SUMMARY

INTRODUCTION:

Nearly 73% of the Chilean population is infected with Helicobacter pylori (H. pylori), a factor predisposing for gastric cancer. Recent studies have demonstrated the presence of this pathogen within yeasts, suggesting that this fact can directly influence the failure of a treatment, transmission, and reinfection.

AIM:

To detect the presence of H. pylori inside oral yeasts isolated from students of the University of Concepción (Chile).

METHODS:

72 samples, obtained from the oral cavity using cotton swabs were incubated in YPD broth for 48h at 37°C and posteriorly seeded in Sabouraud Dextrose agar plus chloramphenicol at the same temperature and for the same time. Yeasts isolated were observed microscopically (wet mounting and Gram-stained) and identified using microbiological techniques. Intracellular H. pylori detection was performed by the amplification of 16S rDNA by PCR.

RESULTS:

Oral yeasts were detected in 24 samples (33.3%), being C. albicans (79.2%) the most frequent species, followed by C. dubliniensis (12.4%), C. krusei (4.2%), and C. tropicalis (4.2%). When analyzed by PCR, 15 of the 24 oral yeasts 62.5 % were positive for H. pylori 16S rDNA. From the 15 individuals positive for yeast harboring H. pylori, 81% of them reported stomach discomfort, and the presence of the bacteria was diagnosed at some moment in 20% of them.

CONCLUSION:

The intracellular presence of the H. pylori in oral yeasts suggests an endosymbiotic relationship of these microorganisms, which could favor H. pylori transmission and reinfection in the gastrointestinal tract.

KEYWORDS:
Helicobacter pylori; Candida; Mouth

RESUMO

INTRODUÇÃO:

Quase 73% da população chilena estão infectadas pelo Helicobacter pylori (H. pylori), fator predisponente ao câncer gástrico. Estudos recentes demonstraram a presença desse patógeno em leveduras, sugerindo que esse fato pode influenciar diretamente a falha de um tratamento, transmissão e reinfecção.

OBJETIVO:

Detectar a presença de H. pylori em leveduras orais isoladas de estudantes da Universidade de Concepción (Chile).

MÉTODOS:

72 amostras, obtidas da cavidade oral utilizando cotonetes, foram incubadas em caldo YPD por 48h a 37°C e posteriormente sementes em ágar Sabouraud Dextrose mais cloranfenicol na mesma temperatura e ao mesmo tempo. Leveduras isoladas foram observadas microscopicamente (montagem úmida e corada por Gram) e identificadas utilizando técnicas microbiológicas. A detecção intracelular de H. pylori foi realizada pela amplificação do 16S rDNA por PCR.

RESULTADOS:

Leveduras orais foram detectadas em 24 amostras (33,3%), sendo C. albicans (79,2%), a espécie mais frequente seguida por C. dubliniensis (12,4%), C. krusei (4,2%) e C. tropicalis (4,2 %) Quando analisadas por PCR, 15 das 24 leveduras orais 62,5% foram positivas para o H. pylori 16S rDNA. Dos 15 indivíduos positivos para leveduras que abrigam H. pylori, 81% deles relataram desconforto estomacal e a presença da bactéria foi diagnosticada em algum momento em 20% deles.

CONCLUSÃO:

A presença intracelular do H. pylori em leveduras orais sugere uma relação endossimbiótica desses microrganismos, o que poderia favorecer a transmissão e a reinfecção do H. pylori no trato gastrointestinal.

