|Year : 2023 | Volume
| Issue : 1 | Page : 17-22
Coexistence of periodontal pathogens and Helicobacter pylori in the oral cavity of a healthy cohort
Josefina Bennett, Patricio Carrasco, Francisca Solis, Rainiero Morroni, Jorge Gallardo, Carolina María Inostroza Silva
Department of Biomedical Sciences, Ethics, Research and Education, Dental School, University of the Andes, Monseñor Alvaro del Portillo 12455, Santiago, Chile
|Date of Submission||18-Jan-2023|
|Date of Acceptance||02-Mar-2023|
|Date of Web Publication||28-Apr-2023|
Dr. Carolina María Inostroza Silva
Dental School, University of the Andes, Monseñor Alvaro del Portillo 12455, Santiago
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The oral cavity has optimal conditions to act as a reservoir of microorganisms. Aims and Objectives: This research corresponds to a descriptive study, which aims to study the presence or absence of periodontopathogens and Helicobacter pylori in oral swab samples from healthy Chilean women between 18 and 26 years old. Materials and Methods: Fifty-four oral samples were recruited and then cultured on 5% blood agar plates for the semiquantitative macroscopic microbiological analysis. Of the 54, only 38 showed regular to abundant bacterial growth. Of these 38 samples, 21 were randomly selected and analyzed by conventional polymerase chain reaction (PCR) to detect periodontopathogens (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) and Helicobacter pylori. Results: 71% of the samples studied were negative for this group of bacteria, while 29% were positive for at least one of the microorganisms studied. Of this group, six samples were positive for Helicobacter pylori, one for Treponema denticola, and three for Tannerella forsythia. These last two microorganisms were presented together with Helicobacter pylori. Conclusion: The buccal swab of the healthy cohort was a matrix that allowed bacterial molecular analysis; however, due to the small sample size, no correlations were found between them.
Keywords: Helicobacter pylori, periodontopathogens, polymerase chain reaction
|How to cite this article:|
Bennett J, Carrasco P, Solis F, Morroni R, Gallardo J, Silva CM. Coexistence of periodontal pathogens and Helicobacter pylori in the oral cavity of a healthy cohort. J Interdiscip Dentistry 2023;13:17-22
|How to cite this URL:|
Bennett J, Carrasco P, Solis F, Morroni R, Gallardo J, Silva CM. Coexistence of periodontal pathogens and Helicobacter pylori in the oral cavity of a healthy cohort. J Interdiscip Dentistry [serial online] 2023 [cited 2023 Jun 2];13:17-22. Available from: https://www.jidonline.com/text.asp?2023/13/1/17/375280
| Clinical Relevance to Interdisciplinary Dentistry|| |
In our study, a trend in the presence of HP was also observed, which can determine the presence of HP in stages and early signs of future infection, which may be relevant in early diagnosis and treatment.
| Introduction|| |
The oral cavity is a significant bacterial reservoir that includes commensal microorganisms and bacterial flora with pathogenic capacity, mainly periodontal pathogens that cause highly prevalent gum diseases in Chile. Periodontal pathogens in the oral mucosa, tongue, saliva, and dental biofilm, among others, are Gram-negative and anaerobic bacilli, such as Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), and Treponema denticola (Td). However, recent studies have shown the presence in saliva and oral fluids of pathogenic bacteria such as Helicobacter pylori (Hp). Hp is a Gram-negative bacterium associated with digestive pathologies, especially gastric-esophageal carcinoma. This bacterium affects 50% of the world population and is identified as a type I carcinogen and the causative agent of peptic ulcers. In Chile, approximately 79% of the population has Hp, with gastric carcinoma being the leading cause of death in men and the third in women. Since Hp from the oral cavity was successfully isolated and cultivated, it is related to it as a potential reservoir of this microorganism. A study on young people between 12 and 21 in Santiago province reported that 69.2% of individuals present damage to at least one dental piece. The prevalence of periodontal disease increases mainly in the female gender, smoking, and age. Another study in Chile described the periodontal sulcus in patients with chronic periodontitis, the prevalence of Pg, Td, Aa, Tf, and additionally, Fusobacterium nucleatum (Fn), which can co-aggregate with the other bacteria and thus facilitate colonization. Other studies have shown that in patients with a morbid history of chronic diseases, precisely type II diabetes, who also have some degree of periodontitis, the prevalence of periodontal pathogens such as Td and Tf is high.
