Grantee Research Project Results

2007 Progress Report: 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. , Farone, Mary B , Berk, Sharon G. , Gunderson, John H.
Institution: Middle Tennessee State University , Tennessee Technological University
EPA Project Officer: Aja, Hayley
Project Period: August 15, 2006 through August 14, 2007 (Extended to August 14, 2010)
Project Period Covered by this Report: August 15, 2006 through August 14,2007
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: Targeted Research , Drinking Water , Water

Objective:

The objectives of this study were designed to address the protection of human health through clean and safe water and healthy communities by the detection of unculturable, novel “amoeba-resistant bacteria” (ARB). The objectives are to:  1) continue the biological clean up of previously collected water samples containing infected amoebae, 2) continue the phylogenetic and phenotypic characterization of the bacteria, and 3) isolate additional ARB from both environmental and human-constructed water sources.

Progress Summary:

1. Clean-up of mixed cultures of ARB: During year one of funding, we successfully removed all contaminating bacteria from three, unculturable ARB being co-cultured with Acanthamoeba polyphaga. Year one efforts also were focused on improving methods used to remove contaminating bacteria from the co-cultures by methods such as dilution and antibiotic treatments. We currently are investigating the use of density gradients and micromanipulators to improve efficiency of isolation.
2. Phylogenetic and Phenotypic Characterization of Bacteria: Following cleanup of the ARB, we also were able to phylogentically characterize two of the bacteria in year one. One of the organisms, isolated from a cooling tower biofilm, belonged to the Rickettsiae and exhibited the greatest similarity (94%) to the previously described ARB Trojanella thessalonices. The other ARB, which was isolated from a fire safety sprinkler system supplied by municipal water, exhibited the greatest similarity (98%) to the ARB Legionella lytica, which unlike this ARB is culturable on buffered charcoal yeast extract agar. Both of these organisms were taken up initally into the amoeba food vacuole; however, the Rickettsial ARB escaped from vacuoles and completed its life cycle in the amoeba cytoplasm. Neither one of these ARB was pathogenic for the human macrophage cell line U937. Another ARB, previously phylogenetically characterized as having less than 93% identity to any cultured bacterium, was found to infect the nuclei of not only A. polyphaga, but also human U937 macrophages, HeLa epithelial cells, and murine L929 fibroblasts.

   The three isolated ARB and four unpurified ARB samples found in infected amoebae from a water distribution system, artificial pond, and a slaughterhouse drain, none of which were culturable on conventional laboratory media, were tested for their length of survival outside of the host and their ability to survive desiccation in 3.8% relative humidity. Two of the strains remained infective for at least 130 days after lysis of the host amoeba, two others for 86-89 days, one at 50 days, and another at 21 days. Two strains, including the Rickettsial ARB, were no longer infective by 14 days after lysis. Of the strains that were dried, two were still infectious at 78 days, and three others remained infectious at 16, 23, and 37 days. One strain and Legionella pneumophila were not infectious at 18 hours after being placed in the desiccation chamber.
   During year one, we also developed real time PCR assays to quantitate the growth of these unculturable bacteria inside of host cells.

3. Isolation of ARB from Environmental and Human-Constructed Water Sources: In year one, three ARB were detected in amoeba from a municipal water distribution system, one from the biofilm of a cooling tower, one from an artificial decorative pond, and another in a slaughterhouse drain. Only one of the ARB was able to be isolated in axenic co-culture with A. polyphaga. The 16S rDNA of this organism has been cloned and sequencing for phylogenetic characterization is in progress. The removal of contaminants from the other cultures is ongoing. It is significant that all of these ARB were detected in amoeba naturally occurring in the water systems and were able to be passed to cultures of A. polyphaga.

Journal Articles:

No journal articles submitted with this report: View all 6 publications for this project

Supplemental Keywords:

aquatic, cooling tower, hot tub, protes, Scientific Discipline, Health, PHYSICAL ASPECTS, Risk Assessments, Physical Processes, Ecological Risk Assessment, amoeba resistant bacteria, inhalation, lung pathogens, Legionella-like bacteria, human health risk, detection, environmental risks, pathogens, infectious organisms, exposure,, Health, Scientific Discipline, Ecological Risk Assessment, Risk Assessments, lung pathogens, environmental risks, infectious organisms, amoeba resistant bacteria, Legionella-like bacteria, pathogens, human health risk

Progress and Final Reports:

Original Abstract

2008 Progress Report

2009 Progress Report

Final Report