Distribution of Helicobacter pylori and Periodontopathic Bacterial Species in the Oral Cavity

The oral cavity may serve as a reservoir of Helicobacter pylori. However, the factors required for H. pylori colonization are unknown. Here, we analyzed the relationship between the presence of H. pylori in the oral cavity and that of major periodontopathic bacterial species. Nested PCR was performed to detect H. pylori and these bacterial species in specimens of saliva, dental plaque, and dental pulp of 39 subjects. H. pylori was detected in seven dental plaque samples (17.9%), two saliva specimens (5.1%), and one dental pulp (2.6%) specimen. The periodontal pockets around the teeth, from which dental plaque specimens were collected, were significantly deeper in H. pylori-positive than H. pylori-negative subjects (p < 0.05). Furthermore, Porphyromonas gingivalis, a major periodontopathic pathogen, was detected at a significantly higher frequency in H. pylori-positive than in H. pylori-negative dental plaque specimens (p < 0.05). The distribution of genes encoding fimbriae (fimA), involved in the periodontal pathogenicity of P. gingivalis, differed between H. pylori-positive and H. pylori-negative subjects. We conclude that H. pylori can be present in the oral cavity along with specific periodontopathic bacterial species, although its interaction with these bacteria is not clear.


Introduction
Evidence indicates that Helicobacter pylori infects the human organism via the oral cavity and subsequently remains in the gastric tissue for the rest of its host's life [1]. Chronic inflammation of the gastric mucosa is induced by ammonia and toxins produced by H. pylori [1], eventually causing gastric mucosal damage and diseases such as peptic ulcer and gastric cancer. H. pylori in the oral cavity may cause reinfection of the stomach after eradication therapy [2,3]. However, H. pylori colonizes the oral cavity through an unknown mechanism.
Among the more than 700 bacterial species that reside in the human oral cavity [4], some contribute to the progression of periodontal disease. For example, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia are called the red complex because of their high pathogenicity [5]. The orange complex comprises bacteria such as Prevotella intermedia, Prevotella nigrescens, and Campylobacter rectus, which are associated with periodontal disease, and provides a link between pathogenic species such as the red complex and commensal bacteria [5]. Furthermore, commensal bacteria known as probe. The deepest level reached was recorded. Extraction of the third molar, medical examination by an interview, and measurement of the depth of the periodontal pockets were performed by a single doctor in the department of oral and maxillofacial surgery, and dental caries diagnosis was performed by a single dentist in the department of pediatric dentistry.

Oral Cavity Specimens
The extracted third molar was placed in a sterile plastic tube containing 2.5 mL of sterile saline, and sonication was used to separate the dental plaque from the tooth. After the tooth was removed, the suspension was centrifuged, and the supernatant was discarded. The resulting suspension served as a dental plaque specimen from the extracted tooth. Next, the pulp chamber of the extracted tooth was opened using a sterilized dental handpiece and diamond point, and the dental pulp specimen was deposited into a sterile plastic tube containing 1 mL of sterile saline, according to a published method [12]. Saliva (1 mL) from each subject was collected into a sterile disposable tube. The dental pulp and saliva specimens were centrifuged, and the supernatant was discarded. Bacterial DNA was extracted from these specimens of dental plaque, dental pulp, and saliva as described below.

H. pylori Strains and Growth Condition
H. pylori reference strain J99 (ATCC 700824) purchased from Summit Pharmaceuticals International corporation (Tokyo, Japan) served as the positive control. Blood agar plates (Becton Dickinson, Franklin Lakes, NJ, USA), incubated at 37 • C for 3-5 days, were used to isolate bacterial colonies [12]. Thereafter, colonies were inoculated into 10 mL of brucella broth (Becton Dickinson) supplemented with 1 mL of horse serum by using a sterilized platinum loop and then incubated at 37 • C for 24 h under microaerophilic conditions. The bacteria were collected using centrifugation at 8000 rpm for 10 min, and genomic DNA was extracted as described below.

Bacterial DNA Extraction
Bacterial DNA was extracted using a published method [12]. Briefly, oral specimens or H. pylori strain J99 were resuspended in 250 µL of 10 mM Tris-HCl (pH 8.0) containing 100 mM NaCl and 1 mM EDTA. The cells were collected using centrifugation, lysed in 600 µL of Cell Lysis Solution (Qiagen, Düsseldorf, Germany), and incubated at 80 • C for 5 min, followed by the addition of 3 µL of RNase A (10 mg/mL; Qiagen) and incubation at 37 • C for 30 min. Protein Precipitation Solution (Qiagen) was added, and the mixture was vigorously vortexed for 20 s and then centrifuged at 10,000× g for 3 min. The supernatant was combined with 600 µL of isopropanol (Wako Pure Chemical Industries, Tokyo, Japan) and centrifuged. The precipitate was then resuspended in 70% ethanol (Wako Pure Chemical Industries), centrifuged, combined with 100 µL of 100 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 8.0)), and used in the next study.