In contrast, bacteria such as Pg and Aa are present, but their prevalence is lower. The presence of Hp in dental plaque and saliva suggests that the oral environment would be a route of infection and that Hp would be present in the oral cavity as a consequence of gastric reflux or as part of the transient oral microbiota and normal oral nonresident. In descriptive studies, it was described that the percentage of women infected with Hp in Chile is three times higher than in men, 72.1% and 26.9%, respectively; in addition, out of a total of 56 patients with an age range between 11 and 30 years, 50% turned out to be positive for the Hp urease activity test. Our team recently published a study in which we demonstrated the concordance between the molecular detection of Hp in the oral mucosa and the urease test, constituting a feasible diagnostic tool for Hp in the oral cavity. Some studies suggest that the risk of acquiring the periodontal disease is 2.83 times greater when Hp is present; it also indicates that the oral cavity has certain reservoir conditions for this microorganism.,, According to the background presented, describing the majority of Hp and periodontal pathogens in a healthy cohort is relevant.
| Materials and Methods|| |
Study design and sample collection
The research corresponds to a descriptive study approved by the Ethics Committee of the Eastern Metropolitan Health Service (SSMO, N°protocol= 17-2017). Participants answered a short, straightforward questionnaire (Supplemental material) about their general health status to be included as healthy donors. Individuals included in the study were women ages 18–26. Healthy donors included in the study had no chronic or acute diseases, were not on permanent pharmacological treatment, had not used antibiotics in the last 6 months, were non-smokers, and had no gastric or cancer illnesses. The volunteers read and signed a specially designed informed consent. Fifty-four samples of buccal swabs from the buccal mucosa were collected according to the inclusion criteria: Chilean women between 18 and 26 years of age, born and resident, healthy according to a brief health survey, systemic health, that is, they have not been diagnosed with previous chronic diseases, or are under permanent or sporadic drug treatment, not living with other study participants and not having eaten food during the last 30 min before sample collection. The exclusion criteria included: consumption of antibiotics in the previous 90 days, drug treatment for major or chronic diseases during the last 12 months, pregnancy, undergoing dental procedures or surgeries during the previous 3 months, donors who, at the time of taking sample present dental mobility and bleeding gums. With a sterile swab (Go Scientific, India) and aseptic technique, previously coded with the sample number, the head of the swab was pressed on the inside of one of the cheeks (jugal mucosa) and rubbed with broad vertical movements rotating the swab for at least 30 s avoiding touching the teeth. The swab was then placed in its container and stored in a double-sealed plastic bag to be transported to the laboratory in an aluminized thermal container (Marea Alta®) with frozen refrigerant gels inside (Tres Osos®), ensuring the cold chain during transport for later cold storage (4°C ± 2°C).
Bacterial culture on a blood agar plate
Before the culture, swabs were dried at 37°C in an oven for 1 h and then were sown in 10 cm diameter culture plates (Insumolab®; Lablinsan®) of 5% base sheep blood agar (tryptic soy agar [TSA]). For this purpose, each dish was divided into six equal parts, resulting in each sextant in an approximate area of 13.08 cm2. The depletion sowing technique was used from the sampling swab, and a labeled agar sextant was assigned. In parallel, a 5% TSA agar plate was seeded as a negative control with a swab without a sample. The plates were incubated under aerobic conditions in an oven (Sanyo®) at 37°C for 18–24 h. Subsequently, a semiquantitative microscopic analysis based on the assignment of crosses was carried out to evaluate the development of bacterial colonies [Figure 1]. It was observed with a stereoscopic binocular magnifying glass (ST 30 L, Arcano®). The photographic recording was carried out with an 8-inch camera. Megapixels (iPhone 6, Apple®) adapted to the magnifying glass.
|Figure 1: Macroscopic images of the observed colonies. The photo on the left: Photograph of the bacterial growth of sample number 174-3 seeded on 5% TSA blood agar. The image on the right is a photograph of the colonies observed in the left photo taken under a microscope, magnifying ×100 the distribution of bacterial development. TSA=Tryptic soy agar. The black arrow indicates the colonies|
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Total DNA extraction
The swabs were cut with sterile scissors, and the cotton tips were placed in 1.6 mL Eppendorf® tubes with 0.5 mL of nuclease-free sterile distilled water. The tubes were vortexed (MX-S Vortex– SCILOGEX®) for 60 s and then incubated at 95°C for 10 min in a dry heater (Termoblock-BioProducts®). The tubes are set at − 20°C for 10 min and then centrifuged at 4°C for 5 min at 10,000 rpm (PrismR–Labnet®). Subsequently, the cotton swab was removed, and the supernatant was introduced into a labeled sterile microtube and stored at −80°C for later analysis. Finally, two total DNA samples (torula DNA: tDNA and agar DNA: aDNA) were obtained for each sample.
