Grantee Research Project Results
Final Report: Protozoa in Risk Assessment of LegionellosisInadequacy of Guidelines and Monitoring
EPA Grant Number: R825352Title: Protozoa in Risk Assessment of LegionellosisInadequacy of Guidelines and Monitoring
Investigators: Berk, Sharon G. , Wells, Martha J.M. , Newsome, Anthony L.
Institution: Tennessee Technological University , Middle Tennessee State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1996 through September 30, 1999 (Extended to September 30, 2000)
Project Amount: $359,300
RFA: Exploratory Research - Environmental Biology (1996) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
Studies on interactions between amoebae and Legionella generally concur that amoebae and possibly other protozoa play roles in maintaining Legionellae in man-made environments such as cooling towers, often implicated as sources of outbreaks of legionellosis. The present study had several objectives that examined interactions in more detail. These objectives included several aspects of vesicle production by protozoa in actual cooling tower water. Such vesicles may contain hundreds of viable Legionella and may be in a form that could be inhaled into the lower spaces of the lungs to produce Legionnaires' disease. Co-culture experiments were designed to determine whether intracellular growth of Legionellae occurs when the host protozoa have alternative bacteria as food sources. The potential for cooling tower ciliates to serve as hosts for various species of Legionella was studied. Another objective was to evaluate a relatively new technique, flow field-flow fractionation, for detection of vesicles in water samples. The use of molecular probes for detecting amoebae infected with Legionella in cooling towers and natural waters also was tested. Finally, biocide efficacy against cooling tower protozoa was evaluated, and one goal was to find a combination of commonly used biocides that together would not confer cross-resistance to the protozoan target.Summary/Accomplishments (Outputs/Outcomes):
Factors that increased the production of free vesicles containing viable Legionella cells by amoebae included a temperature of 30?C (as compared with 25?C), an increase in the ratio of Legionellae to amoebae, increased surface area (lower amoeba population density per area), addition of E. coli to the Legionella suspension, and a pH close to that of a cooling tower. Biocides had no effect on vesicle production by amoebae. Amoebae feeding on biofilms versus bacteria in suspension showed no difference in the number of vesicles released to the medium. All work on factors affecting production of vesicles was done with a strain that was not virulent for the amoebae, L. pneumophila ATCC # 33216. A temperature of 25 or 30?C made no difference in the number of vesicles released by the ciliate, Tetrahymena sp., and exposure of this ciliate to isothiazolines did inhibit production of vesicles. The ciliate produced more vesicles at pH 8 than at pH 7 or 9 when it was originally cultured at pH 8. Most of the cooling tower samples had pH values around 8.2 to 8.5. Studies with ciliates showed that those fed live Legionella species produced significantly more vesicles than those fed heat-killed Legionella cells. The size range of vesicles produced by Tetrahymena sp. isolated from a cooling tower was 1 to 8 m diameter when feeding on L. pneumophila and L. micdadei. The majority of vesicles were approximately 4 m diameter, within the size range for respirable particles.Experiments to determine the lowest infectious dose of L. pneumophila strain AA100 revealed that a ratio as low as 1 bacterium to 1,000 amoebae could initiate an infection that can destroy amoebae and cause a 7,000-fold increase in the number of the bacteria. Addition of other non-Legionellae bacteria in much higher concentrations than the L. pneumophila did not alter the number of recoverable L. pneumophila cells from the co-culture. Also, if the ciliate, Tetrahymena, was added to the suspension after 72 hours, the ciliates filtered the bacteria released from the amoebae and packaged them in vesicles expelled to the medium. The ciliates were not affected by the bacteria.
A digoxygenin-labeled gene probe, LEG 705, was used to demonstrate Legionella within amoebae from water samples. One sample from a showerhead of a suspected outbreak source revealed LEG 705 positive reactions with what appeared to be bacteria in food vacuoles of amoebae or clusters of bacteria. Such clusters may lead to an underestimate of the total number of Legionella in a sample if plate counting techniques are used. Members of the research team found infected amoebae from biofilm of a hospital cooling tower. The infecting agent was eventually isolated and identified as a novel strain of L. anisa, based on the 16S rRNA gene sequence.
Evaluation of the new flow field-flow fractionation technique revealed that this technique would not be appropriate at the present time. The LEG 705 gene probe labeled with a fluorochrome did not work well for suspensions of Legionellae packaged in protozoan vesicles. Furthermore, the detection limits of the flow field-flow fractionation technique required a very high concentration of vesicles in water, and such concentrations would not likely be encountered in cooling tower water, even after concentration of the water samples. Detection limits need to be improved.
The biocide, BCDMH, was effective in reducing numbers of ciliates, but was not as effective against amoebae. One of the goals of this work was to find a combination of biocides that did not result in cross-resistance for protozoa. The combination of glutaraldehyde and BCDMH appears to be a good one, as exposure of protozoa to each of these does not confer resistance to the other. BCDMH was equally effective in killing Legionella within vesicles or free in the medium, whereas vesicles protected the bacteria from effects of other biocides tested.
Results indicate that protozoa may play important roles in maintaining populations of Legionellae in cooling towers, and that conditions of cooling towers may facilitate production and release of vesicles containing Legionellae from protozoa. For virulent strains of L. pneumophila, very low infectious doses can initiate replication of the bacteria in amoeba populations. Amoebae can serve as hosts to several species of Legionella and strains of L. pneumophila. Ciliates from cooling towers appear to be unaffected by virulent strains of the bacteria, but may interact with them to produce more hazardous forms. Certain biocides may be more effective in inhibiting Legionellae within vesicles. All the information obtained from this project indicates that protozoa and their activities should be considered in monitoring and risk assessment of Legionella in cooling towers, and that biocides should target protozoan hosts.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
Other project views: | All 16 publications | 2 publications in selected types | All 2 journal articles |
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Berk SG, Ting RS, Turner GW, Ashburn RJ. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Applied and Environmental Microbiology 1998;64(1):279-286. |
R825352 (1997) R825352 (1999) R825352 (Final) |
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Desai R, Welsh C, Summy M, Farone M, Newsome AL. The potential of in situ hybridization and an immunogold assay to identify Legionella associations with other microorganisms. Journal of Microbiological Methods, Volume 37, Issue 2, August 1999, Pages 155-164. |
R825352 (1999) R825352 (Final) |
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Supplemental Keywords:
ciliates, amoebae, Legionellae, air-conditioning., Health, Scientific Discipline, Environmental Chemistry, Chemistry, Epidemiology, Disease & Cumulative Effects, Biology, health effects, microbiology, risk assessment, Legionellosis, bacteria, infective dose, waterborne disease, airway disease, cooling towers, protozoa, biocidesProgress 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.