United States Environmental Protection Agency Office of Research and Development National Exposure Research Laboratory Research Abstract Government Performance Results Act Goal: Clean and Safe Water Significant Research Findings: Occurrence of CCL-Related Pathogens in Source and Drinking Waters Scientific Problem The Safe Drinking Water Act requires EPA to establish a list of and Policy Issues unregulated microbiological and chemical contaminants to aid in priority setting for the 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 which may possibly pose a significant public health risk to consumers if present in drinking water. There are three bacterial species listed on the CCL (Aeromonas, 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 these species in drinking water in the U.S. Currently, suitable methods for obtaining occurrence data are available only for Aeromonas and Mycobacterium avium complex (MAC) organisms. MAC organisms are members of the nontuberculous mycobacteria (NTM) group in the genus Mycobacterium. Although it was generally thought that Mycobacterium tuberculosis was the only clinically significant species, changes in attitudes toward species of mycobacteria other than M. tuberculosis came from reports in the 1950's and 1960's that acid-fast bacilli had been cultured from pathological material under circumstances that led microbiologists to believe that some may be clinically significant. Searches for mycobacteria in the environment were renewed. Among the many sources of what became known as NTM water was not at first identified as important. Recently, however, there has been increasing evidence that water may be one of the vehicles by which these organisms infect or colonize the human body. In recent years NTM have emerged as a major cause of opportunist infection in individuals with impaired immunity such as AIDS patients, the elderly, or those undergoing immunosuppressive drugs or chemotherapy. Studies have shown that NTM can survive, grow, and colonize drinking water supply systems. Without evidence of person-to-person National Exposure Research Laboratory — October 2001 ------- transmission, there is a strong possibility that humans may be infected from environmental sources. Because NTM disease in immunocompromised hosts is primarily disseminated to many organs, questions have been raised concerning the portal of entry of these mycobacteria. Before the discovery of AIDS and in the absence of evidence of person-to-person spread of NTM, pulmonary infection was thought to be due to aerosol inhalation. However, in AIDS patients and other immunocompromised hosts, infection can occur via the gastrointestinal tract, lungs, or both. The environment is considered a likely source. Thus, a wide range of sources, exposures, and modes of transmission need to be investigated. Having data available showing the occurrence of these important emerging opportunistic pathogens greatly aids in the assessment of risks to susceptible hosts from exposure to drinking water and other environmental samples that may contain these organisms. Aeromonas are Gram negative, facultatively anaerobic, rod-shaped bacteria in the family Aeromonadaceae that can be found in all surface water environments. Many of the recognized species or biotypes have been implicated as causing human disease, although only three, A hydrophila, A. caviae and A. veronii/sobria are considered to be major human pathogens. Primary effects are gastrointestinal disease resulting in diarrhea. However, these bacteria can also cause serious wound infections and bacteremia. Although they are generally susceptible to chlorine disinfection, the presence of Aeromonas in source waters indicates that these bacteria could be present in municipal drinking water. Past published studies have confirmed this possibility, reporting the isolation of Aeromonas bacteria in drinking water from distribution systems. It is also possible that these bacteria could evade disinfection and reproduce as a part of the biofilm material inside distribution system pipes. Research Approach To aid the Agency in assessing the risks associated with contact with drinking water, the objectives of this research are as follows: • To determine the potential for human exposure by measuring the occurrence of MAC and other NTM organisms in drinking water supply system samples and other environmental samples. • To determine the potential for human exposure by measuring the occurrence of Aeromonas bacteria in drinking water samples. Mycobacterium: A total of 139 samples were collected from five National Exposure Research Laboratory — October 2001 ------- different types of sources. Sources included public drinking water utilities, cisterns, bottled waters, drinking water treatment samples, and ice samples. Drinking water samples were collected from geographically dispersed sites (21 states). Samples were collected from cold taps, warm taps, or showers. The temperatures of the samples were recorded, and total and free chlorine analyses were performed. The age of the sample site (residence or hospital), the type of drinking water treatment, and the source water information was provided with the samples. Noncarbonated bottled water samples were collected from multiple bottlers. Ice samples were collected in sterile sample bottles and allowed to melt at room temperature before analyses. Samples were analyzed for NTM by a membrane filtration method using 7H10 agar after treatment of the sample with cetylpyridinium chloride to reduce background organism levels. The plates were incubated for eight weeks at 37° C and the NTM isolates were identified by a polymerase chain reaction (PCR) -mediated sequence analysis of the gene encoding the 16S rRNA. Heterotrophic plate counts were performed by the R2A membrane filtration method. Aeromonas: Utilities were chosen to participate in the survey with regard to known characterisitics of their drinking water distribution systems. Characteristics of particular interest were low free chlorine residuals in the distribution systems, the use of surface waters as the source water, long residence times for water in the system, high assimilable organic carbon values and the use of chloramine or chlorine dioxide disinfection. Usually 4-6 samples were collected by utility