Grantee Research Project Results
2009 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, 2008 through August 14,2009
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:
Progress Summary:
Objective I. Continue the biological clean up of samples containing infected, naturally occurring amoebae and identify the bacteria based on 16S rRNA sequencing.
To date, six cleanups of organisms have been completed so that they are in axenic co-culture with Acanthamoeba polyphaga (if unculturable; 4 organisms) or in pure culture (2 organisms).
The organisms and their sources are as follows:
--Legionella pneumophila—1—natural water source (creek)
--Trojanella thessalonices—1—cooling tower--closest match (97% identity)
--LLAP-2-LLAP-6-L. lytica clade—3 potentially novel bacteria:
--1 fire safety sprinkler
--1 fire hydrant
--1 cooling tower
--LLAP 14—cooling tower
Objective II: Use 16S rRNA gene sequences to generate phlylogenetic trees comparing novel sequences with known sequences of related bacteria and use the novel sequences as probes for the distribution of the bacteria in natural and human-made environments.
Screening/Probing of Fire Hydrant Samples (18) and Cooling Tower samples (10) for AAM DNA (looking for distribution of these organisms in the environment)
--PCR was performed using primers specific for:
eubacteria, LLAP 2-6-L. lytica clade, CC99/HT99, mimivirus
Table 1: Percentages of water samples positive for ARB by DNA analysis.
% positive for DNA
|
Bacteria
|
LLAP 2-LLAP 6-L. lytica
|
CC99/HT99
|
Mimivirus
|
Cooling Towers
|
100%
|
40%
|
20%
|
0%
|
Hydrants
|
88.9%
|
12.2%
|
0%
|
5.5% (1)
|
Objective III: Determine the stability of the novel ARB outside of host cells and whether the ARB have potential for human disease.
5 different ARB isolates from these studies and a Legionella pneumophila control were tested for their infectivity over time and ability to survive desiccation:
Longevity: 2 strains remain infective in the medium at 130 days after lysis of amoeba host,
2 others at 86-89 days, and 1 at 50 days
Resistance to desiccation: Of strains that were dried, 2 were still infectious at 78 days, and 3 others have remained infectious at 16, 23 and 37 days
Objective IV: Continue to search for ARB in both human-made and natural environments.
To date: the following have been screened with corresponding number of ARB found
--Municipal fire hydrant samples—41
41% + for amoebae
63% + for bacteria
34% + for amoeba and bacteria
12.2 % (5 of 41) had ARBs
--Cooling Towers—7
100% + for amoebae
100% + for bacteria
10 ARBs found in 5 of the 7 cooling towers
--Other (human-constructed)
Eye Wash Station Drain—2 ARB
Fire Safety Sprinkler System—1 ARB
Artificial Pond—1ARB
Meat Processing Plant—2 ARB
--Natural samples—4 (2 creeks, 2 state park lakes)—4 ARB
5. Completion Date: August 2010
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.