The spiral bacteria, Helicobacter pylori (H. pylori) was originally discovered by W. Jaworski (1), professor of Jagiellonian University in Cracow at the end of the 19th century. However, the recognition of the pathological aspects of H. pylori inoculation of upper gastro-intestinal tract, originate from the rediscovery of this bacteria by Australians clinicians, Marshal and Warren in 1982 (2). They found that the presence of H. pylori in the stomach is closely associated to gastritis and gastro-duodenal peptic ulcerations. Nevertheless, to understand the pathology of H. pylori, we still need to examine thoroughly: 1. the routes of transmission of the infection and 2. the distribution of the bacteria in the gastro-intestinal tract, including the oral cavity. Since H. pylori is known to be found not only in stomach but also in the oral cavity, the question rises what is the relationship between presence of this bacteria in oral cavity and the infection of stomach. Main question, which should be posed remains, wheather oral cavity could serve as a reservoir of H. pylori for gastric infection. Furthermore, the question remains, what is the characteristics of the H. pylori detected in oral cavity.

It is possible, that H. pylori simply belongs to the normal flora of the oral cavity, maintaining a commensal relationship with the host (3). Song at al. provided evidence that the difference in H. pylori detection in oral cavity in different studies derives from the various sensitivity of bacteria detection methods (4). Some others authors suggest that H. pylori is not consistently present in oral cavity environment, but is only transiently contaminating the oral cavity with ingested food or because of due to bacteria contaminating esophagus due to gastroesophageal reflux (5, 6). Another possibility is that, the presence of H. pylori in oral cavity represents real infection. In that case, the question raises, what could be the consequences of this infection for the oral cavity itself. It is well recognized that stomach is the natural niche for H. pylori, despite the fact that its environment with highly concentrated hydrochloric acid is hostile for most other bacteria including H. pylori. As gastric acid is to serve as defense factor aimed to kill bacteria passing the stomach with contaminating food (2). H. pylori is exceptionally adapted to survive and grow in acid environment because it developed several mechanisms preventing detrimental influence of gastric acid (1). First of all, it hides itself from direct exposure to acid by moving under the mucus layer covering the surface mucosal epithelial cells, which additionally secretes HCO3- to create alkaline environment. Next, H. pylori has high urease activity producing alkaline ammonia to neutralize H+ around the germ. Ammonia creates alkaline milieu around H. pylori cell and shields it from the direct action of acid. Thus, the bacteria is extremely well adapted to acidic milieu of the stomach, this is why the H. pylori infection once aquired, usually during childhood remains for decades or even throughout the lifetime.

The major question arises whether H. pylori outside the stomach, especially in the oral cavity is equally infective as that found in the stomach, considering different environmental conditions such as high pH and lack of acid. In addition to this, enormous amount of other bacteria inhabiting the oral cavity may interfere with the viability of H. pylori in the mouth (7). The next question is, whether such orally originated H. pylori can inoculate the gastric mucosa.

Accordingly, the aim of the present study was firstly to determine the occurrence of H. pylori in the oral cavity (in the saliva and in gingival pockets) and secondly, to study the relationships between gastric H. pylori infection and the presence of this bacteria in the oral cavity.


MATERIALS AND METHODS
Studied group

We studied the presence of H. pylori in the stomach and oral cavity in 100 female subjects. Mean age of the studied group ranged from 19.9 to 51.8 years. Gastro-intestinal symptoms, mainly dyspeptic symptoms, were recorded in 41.8% of studied individuals. Informed consent was obtained from all individuals and study was approved by Local Research Ethics Committee at the Jagiellonian University, Cracow.

