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Journal of Interdisciplinary Dentistry • Volume 13 • Issue 1 • January - April 2023 • Pages ***-***
Original Article

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 Background: The oral cavity has optimal conditions to act as a reservoir of

Abstract
Sciences, Ethics, Research
and Education, Dental
microorganisms. Aims and Objectives: This research corresponds to a descriptive
School, University of the study, which aims to study the presence or absence of periodontopathogens and
Andes, Monseñor Alvaro Helicobacter pylori in oral swab samples from healthy Chilean women between 18
del Portillo 12455, Santiago, and 26 years old. Materials and Methods: Fifty-four oral samples were recruited
Chile 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

Clinical Relevance to Interdisciplinary Dentistry

Received: 18 January 2023 In our study, a trend in the presence of HP was also observed, which can determine
Revised: 01 March 2023 the presence of HP in stages and early signs of future infection, which may be
Accepted: 02 March 2023
Published: 28 April 2023 relevant in early diagnosis and treatment.

Introduction pylori (Hp).[3] Hp is a Gram‑negative bacterium

T he 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.[1] Periodontal pathogens in the oral
mucosa, tongue, saliva, and dental biofilm, among
others,[2] 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
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associated with digestive pathologies, especially
gastric‑esophageal carcinoma. This bacterium affects
50% of the world population[4] and is identified as a
type I carcinogen and the causative agent of peptic
ulcers.[5] 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.[6] Since Hp
Address for correspondence: Dr. Carolina María Inostroza Silva,
Dental School, University of the Andes, Monseñor Alvaro del
Portillo 12455, Santiago, Chile.
E‑mail: cminostroza@uandes.cl
This is an open access journal, and articles are distributed under the terms of the
Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows
others to remix, tweak, and build upon the work non‑commercially, as long as
appropriate credit is given and the new creations are licensed under the identical
terms.
For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com

DOI: How to cite this article: Bennett J, Carrasco P, Solis F, Morroni R, Gallardo J,
10.4103/jid.jid_1_23 Silva CM. Coexistence of periodontal pathogens and Helicobacter pylori in
the oral cavity of a healthy cohort. J Interdiscip Dentistry 2023;13:17-22.

© 2023 Journal of Interdisciplinary Dentistry | Published by Wolters Kluwer - Medknow 17

 Bennett, et al.: Periodontal pathogens and Helicobacter pylori in oral samples
from the oral cavity was successfully isolated and
cultivated, it is related to it as a potential reservoir of
this microorganism.[7] 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.[8] Another study
in Chile[9] 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.[10] 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[11] and normal oral
nonresident.[12] 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;[4] 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.[6] 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.[13] 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.[12,14,15] 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.
18 Journal of Interdisciplinary Dentistry  ¦  Volume 13  ¦  Issue 1  ¦  January-April 2023
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
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
 Bennett, et al.: Periodontal pathogens and Helicobacter pylori in oral samples

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.
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.
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.
Statistical analysis
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

Table 1: Detail of the bacterial genes and the respective sequences of the primers used in the polymerase chain
reaction reaction
Bacteria Gene Primer sequences Size (pb)
Hp cagA2‑1 5’‑TGA TAG GGA TAA CAG GCA AGC TT‑3’ 181
cagA2‑2 5’‑CTG AAA GCT CTT TGT GGA AGA TTC‑3’
Td tden‑1 5’‑TAA TAC CGA ATG TGC TCA TTT ACA T‑3’ 316
tden‑2 5’‑TAC AAG AAG CAT TCC CTC TTC TTC TTA‑3’
Tf for‑1 5’‑TAC AAG GGA ATA AAA TGA GAT ACG‑3’ 745
for‑2 5’‑ACG TCA TCC CCA CCT TCC TC‑3’
Pg pgin‑1 5’‑AGG CAG CTT GCC ATA CTG CG‑3’ 404
pgin‑2 5’‑ACT GTT AGC AAC TAC CGA TGT‑3’
The expected fragment size in the amplification for each bacterium is also indicated in base pairs (bp). Pg=Porphyromonas gingivalis,
Tf=Tannerella forsythia, Td=Treponema denticola, Hp=Helicobacter pylori

Journal of Interdisciplinary Dentistry  ¦  Volume 13  ¦  Issue 1  ¦  January-April 2023 19

