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Introduction

Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that was first isolated from the gastric mucosal biopsy of patients with chronic gastritis by Australian scholars in 1982(1). Since then, numerous studies have shown that H. pylori is an important pathogen associated with the etiology of human chronic gastritis, gastric ulcers and mucosa-associated lymphoid tissue lymphoma (2-4). More recently, it has been designated as a Group 1 carcinogen by the International Agency for Research on Cancer (5). In addition to gastrointestinal diseases, the latest data indicate that this microorganism may be related to certain oral diseases, including halitosis (6), caries (7), recurrent oral ulcers (8), chronic gingivitis (9) and periodontitis (10).

Periodontitis is one of the most common diseases of the human oral cavity. A total of >50% of the global population suffers from periodontitis (11). Dental plaque biofilm is the initiating factor of periodontitis. A wide variety of bacteria are attached to dental plaque. The existence and interactions of these bacteria cause the occurrence and development of periodontitis. In 1989, Krajden et al (12) isolated and cultured H. pylori from dental plaque. In 1993, Ferguson et al (13) obtained viable H. pylori from saliva. The relationship between H. pylori infection and periodontitis gradually garnered increased attention. Studies have since indicated that H. pylori infection is related to periodontitis, and the detection rate of H. pylori in patients with periodontitis is higher than that in periodontally healthy individuals (14,15). However, by contrast, Salehi et al (16) used PCR to detect H. pylori in the gingival crevicular fluid of periodontally healthy individuals and patients with periodontitis, and the results showed that periodontitis was not associated with H. pylori infection. Al-Ahmad et al (17) also failed to detect the presence of H. pylori in the saliva, supragingival plaque, subgingival plaque and tongue mucosa swabs of 15 patients with periodontitis. These studies suggested that H. pylori infection was not associated with periodontitis. Considering the inconsistency of the previous results, the primary aim of the present study was to explore the relationship between the presence of H. pylori and the periodontal condition.

Whether H. pylori can cause disease depends on the virulence factors of different H. pylori strains, and the strength of virulence in association with the expression of virulence genes has been previously established (10). The virulence genes of H. pylori primarily include vacuolating cytotoxin gene A (vacA) and cytotoxin-associated gene A (cagA) (18). VacA encoded by the vacA gene enters the cell by binding to the receptor on the target cell, causing damage to the lysosome and endoplasmic reticulum, resulting in vacuolar degeneration. There is a mosaic structure of alleles in the vacA gene sequence, which includes two important variant regions: The signal peptide region (s region) and the middle region (m region). According to the combination of different s and m regions, the virulence genes of vacA can be divided into 4 subtypes: s1m1, s1m2, s2m1 and s2m2. The differences in the combinations of these signal sequence regions affects the vacuolar cytotoxic activity of H. pylori, which is closely related to its pathogenicity. A previous study showed that the virulence of H. pylori is s1m1 > s1m2 > s2m1 > s2m2 from strong to weak (19). CagA encoded by the cagA gene is highly immunogenic; it enters the host cell through the type IV secretion system and undergoes phosphorylation, which interferes with the EMT-associated signaling pathway, miRNA-584, miRNA-1290 and other gene expression levels to causes inflammation in the host cell; there are no subtypes of cagA (20).

To date, studies have shown that the expression of vacA and cagA genotypes is associated with the severity of gastrointestinal diseases (21,22). However, the relationship between genotype of H. pylori and periodontal status remains unclear. Therefore, the second aim of the present study was to investigate the association between periodontal conditions and H. pylori genotypes.

Materials and methods

Study population

The present study consisted of a cohort of individuals who visited the Department of Periodontics, Stomatological Hospital of China Medical University (Shenyang, China) between April 2020 and May 2021. A total of 53 participants were selected. The exclusion criteria were as follows: i) <20 natural teeth, ii) symptoms of dyspepsia, iii) systemic disease, iv) current history of antibiotic usage or use during the previous 2 months, and v) smokers (23). The demographic information of the participants was recorded, including sex, age, height and weight. The present study was approved by the Ethics Committee of the Affiliated Stomatological Hospital of China Medical University (approval no. 2018-30). Prior to the examination, the subjects were informed of the purpose of the study, and written informed consent was obtained.

