†These authors contributed equally to this work undamental to the pathogenic lifestyle of several intracellular bacteria is their ability to modulate trafficking of the phagosomes in which they reside 1 . The bacterial pathogen Legionella pneumophila resides in a phagosome that restricts its fusion with host endosomes and lysosomes 2 . The intracellular fate of L. pneumophila is determined by a type-IV-related transport system encoded by 24 dot and icm virulence genes 3,4 . L. pneumophila dot and icm mutants reside in phagosomes that rapidly fuse with lysosomes, resulting in decreased intracellular survival and a severe intracellular growth defect 5,6 . Here we show that the primary function of the dot and icm products is to transmit a functionally cis-acting signal to the host macrophage that drives maturation of the L. pneumophila phagosome into a replicative vacuole. We find that L. pneumophila dot and icm mutants are able to grow intracellularly when the bacteria are residing in a phagosome established by L. pneumophila that retains its ability to alter phagosome trafficking. These results show that L. pneumophila tailors the phagosome into a specialized organelle permissive for bacterial growth through the specific actions of the dot/icm genes.We first determined whether macrophages infected with wildtype L. pneumophila are defective in delivering phagosomes containing killed yeast particles to lysosomes. We infected mouse bone-marrow-derived macrophages with wild-type L. pneumophila, and then fed the macrophages a suspension of heat-killed Saccharomyces cerevisiae. S. cerevisiae phagosomes rapidly acquired the lysosomal membrane protein LAMP-1 in macrophages preinfected with L. pneumophila (Fig. 1a). Phagosomes containing S. cerevisiae were uniformly LAMP-1-positive in macrophages that had been infected for 1 h or for as long as 8 h with virulent L. pneumophila (see Supplementary Information). The finding that trafficking of S. cerevisiae phagosomes was unaffected in macrophages containing virulent L. pneumophila indicates that the Dot/Icm transport apparatus does not generate a signal that accumulates and affects the trafficking of newly formed phagocytic compartments in the host cell.To further address whether there is a signal transmitted by virulent L. pneumophila that has the capacity to act in trans, we used a mixed inoculum consisting of a wild-type and a dotA mutant strain of L. pneumophila to infect bone-marrow-derived macrophages. If the wild-type bacteria were secreting a trans-acting factor, it could potentially restore intracellular growth to a dotA mutant that has lost the ability to alter phagosome trafficking. In these studies, we used a dotA mutant carrying a plasmid that produces green fluorescent protein (GFP). In macrophages that had ingested both wildtype and mutant bacteria, we did not detect replication of dotA mutants in phagosomes that were isolated from those containing wild-type L. pneumophila (Fig. 1b).Upon close inspection of all macrophages that had been infected with both wild...

cViridans group streptococci (VGS) are part of the normal flora that may cause bacteremia, often leading to endocarditis. We evaluated daptomycin against four clinical strains of VGS (MICs ‫؍‬ 1 or 2 g/ml) using an in vitro-simulated endocardial vegetation model, a simulated bacteremia model, and kill curves. Daptomycin exposure was simulated at 6 mg/kg of body weight and 8 mg/kg every 24 h for endocardial and bacteremia models. Total drug concentrations were used for analyses containing protein (albumin and pooled human serum), and free (unbound) drug concentrations (93% protein bound) were used for analyses not containing protein. Daptomycin MICs in the presence of protein were significantly higher than those in the absence of protein. Despite MICs below or at the susceptible breakpoint, all daptomycin regimens demonstrated limited kill in both pharmacodynamic models. A reduction of approximately 1 to 2 log 10 CFU was seen for all isolates and dosages except daptomycin at 6 mg/kg, which achieved a reduction of 2.7 log 10 CFU/g against one strain (Streptococcus gordonii 1649) in the endocardial model. Activity was similar in both pharmacodynamic models in the presence or absence of protein. Similar activity was noted in the kill curves over all multiples of the MIC. Regrowth by 24 h was seen even at 8؋ MIC. Postexposure daptomycin MICs for both pharmacodynamic models increased to >256 g/ml for all isolates by 24 and 72 h. Despite susceptibility to daptomycin by standard MIC methods, these VGS developed high-level daptomycin resistance (HLDR) after a short duration following drug exposure not attributed to modification or inactivation of daptomycin. Further evaluation is warranted to determine the mechanism of resistance and clinical implications.