Background
Helicobacter pylori is a Gram-negative microaerophilic bacterium found in the stomach that is responsible for gastric diseases [
1]. Though details regarding its transmission and infection source remain unclear, the most likely route of infection is through the oral cavity [
2]. To detect
H. pylori in oral cavity specimens, molecular biological technique has been applied [
3]. However, it is not easy to estimate the actual infection rate as
H. pylori detected in the oral cavity has been reported to be 0–100% [
4].
To overcome the difficulty of the detection of
H. pylori in the oral specimens, we previously designed novel primer sets based on the information of the complete genome for 48
H. pylori strains in the database [
5]. We searched genes including 16S rRNA,
vacA,
cagA,
glmM (
ureC), and
ureA because these genes were used for
H. pylori detection in published PCR methods with high frequency [
6‐
10]. Among these genes, six sequences of at least 20 consecutive nucleotides conserved among all strains were found only in
ureA, which were selected as primer sets for the detection. These primer sets produced amplicons for genomic DNA across
H. pylori strains, but did not for
Helicobacter pullorum and
Helicobacter felis strains, the closest related species to
H. pylori.
Most studies regarding
H. pylori detection from oral specimens have focused on dental plaque or saliva specimens [
11,
12]. A recent study showed that
H. pylori was isolated from endodontic-infected root canals of primary teeth [
13]. In addition, we detected
H. pylori bacterial DNA in inflamed pulp specimens using our novel PCR method [
5]. In the present study, we refined our system using a nested PCR method, which is much more sensitive than the previous single PCR method. The nested PCR method was then applied to investigate the actual
H. pylori distribution in inflamed pulp specimens as well as correlations between the detection of
H. pylori and clinical information regarding endodontic-infected teeth. Furthermore, the adhesion property of
H. pylori strains to human dental fibroblast cells, which is considered an important virulence factor of
H. pylori detected in inflamed pulp, was also investigated.
Discussion
The reliable PCR system for the
H. pylori detection in oral specimens requires high specificity and sensitivity levels since it is known that there are approximately 700 bacterial phylotypes found in the oral cavity [
17]. To overcome difficulties in detecting
H. pylori in the oral cavity, we previously designed novel primer sets using the complete genome information for 48
H. pylori strains registered in the GenBank database [
5]. In that study, we found six sequences of at least 20 consecutive nucleotides conserved among all strains in the
ureA gene. The
ureA gene is known to encode the urease enzyme, which has been frequently used to develop PCR primers for
H. pylori detection [
7,
10,
18]. However, no previous
ureA-based primers were completely conserved among all strains registered in the database [
5].
We previously constructed five primer sets (
ureA-aF/aR,
ureA-aF/bR,
ureA-bF/aR,
ureA-bF/bR,
ureA-cF/cR) for the detection of
H. pylori based on six sequences of at least 20 consecutive nucleotides [
5]. These primer sets showed an appropriate level of sensitivity at approximately 1–10 c.f.u. per reaction for
H. pylori genomic DNA. However, these primers sets revealed a sensitivity of approximately 10
2–10
3 cells for the detection of
H. pylori DNA from inflamed pulp specimens. It has been reported that some PCR primers sets showed lower detection limits in
H. pylori from gastric tissues or oral specimens compared with that from
H. pylori genomic DNA [
11]. In addition, we compared the sensitivities of detecting
H. pylori from infected pulp specimens versus non-infected pulp specimens by adding a known number of serially diluted
H. pylori to these specimens using the single PCR methods. The sensitivity in inflamed pulp specimens was much lower than that in non-infected pulp specimens, which indicated that numerous bacteria present at more than 10
6 c.f.u. in inflamed pulp may be the main cause for difficulty in detecting
H. pylori in such specimens. Thus, we refined our system using a nested PCR method to improve the sensitivity for detection of
H. pylori DNA from inflamed pulp tissue. Among the five primer sets beased on the
ureA gene designed in our previous study, primer sets
ureA-aF/bR and
ureA-bF/aR were suitable for the nested PCR method.