 Bennett, et al.: Periodontal pathogens and Helicobacter pylori in oral samples

Table 2: Record of bacterial development on a 5% by conventional PCR for Pg, Td, Tf, and Hp from the
Tryptic Soy Agar plate DNA of the spindle. Table 3 shows the results indicating
n° Sample Bacterial n° Sample Bacterial the presence  (+), absence  (−), or no amplification  (0).
code growth code growth After analyzing and evidencing the degradation of the
1 133 + 28 188 +++ PCR products from the torula, we decided to discard
2 134 +++ 29 189 ++ the direct PCR analysis from the torula. It proceeded
3 135 ++ 30 190 +++ to analyze only agar samples. For this, 15 additional
4 136 + 31 192 +++ agar samples were selected randomly. The results were
5 137 +++ 32 197 +++ entirely negative for Pg, one positive for Td, 3 for Tf,
6 138 +++ 33 199 −
and six positives for Hp [Table 4]. Of the total samples
7 139 +++ 34 201 −
analyzed,[16] six samples were amplified for one or
8 141 +++ 35 202 +++
9 148 ++ 36 203 +
more of the four bacteria under study, 29%. However,
10 152 +++ 37 205 +++ 15 (71%) samples were negative for any bacteria studied.
11 153 +++ 38 206 +/− Of the six samples that presented positive results (29%)
12 154 +++ 39 207 ++ in the electrophoresis of the PCR amplified products, it
13 155 ++ 40 209 +++ was found that for Tf, there were three positive results.
14 156 +++ 41 210 ++ For Td, there was only one positive result; for Hp, six
15 162 +++ 42 211 ++ samples with positive results, as indicated, are shown in
16 165 + 43 212 +/− Graphs 1 and 2.
17 166 − 44 213 +++
18 167 ++ 45 215 +/− Discussion
19 168 +++ 46 049‑4 −
Several studies currently link the presence of Tf, Td,
20 174 +++ 47 144‑4 +
and Pg bacteria with Hp in the oral cavity. These studies
21 176 ++ 48 174‑3 +++
22 177 +++ 49 183‑8 +++
were conducted in cohorts of individuals with gingivitis,
23 178 +/− 50 265‑K +++ periodontitis, or chronic noncommunicable diseases,
24 179 +++ 51 292‑1 + such as type II diabetes,[10] arterial hypertension, among
25 185 ++ 52 705‑7 +++ others. However, there are no precedents where the
26 186 +++ 53 823‑5 + presence of these bacteria in a healthy cohort is studied.
27 187 +++ 54 974‑K − Our report detected the presence and/or absence of Tf,
+++=Abundant quantity, ++=Average amount, +=Small Td, Pg, and Hp in a cohort of healthy Chilean women
amount, +/−=Very little quantity and (−)=There was no growth. between 18 and 26 years old. Our results show that these
TSA=Tryptic Soy Agar microorganisms have a low prevalence in the studied
cohort.[17] A descriptive study in Chile[18] found that,
Table 3: Evaluation of the presence or absence of out of 75 Chilean patients with chronic and aggressive
amplification for the 4 bacteria analyzed by polymerase periodontitis, 88% presented Pg, which correlates with
chain reaction+electrophoresis of DNA extracted from
the antecedents described above.[19] These antecedents
swab versus DNA extracted from 5% Tryptic Soy Agar
highlight Pg as the most prevalent periodontal pathogens
Sample code Pg Td Tf Hp
139‑A − − + + of interest. It should be noted that these studies were
139‑T − − − − carried out on individuals with underlying pathologies,
148‑A − − − + chronic periodontitis, unlike this study, where there
148‑T − − − 0 was no information on the periodontal health of the
155‑A − − + + donors. Regarding the periodontal condition of the
155‑T − − 0 0 population in Chile, a high prevalence of periodontal
167‑A − + − + lesions is present in the adult population.[8] The absence
167‑T − − 0 0 of Pg could be the cause of the good oral health of the
210‑A − − + + participants. Variables such as oral hygiene and the time
210‑T − − 0 0 between using toothpaste and/or mouthwash before the
211‑A − − − + sample collection could inhibit PCR detection. Another
211‑T − − − −
possibility is that, with the methodology used, it was
Sample code number-A=Agar sample, “Sample code
impossible to detect Pg because the sample was taken
number”-T=Swab sample, +=Positive result, (−)=Negative
result, 0=There was no amplification. Pg=Porphyromonas using sterile paper cones directly from the gingival
gingivalis, Tf=Tannerella forsythia, Td=Treponema denticola, sulcus,[20] which is the main difference from our study.
Hp=Helicobacter pylori The oral swab is a minimally invasive, low‑cost,

