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CCL Nominations
Environmental Protection Agency
Mail Code: 4607M
1200 Pennsylvania Ave., NW
Washington, D.C. 20460

The American Society for Microbiology (ASM) is submitting nominations for two candidates (Naegleria fowleri and Mimivirus) in response to the Environmental Protection Agency (EPA) notice requesting nominations of chemical and microbial contaminants for possible inclusion in the third drinking water Contaminant Candidate List (CCL 3), in the Federal Register, Vol. 71, No. 199 on October 16, 2006, Docket ID No. EPA-HQ-OW-2005-0039.  The following comments were developed by the ASM Public and Scientific Affairs Board Committee on Environmental Microbiology.

The ASM is the largest single life science society with more than 42,000 members, including scientists in academic, industrial, clinical, and government institutions, working in areas related to basic and applied research, the prevention and treatment of infectious diseases, laboratory and diagnostic medicine, the environment, and water and food safety.

In addition to providing nominations for CCL 3, the ASM strongly encourages EPA to develop and implement a systematic strategy and set of criteria to identify and review all potential CCL organisms.  Further, ASM recommends that in evaluating CCL organisms, EPA should use a formal decision-making process that reflects both the objectives of the CCL and the Safe Drinking Water Act.

The ASM also notes that a more comprehensive strategy and set of evaluation criteria could lead to inclusion of additional organisms of concern, for example, E. coli O157:H7 and various campylobacteria, that do not meet current CCL criteria.  Because of their increasing prevalence within the U.S. and abroad and the difficulties of detecting them, both E. coli O157:H7 and campylobacteria may warrant special consideration.

The ASM’s nominations for the CCL3 are below:

A. Naegleria fowleri (protozoa)

Factors that warrant designation as a priority contaminant for CCL 3.

Naegleria fowleri, a water-based parasite found in soil and warm water, is relatively resistant to chlorine-based disinfection (Cassells et al, 1995).  It has a three-stage lifecycle:  a cystic form in soil that excysts to a flagellate stage in the presence of warm water and a food source (bacteria). The flagellate stage feeds on bacteria at the air-water interface in springs, surface water, and certain stagnant water systems.  Human and animal health risks occur during the third stage, when the organism is inhaled or forced into the nasal passages during recreational water use. N. fowleri can then cause a fatal form of meningitis (Parija and Jaykeerthee, 1999).

In addition, drinking water has been linked to human illness in Australia and more recently the United States (Marciano-Cabral, 2003).  In 2002, deaths of two children in the Phoenix metropolitan area were traced to non-disinfected drinking water obtained from a well used by a utility serving the area (Marciano-Cabral, 2003).  A recent survey has indicated that N. fowleri colonizes at least 8% of the utility groundwater production wells used for drinking water supply in central and southern Arizona (Blair, 2006).  The occurrence of N. fowleri may be related to warm groundwater temperatures (usually above 20oC) and the growth of bacteria in wells due to the use of biodegradable oils used as lubricants for well pumps.  The cysts and oocysts are fairly resistant to chlorine.  Sarker (2006) found Ct (contact time concentration of chlorine) values for trophozoites at pH 7.5 and 23oC with an average dose of 1.29 mg/l free chlorine predicted by the efficiency factor hom model (Thurston-Enriquez et al., 2003) as follows:

99% inactivation = 6 minutes
99.9% inactivation = 9 minutes
99.99% inactivation = 12 minutes

These values are similar to those reported by Cassells et al., 1995.

Ct value of cysts at pH 7.5 and 23oC at an average dose of 1.26 mg/l predicted by the efficiency factor hom model (Thurston-Enriquez et al., 2003) are:

99% inactivation = 31 minutes
99.9% inactivation = 42 minutes
99.99% inactivation = 53 minutes

These results indicate that the cysts are much more resistant to free chlorine than the trophozoites.  The resistance of N. fowleri cysts to chlorine is similar to that for Giardia cysts.

In summary, it appears that at least in wells supplying drinking water at temperatures at 20oC or greater, N. fowleri can grow to large numbers and present a potentially serious health risk. For groundwater systems that do not use disinfectant or do not provide enough contact with chlorine to cause a significant reduction of N. fowleri cysts and trophozoites, exposure will be greater.

Significant Health Effects:

Humans and other mammals contact N .fowleri via swimming, bathing, or in the case of cattle and domesticated animals, drinking from or swimming in water sources where N. fowleri is present.  The organism is inhaled and travels up the nasal passageway to the ethmoid sinuses.  Penetration of the mucosa and invasion of olfactory nerves is followed by movement through the cribiform plate to brain tissue and cerebral spinal fluid.  Once infected the brain tissue produces toxins that attempt to kill the parasite, but end up emulsifying tissue.  The immune response leads to swelling and PAM (Marshall, et al, 1997).  N. fowleri is the only member of the species known to be pathogenic to humans.

Clinical signs of infection include headache, nausea, vomiting, high fever, lethargy, coma, seizures, and eventually death due to infection and swelling of brain tissue (Marshall, et al, 1997).  The average time for onset of symptoms is 4 days.  Mean time between onset of symptoms and death is 6.4 days (Parija and Jaykeerthee, 1999; Marshall, et al, 1997).  The short onset period and symptoms mimic the flu, allergic reaction or hangover from alcohol.  As a result, many cases have been misdiagnosed or not treated in time.  Documented cases of PAM have been noted worldwide, including the United States, England, Czechoslovakia, and Mexico.  The largest number of cases has been observed in the United States (Cabanes, 2001; Rivera, et al, 1993; Kadlec, Cerva, and Skvarova, 1978).  N. fowleri affects the young, the old, and those whose immune systems may be impaired, such as patients with human immunodeficiency virus (Rivera, et al, 1993).  Contact with the organism may be common, as antigens to the organism are often found in the blood of adults (Marshall, et al, 1997).


