Prevalence of fimA genotypes of Porphyromonas gingivalis in adolescent orthodontic patients

Background The placement of fixed orthodontic appliances may alter the composition of oral microbiota and has the potential risk of periodontal complication. Porphyromonas gingivalis fimbriae play a critical role in colonization of P. gingivalis in subgingival regions. In this study, we investigated the association between the prevalence of P. gingivalis-specific fimA genotypes and periodontal health status in adolescent orthodontic patients, to identify the pathogencity of P. gingivalis during orthodontic therapy. Methods Sixty-one adolescent orthodontic patients were enrolled in the case group, while the control group consisted of 56 periodontally healthy adolescents. At baseline (T0), clinical parameter (gingival index) was tested, and subgingival plaque samples were obtained from the lower incisors. The incidences of P. gingivalis and fimA genotypes were detected by polymerase chain reaction. All parameters were reassessed after 1 month (T1), 2 months (T2), 3 months (T3), and 6 months (T4) in the case group and then compared with those of the controls. Results Both microbiological and clinical parameters from orthodontic patients started to increase after placement of fixed appliances. Maximum values were reached at 3 months after placement and followed by their decreases at six months. However, the microbiological and clinical parameters in the case group were significantly higher than those of the control group. The GI of fimA II, IV-positive samples was significantly higher than that of negative samples. Conclusion P. gingivalis carrying fimA II or IV was closely related to orthodontic gingivitis. In addition, proper oral hygiene control could lead to little increase in dental plaque accumulation, and exert a beneficial effect to periodontal tissues.

Introduction Fixed orthodontic therapy is the most effective method in contemporary orthodontics, however, it may cause periodontal complication due to hampering oral hygiene and altering the nature of dental plaque [1]. The overall incidence of gingivitis associated with orthodontic therapy was 56.8% in adolescents and 34.4%in adult in China, respectively [2].
It has been confirmed that anaerobic microbiota in subgingival plaque are the primary agents in the initiation and progression of periodontal diseases [1]. Previous studies by our team and other researchers demonstrated that there were positive associations between periodontal diseases and several pathogens, including A. actinomycetemcomitans , P. gingivalis, P. intermedia, and T. forsythia [1,[3][4][5][6]. P. gingivalis is considered as a putative etiological agent in different types of periodontal diseases, whose virulence attributes to its various surface components, such as lipopolysaccharide, capsule, gingipain, fimbria and so on [6][7][8][9]. Among them, the major fimbrial subunit, fimbrillin, is thought to play critical roles in the colonization of P. gingivalis in periodontal tissues, as it promotes bacterial adhesion to and invasion to subgingival regions [10][11][12][13][14][15]. The P. gingivalis fimA gene, which encodes fimbrillin, is a single copy on the bacterial chromosome and no homologous sequences were detected in other gram-negative oral anaerobe [16]. FimA has been classified into six variants (types I-V, Ib) according to clonal variations of its nucleotide sequences [17][18][19]. Accumulating evidences suggested that P. gingivalis strains with different fimA genotypes displayed distinct virulence features [20,21]. According to the diversity of pathogenicity, there are two groups in P. gingivalis stains, disease related and non-disease related, and the variance of genetic heterogeneity was the base of the differential of pathogenicity in microorganisms [22,23]. Analysis of the different fimA genotypes and their clonal distribution may provide insight into the epidemiology of periodontal diseases.
It is demonstrated that fixed appliances could alter the equilibrium of the microorganism ecosystem, and increase the colonization of pathogenic bacteria which is responsible for periodontal diseases [1,24,25]. Our previous research showed that the prevalence of P. gingivalis detected at two months after orthodontic therapy was as much as 31.11%higher than baseline and the count of P. gingivalis assessed immediately after removing orthodontic appliances was obviously higher than that of healthy group [1,26]. However, there was no report on the variation in distribution of fimA genotypes during the orthodontic therapy.
In this case, we assessed the prevalence of P. gingivalis and fimA genotypes in subgingival plaque samples among different time points after placing orthodontic appliances, to identify the pathogencity of P. gingivalis with different fimA genotypes during orthodontic therapy.

Ethics statement
The human subject protocol was approved by the Ethics Committee of Jinan Stomatological Hospital and School of Stomatology, Shandong University, and written informed consents were provided by the patients of each participant.