PCR Detection of H. pylori
Nested PCR was performed using bacterial DNA extracted from oral cavity specimens with H. pylori-specific primer sets described in our previous study [13] (Table 1). Briefly, PCR assays to detect H. pylori were first performed using the primers ureA-aF and ureA-bR, followed by a second step using the primers ureA-bF and ureA-aR. For first-step PCR, 2 µL of bacterial DNA extracted from oral cavity specimens was amplified in 20 µL of reaction mixture. For second-step PCR, 1 µL of the first PCR product was used as a template for 20 µL reactions containing TaKaRa Ex Taq polymerase (Takara Bio. Inc., Otsu, Japan). In the first-and second-step reactions, PCR amplification was performed as follows: initial denaturation at 95 • C for 4 min, followed by 30 cycles at 95 • C for 30 s, 55 • C for 30 s, 72 • C for 30 s, and final extension at 72 • C for 7 min. The PCR products were fractionated using a 1.5% (w/v) agarose gel containing Tris-acetate-EDTA buffer, stained with ethidium bromide (0.5 µg/mL), and visualized under UV illumination.

PCR Detection of Periodontopathic Bacterial Species
PCR was performed to detect the following periodontitis-related bacterial species: P. gingivalis, T. denticola, T. forsythia, C. ochracea, C. sputigena, P. intermedia, P. nigrescens, C. rectus, Aggregatibacter actinomycetemcomitans, and E. corrodens. A ubiquitous primer set which matches almost all bacterial 16S rRNA genes was used as a positive control [19]. For PCR, 2 µL of bacterial DNAs was amplified using the respective species-specific primer sets and TaKaRa Ex Taq polymerase [20][21][22][23][24] (Table 1). PCR assays were performed using the following cycling parameters: initial denaturation at 95 • C for 4 min, followed by 30 cycles at 95 • C for 30 s, 58 • C for 30 s, and 72 • C for 30 s, with a final extension at 72 • C for 7 min. The PCR products were fractionated using a 1.5% (w/v) agarose gel containing Tris-acetate-EDTA buffer, stained with ethidium bromide (0.5 µg/mL), and visualized under UV illumination.

PCR Detection of fimA Genotypes
The fimA genotypes of P. gingivalis were determined using fimA primers specific for types I to V and type Ib genotypes [25][26][27]. (Table 1). For PCR, 2 µL of bacterial DNAs was amplified using the respective species-specific primer sets and TaKaRa Ex Taq polymerase. PCR assays were performed using the following cycling parameters: initial denaturation at 95 • C for 4 min, followed by 30 cycles at 95 • C for 30 s, 58 • C for 30 s, and 72 • C for 30 s, with a final extension at 72 • C for 7 min. The PCR products were fractionated using a 1.5% (w/v) agarose gel containing Tris-acetate-EDTA buffer, stained with ethidium bromide (0.5 µg/mL), and visualized under UV illumination.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). Comparisons between two groups were performed using the Student's t test. Intergroup differences of each analysis were determined using analysis of variance. The Bonferroni correction was used for post-hoc analysis; p < 0.05 was considered statistically significant.

Clinical Characteristics of H. pylori-Positive and H. pylori-Negative Subjects
Saliva was collected from 39 subjects who underwent extraction of the third molar. Dental plaque and pulp specimens were collected from the extracted teeth. Nested PCR was performed using as a template bacterial DNA obtained from saliva, extracted teeth, and dental pulp specimens, using the previously described H. pylori-specific primers sets [13]. H. pylori was detected in 10 of 39 samples (25.9%), which were defined as H. pylori-positive (Table 2, Figure 1A). Interestingly, 16 out of 39 specimens were impacted teeth, three (18.8%) of which were positive for H. pylori. The percentage of subjects with history of systemic and gastrointestinal diseases, prior H. pylori infection, and previous eradication of H. pylori was higher for H. pylori-positive subjects than for H. pylori-negative subjects, although the differences were not significant ( Table 2).

