Viable Legionella pneumophila Not Detectable by Culture on Agar Media

Abstract

Detection and monitoring of genetically engineered microorganisms released to the environment, as well as pathogens, are primary factors in risk assessment. Culture methods have been proposed for both detection and monitoring. However, microorganisms in natural systems may not always be culturable. We surveyed environmental samples collected from sources implicated in an epidemic of Legionnaires' disease and, although no cultures were recovered from environmental samples, numerous cells were observed by fluorescent microscopy when anti–L. pneumophila group 1 antibody was used. Similar observations have often been made by others. To study this loss of culturability, L. pneumophila strains were maintained in a microcosm (vessels containing sterilized environmental water) and assayed at intervals for growth on appropriate media, and lethality for chick embryos. At 4°C, the decimal rate of decline of colony forming cells was approximately 29 days; at 37°C it was 13 days. When microcosm water samples were injected into embryonated eggs, far greater chick embryo mortality was observed than could be accounted for by the number of culturable cells in the injections. Thus, previously non–culturable Legionella had multiplied once again and become culturable. These results indicate that samples that do not contain culturable cells, may contain cells that are viable, as demonstrated by their pathogenicity for chick embryos. The fluorescent antibody assay may provide a valuable indication of the presence of such viable but non–culturable cells.