Subjects
Subjects of both case and control groups were consecutively recruited from Department of Orthodontics, Jinan Stomatological Hospital during June 2015-October 2015 with same procedure and criteria as described previously [1]. In short, 107periodontally healthy adolescents who referred to our hospital for orthodontic treatment were enrolled in this study and randomly assigned to be the case group and control group. Among them, sixty-one subjects (22 males and 39 females, mean age 13.80±1.61 years, ranging from 11 to 17 years, S1 Table) were classified into the case group and 56 subjects (16 males and 40 females, mean age 14.24±1.30 years, ranging from 12 to 17 years) were grouped as the controls. Both groups matched for age and sex. All participants met the following criteria: (1) a healthy gingival condition defined as a periodontal probing depth of less than 4mm and a gingival index less than 1, (2) no smoking, (3) no known systemic disease, (4) no alveolar bone loss visible on X-ray, (5) absence of fixed restorations or removal partial dentures, (6) no use of antibiotics within 3 months before the study, (7) no periodontal therapy within the previous 6 months. [1].
All subjects in the case group were treated with fixed buccal metal appliances (Victory, 3M Unitek, Calif) on incisors, premolars, and molars. The arch wires were ligated with elastomeric ligature. The brackets were placed on both jaws when the first clinical periodontal examination was done. The oral hygiene instructions (tooth-bushing Bass skill) and correct method of using tooth brush were provided for all case subjects before placing brackets (S1 Appendix).

Bacteria strains
The reference strains of P. gingivalis (ATCC33277), F. nucleatum (ATCC25586) and A. actinomycetemcomitans (ATCC29522) were obtained from The State Key Laboratory of Oral Diseases, Sichuan University (Chengdu, China).

Clinical examination and gingival index
Before sample collection, the dentist evaluated Gingival index (GI) of both groups at the teeth of 31, 32, 41 and 42 with same procedure as described previously [1]. The value of GI for each subject was the mean measurement of the four teeth. In the case group, measurement were made at five time points: Baseline T 0 = before appliances placement, T 1 = one month after bonding, T 2 = two months after bonding, T 3 = three months after bonding, and T 4 = six months after bonding; while in the control group, the GI values were only measured at baseline (T0) and compared with those of the case group.
Periodontal examination and sample collection were carried out by the same dentist.

Microbial sampling and DNA extraction
Sampling procedure was described before [1]. In brief, subgingival microbial samples were collected at the same sites after measuring the GI. Samples were obtained from 10:00 to 12:00 a. m. and at least two hours after eating, drinking, or taking oral hygiene measures, and then careful removal of supragingival plaque and saliva, the sites were isolated with sterile cotton rolls, and gently air dried. A sterile filter paper strip (ISO 30) was softly inserted into the bottom of periodontal pockets for 30 seconds until a minimum of resistance. Four paper points from each subject were immediately placed into a sterile microcentrifuge tube containing 0.5 ml of PBS [1,27,28]. The tubes were stored at -20˚C until analyzed. The bacterial DNA was extracted by the boiling method [29]. Briefly, a 10 μl aliquot of each stored sample was added to 10μl of 2 ×lysis 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 PCR amplification.

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 P. gingivalis (ATCC33277), F. nucleatum (ATCC25586), and A. actinomycetemcomitans (ATCC29522). The amplified products from clinical samples were randomly chosen for sequencing.
The 16S rRNA-based PCR and fimA genotypes specific PCR The 16S rRNA-based PCR was used to determine the prevalence of P. gingivalis in subgingival biofilm. The specific primers of fimA genotypes designed previously [18,30] were used for fimA typing and detection from positive samples of P. The products of six fimA genotypes were randomly sequenced on 3730 sequencer (Applied Biosystems, Foster City, CA, USA) to confirm the validity of the specific fimA PCR in P. gingivalis positive samples. The alignment of the resulting sequences was carried out with BLAST (http://www.ncbi.nlm.nih.gov/BLAST) The primers for amplification of 16S rRNA gene and fimA gene were listed in Table 1.