Polymerase chain reaction
The standard polymerase chain reaction (PCR) technique was used to determine the presence or absence of Hp, Pg, Td, and Tf. For the PCR reaction, each sample was prepared in a final volume of 20 μL, with 10 μL of GoTaq Green Master Mix 2X (Promega®, USA), 2 μL of each primer, and 8 μL of total DNA suspension. [Table 1] describes the sequences of the primers of the genes studied and the expected size of the amplified fragment for each bacterium.
|Table 1: Detail of the bacterial genes and the respective sequences of the primers used in the polymerase chain reaction reaction|
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Electrophoresis of the polymerase chain reaction fragments
Each gel was prepared with a 2% solution of SeaKem LE Agarose (Lonza®, Switzerland) in TAE1x buffer (Fermentas®, USA). Two microliters of DNA intercalator (Syber Safe Life technologies®) was added. Twenty microliters of the samples were loaded onto the gel and placed in a horizontal electrophoresis chamber (Labnet®, USA) for 60 min at 90 volts with a power source (Bio Products®, USA). To visualize the amplified fragments, the gel was loaded into the Enduro™ GDS Photo documentation System (Labnet, USA), with a built-in integrated ultraviolet transilluminator and a 5-megapixel resolution camera acquired in the equipment software on the computer.
An Excel table was created to organize the data. For the analysis, a one-way ANOVA statistical test and a paired t-test were performed using Graph Pad Prism 7 (GraphPad Software, Inc. California, USA) software to calculate significant differences with a 95% confidence interval.
| Results|| |
The average age of the participants was 21.5 years; the age range of 18 years was the least frequent, while the most frequent was 22 years. The semi-quantitative microbiological analysis of the 54 buccal swab samples showed that only five did not show bacterial development (−). The effect of up to two types of morphologically distinct colonies was observed in almost all the plates. The first type of colony, or Type 1, was characterized as small, shiny in appearance, with regular borders, and α-hemolytic. The second or two colony types were medium sized, whitish in color, with standard, convex edges, and γ-hemolytic [Figure 1]. In 54% of the plates, the development of Type 1 colonies was observed, and 2% were only Type 2 colonies. In 35% of the samples, the effect of both types of colonies was observed [Table 2]. Of the 54 samples collected, 16 samples, in which the bacterial growth was less than “++” were discarded. Of the 38 remaining samples, which had a development of “++” or “+++,” 6 samples were randomly chosen and were analyzed by conventional PCR for Pg, Td, Tf, and Hp from the DNA of the spindle. [Table 3] shows the results indicating the presence (+), absence (−), or no amplification (0). After analyzing and evidencing the degradation of the PCR products from the torula, we decided to discard the direct PCR analysis from the torula. It proceeded to analyze only agar samples. For this, 15 additional agar samples were selected randomly. The results were entirely negative for Pg, one positive for Td, 3 for Tf, and six positives for Hp [Table 4]. Of the total samples analyzed, six samples were amplified for one or more of the four bacteria under study, 29%. However, 15 (71%) samples were negative for any bacteria studied. Of the six samples that presented positive results (29%) in the electrophoresis of the PCR amplified products, it was found that for Tf, there were three positive results. For Td, there was only one positive result; for Hp, six samples with positive results, as indicated, are shown in [Graph 1] and [Graph 2].