Relationship between H. pylori and Oral Disease
H. pylori was detected mainly in the dental plaque around the extracted teeth. Therefore, we analyzed the relationship between the presence of H. pylori and major oral diseases affecting the extracted teeth, such as dental caries, as well as periodontal pocket depth. In subjects with dental caries, those with H. pylori were more numerous than those without H. pylori, although the difference was not significant (Supplementary Figure S1). Furthermore, periodontal pockets of 23 of the 39 erupted teeth were measured, because the remaining 16 extracted teeth were impacted. Thus, the average periodontal pocket depths of H. pylori-positive and -negative specimens were 3.8 mm and 3.1 mm, respectively (p < 0.05) (Figure 2A). Similarly, there was a significant difference in average periodontal pocket depth between P. gingivalis-positive and -negative specimens (p < 0.01) ( Figure 2B). In contrast, C. rectus and C. sputigena were more frequent in the periodontal pocket among specimens without detectable bacteria (p < 0.05) ( Figure 2C,D). Furthermore, no significant differences between the presence or the absence of other periodontopathic bacterial species were found ( Figure 2E-G). There were few P. intermedia-positive and A. actinomycetemcomitans-positive specimens and few P. nigrescens-negative and E. corrodens-negative specimens in the extracted teeth. Therefore, these specimens were not suitable for the analysis (data not shown). Furthermore, 23 subjects with a periodontal pocket of 3 mm or less were classified as healthy, and those with a pocket of 4 mm or more were classified as having periodontitis, as defined in a previous study [28]. As a result, only H. pylori and P. gingivalis showed high rates of detection in the periodontitis group, although there were no significant differences between the groups (Supplementary Figure S2).

Relationship between H. pylori and Periodontopathic Bacterial Species
We evaluated the relationship between the presence of H. pylori and the distribution of periodontopathic bacterial species in the oral cavity. At least four periodontopathic bacterial species were detected in most dental plaque specimens, with or without H. pylori, although a few were found in certain H. pylori-negative subjects ( Figure 3A).
We next classified the periodontopathic bacterial species into groups according to decreasing pathogenicity [5] as follows: red complex (P. gingivalis, T. denticola, and T. forsythia), orange complex (P. intermedia, P. nigrescens, and C. rectus), and green complex (C. ochracea, C. sputigena, A. actinomycetemcomitans, and E. corrodens). The number of red-complex species in H. pylori-positive subjects was significantly higher than that in H. pylori-negative subjects (p < 0.01) ( Figure 3B). In contrast, the number of orange complex species in H. pylori-positive subjects was significantly lower than that in H. pylori-negative subjects (p < 0.05) ( Figure 3C); the results were similar for the green complex ( Figure 3D).
We next selected two strains of the red complex and assessed their relationship with H. pylori. We found that P. gingivalis and T. denticola were detected at significantly higher frequencies in H. pylori-positive specimens compared with H. pylori-negative specimens. (p < 0.05) ( Figure 3E). Furthermore, there was a significant association with the combination of P. gingivalis and T. forsythia ( Figure 3F) and with the combination of T. denticola and T. forsythia ( Figure 3G). The relationships between the presence or absence of H. pylori and the detection frequency of each periodontopathic bacterial species are shown in Figure 3H. The detection rates of P. gingivalis, T. forsythia, and P. intermedia in H. pylori-positive subjects was higher than that in H. pylori-negative subjects. Among these bacterial species, 3 of 7 H. pylori-positive specimens were P. gingivalis-positive (42.9%), which was significantly higher than for H. pylori-negative specimens (2 of 32; 6.3%) (p < 0.05).
In contrast, C. ochracea, C. sputigena, and C. rectus were detected at lower rates in H. pylori-positive specimens compared with H. pylori-negative specimens, although the differences were not significant.

Distribution of fimA Genotypes in Subjects with or without H. pylori
The detection rate of P. gingivalis was higher in H. pylori-positive subjects. We therefore analyzed P. gingivalis-positive subjects, focusing on the distribution of fimA genotypes. Six of eight H. pylori-negative subjects were classified as type IV fimA ( Figure 4A). In contrast, type IV fimA was detected in one of five H. pylori-positive subjects, and types I and II fimA genotypes were detected in four and three H. pylori-positive subjects, respectively ( Figure 4B). When the distribution frequencies of fimA genotypes with or without H. pylori were compared, type II fimA was detected at a significantly higher frequency in H. pylori-positive subjects ( Figure 4C). Although type I fimA and type Ib fimA genotypes were detected at high frequencies in H. pylori-positive subjects, no significant difference with respect to H. pylori-negative subjects was found. In contrast, the type IV fimA genotype was detected at high frequency in H. pylori-negative subjects, although no significant difference with respect to H. pylori-positive subjects was found.

