Grantee Research Project Results

2009 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, 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: Targeted Research , Drinking Water , 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 project

Supplemental Keywords:

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

The 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.