Determination of H. pylori infection status in stomach

The active H. pylori infection in stomach was estimated using 13C- Urea Breath Test (UBT) as described previously (8). After overnight fast two baseline (prior to urea administration) breath samples were collected into testing vials from each subject. Next, gelatine capsule containing 38 mg of 13C-urea was swallowed with 25 ml of water by each subject. Breath samples were collected after 10 and 20 min following the 13C-urea administration. Final results of 13CO2/12CO2 ratios were measured with the use of isotope ratio mass-spectrometry (IRMS, Heliview, Medichems Seoul, Korea) and were expressed as d13CO2 (per mil) values. A change of mean 13CO2 value over baseline (DOB) after urea capsule ingestion, of more than 2.5 was considered as positive result.

Determination of H. pylori presence in the oral cavity

The presence of H. pylori in the oral cavity was determined using microbiological cultures as described previously (9). At the same time as UBT test was done material from gingival pockets and saliva was obtained into sterile vials. Microbiological cultures were performed on solid selective, enriched medium for H.pylori growth (H. pylori agar, Becton Dickinson). Gram staining was carried out to show the presence of gram-negative spiral bacteria. H. pylori was identified using a test for the presence of bacterial urease, catalase and oxidase. The API-Campy test (BioMerieux) was performed to confirm diagnosis.

Salivary anti-Hp IgA determination

Salivary anti-Hp IgA was determined using a commercially available ELISA kit (EIAGEN HP, Clone Systems) (9). Briefly saliva specimens were centrifuged for 10 minutes at 2000g. Specimens were stored at -80°C until assay. No diluted supernatant was used for immunoglobulin determinations. Results of anti-Hp IgA >1.0 OD (optical density) were considered positive.

Statistical analysis

The presence of H. pylori in various studied locations were compared by chi-square tests with the Yates correction and population structure analysis tests. P<0.05 was considered statistically significant.


RESULTS
Prevalence of gastric H. pylori infection and oral colonization by H. pylori

H. pylori was found in the stomach in 51% of studied individuals, while oral H. pylori was found in 54% (in saliva) and in 48.3% (in gingival pockets) (Fig.1). There were no significant differences in the occurrence of oral and gastric H. pylori in the studied group of subjects (p=NS). The presence of H. pylori in the saliva was greatly correlated with the presence of bacteria in the gingival pockets (X2=18.4; p=0.0002) (Table 1), which could suggest that gingival pockets might be the source of H. pylori in saliva. Alternatively the colonization may occur in parallel at both locations. Interestingly anti-Hp IgA was found in 84% of studied individuals, which is significantly more common than the occurrence of H. pylori either in the stomach or in the oral cavity (Fig. 1).

Fig. 1. Occurrence of H. pylori in the stomach and in the oral cavity. Left panel: The presence of H. pylori was studied in the stomach by urea breath test (UBT) and in the oral cavity by bacterial culture from saliva and swabs from gingival pockets. Right panel: Additionally anti-Hp IgA was measured in the saliva of studied individuals (right panel). Bars represent % of H.pylori positive individuals in the whole study group (n=100); * - p<0.05 vs gastric and oral H. pylori.

Table 1. Relationship between the presence of H. pylori in the saliva and in the gingival pockets. Values indicate numbers of patients; and % or row and column (col). Statistical significance of this relationship was determined using chi-square test. (X2=18,4; p<0.0002)

Relationships between the presence of oral and gastric H. pylori

Similar occurrence of gastric and oral H. pylori could suggests that there is a close relationship between these two. We therefore, studied the potential relationships between the presence of H. pylori in the oral cavity and in the stomach. No relationship was found between the presence of the bacteria in the oral cavity and the H. pylori gastric infection (Fig. 2). 54.9% of subjects with stomach infection showed concomitant presence of H. pylori in saliva. 53.2% of examined subjects with negative UBT-test revealed the presence of H. pylori in culture from the saliva. The X2 value of relationship between UBT (as an induced of gastric infection) and H pylori saliva culture was 0.029 (p=0.9). Similarly, no relationship was found between the presence of H. pylori in the stomach and in the gingival pockets (X2=0.6; p=0.4).