20 Journal of Interdisciplinary Dentistry  ¦  Volume 13  ¦  Issue 1  ¦  January-April 2023

 Bennett, et al.: Periodontal pathogens and Helicobacter pylori in oral samples

Graph 2: Number of samples positive for Treponema denticola and

Graph 1: Distribution of the number of positive samples according to Tannerella forsythia that are associated with Helicobacter pylori
the four bacteria analyzed

that our results can be contrasted with the saliva study

Table 4: Results obtained after polymerase chain of an adult Japanese population (>40 years) of 2344
reaction individuals from Hisayama, Japan.[11] Regarding the
Sample code Hp Td Tf Hp health of the individuals, no further details are provided;
139 − − + +
however, the authors demonstrated a great abundance of
148 − − − +
Td along with Pg and Tf in saliva and dental biofilm.
152 − − − −
155 − − + + Another study describes the presence of Td and Pg
167 − + − + colonizing the gingival sulcus, indicating some metabolic
179 − − − − dependence between them.[21] This fact could explain
192 − − − − why in the present analysis, Pg was not found in any
162 − − − − of the study samples, since only 1 sample revealed the
168 − − − − presence of Td, and Tf was identified in 14% of the
190 − − − − samples, considering that in addition to being individuals
202 − − − − healthy, should not present these pathogens.[18] Quintero
209 − − − − et  al.[10] reported that 20% of the individuals with mild
213 − − − − periodontitis had Tf at the subgingival level. This finding
138 − − − −
agrees with other articles that showed the presence of
137 − − − −
this bacterium increases according to the periodontitis
154 − − − −
186 − − − − picture. Although the study populations differ, our
210 − − + + results are like those presented by both authors. Tf is
211 − − − + not considered part of the oral flora but is one of the
174‑3 − − − − leading causes of periodontitis. Therefore, its presence
705‑7 − − − − in healthy individuals could be considered a periodontitis
The code "+" means=There was DNA amplification or positive risk factor. Concerning Hp, our results agree with what
result. The code "-" means= There was no amplification, or was postulated by Chumpitaz Conde et  al.,[7] where the
negative result. Tf=Tannerella forsythia, Td=Treponema denticola, oral cavity is suggested as a reservoir of Hp. However,
Hp=Helicobacter pylori. +=Presence, −=Absence
these results contradict the study by Kazanowska carried
out in 2016[16] where patients with some oral pathologies
easy‑to‑use, low‑risk method for the handler and the
were studied. Hp was detected in 21.7% of the
volunteer. In addition, it is a good alternative since it
individuals (126 patients) and was not seen in the healthy
allows the recovery of bacteria, yeasts, and cells, among
control group. There are records of a greater abundance
others.[13] Our group recently published an article in which
of Pg, Fn, and Td together with Hp, compared to samples
we identified and associated the presence of Hp in the
where Hp has not been detected,[22] demonstrating their
oral mucosa with gastric biopsy. Detecting this bacterium
close relationship.
in a noninvasive sample, such as saliva or oral mucosa,
without performing a complex procedure such as an This fact may explain the presence of
endoscopy could provide a tremendous complementary periodontopathogens together with Hp but without
diagnostic tool, low cost, and rapid implementation statistically significant values. We consider that factors
compared to invasive gastric biopsy.[13] Therefore, these not considered in this investigation could have affected
attributes associated with the buccal swab sample make it the bacterial load at the time of sample collection, for
a method with excellent characteristics to consider when example, toothpaste containing triclosan, which inhibits
performing molecular diagnosis. We want to emphasize the bacterial development of Fn.[23] Another factor is 2%

Journal of Interdisciplinary Dentistry  ¦  Volume 13  ¦  Issue 1  ¦  January-April 2023 21

 Bennett, et al.: Periodontal pathogens and Helicobacter pylori in oral samples
mouthwash, which, like toothpaste, contains triclosan
with a moderate inhibitory effect on plaque formation
that can last up to 5 h.[24] 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
Nil.
Conflicts of interest
There are no conflicts of interest.
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