Under similar treatment Screening Library manufacturer conditions, Bouyer et al. (2007) have also observed an enhanced gentamicin resistance after passage into amoebae. The latter authors suggested a possible role of the vesicle membrane in the protection of Legionella, but also considered a partial intrinsic resistance. This resistance was intrinsic to the differentiated MIFs and was not due to physical barriers imposed by the pellet configuration, as we released the MIFs from the pellets and tested them as free bacteria. This resistance was also conserved in MIFs released from pellets aged for 90 days in Osterhout’s buffer. Garduno et al.

(2002) previously observed that MIFs recovered from HeLa cells were also resistant to gentamicin. Taken together, these observations

suggest that MIFs produced in amoeba or in ciliates share a common phenotype regarding gentamicin resistance. Survival of Legionella in the freshwater environment must include an ability to resist starvation Navitoclax for long periods. Thus, we studied the long-term survival in a low-nutrient environment of Legionella pellets and SPFs. For the two types of suspensions, we observed a rapid decrease of culturability in the encystment buffer up to 11 days (Fig. 3). After that, evident differences appeared. Culturability of SPFs legionella continue to decrease strongly until 90 days, when no more culturable bacteria were detected, as previously reported by Bouyer et al. (2007). In contrast, Tetrahymena-derived pellets of MIFs still contained culturable Legionella after 4 months (Fig. 3). It is Liothyronine Sodium therefore clear that pellets protect Legionella from starvation. However, whether the pellet structure itself contributes to starvation resistance is not yet known, as the intrinsic starvation resistance of MIFs that had been released from pellets was not measured separately. We observed by optical microscopy that large aggregates after an aging period of 90 days are still present (data not shown), suggesting that these structures could persist in the environment. MIF obtained from HeLa cells have previously been reported to be highly infectious

in macrophages or HeLa cells (Garduno et al., 2002). We observed here that MIFs derived from Tetrahymena are also infectious in pneumocytes (Fig. 4). Furthermore, our results showed that these MIFs retained their infectivity after an aging period of 90 days, being capable of exhibiting a higher capacity to multiply into pneumocytes, in relation to SPFs freshly grown in vitro. Our results demonstrate that Tetrahymena, as previously reported for amoeba, could participate in determining the environmental fitness and infectivity of Legionella, and thus play a critical role in the dissemination of these bacteria. To our knowledge, this work is the first report concerning the behaviour of Legionella expelled from Tetrahymena, a field of research that should be more studied in more detail.