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

Abstract

BackgroundLegionella 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.ResultsWe 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.ConclusionThese new media could be used to reduce the risk of underestimation of counts of virulent of L. pneumophila cells in environmental samples.

Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria

“…Intracellular human pathogen L. pneumophila is a bacterial parasite of many species of freshwater protozoa. The VBNC cells of this pathogen were resuscitated in a supportive medium with reactive oxygen scavengers such as sodium pyruvate or glutamate or with cultures of Amoebae castellanii (45). Sodium pyruvate restores the biosynthesis of macromolecules such as DNA and proteins, thereby converting the VBNC cells to a culturable state (46).…”

Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR

“…When exposed to stressful conditions such as starvation and low temperatures, many species of bacteria can enter the VBNC state (Oliver, 2010;Pinto et al, 2015). It has been suggested that the VBNC state is an adaptive strategy for long-term survival of bacteria under unfavorable environmental conditions (Ducret et al, 2014). However, while the ability to enter the VBNC state may be advantageous for bacteria, it poses a risk to public health (Li et al, 2014).…”

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

“…VBNC bacteria are considered a public health and food safety risk as they are nondetectable by conventional food and water testing methods . Interestingly, VBNC bacteria can be resuscitated by various mechanisms, including the return to favorable conditions , addition of sodium pyruvate which restores biosynthesis of DNA and protein , or coculture of VBNC Legionella pneumophila with amoebae or VBNC Vibrio cholera with eukaryotic cell lines , resulting in a return to a culturable state. Thus, the ability to rapidly and easily identify VBNC pathogenic bacteria is of utmost clinical importance.…”