PALAVRAS-CHAVE:
Helicobacter pylori; Candida; Boca

INTRODUCTION

H. pylori is a Gram-negative bacterium with several virulence factors, which allow it to selectively colonize the gastric mucosa. It is the etiological agent of several gastrointestinal diseases in humans¹1. Palomino Camargo C, Tomé Boschian E. Helicobacter pylori: rol del agua y los alimentos en su transmisión. An Venez Nutr. 2012;25(2):85-93.^–33. Wroblewski LE, Peek RM Jr, Wilson KT. Helicobacter pylori and gastric cancer: factors that modulate disease risk. Clin Microbiol Rev. 2010;23(4):713-39.. The latter acts as its main reservoir, acquiring an infection mainly during childhood, supposedly in the intrafamily environment. However, how this pathogen spreads is not fully understood; up until now, it is accepted that its transmission occurs through the oral-oral and fecal-oral route. Although H. pylori has been studied for decades, questions remain regarding how it spreads among populations, since H. pylori genes have been amplified in different sources, including food and water, with no success in the isolation of this bacterium through cultivation²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35.^,44. Quaglia NC, Dambrosio A. Helicobacter pylori: a foodborne pathogen? World J Gastroenterol. 2018;24(31):3472-87.^,55. Brown LM. Helicobacter pylori: epidemiology and routes of transmission. Epidemiol Rev. 2000;22(2):283-97.. H. pylori infects more than 50% of the world population and is considered the main predisposing factor to the development of gastric cancer⁶6. Asokan GV, Ramadhan T, Ahmed E, Sanad H. WHO global priority pathogens list: a bibliometric analysis of Medline-PubMed for knowledge mobilization to infection prevention and control practices in Bahrain. Oman Med J. 2019;34(3):184-93.. In Chile, its prevalence is 73% in children older than 17 years and is significantly higher in individuals of lower socioeconomic classes and males⁷7. Ferreccio C, Rollán A, Harris PR, Serrano C, Gederlini A, Margozzini P, et al. Gastric cancer is related to early Helicobacter pylori infection in a high-prevalence country. Cancer Epidemiol Biomarkers Prev. 2007;16(4):662-7..

H. pylori infections are a public health problem worldwide, and it features in the list of pathogens with high priority by WHO⁸8. World Health Organization. Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. [cited 2018 Jan 06]. Available from: http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant-bacteria/en/
http://www.who.int/medicines/publication... , which makes us ask: What makes H. pylori such an effective pathogen? One way to answer this question is by saying that this pathogen has multiple virulence factors, which ensure its effective colonization. In addition, it is a facultative intracellular microorganism able to invade and multiply itself inside vacuoles of eukaryotic cells, including yeasts of the genus Candida²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35.^,99. Lai CH, Huang JC, Cheng HH, Wu MC, Huang MZ, Hsu HY, et al. Helicobacter pylori cholesterol glucosylation modulates autophagy for increasing intracellular survival in macrophages. Cell Microbiol. 2018;20(12):e12947.^,1010. Siavoshi F, Saniee P. Vacuoles of Candida yeast as a specialized niche for Helicobacter pylori. World J Gastroenterol. 2014;20(18):5263-73..

There is evidence that H. pylori and Candida share habits in areas of the human anatomy, such as in the mouth, which is the anatomical site for the entry of microorganisms that colonize the digestive apparatus, in addition to the fact that both microorganisms can survive in the acidic environment of the stomach¹¹11. Medina ML, Medina MG, Merino LA. Correlation between virulence markers of Helicobacter pylori in the oral cavity and gastric biopsies. Arq Gastroenterol. 2017;54(3):217-21.^–1313. Karczewska E, Wojtas I, Sito E, Trojanowska D, Budak A, Zwolinska-Wcislo M, et al. Assessment of co-existence of Helicobacter pylori and Candida fungi in diseases of the upper gastrointestinal tract. J Physiol Pharmacol. 2009;60(Suppl 6):33-9., which would favor their interaction and the internalization of the bacterium in the yeast, which would grant it protection, nutrition, and a transmission medium that would allow the bacteria to survive outside the human gastrointestinal environment since yeast can survive in different environments, including in conditions with a lack of nutrients²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35.^,1010. Siavoshi F, Saniee P. Vacuoles of Candida yeast as a specialized niche for Helicobacter pylori. World J Gastroenterol. 2014;20(18):5263-73.^,1414. Siavoshi F, Taghikhani A, Malekzadeh R, Sarrafnejad A, Kashanian M, Jamal AS, et al. The role of mother's oral and vaginal yeasts in transmission of Helicobacter pylori to neonates. Arch Iran Med. 2013;16(5):288-94.^–1616. Chaieb K, Kouidhi B, Zmantar T, Mahdouani K, Bakhrouf A. Starvation survival of Candida albicans in various water microcosms. J Basic Microbiol. 2011;51(4):357-63..