Measurement of periodontitis

Periodontal examinations were performed by a periodontist who was not involved in the study, and κ tests were performed to ensure consistency (κ>0.75). Probing depth (PD) and clinical attachment loss (CAL) of all the teeth were recorded from 6 points (mesiobuccal, midbuccal, distobuccal and the corresponding points lingually). The mean values of PD and CAL in every subject were calculated and recorded. Periodontitis was diagnosed when the clinical examination met observed ≥2 non-adjacent teeth with CAL; or ≥2 teeth with buccal or lingual CAL ≥3 mm and simultaneous PD of ≥3 mm (24). Patients with periodontitis were graded according to the 2017 AAP/EFP classification (25).

Subgingival plaque collection

The subgingival plaque was collected. Before periodontal treatment, the subgingival plaque was obtained from the index teeth (16/11/26/31/36/46) of each participant (26) and frozen at -80˚C for subsequent use. PD and CAL of the index teeth were recorded from 6 points. For each sextant only the highest score was recorded (27). The PD and CAL according to the 2017 new classification were divided into healthy group, stage I/II periodontitis group and stage III/IV periodontitis group to compare the difference in H. pylori detection rate in subgingival plaque at the tooth level (25).

DNA extraction and PCR amplification

DNA was extracted from subgingival samples using the Magnetic Bead Micro Genomic DNA Extraction Kit [cat. no. B518749; Sangon Biotech (Shanghai) Co., Ltd.] according to the manufacturer's protocol.

To detect the presence of H. pylori, PCR was performed utilizing the primers for the ureC gene (28). All H. pylori-positive samples in the same individual were mixed for further genotyping at the individual level using specific PCR to detect the cagA gene and vacA alleles (s1, s2, m1 and m2), respectively. The sequences of the primers used in this study are shown in Table I. PCR was conducted using a Takara Ex Taq DNA polymerase (Takara Bio, Inc.). The thermocycling conditions were: Initial denaturation at 95˚C for 3 min, followed by 30 cycles of denaturation at 98˚C for 10 sec, annealing at 58˚C for 30 min and extension at 72˚C for 1 min, and then a final extension step at 72˚C for 10 min. The amplified products were analyzed by electrophoresis in a 1% (w/v) agarose gel (Sheng gong Biological Company) and stained with ethidium bromide (0.5 µg/ml). Stained amplicons were visualized on a UV transilluminator at 260 nm, and imaged (Bio-Rad Laboratories, Inc.). H. pylori standard strain ATCC 43504 (gifted by the laboratory of Guizhou University of Traditional Chinese Medicine) was used as a positive control. A PBS solution was used as a negative control. As long as one or more samples from each subject were positive for ureC, the subject was marked as positive for H. pylori.

Table I Sequences of primers used in the present study.

Table I

Sequences of primers used in the present study.

Target gene	Sequence	Product size, bp	(Refs.)
ureC		296	(28)
Forward	5'-GGATAAGCTTTTAGGGGTGTTAGGGG-3'
Reverse	5'-GCTTACTTTCTAACACTAACGCGC-3'
cagA		400	(29)
Forward	5'-AATACACCAACGCCTCCAAG-3'
Reverse	5'-TTGTTGCCGCTTTTGCTCTC-3'
vacA s1		258	(30)
Forward	5'-ATGGAAATACAACAAACACAC-3'
Reverse	5'-CTGCTTGAATGCGCCAAAC-3'
vacA s2		199	(31)
Forward	5'-GCTAACACGCCAAATGATGC-3'
Reverse	5'-CTGCTTGAATGCGCCAAAC-3'
vacA m1		570	(30)
Forward	5'-CAATCTGTCCAATCAAGCGAG-3'
Reverse	5'-GCGTCTAAATAATTCCAAGG-3'
vacA m2		352	(32)
Forward	5'-GGAGCCCCAGGAAACATTG-3'
Reverse	5'-CATAACTAGCGCCTTGCAC-3'

[i] ureC, urease C; cagA, cytotoxin-associated gene A; vacA, vacuolating cytotoxin gene A.

Statistical analysis

SPSS version 21 (IBM Corp.) was used for statistical analysis. A one-way ANOVA followed by Tukey's post-hoc test to compare age, BIM, PD and CAL. A χ2 or Fisher's exact test was used to analyze the categorical data such as the presence of H. pylori, as well as the frequency of each genotype in the different groups. All tests were two-tailed tests, and P<0.05 was considered to indicate a statistically significant difference.

Results

Clinicopathological characteristics of the patients

Among the 53 subjects, there were 20 individuals with stage I/II periodontitis (6 men and 14 women), with a mean age of 32.85±7.74 years (16-48), and 18 individuals with stage III/IV periodontitis (10 men and 8 women), with a mean age 37.50±8.71 years (28~59). The 15 periodontally healthy individuals (5 men and 10 women) had a mean age of 34.53±7.41 years (27~55). There was no significant difference in terms of sex (χ2=2.932, P=0.231), age (F=1.533, P=0.226) or body mass index (F=0.426, P=0.656) among the groups (Table II).

