Quantitative Detection of Helicobacter pylori by Real Time PCR in Drinking Water—Environmental and Public Health Risk Significance
Author(s)
Virginia Montero-Campos1,
Shirley Arias-Cordero2,
Benedicto Valdés-Rodríguez3,
Monserrat Jarquín-Cordero2
Affiliation(s)
1 Research & Chemical and Microbiology Services Center—CEQIATEC, School of Chemistry, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica. 2 Biotechnology Research Center, School of Biology, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica. 3 Water Laboratory Services Physicochemical, University of Chiriqui, Chiriqui City, Panama.
1 Research & Chemical and Microbiology Services Center—CEQIATEC, School of Chemistry, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica. 2 Biotechnology Research Center, School of Biology, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica. 3 Water Laboratory Services Physicochemical, University of Chiriqui, Chiriqui City, Panama.
ABSTRACT
Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms; nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panamá (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriquí were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized; also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler® 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori; removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panamá it was determined that 86% of the samples were found positive for the presence of H. pylori; removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.
Helicobacter pylori (H. pylori) is bacteria considered to be present in half of the population and it is a public health problem worldwide. Most patients infected with H. pylori show no clinical symptoms; nonetheless, approximately 10% to 20% of these patients will develop peptic ulcers and 1% will develop gastric cancer. The International Agency for Research on Cancer has classified H. pylori as a Group 1 carcinogen, recognized as the only bacteria capable of producing cancer. Samples of drinking water (n = 44) from aqueducts with chlorination treatment in selected areas with high prevalence of gastric cancer were analyzed in Costa Rica. Samples of drinking water from Panamá (n = 44) from aqueducts supplying untreated water for human consumption in the province of Chiriquí were also analyzed. The molecular marker of H. pylori, glmM, was used, and to optimize the Real Time PCR (qPCR) technique, annealing temperature, concentration of primers and probe were standardized; also, by analyzing different standard curves, the best reaction conditions that allowed detecting and quantifying the gene were determined. The LightCycler® 480 II (LC480II) equipment from Roche Diagnostics GmbH was used, as well as the Absolute Quantification Analysis by means of the Second Derivative Maximum Method. In the case of the samples from Costa Rica, it was determined that 79.5% were positive for H. pylori; removing outlier high average, quantification of bacteria was determined in 3.6 × 103 copies/100 mL. For Panamá it was determined that 86% of the samples were found positive for the presence of H. pylori; removing outlier high average quantification of bacteria was determined at 3.3 × 102 copies/100 mL. The difference in values between the aqueducts in both countries revealed an environmental distribution of the bacteria of epidemiological interest in each case.
Cite this paper
Montero-Campos, V. , Arias-Cordero, S. , Valdés-Rodríguez, B. and Jarquín-Cordero, M. (2015) Quantitative Detection of Helicobacter pylori by Real Time PCR in Drinking Water—Environmental and Public Health Risk Significance. Open Journal of Medical Microbiology, 5, 118-127. doi: 10.4236/ojmm.2015.53015.
Montero-Campos, V. , Arias-Cordero, S. , Valdés-Rodríguez, B. and Jarquín-Cordero, M. (2015) Quantitative Detection of Helicobacter pylori by Real Time PCR in Drinking Water—Environmental and Public Health Risk Significance. Open Journal of Medical Microbiology, 5, 118-127. doi: 10.4236/ojmm.2015.53015.
References
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[1] Azevedo, N., Guimares, N., Figuereido, C. and Kevil, C. (2007) A New Model for the Transmission of Helicobacter pylori: Role of Environmental Reservoirs as Gene Pools to Increase Strain Diversity. Critical Reviews in Microbiology, 33, 157-169.
http://dx.doi.org/10.1080/10408410701451922 [2] Perez, G., Rothenbacher, D. and Brenner, H. (2004) Epidemiology of Helicobacter pylori Infection. Helicobacter, 9, 1-6.
http://dx.doi.org/10.1111/j.1083-4389.2004.00248.x [3] Megraud, F. (2003) When and How Does Helicobacter pylori Infection Occur? Gastroenterologie Clinique et Biologique, 27, 374-379. [4] Pounder, R. and Ng, D. (1995) The Prevalence of Helicobacter pylori Infection in Different Countries. Alimentary Pharmacology & Therapeutics, 9, 33-39. [5] Goodman, K., O’Rourke, K., Day, R., Wang, C., Nurgalieva, Z., Phillips, C.V., Aragaki, C., Campos, A. and De la Rosa, J.M. (2005) Dynamics of Helicobacter pylori Infection in a US-Mexico Cohort during the First Two Years of Life. International Journal of Epidemiology, 34, 1348-1355.
