Survival of Helicobacter pylori in the wastewater treatment process and the receiving river in Michigan, USA

Helicobacter pylori, a bacterium initially observed in 1893, was not recognized as an infectious agent until 1982 when Warren and Marshall isolated this pathogen (Marshall & Warren 1984). H. pylori is a bacterium that infects the stomach of 80% of people living in low socioeconomic areas of Latin America. By contrast, less than 20% of asymptomatic Caucasians carry H. pylori in the USA. The WHO estimates that in the world there are over 900,000 new diagnoses and 650,000 deaths from gastric cancer each year, mainly in Eastern Europe, Asia and Latin America. H. pylori is a leading cause of gastrointestinal diseases in humans, including gastric and duodenal ulcers, gastric cancer and primary gastric lymphoma (Aziz et al. 2015).

Over the preceding years and to date, the definitive mode of human infection by H. pylori has remained largely unknown and has thus gained the interest of researchers around the world. Fecal-oral and person to person contact have been suggested as possible routes of exposure to this organism. However, many investigations showed that contaminated water may play a key role in the transmission of H. pylori (Baker & Hegarty 2001; Engstrand 2001; Bellack et al. 2006).

Survival of H. pylori in different types of water has been reported to extend from days to weeks at temperatures between 4 and 15°C over a wide pH range (Velazquez & Feirtag 1999; Adams et al. 2003; Gomes & De Martinis 2004). The water transmission possibility studied by Azevedo strongly argues that drinking water can pose a substantial threat of H. pylori infection based on the fulfillment of several essential criteria. These criteria include the ability of H. pylori to adhere to different materials, and to co-aggregate with other bacteria and form complex structures on pipes or other surfaces in contact with water (Azevedo et al. 2006, 2007, 2008). Biofilms in drinking water systems have been reported as possible reservoirs of H. pylori (Watson et al. 2004; Braganca et al. 2007; Linke et al. 2010). Although different types of disinfectants have been used to maintain microbial safety, H. pylori was shown to retain its viability in chlorinated water (Moreno et al. 2007; Giao et al. 2008) and little is known about its viability in UV disinfected water.

Attempts to culture H. pylori cells from environmental water samples have been largely unsuccessful. The cellular morphology of this organism has been demonstrated to change from vegetative rod-shaped cells to a non-culturable coccoid form after storage in water (Keevil 2003; Piqueres et al. 2006). However, clear colony morphology has been rarely reported. Entrance of H. pylori into the viable but non-culturable state allows H. pylori to persist in water (Buck & Oliver 2010). Unsuccessful attempts to culture H. pylori from environmental waters have led to the use of molecular methods to detect and identify this organism (McDaniels et al. 2005; Guimaraes et al. 2007). Several articles have been published using conventional polymerase chain reaction (PCR) to detect H. pylori in different types of water including ground water, surface water, treated and untreated wastewater, marine waters and also biofilms (Liu et al. 2002; Fujimura et al. 2004; Queralt et al. 2005; Voytek et al. 2005). However, these methods could not differentiate between viable and dead cells.

In this paper, the real colony morphology of H. pylori was confirmed by plate cultivation with a selective medium. A molecular detection method for H. pylori by specific primers was established. The survival of H. pylori through a tertiary wastewater treatment process, especially UV disinfection, and in the receiving river, was investigated by plate culture, regular PCR using specific primers and quantitative real-time PCR.

The wastewater treatment process consisted of: (i) preliminary treatment (screening and grit removal); (ii) primary treatment (gravity sedimentation tanks); (iii) secondary treatment (activated sludge process utilizing anoxic/oxic biological nutrient removal in aeration basins (ferric chloride added as required to polish effluent for soluble phosphorus) followed by secondary sedimentation); (iv) tertiary treatment (rapid sand filtration of secondary effluent to remove particulates); and (v) disinfection (UV light). About 45% of BOD₅ in the raw influent was removed after primary treatment, 95% after secondary treatment, and 99.5% after tertiary treatment.

Samples were collected three times from four sites in the plant, namely raw water (RW), primary treated wastewater (PW), secondary treated wastewater (SW) and final effluent (FW) (after UV disinfection and ready for discharge). Samples were also collected from the Huron River site 100 m downstream (HRW) of the wastewater discharge point. There was no other discharge to the river before this sampling site.