Blair, B. 2006. Occurrence of Naegleria fowleri in Well Water in Arizona. M.S. Thesis. Department of Soil, Water and Environmental Science. University of Arizona. Tucson, AZ.

Cabanes,  P., 2001.  Assessing the risk of primary amoebic meningoencephalitis from swimming in the presence of environmental Naegleria fowleri. Appl. Environ. Micobiol. 11:2927-2931

Cassells, J. M., M. T. Yahya, C. P. Gerba anbd J. B/. Rose. 1995. Efficacy of combined system of copper and silver and free chlorine for inactivation of Naegleria fowleri amobas in water. Water. Sci. Tech. 31:119-122.

Kadlec, V., L. Cerva, and J. Skvarova. 1978. Virulent Naegleria fowleri in an indoor swimming pool. Science. 210:1025.

Marciano-Cabral, F., R. MacLean, A. Mensah, and L. LaPat-Polasko 2003. Identification of Naegleria fowleri in domestic water sources by nested PCR. Appl. Environ. Microbiol. 69:5864-9.

Marshall, M., D. Naumovitz, Y. Ortega, and C.R. Sterling 1997. Waterborne protozoan pathogens.  Clin. Microbiol. Rev. 1:67-85.

Sarkar, P. and C. P. Gerba 2006. Occurrence and control of emerging waterborne protozoan parasites in water in Arizona: N. fowleri. Progress Report. National Science Water Quality Center. December 4, 2006.

Thurston-Enriquez, J. A., C. N. Haas, J. Jacangelo and C. P. Gerba. 2003. Chlorine inactivation of adenovirus type 40 and feline calicivirus. Appl. Environ. Microbial. 69:3979-3985.

Parija, S. C., and S. R. Jaykeerthee, 1999. Naegleria fowleri: a free living amoeba of emerging medical importance. Com. Dis. 31:153-159

Rivera, F., E. Ramirez, P. Bonilla, A. Calderon, E. Gallegos, S. Rodríguez, R. Ortiz, B. Zaldivar, P. Ramirez, and A. Duran 1993. Pathogenic and free-living amoebae isolated from swimming pools and physiotherapy tubs in Mexico. Environ. Res. 62:43-52.

B. Mimivirus (virus)

Factors that warrant designation as a priority contaminant for CCL 3.

Mimivirus is a recently discovered giant virus that infects amoeba.  The virus was discovered in studies of cooling tower water in Bradford, England containing the free-living amoeba, Acanthamoeba polyphaga, which was implicated in a pneumonia outbreak in 1992.  Studies of the water revealed a microbe growing in the amoebae that resembled small Gram-positive cocci.  The agent has characteristic viral morphology, an icohsahedral capsid, and contains a double-stranded, circular DNA genome of about 800 kilobase pairs.  The agent has a typical virus developmental cycle, including an eclipse phase, but it resembles a bacterium when Gram-stained.  It has been named Mimivirus for Mimicking microbe, and is the largest known virus.  The ecology of Mimivirus is poorly understood, but it is apparently associated with natural waters containing free-living amoeba, and in that respect it resembles Legionella bacteria in its behavior.  Genetically similar giant viruses have now been discovered to be widespread in ocean waters as well as freshwater aquatic environments, where they play an important role in controlling phyto- and bacterioplankton populations.  Hence, these viruses are quite ubiquitous in aquatic habitats.  Furthermore, Mimivirus has been implicated in cases of human illness, specifically pneumonia.  When Mimivirus is used as an antigen in microimmunofluorescense assays, seroconversion has been documented in patients with both community- and hospital-acquired pneumonia.  Additionally, Mimivirus DNA has been found in respiratory samples of patients with hospital-acquired pneumonia.

These data suggest that Mimiviruses need to be considered as CCL candidates.  They are waterborne microbes and they have been implicated in human illness associated with water exposure, in a manner and natural history similar to that of Legionella, an EPA-regulated pathogen in drinking water.  Because little is known about Mimivirus and analytical methods are available to detect it, it deserves consideration and further study.

Significant Health Effects:

At present, Mimivirus has been implicated in various cases of pneumonia.  However, its recent discovery has not provided sufficient time to fully recognize or understand the scope of its health effects and risks.  Its ecology is also uncertain, so it is not yet possible to ascertain whether it constitutes an emerging risk, or how it may respond to environmental change.  The impact of the Mimivirus on pneumonia warrants further study of it as a CCL 3 organism.


La Scola B, Audic S, Robert C, Jungang L, de Lamballerie X, Drancourt M, et al. (2003) A giant virus in amoebae. Science. 299:2033.

La Scola, B., T.J. Marrie, J.-P. Auffray and D. Raoult (2005) Mimivirus in Pneumonia Patients.  EID, Vol. 11, No. 3, pages

Jean-Michel Claverie (2005) Giant viruses in the oceans: the 4th Algal Virus Workshop. Virol J. 2005; 2: 52.

The ASM is pleased to have this opportunity to provide nominations for the CCL 3, and hopes that the comments and recommendations above are of assistance.


Ruth Berkelman, M.D., Chair, Public and Scientific Affairs Board
Gary King, Ph.D.,Chair, Committee on Environmental Microbiolog

Charles Gerba, Ph.D.
Member, Committee on Environmental Microbiology

Charles Haas, Ph.D.
Member, Committee on Environmental Microbiology

Ian Pepper, Ph.D.
Member, Committee on Environmental Microbiology

Joan Rose, Ph.D.
Member, Committee on Environmental Microbiology

Mark Sobsey, Ph.D.
Member, Committee on Environmental Microbiology