http://dx.doi.org/10.1093/ije/dyi152 [6] Haggerty, T., Perry, S., Sanchez, L., Perez-Perez, G. and Parsonnet, J. (2005) Significance of Transiently Positive Enzyme-Linked Immunosorbent Assay Results in Detection of Helicobacter pylori in Stool Samples from Children. Journal of Clinical Microbiology, 43, 2220-2223.
http://dx.doi.org/10.1128/JCM.43.5.2220-2223.2005 [7] Graham, D. (2007) The Year in Helicobacter. Helicobacter, 12, 14-28. [8] Kanbay, M. (2005) Helicobacter pylori Seroprevalence in Patients with Coronary Artery Disease. Digestive Diseases and Sciences, 50, 2071-2074.
http://dx.doi.org/10.1007/s10620-005-3009-7 [9] Peter, A. and Montenero, A. (2006) Is Helicobacter pylori a Cause of Atrial Fibrillation? Future Cardiology, 2 429-439.
http://dx.doi.org/10.2217/14796678.2.4.429 [10] Migneco, A., Ojetti, V., Specchia, L., Franceschi, F., Candelli, M., Mettimano, M., Montebelli, R., Savi, L. and Gasbarrini, G. (2003) Eradication of Helicobacter pylori Infection Improves Blood Pressure Values in Patients Affected by Hypertension. Helicobacter, 8, 585-589.
http://dx.doi.org/10.1111/j.1523-5378.2003.00180.x [11] Kaptan, K. (2006) Helicobacter pylori and Cobalamin Deficiency. Clinical & Laboratory Haematology, 28, 360-360.
http://dx.doi.org/10.1111/j.1365-2257.2006.00799_1.x [12] Aydemir, S. (2005) The Effect of Helicobacter pylori on Insulin Resistance. Digestive Diseases and Sciences, 50, 2090-2093.
http://dx.doi.org/10.1007/s10620-005-3012-z [13] Ojetti, V. (2005) The Role of H. pylori Infection in Diabetes. Current Diabetes Reviews, 1, 343-347.
http://dx.doi.org/10.2174/157339905774574275 [14] Ojetti, X. and Chen, D. (2006) Helicobacter pylori and Hepatocellular Carcinoma: Correlated or Uncorrelated? Journal of Gastroenterology and Hepatology, 21, 345-347.
http://dx.doi.org/10.1111/j.1440-1746.2006.04245.x [15] Queiroz, D.M., Rocha, A.M., Rocha, G.A., Cinque, S.M., Oliveira, A.G., Godoy, A. and Tanno, H. (2006) Association between Helicobacter pylori Infection and Cirrhosis in Patients with Chronic Hepatitis C Virus. Digestive Diseases and Sciences, 51, 370-373.
http://dx.doi.org/10.1007/s10620-006-3150-y [16] Brawner, K., Morrow, C. and Smith, P. (2014) Gastric Microbiome and Gastric Cancer. Cancer Journal, 20, 211-216.
http://dx.doi.org/10.1097/PPO.0000000000000043 [17] Cao, L. and Yu, J. (2015) Effect of Helicobacter pylori Infection on the Composition of Gastric Microbiota in the Development of Gastric Cancer. Gastrointestinal Tumors, 2, 14-25.
http://dx.doi.org/10.1159/000380893 [18] Ferlay, J., Hai-Rim, S., Bray, F., Forman, D., Mathers, C. and Maxwell, D. (2010) Estimates of Worldwide Burden of Cancer in 2008: GLOBOCAN 2008. International Journal of Cancer, 127, 2893-2917.
http://dx.doi.org/10.1002/ijc.25516 [19] Engstrand, L. and Lindberg, M. (2013) Helicobacter pylori and the Gastric Microbiota. Best Practice & Research Clinical Gastroenterology, 27, 39-45.
http://dx.doi.org/10.1016/j.bpg.2013.03.016 [20] Dicksved, J., Lindberg, M., Rosenquist, M., Enroth, H., Jansson, J. and Engstrand, L. (2008) Molecular Characterization of the Stomach Microbiota in Patients with Gastric Cancer and in Controls. Journal of Medical Microbiology, 58, 509-516.
