Detection of viable Helicobacter pylori inside free-living amoebae in wastewater and drinking water samples from Eastern Spain.

Helicobacter pylori is one of the most concerning emerging waterborne pathogens. It has been suggested that it could survive in water inside free-living amoebae (FLA), but nobody has studied this relationship in the environment yet. Thus, we aimed to detect viable H. pylori cells from inside FLA in water samples. Sixty-nine wastewater and 31 drinking water samples were collected. FLA were purified and identified by PCR and sequencing. For exclusively detecting H. pylori inside FLA, samples were exposed to sodium hypochlorite and assayed by specific PMA-qPCR, DVC-FISH and culture. FLA were detected in 38.7% of drinking water and 79.7% of wastewater samples, even after disinfection. In wastewater, Acanthamoeba spp. and members of the family Vahlkampfiidae were identified. In drinking water, Acanthamoeba spp. and Echinamoeba and/or Vermamoeba were present. In 39 (58.2%) FLA-positive samples, H. pylori was detected by PMA-qPCR. After DVC-FISH, 21 (31.3%) samples harboured viable H. pylori internalized cells. H. pylori was cultured from 10 wastewater samples. To our knowledge, this is the first report that demonstrates that H. pylori can survive inside FLA in drinking water and wastewater, strongly supporting the hypothesis that FLA could play an important role in the transmission of H. pylori to humans.

DVC-FISH and PMA-qPCR techniques to assess the survival of Helicobacter pylori inside Acanthamoeba castellanii.

Free-living amoebae (FLA) are ubiquitous microorganisms commonly found in water. They can act as Trojan Horses for some amoeba-resistant bacteria (ARB). Helicobacter pylori is a pathogenic bacteria, suggested to be transmitted through water, which could belong to the ARB group. In this work, a co-culture assay of H. pylori and Acanthamoeba castellanii, one of the most common FLA, was carried out to identify the presence and survival of viable and potentially infective forms of the bacteria internalized by the amoeba. Molecular techniques including FISH, DVC-FISH, qPCR and PMA-qPCR were used to detect the presence of internalized and viable H. pylori. After 24 h in co-culture and disinfection treatment to kill extra-amoebic bacteria, viable H. pylori cells were observed inside A. castellanii. When PMA-qPCR was applied to the co-culture samples, only DNA from internalized H. pylori cells was detected, whereas qPCR amplified total DNA from the sample. By the combined DVC-FISH method, the viability of H. pylori cells in A. castellanii was observed. Both specific techniques provided evidence, for the first time, that the pathogen is able to survive chlorination treatment in occurrence with A. castellanii and could be very useful methods for performing further studies in environmental samples.

Identification of Viable Helicobacter pylori in Drinking Water Supplies by Cultural and Molecular Techniques.

BACKGROUND: Helicobacter pylori is one of the most common causes of chronic bacterial infection in humans, directly related to peptic ulcer and gastric cancer. It has been suggested that H. pylori can be acquired through different transmission routes, including water. In this study, culture and qPCR were used to detect and identify the presence of H. pylori in drinking water. Furthermore, the combined techniques PMA-qPCR and DVC-FISH were applied for detection of viable cells of H. pylori. RESULTS: Among 24 drinking water samples, 16 samples were positive for the presence of H. pylori, but viable cells were only detected in six samples. Characteristic colonies, covered by a mass of bacterial unspecific growth, were observed on selective agar plates from an only sample, after enrichment. The mixed culture was submitted to DVC-FISH and qPCR analysis, followed by sequencing of the amplicons. Molecular techniques confirmed the growth of H. pylori on the agar plate. CONCLUSIONS: Our results demonstrate for the first time that H. pylori can survive and be potentially infective in drinking water, showing that water distribution systems could be a potential route for H. pylori transmission.

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

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.

Specific detection of viable Listeria monocytogenes in Spanish wastewater treatment plants by Fluorescent In Situ Hybridization and PCR.

Listeria monocytogenes detection in wastewater can be difficult because of the large amount of background microbiota and the presence of viable but non-culturable forms in this environment. The aim of this study was to evaluate a Fluorescent In Situ Hybridization (FISH) assay combined with Direct Viable Count (DVC) method for detecting viable L. monocytogenes in wastewater samples, as an alternative to conventional culture methods. 16S rRNA sequence data were used to design a specific oligonucleotide probe. In order to assess the suitability of the method, the assays were performed on naturally (n=87) and artificially (n=14) contaminated samples and results were compared to those obtained with the isolation of cells on selective media and with a PCR method. The detection limit of FISH and PCR assays was 10(4) cells/mL without enrichment and 10 cells/mL after enrichment. A total of 47 samples, including 3 samples from effluent sites, yielded FISH positive results for L. monocytogenes. Using DVC-FISH technique, the presence of viable L. monocytogenes cells was detected in 23 out of these 47 FISH positive wastewater samples. PCR and culture methods yielded 27 and 23 positive results, respectively. According to these results, FISH technique has the potential to be used as a sensitive method for the detection and enumeration of L. monocytogenes in environmental wastewater samples.

Survival and injury of Arcobacter after artificial inoculation into drinking water.

The aim of this work was to assess the effect of chlorine water treatment on Arcobacter butzleri and to study the survival strategies of this organism in chlorinated and non-chlorinated drinking water. A. butzleri NCTC 12481 was inoculated into chlorinated and non-chlorinated water and samples were removed aseptically, immediately and periodically during the next 2 days (for chlorinated drinking water) or 35 days (for non-chlorinated drinking water). The membrane integrity (Live/Dead BacLight kit), 16S rRNA (FISH technique), DNA content (23S rRNA PCR-RFLPs) and culturability changes in A. butzleri cells were analyzed. Culturability of the cells was lost at 5 min in chlorinated drinking water. At that time the cells showed membrane damage, although fluorescent intensity of 16S rRNA hybridization was constant throughout the chlorine treatment. After 48 h the amplicon specific for the 23S rRNA gene was weakly detected. In non-chlorinated drinking water cells lost their culturability after 16 days but the other factors measured indicated that Arcobacter remained viable throughout the experiment.