|Table 3: Evaluation of the presence or absence of amplification for the 4 bacteria analyzed by polymerase chain reaction*electrophoresis of DNA extracted from swab versus DNA extracted from 5% Tryptic Soy Agar|
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| Discussion|| |
Several studies currently link the presence of Tf, Td, and Pg bacteria with Hp in the oral cavity. These studies were conducted in cohorts of individuals with gingivitis, periodontitis, or chronic noncommunicable diseases, such as type II diabetes, arterial hypertension, among others. However, there are no precedents where the presence of these bacteria in a healthy cohort is studied. Our report detected the presence and/or absence of Tf, Td, Pg, and Hp in a cohort of healthy Chilean women between 18 and 26 years old. Our results show that these microorganisms have a low prevalence in the studied cohort. A descriptive study in Chile found that, out of 75 Chilean patients with chronic and aggressive periodontitis, 88% presented Pg, which correlates with the antecedents described above. These antecedents highlight Pg as the most prevalent periodontal pathogens of interest. It should be noted that these studies were carried out on individuals with underlying pathologies, chronic periodontitis, unlike this study, where there was no information on the periodontal health of the donors. Regarding the periodontal condition of the population in Chile, a high prevalence of periodontal lesions is present in the adult population. The absence of Pg could be the cause of the good oral health of the participants. Variables such as oral hygiene and the time between using toothpaste and/or mouthwash before the sample collection could inhibit PCR detection. Another possibility is that, with the methodology used, it was impossible to detect Pg because the sample was taken using sterile paper cones directly from the gingival sulcus, which is the main difference from our study. The oral swab is a minimally invasive, low-cost, easy-to-use, low-risk method for the handler and the volunteer. In addition, it is a good alternative since it allows the recovery of bacteria, yeasts, and cells, among others. Our group recently published an article in which we identified and associated the presence of Hp in the oral mucosa with gastric biopsy. Detecting this bacterium in a noninvasive sample, such as saliva or oral mucosa, without performing a complex procedure such as an endoscopy could provide a tremendous complementary diagnostic tool, low cost, and rapid implementation compared to invasive gastric biopsy. Therefore, these attributes associated with the buccal swab sample make it a method with excellent characteristics to consider when performing molecular diagnosis. We want to emphasize that our results can be contrasted with the saliva study of an adult Japanese population (>40 years) of 2344 individuals from Hisayama, Japan. Regarding the health of the individuals, no further details are provided; however, the authors demonstrated a great abundance of Td along with Pg and Tf in saliva and dental biofilm. Another study describes the presence of Td and Pg colonizing the gingival sulcus, indicating some metabolic dependence between them. This fact could explain why in the present analysis, Pg was not found in any of the study samples, since only 1 sample revealed the presence of Td, and Tf was identified in 14% of the samples, considering that in addition to being individuals healthy, should not present these pathogens. Quintero et al. reported that 20% of the individuals with mild periodontitis had Tf at the subgingival level. This finding agrees with other articles that showed the presence of this bacterium increases according to the periodontitis picture. Although the study populations differ, our results are like those presented by both authors. Tf is not considered part of the oral flora but is one of the leading causes of periodontitis. Therefore, its presence in healthy individuals could be considered a periodontitis risk factor. Concerning Hp, our results agree with what was postulated by Chumpitaz Conde et al., where the oral cavity is suggested as a reservoir of Hp. However, these results contradict the study by Kazanowska carried out in 2016 where patients with some oral pathologies were studied. Hp was detected in 21.7% of the individuals (126 patients) and was not seen in the healthy control group. There are records of a greater abundance of Pg, Fn, and Td together with Hp, compared to samples where Hp has not been detected, demonstrating their close relationship.
This fact may explain the presence of periodontopathogens together with Hp but without statistically significant values. We consider that factors not considered in this investigation could have affected the bacterial load at the time of sample collection, for example, toothpaste containing triclosan, which inhibits the bacterial development of Fn. Another factor is 2% mouthwash, which, like toothpaste, contains triclosan with a moderate inhibitory effect on plaque formation that can last up to 5 h. The DNA extraction method was a rapid assay without purification steps; it is not pure DNA but rather an extract with cell debris and salts that can affect and inhibit PCR, which is reason enough to rule out samples from swabs for analysis by PCR. Within the study's limitations, it is essential to increase the sample size and include male individuals; likewise, the investigation could be complemented with culture for developing periodontopathogens and Hp in the future. More efficient DNA extraction methods should also be considered for pure samples and better results.
| Conclusions|| |
Bacterial growth was observed in all the samples and allowed a microbiological analysis to be carried out, describing the level of abundance and colony type (1 or 2). PCR detection was positive for Hp alone or with Td and Tf. Therefore, we can conclude that the presence of Hp and periodontal pathogens was evidenced in healthy women; however, due to the small sample size, we could not make correlations between them.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]