Table II Clinicopathological characteristics of participants in the present study.

Table II

Clinicopathological characteristics of participants in the present study.

Group	Male/female patients, n	Age, yearsa	BMI, kg/m²,a	PD, mma	CAL, mma
Periodontally healthy (n)=15	5/10	34.530±7.41	23.05±2.98	2.30±0.72	0.47±0.70
Stage I/II periodontitis (n=20)	6/14	32.85±7.74	22.46±1.18	3.49±0.36	2.50±1.10
Stage III/IV periodontitis (n=18)	10/8	37.50±8.71	22.64±1.23	5.60±0.50	5.44±1.07
χ2/F-value	2.932	1.533	0.426	155.650	98.030
P-value	0.231b	0.226c	0.656c	<0.0001c	0.0001c

[i] ^aMean ± SD;

[ii] ^bFisher's exact test;

[iii] ^cone-way ANOVA. PD, probing depth; CAL, clinical attachment loss; BMI, body mass index.

H. pylori detection rates in periodontal states

Among the 53 subjects, 21 individuals were positive for H. pylori infection, accounting for 39.62% of all subjects. The number of H. pylori-positive individuals in the periodontally healthy group, stage I/II periodontitis group and stage III/IV periodontitis group was 2 (13.33%), 8 (40.00%) and 11 (61.11%), respectively (Table III). The difference between the three groups was significant (χ2=8.760, P<0.0001). The electrophoresis detection results of the ureC gene are shown in Fig. 1. The results in Table IV show that the detection rate of H. pylori in subgingival plaque of index teeth increased with the deepening of PD and CAL. The differences between PD (χ2=41.909, P<0.0001) and CAL (χ2=41.521, P<0.0001) among the three groups were significant.

Figure 1 PCR was performed using specific primers targeting ureC gene to detect the presence of Helicobacter pylori. Lane M, molecular weight marker 0.1 to 0.6 kb DNA marker; lane 1, positive control; lane 2, negative control (without DNA); lanes 3-6, ureC-positive samples (DNA from samples). ureC, urease C.

Table III H. pylori presence in individuals with different periodontal states.

Table III

H. pylori presence in individuals with different periodontal states.

H. pylori detection	Periodontally healthy (n=15)	Stage I/II periodontitis (n=20)	Stage III/IV periodontitis (n=18)	Total (n=53)	χ2-value	P-value
H. pylori-positive, n	2	8	11	21
H. pylori-positive, %	13.33	40.00	61.11	39.62	8.76	<0.0001

[i] H. pylori, Helicobacter pylori.

Table IV H. pylori presence in subgingival plaque with different periodontal conditions.

Table IV

H. pylori presence in subgingival plaque with different periodontal conditions.

Variable	PD ≤3 (n=114)	3< PD ≤5 (n=120)	PD >5 (n=84)	CAL <1 (n=73)	1≤ CAL <5 (n=174)	CAL ≥5 (n=71)
H. pylori-positive, n	9	37	41	6	42	39
H. pylori-positive, %	7.89	30.83	48.81a	8.22	24.14	54.93a
χ2 value			41.91			41.521
P-value			<0.0001			<0.0001

[i] ^aSubgingival plaque was divided according to the PD and CAL values, and there were significant differences in the detection rate of H. pylori in the plaque among the three groups. H. pylori, Helicobacter pylori; PD, probing depth; CAL, clinical attachment loss.

Association between the genotype of H

pylori and periodontal conditions. The genotype of H. pylori-positive individuals is characterized using PCR by detecting cagA and vacA alleles (Fig. 2). In the periodontally healthy group, 2 individuals tested positive for H. pylori and both of these were negative for cagA. The detection rates of cagA in the stage I/II periodontitis group and the stage III/IV periodontitis group were 87.50 and 90.91%, respectively (Table V). The positive rate of the cagA genotype in the periodontitis group was markedly higher than that in the healthy group.

Figure 2 Genotypes of Helicobacter pylori-positive individuals were characterized using PCR by detecting the presence of the cagA gene and vacA alleles. Lane M, molecular weight marker 0.1 to 0.6 kb DNA marker; lanes 1-3, cagA-positive samples (DNA from samples); lanes 4-6, s1 allele-positive samples (DNA from samples); lanes 7-9, s2 allele-positive samples (DNA from samples); lanes 10-12, m1 allele-positive samples (DNA from samples); lanes 13-15, m2 allele-positive samples (DNA from samples); cagA, cytotoxin-associated gene A; vacA, vacuolating cytotoxin gene A.