The high prevalence of gastrointestinal diseases associated with H. pylori, the increased resistance of this pathogen to antibiotic treatment¹⁷17. Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology. 2017;153(2):420-9.^.1818. Parra-Sepúlveda C, Merino JS, Sáez-Carrillo K, González C, García-Cancino A. Antibiotic resistance surveillance of Helicobacter pylori at the Biobío region (Chile) in a decade. Arq Gastroenterol. 2019;56(4):361-66., and the lack of knowledge regarding its means of transmission make it necessary to obtain evidence that allows developing effective strategies to reduce its spread and, consequently, the prevalence of pathologies associated to its infection. For this reason, the goal of this study was to detect the presence of H. pylori in intracellular yeasts of the genus Candida of oral origin isolated from young graduates of the University of Concepción, Chile.

METHODS

This study was approved by the ethics committee of the University of Concepción, under number E-224-18. Prior to the sample collection, each participant received information, oral and written, about the importance of the study and how the sample would be collected. In addition, we explained they could leave the study at any stage. We carried out a quantitative, observational, transversal study. Our population consisted of undergraduate students of the University of Concepción.

Inclusion criteria

Signing the informed consent form.

Exclusion criteria

Treatment with antifungal agents a month before or at the time of sample collection.

Questionnaire

The people who participated in the study individually filled out a questionnaire with the questions: 1) Have you presented or do you present stomach discomfort in the last 30 days? 2) What kind of discomfort is/was that? (Gastritis/Reflux/Abdominal pain/Nausea/Vomiting/Diarrhea), 3) What was the duration of the discomfort? (Hours/A day/ More than a day/More than a week), 4) Do you wash your hands before and after using the restroom?, 5) Do you drink potable water, bottled water, or non-potable water?, 6) Were you diagnosed with any gastrointestinal pathology? 7) Have you ever been diagnosed with H. pylori?, 8) Has any of your relatives ever been diagnosed H. pylori?

Sample collection

Participants were instructed not to brush their teeth for at least two hours before the sample collection. The sample was collected orally with a cotton swab, which was scraped on the cheeks, soft palate, tongue's surface, underneath the tongue, and gums, according to Matamala-Valdés et al.²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35.. Subsequently, each swab was placed in test tubes with 5 mL of liquid YPD (Difco, USA), which were incubated in aerobic conditions for 48 hours. After that, each sample was seeded on Sabouraud Dextrose Agar plates (Difco, USA) supplemented with Chloramphenicol (Oxoid, United Kingdom) rubbing the swab across the entire surface of the culture medium; the plates were incubated at 37°C for 48 hours under aerobic conditions. Subsequently, we confirmed the growth of pure yeasts through wet Gram staining. From the primary culture, two consecutive seedings were carried out in Sabouraud Dextrose Agar (Difco, USA), supplemented with Chloramphenicol (Oxoid, United Kingdom) to ensure the purity of the sample; each re-seeding was followed by wet Gram staining examination.

Detection of bacterium-like organelles (BLOs) on the inside of fungal cells by optical microscopy.

We carried out a wet examination in yeast colonies randomly chosen in search of BLOs inside the fungal cells, using as a negative control C. albicans ATCC 90028. Each colony was placed in 20 µL of saline solution and observed using an optical microscope, with a 100X objective lens.

Tests for the identification of Candida spp.

We took an inoculum from the crops of pure yeast in Sabouraud Agar, which was then sown on CHROMagar Candida (Difco, USA) and incubated under aerobic conditions at 37°C for 24-48 hours. The interpretation was performed according to the manufacturer's instructions. Subsequently, the results were confirmed using the identification system API®Candida, following the manufacturer's recommendations (BIOMÉRIEUX, France).

DNA extraction

The total DNA from the yeast isolated from the oral swab samples and controls was extracted from pure cultures obtained in Sabouraud Agar, using the commercial kit UltraClean® Microbial DNA Isolation (MO BIO lab) and following the manufacturer's instructions. The quantification and degree of purity of the DNA extracted was obtained by spectrophotometry (TECAN, Switzerland)

Genotyping of Helicobacter pylori

The amplification of the 16S rDNA of the H. pylori and the electrophoresis of the amplified products was carried out according to the protocol described by Matamala-Valdés et al.²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35..

Statistical analysis

The data obtained in this study were processed using the XLSTAT software 2017. We used Fisher's exact test to determine the relationship between the categorical variables. The level of significance used was 0.05, considering a value significant whenever p <0.05.

