TRANSMISIÓN DE HELICOBACTER PYLORI A TRAVÉS DEL AGUA: ESTUDIO DE LA PRESENCIA DEL PATÓGENO E IDENTIFICACIÓN DE FORMAS VIABLES MEDIANTE TÉCNICAS MOLECULARES
Abstract Helicobacter pylori occupies a prominent position among emerging human pathogens, one of the most common causes of chronic bacterial infection in humanity and closely linked to peptic ulcers and gastric cancer. It has been suggested that H.pylori can be acquired by different routes of trans...
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| Tipo de recurso: | tesis doctoral |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | Universitat Politècnica de València (UPV) |
| Repositorio: | RiuNet. Repositorio Institucional de la Universitat Politécnica de Valéncia |
| Idioma: | español |
| OAI Identifier: | oai:riunet.upv.es:10251/75086 |
| Acceso en línea: | https://riunet.upv.es/handle/10251/75086 |
| Access Level: | acceso abierto |
| Palabra clave: | Helicobacter pylori agua residual potable qPCR FISH PMAqPCR DVCFISH bacterias técnicas moleculares MICROBIOLOGIA |
| Sumario: | Abstract Helicobacter pylori occupies a prominent position among emerging human pathogens, one of the most common causes of chronic bacterial infection in humanity and closely linked to peptic ulcers and gastric cancer. It has been suggested that H.pylori can be acquired by different routes of transmission, including water. The growing demand for water due to increasing world population and the concurrent expansion of the industry necessitates the use of treated wastewater. However, H. pylori resistance to water disinfection treatment constitutes a risk to health. Similarly, H.pylori has resilience to water purification treatments and at high concentrations of free chlorine in distribution systems by biofilm formation. The VBNC state of H.pylori in both matrices is the usual in stress conditions, necessitating the application of molecular techniques for detection and identification. In this thesis we have studied the presence and viability of this important pathogen in both wastewater and drinking water. A total of 60 wastewater samples, including samples of the biological reactor inlet, the outlet of this one and samples after tertiary ultraviolet disinfection treatment, were analyzed for the presence of H.pylori by PCR, q-PCR and FISH and the traditional cultivable technique. The FISH technique proved to be an effective and rapid method for the detection of H. pylori in waste and debugged water (61.2 %), without the need for a preliminary step enrichment of samples. The PCR and q-PCR techniques determined the presence of H. pylori in the samples, with detection rates of both 15 %. It was possible to quantify only in 3 direct samples belonging to the reactor inlet and outlet (1.8x10; 1.5x10 and 6.3x10 GU/mL), the other samples had the Ct above the threshold of reliability (> 35 cycles). Although the culture turned out to be a not fully resolutive method, the method of isolation by 0.65 µm membrane, tuned in this thesis, was effective in reducing the accompanying microbiota and to obtain cultivable H. pylori for the first time from debugged water. Similarly, 63 drinking water samples from 51 public drinking water sources located on the coastal area in Eastern Spain, were analyzed and could be confirmed for the first time the presence of viable cells of the pathogen in drinking water. FISH and q-PCR techniques were effective for detecting H. pylori with detection rates of 46 % and 50.8 % respectively. For detection of viable cells of H. pylori in drinking water DVC-FISH technique it proved to be a more effective tool than the PMA-qPCR, with a detection rate of 38 % viable cells. Quantitation of viable cells was possible by obtaining values between 4x102 and 1.6x103 viable cells of H.pylori/mL. Despite the difficulty of culturing the pathogen H. pylori it was identified in a sample after 24 hours enrichment. Detection of mutations in the 23S, in specific resistance to clarithromycin, indicated that 41.6% of the samples of wastewater and 17.4% of drinking water samples had H.pylori with this potential resistance. The results confirm the ability of H.pylori to survive the water purification treatments in a wastewater treatment plant, it should be considered when evaluating the potential risk in reuse for irrigation. Moreover, H.pylori is found in the distribution systems of drinking water, so its consumption may be a risk to human health, since they are a possible route of transmission of the pathogen. The presence of clarithromycin-resistant H.pylori in the environment, show the need to monitor effective control of the pathogen, since water can pose a risk to the expansion of resistance and consequent therapeutic failures in clinical practice. Given the role of water in the transmission of H.pylori, confirmed in this thesis, the established protocols are proposed, mainly the DVC-FISH method, as they can be validated for an efficient environmental control of H.pylori. |
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