Table V Expression of each genotype in H. pylori-positive individuals.

Table V

Expression of each genotype in H. pylori-positive individuals.

H.pylori genotype	Periodontally healthy (n=2)	Stage I/II periodontitis (n=8)	Stage III/IV Periodontitis (n=11)	Total (n=21)
cagA, n (%)
Positive	0 (0.0)	7 (87.5)	10 (90.9)	17 (81.0)
Negative	2 (100.0)	1 (12.5)	1 (9.09)	4 (19.0)
vacA, n (%)
s1a	0 (0.0)	4 (50.0)	7 (63.6)	11 (52.4)
s2a	2 (100.0)	1 (12.5)	1 (9.1)	4 (19.0)
s1s2a	0 (0.0)	3 (37.5)	3 (27.3)	6 (28.6)
m1a	0 (0.0)	4 (50.0)	8 (72.7)	12 (57.1)
m2a	2 (100.0)	3 (37.5)	3 (27.3)	8 (38.1)
m (-)a	0 (0.0)	1 (12.5)	0 (0.0)	1 (4.8)
s1m1b	0 (0.0)	1 (12.5)	4 (36.3)	5 (23.8)
s1m2b	0 (0.0)	3 (37.5)	3 (27.3)	6 (28.6)
s2m1b	0 (0.0)	1 (12.5)	1 (9.1)	2 (9.5)
s2m2b	2 (100.0)	0 (0.0)	0 (0.0)	2 (9.5)
s1s2m1b	0 (0.0)	2 (25.0)	3 (27.3)	5 (23.8)
s1s2m(-)b	0 (0.0)	1 (12.5)	0 (0.0)	1 (4.8)
cagA+/vacA s1m1c	0 (0.0)	1 (12.5)	4 (36.3)	5 (23.8)
cagA+/vacA s1m2c	0 (0.0)	3 (37.5)	3 (27.3)	6 (28.6)
cagA+/vacA s2m1c	0 (0.0)	1 (12.5)	1 (9.1)	2 (9.5)
cag+/vacA s1s2m1c	0 (0.0)	1 (12.5)	2 (18.2)	3 (14.3)
cag+/vacA s1s2m(-)c	0 (0.0)	1 (12.5)	0 (0.0)	1 (4.8)
cagA-/vacA s2m2c	2 (100.0)	0 (0.0)	0 (0.0)	2 (9.5)
cagA-/vacA s1s2m1c	0 (0.0)	1 (12.5)	1 (9.1)	2 (9.5)

[i] ^aAlleles of vacA;

[ii] ^bgenotypes of vacA;

[iii] ^ccombination of two virulence genotypes of cagA and vacA. The types of gene combinations not shown in the table were all undetected.

The presence of cagA and vacA combination genotypes varied with periodontal status. The cagA-/vacAs2m2 genotype was detected in 100% of H. pylori-positive individuals without periodontitis. The cagA+/vacAs1m2 (37.5%) and cagA+/vacAs1m1 (36.3%) were the most frequently detected allele combinations in the stage I/II periodontitis and stage III/IV periodontitis groups, respectively (Table V).

Discussion

Whether H. pylori, colonized in subgingival plaque, influences the occurrence and development of periodontitis has been contested for decades. The present study showed that as the degree of aggravation of periodontal inflammation increased, the positive rate of H. pylori detection increased significantly. This suggested that the severity of periodontitis may be aggravated by the colonization of H. pylori. This is consistent with the results of studies by Anand et al (14) and Zheng and Zhou (33). Conversely, successful eradication of H. pylori can reduce the risk of developing periodontitis (34). This suggests that in addition to conventional treatments for periodontitis, a periodontist should be cognizant of H. pylori infections. Patients with periodontitis plus H. pylori infection should be treated with the aim of H. pylori eradication to reduce the risk of recurrence and aggravation of periodontitis.

Studies have shown that H. pylori in dyspeptic diseases may enter the mouth through acid reflux and colonize in the oral cavity (35-37). This may affect the accuracy of the detection rate of oral H. pylori. Therefore, participants with symptoms of dyspepsia were excluded. However, the relationship between subgingival plaque and gastric H. pylori is still contested. Several studies reported that there was a close relationship between gastroesophageal disease and oral status (37,38). Conversely, other studies found that the occurrence of H. pylori in the oral cavity was not correlated with an infected stomach or with the oral dental status of patients (15,17). Based on the aforementioned contrasting results, it is suggested that the genotype of H. pylori should be considered when assessing the differences between subgingival plaque and gastric H. pylori.

