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 b y legionellae and provide the intracellular environment required for t he replication of this bacterium, In low-nutrient environments, howeve r, L. pneumophila is able to enter a nonreplicative viable but noncult urable (VBNC) state, In this study, L. pneumophila Philadelphia I JR 3 2 was suspended in sterilized tap water at 10(4) cells/ml. The decreas ing number of bacteria was monitored by CFU measurements, acridine ora nge direct count (AODC), and hybridization with 16S rRNA-targeted olig onucleotide probes, After 125 days of incubation in water, the cells w ere 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 resu scitation of L. pneumophila JR 32 to a culturable state, A comparison of plate-grown legionellae and reactivated cells showed that the capac ity for intracellular survival in human monocytes and intraperitoneall y infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells, However, reactivation of do rmant legionellae was not observed in the animal model.