United States Environmental Protection Agency Office of Research and Development
National Exposure Research Laboratory
Research Abstract
Government Performance Results Act (GPRA) Goal #2.1.7
Annual Performance Measure #101
Significant Research Findings:
A Method to Detect Helicobacter pylori Bacteria in Water
Scientific The Safe Drinking Water Act requires the EPA to establish a list of unregulated
Problem and microbiological and chemical contaminants to aid in setting priorities for the
Policy Issues Agency's drinking water regulation development program. The list, referred to as
the Contaminant Candidate List (CCL), identifies chemical and microbiological
contaminants the EPA has selected as potential regulatory candidates from the
universe of contaminants that may possibly pose a significant public health risk to
consumers if present in drinking water. There are three bacterial species listed on
the CCL (Aeromoncts, Mycobacterium avium complex, and Helicobacter pylori)
that was published in the Federal Register on March 2, 1998. More information is
needed on the occurrence of all of these species in drinking water in the U.S.
Currently, suitable methods for obtaining occurrence data are available only for
Aeromonas bacteria.
H. pylori is a newly recognized human pathogen. It is known to be an etiologic
agent of gastric ulcers and is thought to be a contributing factor in gastric cancer.
There are reports of H. pylori being cultured from the feces of infected individuals
and there are several reports of polymerase chain reaction (PCR) detection of H.
pylori in surface waters and sewage. To date, however, H. pylori has not been
isolated in culture from an environmental source. The most likely route of
transmission in humans is the oral route; however, the natural reservoirs for H.
pylori and the vehicles of transmission have not been determined.
Research The objective of this research was to develop a method capable of detecting viable
Approach and culturable H. pylori bacteria in potable waters, including treated municipal
drinking water and ground water. This method would then be used to determine
the occurrence of H. pylori in environmental samples and to inform the Agency
about the route of transmission and the potential risk from this pathogen.
The method developed to detect viable H. pylori in water is based on the idea that
viable cells will be able to amplify in number during an enrichment step. Such an
assay is used to analyze samples before and after enrichment. In the current
method, a water sample is filtered through a nylon membrane filter, trapping all
bacterial cells present in the sample. The filter is then placed on an agar medium
capable of supporting the growth of any present H. pylori bacteria. Over a 3-day
enrichment step, viable individual H. pylori cells will reproduce and form micro
colonies. The total bacterial biomass from the filter is then removed and subjected
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Research to cellular lysis and nucleic acid extraction. This extract is then analyzed by
Approach polymerase chain reaction technology to detect any H. pylori DNA present. An
(Continued) additional aliquot of the original sample is also filtered and subjected to the
extraction process, without enrichment. A positive analysis would be one in which
the enriched filter yielded a high PCR signal compared to the non-enriched filter.
Results and This method can detect as few as 10 seeded H. pylori cells in a 100-mL sample of
Impact ground water. The number of nontarget bacteria in the sample has a great influence
on the ability to detect H. pylori bacteria growing on the filter. Other bacteria will
overgrow the slow growing H. pylori bacteria and prevent the formation of micro
colonies. The method was originally developed using sheep blood agar, which
supports the growth of H. pylori but does not suppress the growth of nontarget
bacteria. The ability to demonstrate the growth of 10 seeded H. pylori is
impressive given these conditions. Currently, a more selective medium is being
adapted for use in the enrichment step, which should improve the sensitivity of the
method.
Also, this methodology was used to demonstrate the presence of viable H. pylori
bacteria in a ground water sample from the Cincinnati area. A description of this
work has been published in the journal Acta hydrochimica et hydrobiologica. This
appears to be one of only a few reports in the literature that suggests the possibility
that viable H. pylori bacteria exist in environmental waters.
This research project directly supports ORD's efforts to improve the scientific
foundation for safe drinking water. The results of this research support the
Government Performance and Results Act (GPRA) Goal 2 ("Clean and Safe
Water"), Objective 2.1 ("Ensure Safe Drinking Water and Recreational Waters"),
and Sub-Objective 2.1.7, Long Term Goal 2 ("By FY 2010, develop new data,
innovative tools, and improved technologies to support decision making by the
Office of Water on the Contaminant Candidate List and other regulatory issues,
and implementation of rules by states, local authorities, and water utilities"). It was
done in support of an FY03 GPRA annual performance goal ("The Office of
Water will have data, methods, assessments, and technology evaluations necessary
to support scientifically sound risk assessment and risk management decisions on
unregulated contaminants of potential public health concern") and the annual
performance measure #101 ("Develop methodology to identify and characterize H.
pylori, caliciviruses, and sources of human pathogens in water").
Research The National Exposure Research Laboratory (NERL) has funded a cooperative
Collaboration and agreement with the University of North Carolina to study the possibility of
Research resuscitating the non-culturable coccoid form of H. pylori. This project is due to
Products encj jn june 2004.
Flanigan, D. and M. Rodgers. 2003. A Method to Detect Viable Helicobacter pylori Bacteria in
Groundwater. Acta hydrochimica et hydrobiologica 31(1): 45-48.
Future Research Scientists at the EPA's NERL are now using this enrichment method to collect
occurrence data of H. pylori in a variety of ground and drinking water samples.
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Contacts for Questions and inquires concerning the H. pylori study can be directed to:
Additional
Information Mark R0dgers, Ph.D.
U.S. EPA
National Exposure Research Laboratory
Microbiological & Chemical Exposure Assessment
Research Division
26 W. Martin Luther King Drive
Cincinnati, Ohio 45268
Phone:513/569-7225
E-mail: rodgers.mark@epa.gov
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