Fig. 2. Presence of oral H. pylori in patients in whom gastric infection was identified (UBT +) and in subjects without gastric infection (UBT -). Oral H. pylori was studied by culture from saliva and gingival pockets. Bars represent % of positive individuals in each group. Numbers above bars indicate values of chi-square test (X2) and p values of relationships between gastric and salivary/gingival H. pylori infections.

Toothlessness as a factor in H. pylori colonization in the oral cavity

We also compared the presence of H. pylori in the saliva of patients with and without teeth. H. pylori was found in 63.7% of patients without teeth and in 52.9% of patients with teeth (Fig. 3; p=NS). This may support the thesis that the presence of oral H. pylori is rather incidental and does not seem to be related to the presence of natural teeth.

Fig. 3. Relationships between the toothlessness and the presence of H. pylori in the oral cavity. Toothlessness was judged by clinical finding of complete loss of teeth. Oral H. pylori was studied by bacterial culture from saliva.

DISCUSSION
Gastric H. pylori infections is a common disease in the general population (2). The sources and routes of transmission have been definitively established despite numerous studies which suggested that "oral-fecal" and "oral-oral" transmission of H. pylori is most likely (10). It is, however, unclear whether oral cavity creates sufficiently favorable conditions for H. pylori growth (5) if the oral cavity is to be an important source of gastric infections (11). Accordingly, we studied the relationships between gastric and oral presence of H. pylori. We found that while both gastric and oral H. pylori is very common and occurs roughly in about 50% of population, there was no relationship between gastric infection and oral presence of bacteria. This finding may suggest that oral presence of H. pylori may be only intermittent and transient and other factors like susceptibility associated with gastric pH, concomitant inflammation or loss of barrier function may be critical in the gastric infection with this bacteria. In the absence of the above abnormalities, transient presence of H. pylori in oral cavity may be irrelevant for gastric infections. It may be that H. pylori is merely a standard component of dental film and does not cause infection and is not related to gastric pathologies and symptoms. The transient presence of H. pylori in oral cavity is supported by our observation that the detection of anti-Hp IgA exceeds greatly the presence of bacteria themselves indicating that many patients who were recently exposed to H. pylori do not seem to have the bacteria any more.

Another method to address an importance of H. pylori in gastric colonization, uses the model of H. pylori eradication. The persistence of H. pylori following the eradication therapy may lead to numerous pathological consequences (12, 13). The eradication therapy usually removes the gastric infection (12), while it does not necessarily affect oral colonization (9, 14). This approach was used in a previous study by Karczewska et al. who found that while H. pylori is capable of colonizing oral cavity, it does not seem to be an important source of gastric reinfection after effective H. pylori eradication therapy (9). These results are in direct agreement with our findings in the present study. However, some other studies suggested that H. pylori in dental plaque may represent a risk factor for gastrointestinal re-infection and ulcer relapse after antibiotic therapy (15, 16), though these data were obtained on relatively small study sample. Other studies, do not support the possibility of oral cavity as a reservoir for gastric infections (3, 9, 16-19). These discrepancies indicate that potential relationship between oral and gastric H. pylori infections is more complex. Large epidemiological investigation using UBT performed in over 10 000 subjects revealed that periodontosis significantly increases the risk of gastric H. pylori infection (5). Unfortunately, oral H. pylori was not studied in the above study, but it may be interesting to speculate that in certain oral pathologies H. pylori may be more abundant in the oral cavity making gastric infection possible. We have not performed the quantitative or quantitative microbiological and genetical analysis. It is, therefore, also possible that the numbers of bacteria present in the saliva and/or gingival pockets are not sufficient to infect gastric mucosa. In the conditions of periodontitis, mucosal ulcers and other oral pathologies number of H. pylori may increase and reaches levels sufficient to cause gastric infections. This hypothesis is attractive, as it would indicate that periodontal health would determine whether oral H. pylori may or may not cause gastric infection (20). Direct quantitative microbiological studies are needed to address this possibility. Factors leading to increased H. pylori adherence in the oral cavity may be related to changes of extracellular matrix proteins, as well as functions of various cells in the oral mucosa (21 - 23), in relation to oral mucosal ulcerations etc. (24). Similarly changes in intracellular signaling systems may be important for oral pathologies (25).