Each time, five samples were collected in sequence on the same day. Each sample consisted of 1,000 mL water collected in a sterile bottle, which was then kept in an ice bath during transportation to the laboratory and processed within 12 h of collection.

Helicobacter pylori (ATCC 43504) was grown on Tryptic Soy Agar (TSA, ATCC 260 Agar) plates with 5% defibrinated sheep blood and a selective culture medium under microaerophilic conditions. Colonies were collected from the media after 7 days' growth at 37°C.

TSA consists of 1 L water, 15 g Tryptone, 5 g Soytone, 5 g sodium chloride and 15 g agar. Culture conditions: temperature: 37°C, 7 days; atmosphere: microaerophilic (3–5% O₂, 10% CO₂).

The selective culture medium for H. pylori required sequential addition of components. The mixture, containing special peptone, beef extract, yeast extract, NaCl, phenol red, agar and water, was autoclaved for 20 min at 121°C and then tempered to 50°C. Then calf serum with iron, antibiotics (vancomycin, trimethoprim, cefsulodin, amphotericin B, and polymixin B), and urea were aseptically added with constant stirring. Finally, 0.8 ml of 1 N HCl was added dropwise to the medium as the color changed from red to yellow-orange (final pH at 45°C, 5.7; final pH at 22°C, 6.0). The medium was then poured into Petri plates. The Helicobacter cultures were placed in a BBL anaerobe jar with a BBL CampyPak™ Plus sachet, which creates a microaerophilic atmosphere of 5–10% oxygen and 10% carbon dioxide, and incubated for one week at 37°C (Degnan et al. 2003).

Because of the high density of suspended solids in raw wastewater and primary treated wastewater, water samples were centrifuged first and then transferred for DNA extraction by different kits. For secondary clarifier wastewater and final effluent, they were first passed through a cellulose filter (pore size = 0.45 μm) to retain all bacteria on the membrane, and then transferred for DNA extraction. At the same time, isopropanol treatment for bacterial concentration was compared with centrifugation and filtration methods. As there is much suspended solids in the raw wastewater and the primary clarifier wastewater, Ultraclean Soil DNA isolation kits (MOBIO laboratories, Inc., USA) were used to compare the effect of DNA extraction methods on DNA quality with the metagenomics DNA isolation kit for water (Epicentre Biotechnologies, USA). For the cells on the culture plates, the DNA was extracted using the Wizard Genomic DNA purification kit (Promega Corporation, USA).

As Table 1 shows, three pairs of specific primers were tested and verified for H. pylori DNA. HS primers were selected to detect the survival of H. pylori in the Ann Arbor wastewater treatment process by regular PCR. Thermal cycling of the PCR reaction was performed with an initial melting at 94°C for 15 min, and 30 cycles of denaturation at 94°C for 1 min, annealing at 65, 60, and 62°C for 1 min and extension at 72°C for 1 min with a final extension step at 72°C for 10 min.

Specific H. pylori real-time PCR based on SYBR green I fluorescence was carried out using HPF and HPR primers to amplify a 180 bp fragment in a Mastercycler pro Instrument (Eppendorf AG, Germany) to measure H. pylori copies in the wastewater treatment process and the receiving river. In the meantime, 338F and 518R were used to measure total bacterial copies by q-PCR. A positive control with H. pylori DNA was added to the q-PCR analysis.

All PCR products were analyzed by gel electrophoresis in a 1% (w/v) agarose gel prepared in 1.0x Tris acetate EDTA (TAE) buffer. Gels were run for 30 min at 90 V (BIO-RAD Sub-cell GT Agarose Gel Electrophoresis system; BIO-RAD Laboratories, California) and DNA bands examined under ultraviolet illumination.

The real colony morphology of H. pylori was confirmed by culture plates with TSA and selective medium. H. pylori was found in all processed wastewater samples in Ann Arbor WWTP and the receiving Huron River. There are many antibiotic- and UV-resistant bacteria, including H. pylori, surviving in the final effluent of Ann Arbor WWTP and flowing into the Huron River.

This work was supported by the State Scholarship Fund of China Scholarship Council and Dr Chuanwu Xi's laboratory in the Department of Environmental Health, University of Michigan.