Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans, Free living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium, In low-nutrient environments, however, L. pneumophila is able to enter a nonreplicative viable but nonculturable (VBNC) state, In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes, After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state, A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells, However, reactivation of dormant legionellae was not observed in the animal model.