RESULTS

Prevalence of oral yeasts

A total of 72 students participated in the study, 30 of whom were women and 42 men. Of the 72 oral swab samples, there was a prevalence of 33.3% (24/72) of oral yeasts. The yeast species isolated with greater frequency in the oral mucosa was C. albicans with 79.2%, followed by C. dubliniensis with 12.4%, C. krusei with 4.2%, and C. tropicalis with 4.2%.

Observation by optical microscopy

The observation of the samples by optical microscopy revealed the presence of BLOs in 15/24 (63%) of the isolated yeast samples; the BLOs were observed quickly and continuously detaching themselves inside the yeast vacuoles (Figure 1).

Amplification of the 16S rDNA gene

We amplified the 16S rDNA gene of the H. pylori in 63% of the oral yeasts (15/24), which was in line with the 15 samples of yeast in which the BLOs were observed (Figure 2).

Results of the applied research

Regarding the results obtained from the questionnaire with the 72 participants, we found that 80% of the 15 participants from whom oral yeasts with H. pylori were isolated had had some stomach discomfort in the thirty days prior to the sample collection, and the most frequent type of discomfort was abdominal pain and gastritis, with 33% and 27%, respectively (Table 1). In addition, out of the 15 participants with oral yeasts positive for H. pylori, 20% had been diagnosed at some time with H. pylori, and 33.3% had a relative with a diagnosis of H. pylori infection at some time in their lives (Tables 2 and 3).

Association of intracellular H. pylori in yeasts and information obtained from the research.

Fisher's exact test showed that there was no statistically significant association between the presence of stomach discomfort and the amplification of the 16S rDNA gene of the H. pylori in the DNA extracted from the oral yeasts (p=0.569). No statistically significant association was observed between the positive diagnosis for H. pylori, and the amplification of the 16S rDNA gene for the H. pylori, also based on the total DNA from oral yeasts (p=0.154). Finally, when applying Fisher's exact test for the association of the diagnosis of a patient's family member with H. pylori and the amplification of the 16S rDNA gene for H. pylori, we obtained a p-value of 1.0, indicating that there was no statistical significance for this association.

DISCUSSION

The coexistence of H. pylori and Candida spp. has existed for a long time; however, the benefits the bacteria receive when entering the yeast remain unknown²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35.^,1010. Siavoshi F, Saniee P. Vacuoles of Candida yeast as a specialized niche for Helicobacter pylori. World J Gastroenterol. 2014;20(18):5263-73.^,1414. Siavoshi F, Taghikhani A, Malekzadeh R, Sarrafnejad A, Kashanian M, Jamal AS, et al. The role of mother's oral and vaginal yeasts in transmission of Helicobacter pylori to neonates. Arch Iran Med. 2013;16(5):288-94.^,1919. Saniee P, Siavoshi F, Nikbakht Broujeni G, Khormali M, Sarrafnejad A, Malekzadeh R. Immunodetection of Helicobacter pylori-specific proteins in oral and gastric Candida yeasts. Arch Iran Med. 2013;16(11):624-30.. Furthermore, cases have been reported of bacteria that enter fungi to protect themselves from environmental stress. An example of that is the endosymbiotic relationship between the Candidatus glomeribacter and the Micorriza arbuscular²⁰20. Hogan DA, Kolter R. Pseudomonas-Candida interactions: an ecological role for virulence factors. Science. 2002;296(5576):2229-32.^,2121. Bianciotto V, Genre A, Jargeat P, Lumini E, Bécard G, Bonfante P. Vertical transmission of endobacteria in the arbuscular mycorrhizal fungus Gigaspora margarita through generation of vegetative spores. Appl Environ Microbiol. 2004;70(6):3600-8..

