The oral cavity has been implicated as a source of Helicobacter pylori infection in childhood. Various PCR methods have been used to detect H. pylori DNA in oral specimens with various detection rates reported. Such disparity in detection rates complicates the estimation of the true infection rate of H. pylori in the oral cavity. In the present study, we constructed a novel PCR system for H. pylori detection and used it to analyse oral specimens. Firstly, the nucleotide alignments of genes commonly used for H. pylori detection were compared using the complete genome information for 48 strains registered in the GenBank database. Candidate primer sets with an estimated amplification size of approximately 300-400 bp were selected, and the specificity and sensitivity of the detection system using each primer set were evaluated. Five sets of primers targeting ureA were considered appropriate, of which a single primer set was chosen for inclusion in the PCR system. The sensitivity of the system was considered appropriate and its detection limit established as one to ten cells per reaction. The novel PCR system was used to examine H. pylori distribution in oral specimens (40 inflamed pulp tissues, 40 saliva samples) collected from Japanese children, adolescents and young adults. PCR analysis revealed that the detection rate of H. pylori in inflamed pulp was 15 %, whereas no positive reaction was found in any of the saliva specimens. Taken together, our novel PCR system was found to be reliable for detecting H. pylori. The results obtained showed that H. pylori was detected in inflamed pulp but not saliva specimens, indicating that an infected root canal may be a reservoir for H. pylori. INTRODUCTIONHelicobacter pylori is a Gram-negative microaerophilic bacterium responsible for chronic gastritis, peptic ulcers and gastric cancer (Fennerty, 1994). Details regarding transmission and infection source are controversial, and it has been suggested that most infections are acquired in childhood, mainly via the oral cavity (Prasanthi et al., 2011). H. pylori has been isolated from, and its DNA detected in, gastric biopsy and faecal samples (Momtaz et al., 2012). In addition, several studies have detected H. pylori bacterial DNA in saliva and dental plaque specimens (Song et al., 1999;Kignel et al., 2005).Clinical detection of H. pylori infection in gastric tissue is achieved using the urea breath test, a urease test, histology, culture and serology results (Goodwin et al., 1997); whilst molecular biological methods using PCR assays are widely used for the detection of H. pylori in the oral cavity with high sensitivity (Westblom & Bhatt, 1999). Published PCR methods used for the detection of H. pylori have targeted genes including 16S rRNA, vacA, cagA, glmM (ureC) and ureA genes (Mapstone et al., 1993;Miyabayashi et al., 2000;Wang et al., 2002;Park et al., 2003;Smith et al., 2004). Reports of PCR detection of H. pylori have described good sensitivity and/or specificity of tested primer sets, although detection rates for the ora...

BackgroundRecently, dental pulp has been considered a possible source of infection of Helicobacter pylori (H. pylori) in children. We previously developed a novel PCR system for H. pylori detection with high specificity and sensitivity using primer sets constructed based on the complete genome information for 48 H. pylori strains. This PCR system showed high sensitivity with a detection limit of 1–10 cells when serial dilutions of H. pylori genomic DNA were used as templates. However, the detection limit was lower (102–103 cells) when H. pylori bacterial DNA was detected from inflamed pulp specimens. Thus, we further refined the system using a nested PCR method, which was much more sensitive than the previous single PCR method. In addition, we examined the distribution and virulence of H. pylori in inflamed pulp tissue.MethodsNested PCR system was constructed using primer sets designed from the complete genome information of 48 H. pylori strains. The detection limit of the nested PCR system was 1–10 cells using both H. pylori genomic DNA and bacterial DNA isolated from inflamed pulp specimens. Next, distribution of H. pylori was examined using 131 inflamed pulp specimens with the nested PCR system. In addition, association between the detection of H. pylori and clinical information regarding endodontic-infected teeth were investigated. Furthermore, adhesion property of H. pylori strains to human dental fibroblast cells was examined.ResultsH. pylori was present in 38.9% of inflamed pulp specimens using the nested PCR system. H. pylori was shown to be predominantly detected in primary teeth rather than permanent teeth. In addition, samplings of the inflamed pulp were performed twice from the same teeth at 1- or 2-week intervals, which revealed that H. pylori was detected in most specimens in both samplings. Furthermore, H. pylori strains showed adhesion property to human dental fibroblast cells.ConclusionOur results suggest that H. pylori colonizes inflamed pulp in approximately 40% of all cases through adhesion to human dental fibroblast cells.

BackgroundHelicobacter pylori (H. pylori) colonize the stomach and are considered an etiological agent of gastric cancer. The oral cavity is a transmission route to the stomach, but the exact site of colonization has not yet been explicated. Our study investigated the association between H. pylori infection and presence in oral samples.MethodsDental pulp, supragingival plaque, and saliva from 192 patients visiting the Dentistry’s outpatient clinic were collected for testing. The H. pylori ureA gene was identified via Nested PCR. Urine anti-H. pylori antibody test was utilized to detect infection.ResultsTwenty-five subjects were found to be antibody-positive. PCR analysis of dental pulp revealed that 23 subjects possessed the ureA gene. Twenty-one subjects were positive for both antibodies and genes in dental pulp. PCR testing revealed that 2 subjects were positive in dental plaque but negative for saliva. The subjects positive for H. pylori in dental pulp expressed clinical signs of severe dental caries.ConclusionsH. pylori infected subjects expressed H. pylori in samples from the oral cavity. The main reservoir for infection within the oral cavity was determined to be dental pulp. Moreover, H. pylori are likely transmitted from dental caries to the root canal.

Streptococcus mutans is a major pathogen of dental caries. Collagen-binding proteins (CBPs) (approximately 120 kDa), termed Cnm and Cbm, are regarded as important cell surface antigens related to the adherence of S. mutans to collagenous tissue. Furthermore, CBP-positive S. mutans strains are associated with various systemic diseases involving bacteremia, such as infective endocarditis. Endodontic infection is considered to be an important cause of bacteremia, but little is known regarding the presence of S. mutans in dental pulp tissue. In the present study, the distribution and virulence of S. mutans in dental pulp tissues were investigated by focusing on CBPs. Adhesion and invasion properties of various S. mutans strains were analyzed using human dental pulp fibroblasts (HDPFs). CBP-positive strains had a significantly higher rate of adhesion to HDPFs compared with CBP-defective isogenic mutant strains (P<0.001). In addition, CBP-positive strains induced HDPF proliferation, which is a possible mechanism related to development of hyperplastic pulpitis. The distribution of S. mutans strains isolated from infected root canal specimens was then analyzed by PCR. We found that approximately 50% of the root canal specimens were positive for S. mutans. Approximately 20% of these strains were Cnm-positive, while no Cbm-positive strains were isolated. The Cnm-positive strains isolated from the specimens showed adhesion to HDPFs. Our results suggest that CBP-positive S. mutans strains exhibit high colonization in dental pulp. This could be a possible virulence factor for various systemic diseases.

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.