Statistical analysis
The data were analyzed by using the SPSS statistical software (version 20.0, SPSS, Chicago, IL). The Mann-Whitney U test and the Wilcoxon signed rank test were used to detect significant intergroup and intragroup differences regarding GI. Chi-squared test was used to compare detection rates of P. gingivalis and fimA genotypes between two groups. The McNemar test was utilized to compare the prevalence of P. gingivalis and fimA genotypes in case group. The Mann-Whitney U test was used to determine the correlation between prevalence of fimA genotypes and GI in orthodontic patients. The significance for all statistical tests was predetermined at P<0.05.

Results
Detection and confirmation of 16S rRNA-based PCR for P. gingivalis The reference stains were first amplified by the16S rRNA-based PCR to evaluate its specificity. The positive product appeared only from P. gingivalis (ATCC33277), not from F. nucleatum (ATCC25586) and A. actinomycetemcomitans (ATCC29522). At baseline (T 0 ), the frequency of P. gingivalis in from 117 subgingival samples of the two groups was twenty-nine (24.79%), of which thirteen (23.21%) from the control group and sixteen (26.23%) from the case group. From T 0 to T 3 , the detection rate of P. gingivalis increased significantly in the case group: 25 (42.62%) at T 1 , 31 (50.82%) at T 2 and 51 (83.61%) at T 3 . At T 4 , the detection of P. gingivalis decreased to 26 (42.62%). The detection rate of P. gingivalis at different time points after wearing appliances was significantly higher than that of T 0 in the case group and control group: T 1 (P<0.05), T2 (P<0.01), T 3 (P<0.01) and T4 (P<0.05) ( Table 2).
Ten P. gingivalis 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.

Detection and confirmation of 16S rRNA-based PCR for fimA genotypes
The distribution of fimA genotypes among P. gingivalis-positive samples was subsequently analyzed ( Table 2). The distribution of fimA genotypes in the case group showed no significant change over time from T 0 to T 2 , and there was no difference between two groups (P>0.05). At T 3 , the detection rate was 14.75% for fimA type II (P<0.05), 24.5% for fimA type IV (P<0.01), and 36.07% for fimA type V (P<0.01), which was found at higher level than that of the controls

Association of fimA genotypes and GI
The Mann-Whitney U test was used to determine the correlation between prevalence of fimA genotypes and GI in case group. As P. gingivalis-positive and fimA-positive samples at T 3 were the largest than those of other time points. We used the data of T 3 to assess the correlation of fimA genotypes and GI (shown in Table 3), regarding GI, there was no significant difference between fimA I/ III/ V/ Ib-positive samples and negative samples (P>0.05). However, the GI of fimA II, IV-positive samples was significantly higher than that of negative samples (P<0.01).

