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Applied and Environmental Microbiology, January 2003, p. 600-606, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.600-606.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Survival and Growth of Francisella tularensis in Acanthamoeba castellanii

Hadi Abd,1,2 Thorsten Johansson,1 Igor Golovliov,3 Gunnar Sandström,2 and Mats Forsman1*

Swedish Defence Research Agency, SE 901 82 Umeå,1 Clinical Bacteriology, Department of Clinical Microbiology, Umeå University, SE 901 85 Umeå,3 Division of Clinical Bacteriology, Department of Microbiology, Pathology and Immunology, Karolinska Institute, Huddinge University Hospital, SE 141 86 Stockholm, Sweden2

Received 10 June 2002/ Accepted 15 October 2002

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.

* Corresponding author. Mailing address: Swedish Defense Research Agency, SE 901 82, Umeå, Sweden. Phone: 46 90 106669. Fax: 46 90 106800. E-mail: mats.forsman{at}foi.se. {dagger} Present address: Division of Clinical Bacteriology, Department of Microbiology, Pathology and Immunology, Huddinge University Hospital, SE 141 86 Stockholm, Sweden.

Applied and Environmental Microbiology, January 2003, p. 600-606, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.600-606.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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