The resistance of Acinetobacter baumannii strains to carbapenems is a worrying problem in hospital settings. The main mechanism of carbapenem resistance is the expression of ␤-lactamases (metalloenzymes or class D enzymes). The mechanisms of the dissemination of these genes among A. baumannii strains are not fully understood. In this study we used two carbapenem-resistant clinical strains of A. baumannii (AbH12O-A2 and AbH12O-CU3) expressing the plasmid-borne bla OXA-24 gene (plasmids pMMA2 and pMMCU3, respectively) to demonstrate that A. baumannii releases outer membrane vesicles (OMVs) during in vitro growth. The use of hybridization studies enabled us to show that these OMVs harbored the bla OXA-24 gene. The incubation of these OMVs with the carbapenem-susceptible A. baumannii ATCC 17978 host strain yielded full resistance to carbapenems. The presence of the original plasmids harboring the bla OXA-24 gene was detected in strain ATCC 17978 after the transformation of OMVs. New OMVs harboring bla OXA-24 were released by A. baumannii ATCC 17978 after it was transformed with the original OMV-mediated plasmids, indicating the universality of the process. We present the first experimental evidence that clinical isolates of A. baumannii may release OMVs as a mechanism of horizontal gene transfer whereby carbapenem resistance genes are delivered to surrounding A. baumannii bacterial isolates.

When cultured in broth to the transmissive phase, Legionella pneumophila infects macrophages by inhibiting phagosome maturation, whereas replicative-phase cells are transported to the lysosomes. Here we report that the ability of L. pneumophila to inhibit phagosome-lysosome fusion correlated with developmentally regulated modifications of the pathogen's surface, as judged by its lipopolysaccharide profile and by its binding to a sialic acid-specific lectin and to the hydrocarbon hexadecane. Likewise, the composition of membrane vesicles shed by L. pneumophila was developmentally regulated, based on binding to the lectin and to the lipopolysaccharidespecific monoclonal antibody 3/1. Membrane vesicles were sufficient to inhibit phagosome-lysosome fusion by a mechanism independent of type IV secretion, since only ϳ25% of beads suspended with or coated by vesicles from transmissive phase wild type or dotA secretion mutants colocalized with lysosomal probes, whereas ϳ75% of beads were lysosomal when untreated or presented with vesicles from the L. pneumophila letA regulatory mutant or E. coli. As observed previously for L. pneumophila infection of mouse macrophages, vesicles inhibited phagosome-lysosome fusion only temporarily; by 10 h after treatment with vesicles, macrophages delivered ϳ72% of ingested beads to lysosomes. Accordingly, in the context of the epidemiology of the pneumonia Legionnaires' disease and virulence mechanisms of Leishmania and Mycobacteria, we discuss a model here in which L. pneumophila developmentally regulates its surface composition and releases vesicles into phagosomes that inhibit their fusion with lysosomes.To exploit macrophages as a replication niche, L. pneumophila, Coxiella burnetii, and Leishmania spp. apply a similar strategy (69). When ingested, each pathogen differentiates from a transmissive form that inhibits phagosome-lysosome fusion for several hours to a cell type fit for intracellular replication (20, 35, 37, 63, 67). Differentiation of L. pneumophila can be modeled in broth, where exponential (E)-phase bacteria switch to the transmissive phenotype as their nutrient supply wanes (11). As L. pneumophila enters the postexponential (PE) phase, the alarmone ppGpp triggers a regulatory cascade mediated in part by the two component system LetA/LetS and the sigma factors RpoS and FliA to coordinate expression of motility, cytotoxicity, stress resistance, factors that inhibit phagosome-lysosome fusion, and other traits likely to promote transmission to a new host cell (55, 56).To establish a replication niche in macrophages, L. pneumophila utilizes type IV secretion to deliver substrates that alter host membrane traffic (14, 75). Instead of merging with lysosomes, the L. pneumophila vacuole becomes surrounded by membranes derived from the early secretory pathway by a process that in A/J mouse macrophages resembles autophagy (3,19,34,42,70,72). The pathogen persists for several hours within an ER-like compartment, where it differentiates to the replicative form. Nevertheless, muta...

Acinetobacter baumannii is an opportunistic pathogen that has been associated with severe infections and outbreaks in hospitals. At present, very little is known about the biology of this bacterium, particularly as regards mechanisms of adaptation, persistence and virulence. To investigate the growth phase-dependent regulation of proteins in this microorganism, we analyzed the proteomic pattern of A. baumannii ATCC 17978 at different stages of in vitro growth. In this study, proteomics analyses were conducted using 2-DE and MALDI-TOF/TOF complemented by iTRAQ LC-MS/MS. Here we have identified 107 differentially expressed proteins. We highlight the induction of proteins associated with signaling, putative virulence factors and response to stress (including oxidative stress). We also present evidence that ROS (O(2)(-) and OH(-)) and RNI (ONOO(-)) accumulate during late stages of growth. Further assays demonstrated that stationary cells survive at high concentrations of H(2)O(2) (30 mM), the O(2)(-) donor menadione (500 muM) or the NO donor sodium nitroprusside (1 mM), and showed a higher survival rate against several bactericidal antibiotics. The growth phase-dependent changes observed in the A. baumannii proteome are discussed within a context of adaptive biological responses, including those related to ROS and RNI stress.