engleski

The growth of different Legionella species on BCYE agar plates in the presence of Acanthamoeba castellanii

Legionella species are recognized as a common cause of community-acquired and nosocomial bacterial pneumonia. There are 52 species and 70 distinct serogroups in the genus Legionella, of which 25 species have been associated with human disease. L. pneumophila is the most common cause of Legionnaries' disease in the world. Most Legionella species are found in aquatic environments as they have the ability to reside and multiply in aquatic free-living amoeba. In this study we used a novel assay, the amoeba plate test to analyse the growth of different Legionella species on agar plates in the presence of Acanthamoeba castellanii. The influence of temperature and the number of amoeba on the plates were also analysed. In our study we used different Legionella species: L. pneumophila (clinical isolate, sg 1), L. micdadei, (clinical isolate), L. longbeachae serogrupe 1 (clinical isolate), L. longbeachae serogrupe 2 (clinical isolate), L. steigerwaltii (environmental isolate), L. gormanii (environmental isolate). The amoebae were spread on BCYE agar plates prior to spotting bacteria in serial dilution on the plates. Different number of amoeba: 2, 0 x 10000, 2, 0 x 100000 and 2, 0 x 1000000 , were applied on agar plates and left at room temperature overnight. The bacterial cultures were adjusted to an identical OD600 (~1000000000 cfu) and 3 µl of each tenfold dilution were spotted onto the agar plates and incubated at 25oC and 37oC. L. pneumophila formed robust colonies in the presence of A. castellanii even at a 100000 dilution, despite the number of amoeba. L. micdadei showed a growth defect at 25oC but formed small colonies at 37oC. Growth of L. micdadei was also affected by the number of amoeba on the plates. L. longbeachae sg 1 and L. gomanii formed robust colonies in the presence of amoeba at a 10000 dilution and both bacteria grew equally well at 25°C and 37°C. The growth of L. longbeachae sg 2 and L. steigerwaltii was strongly affected by the presence of amoeba on the plates and both species formed very small colonies only at the highest concentration (10 dilution). The amoeba plate test showed to be a useful assay for screening of bacteria that have the ability to multiply in A. castellanii. The presence of amoeba on the plates did not affect the growth of L. pneumophila and L. longbeachae sg 1, while the growth of L. micdadei and L. gormanii was only partially affected. The growth of L. longbeachae sg 2 and L. steigerwaltii was strongly affected by the presence of amoeba.

Legionella; Acantamoeba; amoeba plate test

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