Specific detection of cultivable Helicobacter pylori cells from wastewater treatment plants.

Helicobacter. 2012 Oct;17(5):327-32.

Moreno Y, Ferrús MA.

Instituto Universitario de Ingeniería del Agua y Medio Ambiente, Universitat Politècnica de València, 46022, Valencia, Spain.


BACKGROUND: Helicobacter pylori is present in surface water and wastewater, and biofilms in drinking water systems have been reported as possible reservoirs of H. pylori. However, its ability to survive in an infectious state in the environment is hindered because it rapidly loses its cultivability. The aim of this study was to determine the presence of cultivable and therefore viable H. pylori in wastewater treatment plants to understand the role of wastewater in the pathogen’s transmission.

MATERIALS AND METHODS: A modified filter technique was used to obtain a positive H. pylori culture, and specific detection of this pathogen was achieved with FISH and PCR techniques.

RESULTS: A total of six positive H. pylori cultures were obtained from the water samples, and molecular techniques positively identified H. pylori in 21 culture-negative samples.

CONCLUSIONS: The combination of a culturing procedure after sample filtration followed by the application of a molecular method, such as PCR or FISH, provides a specific tool for the detection, identification, and direct visualization of cultivable and therefore viable H. pylori cells from complex mixed communities such as water samples.

PMID: 22967115



Helicobacter pylori is one of the most common infective agents worldwide. It is an etiological agent of gastritis, peptic and duodenal ulcer disease, and infection with this organism is a recognized risk factor in the development of gastric cancer. The public health relevance of this infection is high. Although the prevalence of H. pylori infection in the world is decreasing, it is assumed to be near 50%, with higher prevalence in developing countries than in developed countries.

There is a growing consensus in considering the bacterium to be a waterborne pathogen. Epidemiological studies always point out low socioeconomic, crowded homes and difficult access to sanitized water as the main risk factors for the infection.

Dr. Yolanda Moreno and I have been interested in the possibility that Helicobacter pylori could be acquired from faecal-contaminated water since more than ten years ago. But our main challenge was the fact that, to assess the infectivity of H. pylori cells and thus the possibility of infection via a fecal–oral route, culture of the bacteria from these samples is strictly needed.

Previous attempts to culture H. pylori cells from environmental water samples have largely been unsuccessful, and only in one occasion it was recovered from a highly contaminated wastewater collection system in Mexico. Therefore, most methods used to detect H. pylori in environmental samples are based on culture-independent molecular techniques. However, these methods rely on the detection of nucleic acids, which can be present in a sample although the cells are dead. Thus, as molecular detection of H. pylori in environmental samples does not indicate that the bacteria are infective, the concept of waterborne transmission was likely to remain in question until this organism was cultured from natural sources.

Our research team focused on wastewater because, in many countries, treated sewage effluents are increasingly being discharged into the environment and used for irrigation. Moreover, in industrialized countries, the use of treated wastewater for domestic, industrial, and agricultural purposes is currently the most common method of reusing wastewater. This fact can lead to an increased risk of human infection. The presence of H. pylori infective cells in reused water is a possible way by which the organisms reenter the water chain, which is a public health concern

In our previous work, we used the DVC-FISH method to demonstrate that living (viable) H. pylori cells are present in wastewater samples. Direct Viable Count method, DVC, is based on the incubation of samples in the presence of nutrients and an antibiotic which prevents cell division. Cells can continue metabolizing nutrients and become elongated after incubation. Viable cells can then easily be discriminated from non-viable cells, due to differences in their respective sizes. In our work, we developed a modified DVC step prior to in situ hybridization (FISH) of the samples with H. pylori highly specific fluorescent oligonucleotide probes. Field work enabled us to confirm the presence of VBNC H. pylori cells in 16 out of 45 wastewater samples. However, the organism could not be cultured from any sample.

So, our next goal was to culture the bacteria from wastewater samples. In our last work, we used a modified filter method to isolate H. pylori. This technique eliminates large amounts of competitive microbiota. Although it did not allow us for getting a pure culture, presumptive colonies were much more easily detected.

What we made then was to submit those mixed cultures to H. pylori specific FISH assay, vacA PCR and 16S rDNA sequencing. Six cultures were so confirmed to contain H. pylori cells. This way, we demonstrated certainly that cultivable H. pylori cells are present in wastewater, confirming that fecal-contaminated water may act as a transmission vehicle for the bacteria.

Our next work will focus on drinking water. We have already showed that H. pylori can remain viable until 3 hours in chlorinated water. The organism has also been detected in domestic and school drinking water distribution systems, both in biofilms and in water samples. So, we expect that the application of our techniques to drinking water can help us to solve the water transmission controversy, as well as help to establish preventive Public Health measures.


Maria Antonia Ferrus Perez-1




Fig 1: Growth of bacteria from water samples on agar plates after a previous filtration step.

Maria Antonia Ferrus Perez-2











Fig. 2: Viable (elongated) cells of H. pylori in wastewater samples, detected by DVC-FISH procedure



Prof. María Antonia Ferrús Pérez, PhD, MD; Chair

Chief of Biotechnology Department

Universitat Politecnica de Valencia

Cno. Vera s.n.

46022 Spain

+34 963877423


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