Nested PCR is one of the most sensitive methods to detect a small number of bacteria from clinical specimens [
19]. The specificity and sensitivity of target DNA amplification is estimated to be approximately 1000–10,000 times more sensitive than standard PCR [
20]. Some researchers previously reported
H. pylori detection using nested PCR methods [
21]. However, complete genome information was unavailable when these nested PCR methods were reported.
H. pylori 26,695 (ATCC 700392) is the first strain reported to identify the complete genome sequence in 1997, then only several complete genome sequences of
H. pylori strains were reported until 10 years ago [
22]. To our knowledge, this is the first study to propose a nested PCR method designed based on a large amount of genome information.
The detection rate of
H. pylori from inflamed pulp specimens detected by single PCR with primer sets
ureA-aF/
ureA-bR were 3.0%, whereas that with primer set
ureA-aF/
ureA-aR was 15% [
5]. However, the detection rate using primer set
ureA-aF/
ureA-aR was based on the results from only 40 specimens. Thus, the detection rate of the primer set
ureA-aF/
ureA-aR in 131 specimens was investigated in the present study, resulting in a detection rate of 5.3% (data not shown). Though the detection rate of
H. pylori with primer set
ureA-aF/
ureA-aR was higher than that with primer set
ureA-aF/
ureA-bR, which may be because the primer set
ureA-aF/
ureA-aR was the most sensitive of all primer combinations [
5], the detection limits of both primer sets were similar (approximately 1–10 cells using
H. pylori genomic DNA). The single PCR method using primer set
ureA-aF/
ureA-aR is a simple and highly sensitive method; however, nested PCR is the most appropriate method for
H. pylori detection because it showed highest sensitivity (1–10 cells) and detection rate (38.9%) in inflamed pulp specimens.
To determine the detailed distribution of
H. pylori in inflamed pulp tissue, we analyzed the detection rate of
H. pylori in 16 subjects who received root canal treatment in different teeth. In addition,
H. pylori detection in 20 subjects with two samplings from the same tooth was performed.
H. pylori was not detected from all inflamed pulp specimens in each individual, indicating that
H. pylori was not widely distributed in teeth with infected root canals. However, most
H. pylori-positive specimens were positive in the second sampling, suggesting that
H. pylori was not transiently present but rather colonized the inflamed pulp tissue. Since the intervals between first and second samplings were approximately 1–2 weeks in most subjects, the precise colonization period of
H. pylori in infected root canal remains unknown. Further studies should be performed to clarify this point. Comparison of the detection rates of
H. pylori from specimens obtained from primary teeth versus permanent teeth revealed that higher detection rate of
H. pylori was observed in specimens obtained from primary teeth. The result may be reasonable because
H. pylori infections seem to be acquired in childhood [
2].
Since
H. pylori was not transiently present in infected root canal, we investigated the mechanism by which
H. pylori colonizes pulp tissue by analyzing the adhesion property of
H. pylori to HDPFs. Bacterial adhesion to host cells is considered an important virulence factor for many bacterial species [
23]. For
H. pylori, bacterial adhesion to gastric epithelium is a critical factor for
H. pylori colonization [
24]. However, there have been few reports focusing on
H. pylori adhesion to cells obtained from oral origin, and, to our knowledge, this is the first study to investigate the adhesion properties of
H. pylori to HDPFs. All three tested
H. pylori strains showed adhesion to HDPFs, which is likely one of the reasons why
H. pylori DNA was detected in inflamed pulp specimens. Among these strains,
cagA-positive
H. pylori strains 26,695 and J99 showed higher adhesion rates compared with
cagA-negative
H. pylori strain ATCC 51932. The
cagA gene belongs to the
cag pathogenicity island (PAI) [
25]. The
cag PAI contains several virulence genes, some of which were reported to be related to
H. pylori adhesion to gastric epithelial cells [
26,
27]. Though
cagA is one of the possible genes related to
H. pylori adhesion, various
H. pylori adhesins have also been reported [
28], and the adhesion mechanisms to HDPFs remain to be elucidated. Further studies should be performed to investigate bacterial adhesion induced by
H. pylori virulence genes.