Recovery of Viable Legionella pneumophilia from Desiccated Food Vacuoles of the Ciliate

Tetrahymena

Jonathan ThomasDepartment of Biology

Tennessee Technological UniversityCookeville, 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 MaterialsNumber of Bacteria vs. Optical Density

0

12

3

4

56

7

8

0 0.05 0.1 0.15 0.2 0.25 0.3

Optical Density

Nu

mb

er

of

Bacte

ria (

x10^

8)

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

Legionella within vesicles Legionella not within vesiclesTime ZeroDay 1Day 2Day 3Day 4Day 5Day 6Day 7Day 8 Day 9Day 10Day 11Day 12Day 13Day 14 Day 15Day 16Day 17Day 18Day 19Day 20Day 21Day 22Day 23Day 24Day 25Day 26Day 27Day 28

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)

Literature Cited• Barker, J., T. Humphrey, and M. Brown. 1999. Survival of Escherichia coli 0157 in a Soil Protozoan: Implications

for Disease. FEMS Microbiology Letters 173:291-295.

• Berk, S., R., Ting, G. Turner, and R Ashburn. 1998. Production of Respirable Vesicles Containing Live Legionella pneumophilia Cells by Two Acanthamoeba spp. Applied and Environmental Biology 64:279-286.

• Cirillo, J.  1999.  Exploring a Novel Perspective on Pathenogenic Relationships.  Trends in Microbiology 7:96-97.  • Kwaik, Y., L. Gao, B. Stone, C. Venkataraman, and O. Harb. 1998. Invasion of Protozoa by Legionella

pneumophilia and its Role in Bacterial Ecology and Pathenogenesis. Applied and Environmental Biology 64:3127-3133.  

• McNealy, T., A. L. Newsome, R. A. Johnson, and S. Berk. 2000. Impact of Amoebae, Bacteria, and Tetrahymena on Legionella pneumophilia Multiplication and Distribution in an Aquatic Environment. In: Marre, R. et al editors. Legionella. Washington D.C.:ASM Press; 2002. p 170-175.  

• Michel, R.  Legionella-Like Slender Rods Multiplying Within a Strain of Acanthamoeba sp. Isolated from Drinking Water.  1997.  Parasitol Res 60:84-88.

• Michel, R., B. Hauroder-Philippczyk.  Acanthamoeba from Human Nasal Mucosa Infected with an Obligate Intracellular Parasite.  1994.  European Journal of Protistology 30:104-110.  

• Neumeister, B., G. Reiff, M. Faigle, K. Dietz, H. Northoff, and F. Lang. 2000. Influence of Acanthamoeba castellanii on Intracellular Growth of Different Legionella Species in Human Monocytes. Applied and Environmental Microbiology 66:914-919.