Non-culturable Legionella pneumophila associated with Acanthamoeba castellanii: detection of the bacterium using DNA amplification and hybridization.

The intracellular localization of Legionella pneumophila serogroup 1 within Acanthamoeba castellanii rendered the bacteria non-culturable on supplemented BCYE agar. DNA amplification, using two 19-mer primers, and hybridization using a 25-mer oligonucleotide probe, permitted detection of Leg. pneumophila in approximately 81% (29/36) of samples where the bacteria could not be detected using culture. A combination of co-cultivation of samples with Leg. pneumophila-naive A. polyphaga or Hartmannella vermiformis, incubation in a defined liquid medium or use of catalase indicated that approximately 31% (9/29) of the samples contained Leg. pneumophila which were viable although not culturable.