Characterization and resuscitation of ‘non-culturable’ cells of Legionella pneumophila

BACKGROUND: Legionella pneumophila is a waterborne pathogen responsible for Legionnaires’ disease, an infection which can lead to potentially fatal pneumonia. After disinfection, L. pneumophila has been detected, like many other bacteria, in a “viable but non culturable” state (VBNC). The physiological significance of the VBNC state is unclear and controversial: it could be an adaptive response favoring long-term survival; or the consequence of cellular deterioration which, despite maintenance of certain features of viable cells, leads to death; or an injured state leading to an artificial loss of culturability during the plating procedure. VBNC cells have been found to be resuscitated by contact with amoebae. RESULTS: We used quantitative microscopic analysis, to investigate this “resuscitation” phenomenon in L. pneumophila in a model involving amending solid plating media with ROS scavengers (pyruvate or glutamate), and co-culture with amoebae. Our results suggest that the restoration observed in the presence of pyruvate and glutamate may be mostly due to the capacity of these molecules to help the injured cells to recover after a stress. We report evidence that this extracellular signal leads to a transition from a not-culturable form to a culturable form of L. pneumophila, providing a technique for recovering virulent and previously uncultivated forms of L. pneumophila. CONCLUSION: These new media could be used to reduce the risk of underestimation of counts of virulent of L. pneumophila cells in environmental samples.