Impact/Purpose:

Investigate the occurrence of Aeromonas bacteria in potable water derived from both surface and ground water sources. Develop improved method(s) for the detection of culturable H. pylori in environmental samples and investigate the occurrence of H. pylori in potable waters.

Description:

Recent attention has focused on the potential transmission via drinking water of two bacterial pathogens, Aeromonas and Helicobacter pylori, both of which are included in the current Contaminant Candidate List. Aeromonas bacteria occur naturally in surface waters and have been isolated from soil and food. In humans, Aeromonas bacteria have been associated with soft tissue infections and gastroenteritis. The exact mechanisms of pathogenesis are not clearly understood for Aeromonas infections, nor are the primary routes of transmission. H. pylori is a newly recognized human pathogen. H. pylori 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 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 natural reservoir(s) for H. pylori has not been determined, nor has the route of transmission in humans. This study proposes to identify and/or develop methods for detecting viable Aeromonas and H. pylori bacteria in environmental waters and to use these methods to explore the occurrence of these bacteria in potable waters. For Aeromonas the performance of one published method, ampicillin-dextrin agar, will be documented using cultures of all the available Aeromonas species. The evaluation and modification of this method forms the basis for EPA Method 1605 (Fed. Reg., October 29, 2002). For H. pylori, a method will be developed for the detection of culturable cells in environmental water samples. Reliable detection methods and subsequent occurrence data will help public health officials determine the importance of potable waters in the transmission of these pathogens. Results from this study will be disseminated in presentations at national and regional scientific meetings and in submission of articles to peer-reviewed scientific journals.

Record Details:

Record Type:PROJECT

Start Date:05/01/1998

Projected Completion Date:09/01/2004

Record ID: 15806

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Project Information:

Progress : SubTask 1- Method development and occurrence study on Aeromonas bacteria in drinking water- Ampicillin-Dextrin agar (ADA) was evaluated as an Aeromonas recovery medium in an exploratory occurrence study on aeromonads in Cincinnati area tap, well and cistern waters. Reference cultures representing 15 different Aeromonas species/groups were tested; of these, 13 were able to grow on ADA at 35oC. It was determined that all yellow colonies from ADA needed to be confirmed as Aeromonas. A protocol was developed involving testing for cytochrome c oxidase and trehalose fermentation. Aeromonas should be positive for both tests. An initial study included testing 142 yellow colonies from ADA. 7.7% were oxidase negative. 16.7% were oxidase positive but trehalose negative. 109 (76.7%) fit the definition for Aeromonas. Aeromonas bacteria were not detected in 40 tap water samples, but over half of the well and cistern waters were positive for Aeromonas. A majority of the strains were identified using a restriction fragment length polymorphism method. 47% were identified as A. bestiarum, a relatively non-virulent species. We are currently near the end of an occurrence study involving 16 different water utility distribution systems. To date, 1085 water samples have been analyzed for Aeromonas bacteria. Of these, only 28 samples were positive for Aeromonas. Each sample is also characterized with respect to total and free chlorine residuals, temperature, turbidity, presence or absence of total coliforms and E. coli and levels of heterotrophic bacteria. Also in this study we have modified the ADA medium to include the antibiotic vancomycin in order to inhibit the growth of Bacillus bacteria, which produce Aeromonas-like colonies on ADA. This minor modification will significantly decrease the time spent on biochemically confirming suspected Aeromonas-like colonies. Isolates from this study are being archived for future identification to the biotype level, as well as for any additional characterizations (such as virulence).

SubTask 2- Method development and occurrence study of Helicobacter pylori in environmental waters- Several strains of H. pylori have been established in culture in the EPA lab. Preliminary experiments indicate that the use of cumbersome anaerobic chambers can be avoided by using a media component which by itself produces the proper microaerophilic environment. Using PCR we have screened sewage samples from several large metropolitan waste facilities for H. pylori DNA, but have only found its presence sporadically. Little is known concerning the numbers of H. pylori shed by infected persons or the frequency at which cells are shed in the feces. The most useful information to be determined is the presence of viable H. pylori in water. PCR alone can not easily provide this information. No differential and/or selective medium presently exists for H. pylori. And no enrichment procedure has been published. Typically cultured on blood agar, H. pylori produces a slow growing, small, colorless non-hemolytic colony. H. pylori likely occurs in low numbers, therefore an extremely sensitive method or an enrichment medium is needed for detection. Because PCR can provide both good sensitivity and specificity, we have sought to couple PCR with enrichment on blood agar. Potable water is filtered though a 0.45um membrane filter, which is placed onto the surface of a blood agar plate. After incubation for 3 days at 35oC under microaerophilic conditions, during which time any viable and culturable H. pylori grow to form a colony, the cells are removed from the filter, lysed and the DNA amplified with H. pylori specific primers. To date, the results continue to be promising. We have concentrated our efforts so far to analyzing groundwater sample from local wells found previously to be contaminated with fecal coliform bacteria. Similarly contaminated wells in Pennsylvania have

Relevance :The projects included in this task are focused specifically on meeting information needs outlined in the current Contaminant Candidate List. Occurrence information on the bacteria Aeromonas and Helicobacter pylori in drinking water distribution systems is needed before accurate risk assessments can be developed with regards to exposures of the human population to drinking water. In order to produce accurate occurrence data, specific and sensitive methods must be developed and validated. Risk assessment analysis will be the tool used to determine whether Aeromonas or Helicobacter pylori bacteria pose a significant public health risk due to exposure to drinking water and whether the levels of these organisms in drinking water need to be regulated. It is anticipated that the Office of Water will be the primary customer for this occurrence information. Significant outcome(s): The promulgation of an EPA method for Aeromonas in drinking water (EPA Method 1605, Federal Register Vol 67, October 29, 2002) and the availability of this method for use by the EPA Office of Water in the Unregulated Contaminant Monitoring Rule; and the development of novel detection methods for Helicobacter pylori in environmental waters.

