[Establishment of a method for determining the association between Legionella sp. and Amoeba sp. using polymerase...

With two pairs of primers for the amplification of the MIP- (macrophage infectivity potentiator) and the 5S rDNA-fragment, it was possible to establish a DNA extraction and a PCR method for the detection of Legionella sp. in water-samples and, after cultivation, in Amoeba sp. Therefore, water-samples from a warm water-system in a hospital were taken. In all samples, legionellae were detected by the PCR method and identified by cultivation and a direct immunofluorescence-method as L. pneumophila (serogroup 1). Legionellae and amoebae of the same water sample were cocultured. Legionellae were also adherent at the outer-membrane. To separate the amoebae from the legionellae, the amoebae were sedimented selectively by centrifugation at 200 x g. This washing procedure had to be repeated seven times in order to eliminate the extraamoebale legionellae for sure. After DNA-extraction of water samples and heat treatment of the intraamoebale legionellae respectively, the amplification was performed with the MIP- and 5S rDNA-primers. In 14 of 16 cocultivations growth of legionellae was found. This result and the detection of legionellae and amoebae in the same water samples suggest that an infection of amoebae may also take place in the water system of the hospital. This is important for the disinfection as a procedure to eliminate legionellae, since intraamoebale bacteria are more resistant to environmental manipulation. Because in two of the cocultivations no growth of legionellae in amoebae was found, it can be assumed that only specific subtypes of legionellae can infect amoebae species.