engleski

The culturability and virulence of Legionella cells during desiccation

Objectives: Legionellosis is a serious and sometimes fatal form of pneumonia caused by the Legionella species. Most Legionella species are found in aquatic environments where they have the ability to reside and multiply in aquatic free-living amoeba. Some of species, such as Legionella longbeachae, have the ability to grow in soil and potting composts. The bacteria are transmitted to humans by aerosols from natural and human-made aquatic environments or, in the case of L. longbeachae infection, through exposure to contaminated potting soil. The objective of this study was to assess the culturability and viability of Legionella expossed to desiccation. Also, the role of Acanthamoeba castellanii in possible resuscitation of viable but non-culturable (VBNC) Legionella after desiccation was tested. Methods: For desiccation study, bacteria were prepared in sterile water and 5 x 20µl of bacterial suspension (~10^8 cfu/ml) were transferred in 96 wells plates and dried for 1 hour under laminar flow hood. The plates with dried Legionella, as well as plates with bacterial suspensions (wet conditions) were stored at room temperature. Bacteria were rehydrated and cultivated at different time points on BCYE agar. Bacterial viability was assessed with the Bacterial Viability Kit LIVE/DEAD® BacLight™ dying before fluorescent microscopy. At the same time, Legionella cells exposed to desiccation were added to A. castellanii monolayers and incubated for 48 hours. Results: Both Legionella species could be cultivated on BCYE agar only 24 hours after desiccation. Using fluorescent microscope viable Legionella cells could be detected up to 15 days of desiccation so bacteria entered viable but non-culturable (VBNC) state. Our data show that although L. pneumophila become non-culturable after desiccation, co-culture with amoeba resuscitates the VBNC bacteria, with subsequent intracellular proliferation within A. castellanii. In conclusion, L. pneumophila and L. longbeachae exposed to desiccation loss their culturability but remain viable and are able to infect and proliferate in Acanthamoeba castellanii.

amoeba; bacteria; desiccation

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