Clients :Dr. Paul Berger, OGWDW

Research Component :M/DBP (MICROBIAL)

Risk Paradigm :EXPOSURE

Progress : SubTask 1- Method development and occurrence study on Aeromonas bacteria in drinking water- Ampicillin-Dextrin agar (ADA) was evaluated as an Aeromonas recovery medium in an exploratory occurrence study on aeromonads in Cincinnati area tap, well and cistern waters. Reference cultures representing 15 different Aeromonas species/groups were tested; of these, 13 were able to grow on ADA at 35oC. It was determined that all yellow colonies from ADA needed to be confirmed as Aeromonas. A protocol was developed involving testing for cytochrome c oxidase and trehalose fermentation. Aeromonas should be positive for both tests. An initial study included testing 142 yellow colonies from ADA. 7.7% were oxidase negative. 16.7% were oxidase positive but trehalose negative. 109 (76.7%) fit the definition for Aeromonas. Aeromonas bacteria were not detected in 40 tap water samples, but over half of the well and cistern waters were positive for Aeromonas. A majority of the strains were identified using a restriction fragment length polymorphism method. 47% were identified as A. bestiarum, a relatively non-virulent species. We are currently near the end of an occurrence study involving 16 different water utility distribution systems. To date, 1085 water samples have been analyzed for Aeromonas bacteria. Of these, only 28 samples were positive for Aeromonas. Each sample is also characterized with respect to total and free chlorine residuals, temperature, turbidity, presence or absence of total coliforms and E. coli and levels of heterotrophic bacteria. Also in this study we have modified the ADA medium to include the antibiotic vancomycin in order to inhibit the growth of Bacillus bacteria, which produce Aeromonas-like colonies on ADA. This minor modification will significantly decrease the time spent on biochemically confirming suspected Aeromonas-like colonies. Isolates from this study are being archived for future identification to the biotype level, as well as for any additional characterizations (such as virulence).

SubTask 2- Method development and occurrence study of Helicobacter pylori in environmental waters- Several strains of H. pylori have been established in culture in the EPA lab. Preliminary experiments indicate that the use of cumbersome anaerobic chambers can be avoided by using a media component which by itself produces the proper microaerophilic environment. Using PCR we have screened sewage samples from several large metropolitan waste facilities for H. pylori DNA, but have only found its presence sporadically. Little is known concerning the numbers of H. pylori shed by infected persons or the frequency at which cells are shed in the feces. The most useful information to be determined is the presence of viable H. pylori in water. PCR alone can not easily provide this information. No differential and/or selective medium presently exists for H. pylori. And no enrichment procedure has been published. Typically cultured on blood agar, H. pylori produces a slow growing, small, colorless non-hemolytic colony. H. pylori likely occurs in low numbers, therefore an extremely sensitive method or an enrichment medium is needed for detection. Because PCR can provide both good sensitivity and specificity, we have sought to couple PCR with enrichment on blood agar. Potable water is filtered though a 0.45um membrane filter, which is placed onto the surface of a blood agar plate. After incubation for 3 days at 35oC under microaerophilic conditions, during which time any viable and culturable H. pylori grow to form a colony, the cells are removed from the filter, lysed and the DNA amplified with H. pylori specific primers. To date, the results continue to be promising. We have concentrated our efforts so far to analyzing groundwater sample from local wells found previously to be contaminated with fecal coliform bacteria. Similarly contaminated wells in Pennsylvania have

Relevance :The projects included in this task are focused specifically on meeting information needs outlined in the current Contaminant Candidate List. Occurrence information on the bacteria Aeromonas and Helicobacter pylori in drinking water distribution systems is needed before accurate risk assessments can be developed with regards to exposures of the human population to drinking water. In order to produce accurate occurrence data, specific and sensitive methods must be developed and validated. Risk assessment analysis will be the tool used to determine whether Aeromonas or Helicobacter pylori bacteria pose a significant public health risk due to exposure to drinking water and whether the levels of these organisms in drinking water need to be regulated. It is anticipated that the Office of Water will be the primary customer for this occurrence information. Significant outcome(s): The promulgation of an EPA method for Aeromonas in drinking water (EPA Method 1605, Federal Register Vol 67, October 29, 2002) and the availability of this method for use by the EPA Office of Water in the Unregulated Contaminant Monitoring Rule; and the development of novel detection methods for Helicobacter pylori in environmental waters.

Clients :Dr. Paul Berger, OGWDW

Research Component :CCL (MICROBIAL)

Risk Paradigm :EXPOSURE

Project IDs:

ID Code :EX.M.21

Project type :ORD-DW Plan

ID Code :509

Project type :OMIS