Grantee Research Project Results
2008 Progress Report: The Isolation and Characterization of Naturally-Occurring Amoeba-Resistant Bacteria from Water Samples
EPA Grant Number: R833102Title: 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, 2007 through August 14,2008
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
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 1) to 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 were also focused on improving methods used to remove contaminating bacteria from the co-cultures by methods such as dilution and antibiotic treatments. In year two, we are currently investigating the use of density gradients and micromanipulators to improve efficiency of isolation and are making good progress with density gradients; however, our collaborators at TTU are just acquiring the micromanipulator system. We are enthusiastic about characterizing isolates from these isolation procedures.
2) Phylogenetic and Phenotypic Characterization of Bacteria: Following cleanup of the ARB, we were also able to phylogenetically 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 initially 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 percent 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 percent 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, we presented results from these studies at the General ASM meeting in Toronto, and currently have a poster presentation scheduled for the upcoming ASM meeting in Boston. We plan to complete data analysis and submit a manuscript based on these studies by the end of the summer, which will also allow for further work to meet reviewer’s requests for successful acceptance of the manuscript. In addition, because we still need to acquire these sequences, we have yet to fulfill the proposed screening for the prevalence of these microbes that will be conducted in the upcoming year.
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. The two additional microorganisms will be similarly characterized.
Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental Keywords:
Scientific Discipline, Health, Risk Assessments, Ecological Risk Assessment, pathogens, environmental risks, Legionella-like bacteria, amoeba resistant bacteria, infectious organisms, inhalation, lung pathogensProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.