personnel and shipped cold overnight for filtration and Aeromonas analysis using ampicillin-dextrin agar (ADA). In addition, turbidity levels, the presence of total coliforms, E. coli and heterotrophic plate counts were also determined. Water temperature was measured at the tap by the utility. Presumptive Aeromonas colonies on ADA were confirmed by testing for cytochrome c oxidase and the ability to ferment trehalose. Confirmed isolates were archived for future analyses, including identification to the species level and presence of virulence factors. Results and Implications Mycobacterium: Drinking water samples were collected from 42 drinking water supply systems from 21 states. NTM were isolated from 16 (38%) of these systems. In all there were eight different NTM species recovered from surface water drinking water supplies. The most frequently occurring isolate wasM mucogenicum followed by M intracellulare, and M. gastri/kansasii. MAC organisms were found in 9% of the drinking water supplies examined. NTM or MAC organisms were not found predominately in hot water versus cold water. National Exposure Research Laboratory — October 2001 ------- Heterotrophic bacterial levels, chlorine concentration, or the age of the sampling site were not found to correlate with the occurrence of NTM. A number of outbreaks of nosocomial (i.e. hospital-acquired) disease caused by rapidly growing NTM have been reported. In this study M mucogenicum was isolated from 41% of the NTM-positive samples. The most frequent disease associated with M. mucogenicum are catheter sepsis and posttraumatic skin infections. M. mucogenicum was the most frequently occurring NTM in this study and was isolated from all geographic areas. This indicates that the organism may be ubiquitous in drinking water and may pose a potential health risk. In summary, NTM were isolated from 38% of the drinking water supplies examined from a wide geographic area and 33% of all samples. Not only were MAC organisms isolated from 19% of the NTM - positive samples, but other clinically significant mycobacterial opportunist pathogens, such asM kansasii andM mucogenicum, were also present. These organisms were isolated from well-operated, well- maintained drinking water utilities with heterotrophic plate count (HPC) levels of 500 colony forming units (CFU) /ml or less and chlorine residuals of greater than 0.5 mg/L. This study, along with other studies, indicates that exposure to drinking water containing NTM could pose a health risk to immunocompromised hosts and, to a lesser degree, to immunocompetent individuals. Aeromonas: To date 16 utilities have participated by providing water samples for analysis, totaling over 900 samples. Of these, 1.8% have contained Aeromonas bacteria, ranging in density from 1 colony forming unit (CFU)/500ml to >300 CFU/500ml. The occurrence of these bacteria were not randomly distributed among the samples. One utility accounted for half of the positive samples, and one tap at this utility accounted for 30% of the total overall positive samples. Research Collaboration and Publications This research supplies critical information to the EPA Office of Water and will be used in determining whether the MAC group of organisms and Aeromonas bacteria, included on the current Contaminant Candidate List, should be regulated as contaminants in drinking water. This Annual Performance Measure (APM 46) supports FY01 Annual Performance Goal 007, "Reduce uncertainties and improve methods to assess and control risks of microbes, especially CCL." The NTM study was conducted by the EPA's National Exposure Research Laboratory. Publications and presentations from this research include: National Exposure Research Laboratory — October 2001 ------- Covert, T. C., Rodgers, M. R., and Reyes, A. L. "Nontuberculous mycobacteria." Presented at the Annual Water Quality and Measurement Conference, Dallas, Texas. September 10, 1996. Covert, T. C., Rodgers, M. R., Reyes, A. L. and Stelma, G. N. "Occurrence of nontuberculous mycobacteria in environmental samples." Presented at the Annual Meeting of the American Society for Microbiology, Miami Beach, Florida. May 4-8, 1997. Kralovic, S. M., Cannon, C. G., Covert, T. C., Pennington, J. L., Cushion, M. T., Hagland, L. A., Perlada, D. A., Reyes, A. L., Rodgers, M. R, and Rhodes J. C. "Increased isolation of Mycobacterium fortuitum from patients associated with a common nursing unit." Presented at the Annual Meeting of the American Society for Microbiology, Miami Beach, Florida. May 4-8, 1997. Covert, T. C. "Mycobacterium avium - intracellulare." Presented at the AWWA Water Quality Technology Conference. Emerging Waterborne Pathogens Seminar (ST8). San Diego, California. November 4, 1998. Covert, T. C., Rodgers, M. R., Reyes, A. L. and Stelma, G. N. "Occurrence of nontuberculous mycobacteria in environmental samples." Appl. Environ. Microbiol. 65:2492-2496, 1999. The Aeromonas occurrence project is being conducted as a collaboration between the National Exposure Research Laboratory (ORD) and the Technical Service Center (OGWDW). Publications and presentations from this research include: Rodgers, M., Shadix, L. and Feige, M. "Occurrence ofAeromonas bacteria in potable waters." Presented at the Water Quality Technology Conference, Salt Lake City, Utah. November 5-9, 2000. Rodgers, M., Shadix, L.and Feige ,M. "The use of Ampicillin-dextrin agar for the recovery of Aeromonas bacteria from drinking water. " (In preparation). Future Research Scientists at the EPA's National Exposure Research Laboratory are now in the process of developing improved cultural and rapid molecular methods for detection of NTM in drinking water. National Exposure Research Laboratory — October 2001 ------- Questions and inquires concerning the NTM study can be directed to: Terry C. Covert US EPA, Office of Research and Development National Exposure Research Laboratory Cincinnati, Ohio 45268 Phone: 513/569 - 7318 E-mail: covert.terry@epa.gov Questions and inquires concerning the Aeromonas study can be directed to: Mark Rodgers US EPA, Office of Research and Development National Exposure Research Laboratory Cincinnati, Ohio 45268 Phone: 513/569 -7225 E-mail: rodgers.mark@epa.gov Contacts for Additional Information National Exposure Research Laboratory — October 2001 ------- |