culbertsoni or A. castellanii supernatants obtained in PAS may likewise lead to increased bacterial counts (Fig. 3). The results were similar with supernatants obtained from filtered tap water (data not shown). This could be due, particularly for A. culbertsoni, to the death and the lysis of amoeba, especially when they were co-cultivated with A. baumanii, which could provide nutrients for the bacteria to grow. Among microorganisms related to amoebae, some bacteria

that may be human pathogens have evolved in a way that allows them to resist destruction by protozoa either because they are not internalized or else because they are able to survive, grow and exit amoebae following internalization (Greub & Raoult, 2004). We have evaluated the growth and survival of the bacterium in a poor medium such as encystment medium with and without amoebae. The bacterial count showed that the presence of amoebae (A. castellanii or A. culbertsoni) BI 6727 manufacturer allows for increased bacterial PD98059 mouse growth, while A. baumanii in the same medium without amoebae is able to survive, but at lower concentrations. After 60 days in this medium, the survival of the bacteria is favored by the presence of amoebae (Fig. 4) In electron microscopy after 11 days of incubation, some cysts already contained intracellular

A. baumanii, located only in the space between the double walls (Fig. 2), which is similar to the location of Pseudomonas in Acanthamoeba astronyxis (Marciano-Cabral & Cabral, 2003), Mycobacterium avium in A. polyphaga (Steinert et al., 1998), Mycobacterium sp. (Sharbati-Tehrani et al., 2005; Ben Salah & Drancourt, 2010), V. cholerae Docetaxel order (Abd et al., 2005) or Vibrio mimicus (Abd et al.,

2010) in A. castellanii. The significance of both this location within the cyst structures, but outside of the cytoplasm and the fact that the bacteria seem clustered together remain to be determined. The survival of other bacteria such as F. tularensis or Shigella sp. in A. castellanii cysts has also been reported, but the bacteria are intracellular and not associated with the outer surface (Abd et al., 2003; Saeed et al., 2009). According to Ben Salah & Drancourt (2010), the cellulase encoded by some bacteria may play a role in their exocyst location. Moreover, this location could allow the bacterium to more rapidly escape from the cyst. In this study, we have shown that the presence of A. castellanii or A. culbertsoni may allow increase of A. baumanii growth, whatever the co-culture medium, PAS or filtered water. The presence of A. baumanii did not influence the viability of A. castellanii, but did dramatically decrease the viability of A. culbertsoni. When the cells were incubated in a hostile medium such as the encystment medium, the presence of the amoebae (A. castellanii or A. culbertsoni) increased the viable counts of bacteria, even after 60 days of incubation.