Discussion Before the advent of single-cell based analytical methods, researchers worked mostly with pure cultures assuming that the behavior of each single cell in a population is consistent with the average behavior of all cells. However, it has been demonstrated that cell behavior in a bacterial population is Alectinib ic50 divergent even under identical micro-environmental conditions. Complex phenomenon such as stochasticity in gene expression [41], asymmetrical aging [42], asymmetrical division [43], bi-stability

[44] and cell differentiation [7] can lead to the formation of sub-populations with different cellular physiologies and/or morphologies. Unfortunately, the link between the cellular physiology and culturability of each sub-population is not always clear, and as a consequence the characterization of VBNC cells of some organisms is complex. VBNC L. pneumophila cells have been observed by many groups [16, 18, 36, 37, 40] but the mechanisms leading to this physiological state remain unknown. It could be part of an adaptive response (A-VBNC cells), and/or a consequence of cellular deterioration (D-VBNC cells) and/or a consequence of cell death during the plating procedure (injured cells), all leading to the inability of the L. pneumophila cell affected to form a colony. In our study,

we assessed the viability of L. pneumophila at the single-cell level using the ERK inhibitor CV6 procedure. By using high efficiency cell-counting procedures (n > =3000), VBNC cells were detected after, but also in the absence of, biocide treatment. Interestingly, two subpopulations of cells with different levels of metabolic activity

were identified among VBNC cells. These two populations displayed different resistance to the biocide treatment, suggesting that they have different physiological characteristics. We also found that pyruvate and/or glutamate were able to restore the culturability to a large proportion of the non-culturable cells observed both after, but also in the absence of, biocide treatment. Importantly, we demonstrate that the restored population was able to invade amoeba and then replicate, and that this was responsible for the “resuscitation” enough phenomenon. These observations strongly suggest that a suspension of L. pneumophila cells harvested at the beginning of stationary phase is composed of different sub-populations, with different physiological characteristics, susceptibility to stress, culturability and ability to be restored by pyruvate and/or glutamate. It remains unclear exactly how pyruvate and glutamate promote restoration. Pyruvate is an antioxidant that neutralizes or prevents the formation of ROS in rich medium [26, 27, 29, 32, 34]. When pyruvate is converted to alanine, glutamate is concomitantly converted to α-ketoglutarate [45], a substrate already present in the medium used for L.