http://dx.doi.org/10.1099/jmm.0.007302-0 [21] Fernández-Delgado, M., Contreras, M., García-Amado, M., Michelangeli, F. and Suárez, P. (2007) Evidencias de la transmisión acuática de Helicobacter pylori. Interciencia, 33, 412-417. [22] Israel, D. (2001) Genetic Exchange. In: Mobley, H.L.T., Menz, G.L. and Hazell, S.L., Eds., Helicobacter pylori: Physiology and Genetics, American Society for Microbiology Press, Washington DC, Chapter 28.
http://www.ncbi.nlm.nih.gov/books/NBK2430/ [23] Dewhirst, F., Shen, Z., Scimeca, M., Stokes, L., Boumenna, T., Chen, T., Paster, B. and Fox, J. (2005) Discordant 16S and 23S rRNA Gene Phylogenies for the Genus Helicobacter: Implications for Phylogenetics Interference and Systematics. Journal of Bacteriology, 187, 6106-6118.
http://dx.doi.org/10.1128/JB.187.17.6106-6118.2005 [24] Shahamat, M., Alavi, M., Watts, J.E., González, J.M., Sowers, K.R., Maeder, D.W. and Robb, F.T. (2004) Development of Two PCR-Based Techniques for Detecting Helical and Coccoid Forms of Helicobacter pylori. Journal of Clinical Microbiology, 42, 3613-3619.
http://dx.doi.org/10.1128/jcm.42.8.3613-3619.2004 [25] Ogura, M., Perez, J.C., Mittl, P.R.E., Lee, H.-K. and Dailide, G. (2007) Helicobacter pylori Evolution: Lineage-Specific Adaptations in Homologs of Eukaryotic Sel1-Like Genes. PLoS Computational Biology, 3, e151.
http://dx.doi.org/10.1371/journal.pcbi.0030151 [26] UNIPROT CONSORTIUM (2011) P55980 (CAGA_HELPY) Version 60. (en línea). Consultado 11 marzo de 2015.
http://www.uniprot.org/uniprot/P55980 [27] Fux, C., Costerton, J.W., Stewart, P. and Stoodley, P. (2005) Survival Strategies of Infectious Biofilms. Trends in Microbiology, 13, 34-40.
http://dx.doi.org/10.1016/j.tim.2004.11.010 [28] Azevedo, N., Pacheco, A., Keevil, C. and Vieira, M. (2006) Adhesion of Water Stressed Helicobacter pylori to Abiotic Surfaces. Journal of Applied Microbiology, 101, 718-724.
http://dx.doi.org/10.1111/j.1365-2672.2006.03029.x [29] Touati, E., Michel, V., Thiberge, J. and Wuscher, N. (2003) Chronic Helicobacter pylori Infections Induce Gastric Mutations in Mice. Gastroenterology, 124, 1408-1419.
http://dx.doi.org/10.1016/S0016-5085(03)00266-X [30] She, F.F., Lin, J.Y., Liu, J.Y., Huang, C. and Su, D.H. (2003) Virulence of Water-Induced Coccoid Helicobacter pylori and Its Experimental Infection in Mice. World Journal of Gastroenterology, 9, 516-520. [31] Yañez, M., Barberá, V., Soria, E. and Catalán, V. (2009) Quantitative Detection of Helicobacter pylori in Water Samples by Real Time PCR Amplification of the Cag Pathogenicity Island Gene, cagE. Journal of Applied Microbiology, 107, 416-424.
http://dx.doi.org/10.1111/j.1365-2672.2009.04219.x [32] Santiago, P., Moreno, Y. and Ferrus, M.A. (2015) Identification of Viable Helicobacter pylori in Drinking Water Supplies by Cultural and Molecular Techniques. [33] Pérez, L.M., Codony, F., López, D., Fittipaldi, M., Adrados, B. and Morató, J. (2010) Quantification of Helicobacter pylori Levels in Soil Samples from Public Playgrounds in Spain. Journal of Zhejiang University SCIENCE B, 11, 27-29.
http://dx.doi.org/10.1631/jzus.B0900238 [34] Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York. [35] Bio-Rad Laboratories, Inc. (s.f.). Real-Time PCR Application Guide. En línea. el 5 de febrero de 2015.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5279.pdf [36] Lascols, C., Lamarque, D., Costa, J., Copie-Bergman, C., Le Glaunec, J., Deforges, L., Soussy, C., Petit, J., Delchier, J. and Tankovic, J. (2003) Fast and Accurate Quantitative Detection of Helicobacter pylori and Identificaction of Clarithromycin Resistance Mutations in H. pylori Isolates from Gastric Biopsy Specimens by Real-Time PCR. Journal of Clinical Microbiology, 41, 4573-4577.
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