Discussion
H. pylori, a Gram-negative microaerophilic bacterium, causes gastric diseases and may be transmitted via the oral cavity [1]. A relationship is considered to exist between the presence of H. pylori in the oral cavity and the severity of periodontal disease [17,18]. Previous studies mainly focused on the relationship between H. pylori infection and clinical periodontal status by comparing the distribution frequency of H. pylori in healthy subjects with that in periodontal disease patients [17,18]. However, differences in the periodontopathic bacterial species between H. pylori-positive and H. pylori-negative subjects in the oral cavity remain unknown. Therefore, we investigated the relationship between the presence of H. pylori in the oral cavity and periodontal disease by focusing on the distribution of periodontopathic bacterial species. Recent reviews suggest that periodontopathic bacterial species may affect human health [6,29]. Highly pathogenic bacteria involved in periodontal disease, such as P. gingivalis, can also be associated with systemic diseases such as diabetes and arteriosclerosis [6]. Interestingly, abnormal oral microbiota or periodontal disease are also associated with an increased risk of gastric cancer [7,30]. Nevertheless, the periodontopathic bacterial species that co-exist with H. pylori in the oral cavity remain unknown. In the present study, we were able to detect not only species that confer periodontal disease but also H. pylori in the oral cavity and to clarify the relationship between these bacteria. If the coexistence of specific periodontopathic bacterial species and H. pylori can be shown to be associated with gastric disease, these bacterial species may be used as diagnostic markers for gastric disease or as targets for therapeutic interventions.
Although most methods used to detect H. pylori in the oral cavity employ PCR assays, it is difficult to detect H. pylori with high specificity and high sensitivity in the presence of approximately other 700 oral bacterial species [4,13]. Therefore, we developed a reliable nested PCR method based on the complete genome sequences of approximately 50 H. pylori strains [12,13]. Using this method, we accurately detected H. pylori in different types of oral specimens. Similarly, we used a PCR method to detect periodontopathic bacterial species in clinical specimens [31][32][33]. No previous study has attempted to detect both H. pylori and periodontopathic bacterial species at the same time using these molecular analyses. Here, we used methods from previous studies to determine the effects of periodontopathic bacterial species on the colonization of the oral cavity by H. pylori.
We used a novel nested PCR method to detect H. pylori in saliva, dental plaque, and dental pulp. We found that H. pylori was detected at the highest frequency in dental plaque. This result is consistent with previous reports showing that dental plaque harbors H. pylori [34,35]. In contrast, H. pylori was less frequent in saliva, which is consistent with our previous study [12]. Although only a few studies have focused on H. pylori in dental pulp specimens [12,16,36], dental pulp may serve as a reservoir for H. pylori only when the pulp is exposed by dental caries or traumas. Here, more than half of the extracted teeth did not exhibit dental caries, and only a few had severe dental caries extending to the pulp, likely explaining the low detection frequency of H. pylori. These results suggest that H. pylori does not easily enter the dental pulp, unless it is exposed because of a dental problem.
Periodontal pockets are caused by the inflammatory response to periodontopathic bacteria in the dental plaque accumulated in periodontal tissues [37]. A deep periodontal pocket has a low oxygen concentration and is hemorrhagic [37,38], which accelerates the growth of periodontopathic bacterial species. In the present study, periodontal pockets were significantly deeper in H. pylori-positive specimens. Among the 11 bacterial species studied, only H. pylori and P. gingivalis were associated with other bacteria and increased periodontal pocket depth. Therefore, the environment of the periodontal pocket may favor the colonization by H. pylori. Furthermore, H. pylori may be involved in the formation of periodontal pockets. H. pylori as well as Campylobacter species are major microaerobic periodontopathic bacteria [39]. Therefore, periodontal pockets with low oxygen concentrations may serve as reservoirs of H. pylori.
Pericoronitis is a gingival inflammation caused by bacterial infection of the periodontal tissues surrounding tilted or impacted third molar or unerupted teeth [40]. Thus, we detected H. pylori from third molar that was diagnosed as impacted. As a result, H. pylori was detected in 3 of 16 (18.8%) impacted teeth. Therefore, impacted third molar may be a possible reservoir for H. pylori colonization.
To the best of our knowledge, only one study has investigated the correlation between the presence of H. pylori and that of periodontopathic bacterial species in the oral cavity, examining 14 Chinese subjects [8]. In this study, the detection rates of three of five periodontopathic bacterial species, including P. gingivalis, were increased in H. pylori-positive dental plaque specimens. By referring to this paper, we decided to analyze three samples consisting of dental plaque, saliva, and dental pulp from 39 subjects. However, the sample sizes in both the Chinese study and our present study are small. Larger-scale studies with greater numbers of such subjects as well as other populations should be performed in the future, because half of the world's population is infected by H. pylori [41].