The results of the present study were consistent with those in the study by Falsafi et al (39), which found that H. pylori was detectable in the oral cavity of 45% of the population, but that only a minority of affected individuals demonstrated symptoms of the disease. It was speculated that this may be attributed to differences in virulence among different H. pylori strains. A study by Miehlke et al (40) showed that there was a robust association between the vacAs1m1 and cagA genotypes of H. pylori in patients with gastric cancer. It was shown that the detection of virulence genes may assist in identifying patients with an increased risk of gastric cancer from the population. Research by Hu et al (41) found that individuals infected with the vacAs1m2 strain had a significantly increased risk of peptic ulcers, while the vacAs1m1 genotype significantly increased the risk of active gastritis. Based on the aforementioned findings, we hypothesized that different diseases may be associated with H. pylori strains with different dominant genes and different genotypes, and thus different clinical outcomes.

In the H. pylori-positive samples in the present study, the detection rate of cagA was 80.95%, which meant that most of the H. pylori strains carried the cagA gene. This was consistent with the results of the studies by Link et al (42) and Falsafi et al (39). In another study, a biopsy of stomach tissues was performed. The results showed that the positive rate of H. pylori cagA was associated with the degree of inflammation of the gastric mucosa (43). Ferreira et al (44) found that after infection of the stomach mucosa with cagA+ H. pylori, the concentration between IL-8 and neutrophils increased. Mendoza-Cantú et al (45) assessed 100 samples of supragingival plaque from patients with chronic gingivitis and showed that the detection rate of cagA was 16.7 and 80.8% in the healthy and chronic gingivitis groups, respectively. It was suggested that the detection rate of the cagA gene was related to gingival inflammation. The results of the present study indicated that the detection rate of the cagA gene was related to the extent of periodontal inflammation. In general, a cagA-positive strain of H. pylori in the stomach tissues, gingiva or subgingival plaque is more likely to be associated with a state of disease.

In the present study, the most frequently detected genotype was cagA-/vacAs2m2 in the periodontally healthy group, present in 100% of patients in this group, whereas the cagA+/vacAs1m2 genotype was the predominant genotype in stage I/II patients, and the cagA+/vacAs1m1 was most common in patients with stage III/IV periodontitis, accounting for 37.5 and 36.3%, respectively. The aforementioned results illustrated that the cagA-/vacAs2m2 genotype is the dominant genotype in the periodontally healthy individuals, while the cagA+/vacAs1m1 and cagA+/vacAs1m2 genotypes are dominant in those with periodontitis. This further confirmed the theory that different genotypes of H. pylori strains are associated with different periodontal conditions. Furthermore, a previous study showed that the majority of H. pylori strains carrying the cagA+/vacAs1m1 genotype could be isolated from patients with severe gastric diseases, including duodenal and gastric ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma (46). Therefore, it is safe to assume that different diseases may be associated with different dominant genes. Periodontologists should pay more attention to the cagA+/vacAs1m1 and cagA+/vacAs1m2 genotypes, which were frequently detected in the periodontitis groups in the present study. In addition, the results showed that the detection rates of the cagA+/vacAs1m1 genotype also increased with the aggravation of periodontal inflammation. However, further studies are needed to further confirm this speculation to provide a clinical reference for the treatment of oral H. pylori infection and periodontitis.

In conclusion, the detection rate and genotypes of H. pylori in subgingival plaques are associated periodontal conditions. H. pylori infection with the genotype of cagA+/vacAs1m1 and cagA+/vacAs1m2 may be predictive of a worse periodontal status. However, due to the small sample size in the present study, a larger scale and high-quality clinical study is required to confirm the findings.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by a grant from the National Natural Science Foundation of China (grant no. 81970943).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Authors' contributions

RL, DZ and YP designed the study. YL, QD and JP acquired and analyzed the data. YY and YM obtained the clinical samples and revised the manuscript. RL and JP wrote and revised the manuscript. All authors have read and approved the final manuscript. JP and DZ confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the Affiliated Stomatological Hospital of China Medical University (Shenyang, China; approval no. 2018-30). Prior to the examination, the subjects were informed of the purpose of the study, and written informed consent was obtained.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References