic patients must receive oral hygiene
instruction and professional prophylaxis to maintain gingival
health. Moreover, further research is needed to verify the
periodontal potential health risks and to find the most effective
way of controlling periodontal pathogenic anaerobic bacteria
during orthodontic treatment.
Conceived and designed the experiments: SX YL. Performed the
experiments: PL YG YZ. Analyzed the data: PL JW YG. Contributed
reagents/materials/analysis tools: JW YG YZ. Wrote the paper: PL YL
1. Alexander SA ( 1991 ) Effects of orthodontic attachments on the gingival health of permanent second molars . Am J Orthod Dentofacial Orthop 100 ( 4 ): 337 - 340 .
2. Kim SH , Choi DS , Jang I , Cha BK , Jost-Brinkmann PG , et al. ( 2012 ) Microbiologic changes in subgingival plaque before and during the early period of orthodontic treatment . Angle Orthod 82 ( 2 ): 254 - 260 .
3. Edwards AM , Grossman TJ , Rudney JD ( 2006 ) Fusobacterium mucleatum transports noninvasive Streptococcus cristatus into human epithelial cells . Infect Immun 74 ( 1 ): 654 - 662
4. Jewett A , Hume WR , Le H , Huynh TN , Han YM , et al. ( 2000 ) Induction of apoptotic cell death in peripheral blood mononuclear and polymorphonuclear cells by an oral bacterium, Fusobacterium nucleatum . Infect Immun 68 ( 4 ): 1893 - 1898 .
5. Hall ER , Marin SA , Suzuki JB , Falkler WA Jr ( 1994 ) The gingival immune response to periodontal pathogens in juvenile periodontitis . Oral Microbiol Immunol 9 ( 6 ): 327 - 334 .
6. Gursoy UK , Kononen E , Uitto VJ ( 2008 ) Stimulation of epithelial cell matrix metalloproteinase (MMP-2, -9 , - 13 ) and interleukin-8 secretion by fusobacteria . Oral Microbiol Immunol 23 ( 5 ): 432 - 434
7. Gursoy UK , Pollanen M , Kononen E , Uitto VJ ( 2012 ) A novel organotypic dento-epithelial culture model: effect of Fusobacterium nucleatum biofilm on Bdefensin-2, -3, and LL-37 expression . J Periodontol 83 ( 2 ): 242 - 247 .
8. Han YM , Ikegami A , Rajanna C , Kawsar HI , Zhou Y , et al. ( 2005 ) Identification and characterization of a novel adhesin unique to oral fusobacteria . J Bacteriol 187 ( 15 ): 5330 - 5340
9. Feng X , Zhang L , Xu L , Meng H , Lu R , et al. ( 2013 ) Detection of 8 periodontal microorganisms and distribution of Porphyromonas gingivalis fimA genotypes in Chinese patients with aggressive periodontitis . J Periodontol May 7 . [Epub ahead of print]
10. Stingu CS , Jentsch H , Eick S , Schaumann R , Knofler G , et al. ( 2012 ) Microbial profile of patients with periodontitis compared with healthy subjects . Quintessence Int 43 ( 2 ): e23 - 31 .
11. Joshi VM , Vandana KL ( 2007 ) The detection of eight putative periodontal pathogens in adult and rapidly progressive periodontitis patients: an institutional study . Indian J Dent Res 18 ( 1 ): 6 - 10 .
12. Liu Y , Zhang YJ , Wang L , Guo Y , Xiao SQ ( 2013 ) Prevalence of Porphyromonas gingivalis Four rag Locus Genotypes in Patients of Orthodontic Gingivitis and Periodontitis . PLoS One 8 ( 4 ): e61028 .
13. Loe H , Silness J ( 1963 ) Periodontal disease in pregnancy I: Prevalence and severity . Acta Odont Scand 21 : 533 - 551 .
14. Belibasakis GN , Schimidlin PR , Sahrmann P ( 2013 ) Molecular microbiological evaluation of subgingival biofilm sampling by paper point and curette . APMIS , doi:10.1111/apm.12151. [Epub ahead of print]
15. Lee SM , Yoo SY , Kim HS , Kim KW , Yoon YJ , et al. ( 2005 ) Prevalence of putative periodontopathogens in subgingival dental plaques from gingivitis lesions in Korean orthodontic patients . J Microbol 43 ( 3 ): 260 - 265 .
16. Liu X , Xiao S , Liu Y ( 2009 ) Multiplex PCR detecting four species of bacteria in oral specimens from orthodontic patients . Chin J Lab Diagn 13 ( 11 ): 1579 - 1582 .
17. Sallum EJ , Nouer DF , Klein MI , Goncalves RB , Machion L , et al. ( 2004 ) Clinical and microbiologic changes after removal of orthodontic appliances . Am J Orthod Dentofacial Orthop 126 ( 3 ): 363 - 366 .
18. Conrads G , Gharbia SE , Gulabivala K , Lampert F , Shah HN ( 1997 ) The use of a 16s rDNA directed PCR for the detection of endodontopathogenic bacteria . J Endod 23 ( 7 ): 433 - 8 .
19. Moore WE , Moore LV ( 2000 ) The bacteria of periodontal diseases . Periodontol 5 : 66 - 77
20. Signat B , Roques C , Poulet P , Duffaut D ( 2011 ) Fusobacterium nucleatum in periodontal health and disease . Curr Issues Mol Biol 13 ( 2 ): 25 - 36 .
21. Bollen AM , Cunha-Cruz J , Bakko DW , Huang GJ , et al. ( 2008 ) The effects of orthodontic therapy on periodontal health: a systematic review of controlled evidence . J Am Dent Assoc 139 ( 4 ): 413 - 422 .
22. Liu H , Sun J , Dong Y , Lu H , Zhou H , et al. ( 2011 ) Periodontal health and relative quantity of subgingival Porphyromonas gingivalis during orthodontic treatment . Angle Orthod 81 ( 4 ): 609 - 615 .
23. Polson AM , Subtelny JD , Meitner SW , Polson AP , Sommers EW , et al. ( 1988 ) Long-term periodontal status after orthodontic treatment . Am J Orthod Dentofacial Orthop 93 ( 1 ): 51 - 58 .
24. Mombelli A , Gusberti FA , van Oosten MA , Lang NP ( 1989 ) Gingival health and gingivitis development during puberty. A 4-year longitudinal study . J Clin Periodontol 16 ( 7 ): 451 - 456 .