Evaluation of clinical parameter
It was showed that the value of GI (Table 4) in case group increased after placing appliances and reached to the maximum at T 3 , then decreased at T 4 . The GI at T 4 was found higher than that of baseline in two groups (P<0.01), but obviously lower than that at T 3 (P<0.01). The Mann-Whitney U test was used to determine the correlation between prevalence of fimA genotypes and GI in case group.
https://doi.org/10.1371/journal.pone.0188420.t003  [31,32]. Morinushi et al [33] investigated the association between gingivitis and colonization of P. gingivalis in children of different ages demonstrated that the children carrying detectable levels of P. gingivalis had significantly higher inflammation scores than those with negative P. gingivalis. Furthermore, they demonstrated that P. gingivalis was related to the progression of gingivitis and the onset of the periodontitis in adolescents (12 years and older). There were few documents examining the colonization by P. gingivalis in a long term utilization of fixed orthodontic appliances (extending to six months) in adolescents. A previous research showed various periodontal pathogens increased significantly after 6 months of fixed appliance treatment, but returned to pretreatment levels by 12 months of orthodontic treatment [25]. Ristic et al. found the detection rate of P. gingivalis increased from baseline to three months after bonding, then decreased at six months, which varied with the change of GI [34]. In this case, not only the incidence of P. gingivalis, but also fimA II, IV and V continuously increased after appliances placement, and reached the maximum at T 3 , and then followed by a decrease at T 4 , which was consistent with the change of GI. This could be explained by improvement of oral hygiene and reestablishment of host-microorganisms balance. It was demonstrated that periodontal diseases resulted from a rupture of the dynamic balance between the pathogens and host defense system [1,34]. Scientific investigations indicated that the presence of fixed orthodontic appliances would hamper effective oral hygiene and result in an ecological environment favorable to accumulate gram-negative anaerobic bacteria in subgingival regions, especially for adolescents [34,35]. For this reason, the host-microorganisms balance is broken, which leads to periodontal diseases. Guo [35] investigated the differences of the impact of orthodontic appliances on periodontal tissues between adults and children and found that the frequency of P. gingivalis presented stable trends among adults during the first three months, but increased significantly among children in the first three months. This article illustrated that the effect of fixed orthodontic appliance on periodontal tissues was more apparent in children than in adults. It seems that 3 months following placing orthodontic appliances, most adolescent patients raised awareness of oral hygiene and mastered tooth-brushing skills. Accordingly, the rebalance of host and microorganisms started to establish. This resulted in a reduction of periodontal pathogens, which was consistent with better periodontal status. It should also be addressed that proper hygiene controls during the therapy contributed to little increase in plaque accumulation and the rebalance of host-microorganisms, consequently [34]. It was reported that P. gingivalis was detected not only at a high frequency in patients with periodontitis, but also at a low frequency in periodontally healthy individuals without obvious gingival inflammation [36,37]. Therefore, specific virulent genotypes of the pathogens might be a cause of periodontal diseases [20]. Accumulating evidences suggested that P. gingivalis harboring Type II and IV (Pg-II, IV) fimbriae were disease-related isolates, while Pg-I and III were non-disease-related. Some researchers evaluated fimA genotypes in animal trials, demonstrating that fimA genotype II, Ib, and IV could cause stronger infections and inflammatory changes compared to strains with fimA genotype I and III [22,38,39]. Results of several clinical researches also believed that nucleotide genetic variation was likely related to virulence. In chronic periodontitis, P. gingivalis strains harboring fimA genotype II, IV, and Ib were obviously more common than isolates with other genotypes [18,20,22,30,[40][41][42]. Also, P. gingivalis carrying type II fimbriae was found to be the predominant fimA genotype in patients with periodontitis in several epidemiological surveys, while fimA genotype IV was more prevalent in the gingivitis group [8,42]. Additionally, the previous views that genotype II, IV, and Ib were related to virulence were supported by fimA genotyping of cultured clinical isolates of P. gingivalis sampled from patients with chronic periodontitis [20,41]. On the contrary, strains with fimA genotype I were the most prevalent among P. gingivalis positive in periodontally healthy subjects, followed by genotype V [20,30].
We detected the prevalence of fimA genotypes during the orthodontic therapy. At T 3 , consistent with the maximum GI, there was a statistically higher incidence of fimA II, IV and V than that at T 0 and periodontally healthy individuals, demonstrating that they present stronger virulence of P. gingivalis. Subsequently, fimA II and V returned to be normal at T 4 , while the incidence of fimA IV was significantly higher than that at T 0 and periodontally healthy individuals, meanwhile the periodontal clinical parameter was improved, implying the virulence of P. gingivalis tended to be weakened. We inferred that this might also due to reestablishment of host-microorganisms balance and improvement of oral hygiene.
According to previous reports, fimA V was detected in 0~29.7% of healthy subjects and 0~17.4% of periodontitis patients. In this study, we found fimA V appeared in 8.93% of healthy subjects, similar to data obtained from other groups, and in 36.07% of patients at T 3 , which was higher than data from previous reports. Moreover, the incidence of fimA V at T 3 was statistically higher than that at T 0 and healthy subjects. In order to further explore whether fimA V was associated with orthodontic gingivitis, we analyzed the correlation of GI and incidence of fimA V. The data showed that the GI levels of fimA II or IV positive groups were significantly higher than that of negative groups, while there was no difference in the GI levels between fimA V positive and negative groups, demonstrating that only fimA II and IV were associated with orthodontic gingivitis. However, it is unclear why Pg-fimA V significant increase during fixed orthodontic therapy. Longitudinal studies with larger sample size of orthodontic patients are needed to disclose it.

Conclusion
The incidence of P. gingivalis, fimA II, IV and V increased after appliances placement and reached the maximum at the first three months, and followed by a decrease. P. gingivalis carrying fimA II or IV was associated with gingivitis during orthodontic therapy. In addition, appropriate oral hygiene could lead to decrease accumulation of dental plaque, and exert a beneficial effect on periodontal tissues.
Supporting information S1 Table. Demographic characteristics of subjects in this study. (DOCX) S1 Appendix. The oral hygiene protocol. (DOCX)