In addition to the known associations of oral bacterial species with diabetes and arteriosclerosis, recent analyses of oral bacteria, including periodontopathic bacteria, have led to important findings related to systemic diseases, such as gastric cancer [7]. Many of these findings have come from studies with small sample sizes; however, systematic reviews and meta-analyses based on these papers have indicated possible relationships [7]. Therefore, we consider that our research has the potential to lead to new clinical findings.
Periodontal disease is caused by diverse periodontopathic bacterial species, which are classified according to differences in pathogenicity determined using molecular biological analysis [5]. The most pathogenic group is the red complex composed of P. gingivalis, T. denticola, and T. forsythia, which mainly colonizes the adult oral cavity [5]. Here, we showed that H. pylori-positive specimens contained multiple red-complex members that are involved in coaggregation or use mutual metabolites to enhance their pathogenicity [42][43][44]. Furthermore, immunohistochemical analyses showed that red-complex bacteria coexist in dental plaque [45].
H. pylori may be involved in a symbiotic relationship with these bacterial species. Although deep periodontal pockets may represent an important factor required for colonization of the oral cavity by H. pylori, the detection of H. pylori correlated with the presence of red-complex species such as T. denticola and T. forsythia, which are not involved in the formation of periodontal pockets. Therefore, the presence of these bacteria in the absence of periodontal pockets may represent a risk factor for H. pylori colonization.
Members of the orange complex such as P. intermedia and P. nigrescens, which are present during adolescence, act as a link between the red complex and commensal oral bacteria [5]. Members of the green and yellow complexes, including C. ochracea and C. sputigena, are commensal species with low pathogenicity [5]. These commensal bacteria are established during childhood in the absence of periodontal disease, which may be required for the establishment of the red and orange complexes. Interestingly, we frequently detected members of the orange and green complexes in specimens without H. pylori. Therefore, the presence of commensal bacteria with low periodontal pathogenicity suppress the colonization of the oral cavity by H. pylori.
The detection rate of P. gingivalis was significantly higher in H. pylori-positive subjects than in the H. pylori-negative subjects. However, the presence or absence of H. pylori did not affect the detection rate of other periodontopathic bacterial species. Therefore, we further analyzed the relationship between the presence or absence of H. pylori and the fimA genotypes of P. gingivalis. In general, genotypes II and IV are found in oral cavity specimens collected from patients with severe periodontal disease [10], and genotype I is found in oral samples of healthy individuals [10]. Here, we showed that genotypes I and II were frequently detected in H. pylori-positive specimens, and genotype IV was frequently detected in H. pylori-negative specimens. Thus, P. gingivalis with a specific fimA genotype may be involved in the colonization by H. pylori, which is not necessarily highly virulent in periodontal disease.
This study has certain limitations. First, the number of specimens was small, although we established a relationship between H. pylori and specific periodontopathic bacterial species. In addition, the incidences of H. pylori and many periodontopathic bacterial species increase with age. To overcome these limitations, larger scale studies with more subjects and samples taken from various teeth are needed. Furthermore, we were unable to determine if H. pylori was present in the periodontal pocket, because the dental plaque specimens were collected from around the extracted teeth. Thus, future studies should compare the detection rates of H. pylori in dental plaques collected from different parts of a tooth. Moreover, whether H. pylori can affect periodontal disease is unknown. P. gingivalis is a keystone pathogen that induces immunological abnormalities followed by a drastic increase in the population of oral bacteria that cause the damage of periodontal disease [9]. Thus, using H. pylori, P. gingivalis creates an oral microbiome more favorable for its own growth. In contrast, one hypothesis states that H. pylori may change the subgingival flora that contributes to the exacerbation of periodontal disease [8].
In summary, we frequently detected H. pylori in dental plaque around an extracted tooth. Furthermore, H. pylori was frequently detected in dental plaque specimens with deep periodontal pockets and characterized by the presence of by P. gingivalis with a specific fimA genotype. H. pylori was also detected in dental plaque specimens with multiple periodontopathic bacterial species of the red complex, even when the periodontal pocket was shallow. Taken together, these results suggest that H. pylori can co-exist with specific periodontopathic bacterial species, although interactions among the bacteria are not clear.