Other clinical conditions than periodontosis or oral ulcers may also lead to an increase in the number of H. pylori. These may be related to the loss of normal oral bacterial flora, as it has been shown to affect the H. pylori growth by producing bacteriocin-like inhibitory proteins against H. pylori strains (7). The antagonistic effects of oral bacteria against H. pylori may restrain colonization by this organism in the oral cavity (7). The opposite situation, that H. pylori may play important role in the pathogenesis of oral ulcers is also likely. The data suggest that H. pylori may be associated frequently with recurrent oral aphthous ulcers, and are consistent with previous studies indicating that saliva and plaque are not likely sources of contamination with this microorganism (26). In this clinical situation the use of medical and other treatments such as probioties to suppress oral H. pylori could improve periodontal hygiene and condition (27). Chemical plaque control is a useful aid in mechanical oral hygiene, and various chemical agents have been evaluated as antiplaque agents. It has been shown that mastic chewing gum has antibacterial effects on H. pylori (27). If we assume that oral H. pylori colonization is intermittent its origin remains not entirely clear. Unquestionably food may be an important source (2). One should also remember that gastro-esophageal reflux and vomiting may be important risk factors for oral and persistent gastric H. pylori infections (28).

It is very important to determine possible risk factors, which would enable oral H. pylori to colonize and infect stomach. These could include disturbed gastric mucosal barrier function, increased oxidative stress in gastric mucosa leading to increased inflammatory adhesion molecule expression or loss of nitric oxide dependent anti-Hp properties. Finally, numerous genetic factors may predispose to gastric infections with H. pylori. To date, the associations with the polymorphisms of fucosyl transferase 2 (FUT2 or secretor gene), FUT3 (Lewis gene), interleukin 1aA (IL-1alpha), IL-1ß, IL-1RN, IL-8, IL-10, myeloperoxidase (MPO), and tumor necrosis factor A (TNF-alpha) and TNF-ß have been reported (29, 30). Polymorphisms of other related genes, CD14, CXC chemokine receptor 2 (CXCR2), IL-1RI, nuclear factor KB2 (NF-KB2), and Toll-like receptor 4 (TLR4), have the potential to influence persistent infections (28). Gene-environment interactions between these genotypes and smoking or use of non steroidal anti-inflammatory drugs may further increase susceptibility to gastric H. pylori infection (31). As nitric oxide (NO) and superoxide appear to play important roles in the regulation of gastric protection (32) and response to H. pylori (33) it may be interesting to speculate that polymorphisms of NO synthase (34) or NAD(P)H oxidase (35 - 37) (major source of superoxide in human gastric mucosa) may be of potential, although yet uninvestigated importance. Morover, importance of neuro-pharmacological modulation of gastric functions may be important for the regulation of susceptibility to H. pylori infections (13, 38, 39). However, gastric H. pylori infection occurring only in some individuals with oral colonization may also indicate that pathogen specific properties may play a role. Molecular typing experiments revealed that polymorphic variations of H. pylori genome may be important (40). For example strains expressing CagA are known to have higher pathogenecity than the CagA negative bacteria. Other antigens like VacA or IceA may also be important in modulating properties of bacteria itself (41).

Finally, it is important to emphasize that possible intermittent oral H. pylori colonization may have implications in dentistry (18). Our study shows that H. pylori is found in 50% of studied individuals. Continuous exposure of the dentist to the aerosols derived from patient's oral cavity are in fact associated with continuous exposure to H. pylori which in the appropriate conditions could possibly lead to gastric infection. The studies of he occurrence of H. pylori gastric infections in dentists are however inconclusive (41 - 43).


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