The yeasts of the Candida genus are part of the human microbiota and are distributed in greater volume in the oral cavity on the tongue's surface, the palate and in the oral mucosa²²22. Webb BC, Thomas CJ, Willcox MD, Harty DW, Knox KW. Candida-associated denture stomatitis. Aetiology and management: a review. Part 1. Factors influencing distribution of Candida species in the oral cavity. Aust Dent J. 1998;43(1):45-50..²³23. Lynch DP. Oral candidiasis. History, classification, and clinical presentation. Oral Surg Oral Med Oral Pathol. 1994;78(2):189-93.; on the other hand, the presence of H. pylori in the oral cavity was detected by molecular tests ²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35.^,1010. Siavoshi F, Saniee P. Vacuoles of Candida yeast as a specialized niche for Helicobacter pylori. World J Gastroenterol. 2014;20(18):5263-73.^,1414. Siavoshi F, Taghikhani A, Malekzadeh R, Sarrafnejad A, Kashanian M, Jamal AS, et al. The role of mother's oral and vaginal yeasts in transmission of Helicobacter pylori to neonates. Arch Iran Med. 2013;16(5):288-94.^,1919. Saniee P, Siavoshi F, Nikbakht Broujeni G, Khormali M, Sarrafnejad A, Malekzadeh R. Immunodetection of Helicobacter pylori-specific proteins in oral and gastric Candida yeasts. Arch Iran Med. 2013;16(11):624-30.^,2424. Wang XM, Yee KC, Hazeki-Taylor N, Li J, Fu HY, Huang ML, et al. Oral Helicobacter pylori, its relationship to successful eradication of gastric H. pylori and saliva culture confirmation. J Physiol Pharmacol. 2014;65(4):559-66.. However, there are scarce cases of these bacteria being isolated on this anatomical site, assuming that DNA fragments of H. pylori can become lodged in the mouth through the reflux in patients infected at the gastric level²⁵25. Al-Ahmad A, Kürschner A, Weckesser S, Wittmer A, Rauberger H, Jakob T, et al. Is Helicobacter pylori resident or transient in the human oral cavity? J Med Microbiol. 2012;61(Pt 8):1146-52. or that it is temporarily lodged in the mouth, indicating that the oral microenvironment does not provide the conditions necessary for the survival of the bacteria, which would force H. pylori to create strategies to protect itself from unfavorable environments, such as entering oral yeasts, a phenomenon demonstrated by research conducted by Siavoshi et al.¹⁰10. Siavoshi F, Saniee P. Vacuoles of Candida yeast as a specialized niche for Helicobacter pylori. World J Gastroenterol. 2014;20(18):5263-73..¹⁴14. Siavoshi F, Taghikhani A, Malekzadeh R, Sarrafnejad A, Kashanian M, Jamal AS, et al. The role of mother's oral and vaginal yeasts in transmission of Helicobacter pylori to neonates. Arch Iran Med. 2013;16(5):288-94. and Matamala-Valdés et al.²2. Matamala-Valdés L, Sánchez-Alonzo K, Parra C, Sáez K, Aguayo-Reyes A, García A. Detection of intracellular Helicobacter pylori in Candida spp from neonate oral swabs. Rev Assoc Med Bras. 2018;64(10):928-35..

In the general population, the percentage of people with oral Candida spp. varies between 17-75%¹⁷17. Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global prevalence of Helicobacter pylori infection: systematic review and meta-analysis. Gastroenterology. 2017;153(2):420-9..²⁴24. Wang XM, Yee KC, Hazeki-Taylor N, Li J, Fu HY, Huang ML, et al. Oral Helicobacter pylori, its relationship to successful eradication of gastric H. pylori and saliva culture confirmation. J Physiol Pharmacol. 2014;65(4):559-66., and 75% of the isolates from the oral mucosa correspond to C. albicans, followed by C. tropicalis (8%), and C. krusei (3-6%)²³23. Lynch DP. Oral candidiasis. History, classification, and clinical presentation. Oral Surg Oral Med Oral Pathol. 1994;78(2):189-93.. In the present study, the isolated species in order of frequency were C. albicans with 79.2%, followed by C. dubliniensis with 12.5%, C. krusei (4.2%), and C. tropicalis with 4.2%, in agreement with the data presented by the bibliography reviewed.

Regarding the observation by optical microscopy of the wet examination of the oral yeasts with H. pylori, we found that the bacteria movement was limited to the size of the vacuole; in addition, the presence of intravacuolar H. pylori was observed both in yeast obtained from primary culture and on re-seedings, which suggests that this microorganism is not only capable of entering the yeast, but also possibly of reproducing inside the fungal cell. This same essay allowed us to observe that there was an increase in the size of the yeast vacuoles with H. pylori when compared to those without the presence of the bacteria; (Figure 1).

Acknowledgments

We thank all the students who collaborated in the study by donating their oral samples.

REFERENCES

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