Recovery of Viable Legionella pneumophilia from Desiccated Food Vacuoles of the Ciliate Tetrahymena Jonathan Thomas Department of Biology Tennessee Technological University Cookeville, TN 38505

Project Abstract The objective of this study was to test the hypothesis that there is no difference in the number of recoverable viable Legionella pneumophilia between those encased within food vesicles after ingestion and release by Tetrahymena and those living free in the water of cooling towers. Two stock solutions of L. pneumophilia were prepared, using water obtained from a cooling tower at Tennessee Tech University. The two groups consisted of one with the L. pneumophilia within vesicles and one with the L. pneumophilia not in vesicles. Both groups of L. pneumophilia were dried out in a desiccator. Viable L. pneumophilia from both groups were successfully recovered at 24 hours, 72 hours, and 22 days, with a percent recovery of 2% for vesicle-enclosed L. pneumophilia and 1.4% for free-living L. pneumophilia at 22 days. Although the percent recovery does not vary significantly, this experiment showed the virulence of L. pneumophilia when subjected to desiccation. It also proved that the food vacuoles secreted by Tetrahymena may serve to protect L. pneumophilia from desiccation. The results may also explain in part how Legionnaire's Disease is spread from the result of interactions between L. pneumophilia and Tetrahymena in cooling towers. Finally, the results may suggest that L.pneumophilia can travel long distances within these vacuoles as aerosol particles, surviving extreme desiccation, and eventually infecting a new host miles away.

Key Words: Legionella pneumophilia, Tetrahymena, vesicles, vacuoles, desiccation

Introduction Legionnaire’s Disease Robotham studied Legionella-amoebae interactions in cooling towers (Neumeister et al 2000) Berk et al (1998) currently researching on vesicles secreted by amoebae

Introduction Kwaik (1996) and McNeely et al (2000) sparked new interest in the ciliate Tetrahymena secreting vesicles containing Legionella Barker (1999) Demonstrated that Legionella within vesicles exhibit resistance to biocides and high temperatures What about resistance to desiccation?

Introduction The objective of this study is to test the hypothesis that there is no difference in the number of recoverable viable Legionella pneumophilia between those enclosed within food vacuoles released from the ciliate Tetrahymena and those living free in water of cooling towers

Methods and Materials Create graph of number of Legionella vs. optical density Dilutions in microwells Optical density reader Acridine orange and fluorescence microscopy

Methods and Materials Figure 1. Number of Legionella bacteria present for a given optical density

Methods and Materials Feed Legionella to Tetrahymena Allow Tetrahymena to secrete vesicles Use Gunderson’s method to separate vesicles from the Tetrahymena Divide into two sets Dry out both sets using desiccator Attempt to resuscitate over three time periods: 24 hours, 72 hours, 22 days

Table 1. Observed recovery of Legionella at measured times

Results Surprisingly, recovery of both vesicle- bound and free-living at 24 hours, 72 hours, and 22 days 2% recovery for vesicle-bound L. pneumophilia 1.4% recovery for free-living L. pneumophilia

Conclusions No significant difference in percent recovery between the two groups Viable Legionella were recovered in both groups Shows importance of Legionella- Tetrahymena interactions

Conclusions Legionella pneumophilia within food vacuoles are resistant to desiccation, agreeing with current theories of increased virulence (Barker 1999) Recovery of free-living L. pneumophilia is surprising, contradicting current theories (Kwaik 1996) Repeat experiment, use E. coli as a control, see how long L. pneumophilia may remain recoverable after desiccation

Summary This may explain many strange case histories of Legionnaire’s Disease (Berk et al 1998) Workers in factories with cooling towers are at risk for acquiring Legionnaire’s Disease Need to develop new methods of prevention for Legionnaire’s Disease. Current methods are minimally effective (Cirillo 1999)

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