The Isolation and Characterization of Naturally-Occurring Amoeba-Resistant Bacteria from Water Samples

EPA Grant Number: R833102
Title: The Isolation and Characterization of Naturally-Occurring Amoeba-Resistant Bacteria from Water Samples
Investigators: Farone, Anthony L. , Berk, Sharon G. , Gunderson, John H.
Current Investigators: Farone, Anthony L. , Berk, Sharon G. , Farone, Mary B , Gunderson, John H.
Institution: Middle Tennessee State University , Tennessee Technological University
EPA Project Officer: Klieforth, Barbara I
Project Period: August 15, 2006 through August 14, 2007 (Extended to August 14, 2010)
Project Amount: $200,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text |  Recipients Lists
Research Category: Drinking Water , Targeted Research , Water


Bacteria that are able to replicate and survive within amoeba hosts have been termed “amoeba-resistant bacteria” (ARB). Some of the bacteria are lytic for their amoebal hosts. These bacteria include members of the genus Legionella as well as other Legionella-like amoebal pathogens (LLAPs). Serological evidence suggests that these LLAPs may be a significant cause of respiratory disease and, because many do not grow on conventional laboratory media, they may be overlooked.

The objectives of this study are designed to address the protection of human health through clean and safe water and healthy communities. The objectives are to continue the biological clean up of previously collected water samples containing infected amoebae, continue the phylogenetic and phenotypic characterization of the bacteria, and isolate additional ARB from both environmental and human-constructed water sources.


The clean up of the samples to remove contaminating bacteria will involve axenic co-culture with amoebae and extensive washing and dilution of these cultures. The 16S rRNA genes of the bacteria will be sequenced for phylogenetic comparisons and construction of phylogenetic trees. We will also use unique sequences from the bacteria to probe additional water samples to determine the distribution of the ARB in the environment. Phenotypic studies of these organisms will include determining how long bacteria released from infected amoebae can remain infectious and whether these bacteria can survive dessication and chemical treatment when protected by vesicles from ciliates. We will also use histochemical and viability staining to determine whether these bacteria are cytopathogenic for human cell lines. We will continue to use methods developed in our laboratories, which have lead to the successful isolation of ARB, to screen additional water samples and collect more of these novel organisms.

Expected Results:

Preliminary results have already identified novel ARB with characteristics, such as replication in the nucleus, which have not been previously described. The results of this work will not only expand knowledge of the types or organisms that are ARB but also provide genetic sequences that will allow assessment of the distribution of these organisms in both natural and human-constructed water environments. Studying the survivability of these organisms in the environment and their ability to infect human cells will also help to identify previously undescribed organisms potentially pathogenic for humans.

Publications and Presentations:

Publications have been submitted on this project: View all 6 publications for this project

Supplemental Keywords:

aquatic, cooling tower, hot tub, protes,, Health, Scientific Discipline, Risk Assessments, Ecological Risk Assessment, pathogens, environmental risks, Legionella-like bacteria, amoeba resistant bacteria, inhalation, infectious organisms, lung pathogens

Progress and Final Reports:

  • 2007 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • Final Report