New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.

Acinetobacter species are defined on the basis of several phenotypic characters, results of DNA-DNA homology, and more recently, similarities or dissimilarities in 16S rRNA gene sequences. However, the 16S rRNA gene is not polymorphic enough to clearly distinguish all Acinetobacter species. We used an RNA polymerase ␤-subunit gene (rpoB)-based identification scheme for the delineation of species within the genus Acinetobacter, and towards that end, we determined the complete rpoB gene and flanking spacer (rplL-rpoB and rpoB-rpoC) sequences of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. By using complete gene sequences (4,089 bp), we clearly separated all species and grouped them into different clusters. A phylogenetic tree constructed using these sequences was supported by bootstrap values higher than those obtained with 16S rRNA or the gyrB or recA gene. Four pairs of primers enabled us to amplify and sequence two highly polymorphic partial sequences (350 and 450 bp) of the rpoB gene. These and flanking spacers were designed and tested for rapid identification of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. Each of these four variable sequences enabled us to delineate most species. Sequences of at least two polymorphic sequences should be used to distinguish Acinetobacter grimontii, Acinetobacter junii, Acinetobacter baylyi, and genomic species 9 from one another. Finally, 21 clinical isolates of Acinetobacter baumannii were tested for intraspecies relationships and assigned correctly to the same species by comparing the partial sequences of the rpoB gene and its flanking spacers.Acinetobacter species are ubiquitous in the environment and have emerged as important nosocomial pathogens (3). A propensity to tolerate drying (29, 30) and resistance to many commonly used antibiotics (28) are key factors in enabling the organism to survive and spread in the nosocomial environment. Furthermore, Acinetobacter species may serve as reservoirs of antibiotic-resistant genes, particularly in hospital environments (22).The genus Acinetobacter, originally proposed by Brisou and Prevot (6), comprises a collection of bacteria which show a great deal of phenotypic similarities. Currently, the genus Acinetobacter comprises many taxons based on hybridization groups and classified as genomovars and genomospecies. From an official taxonomic point of view, there are currently 17 Acinetobacter nomenspecies with standing in nomenclature. However, several other genomospecies have been proposed, mainly on the basis of DNA hybridization studies (7,20,21,28).Delineation of species within the genus Acinetobacter is often problematic (22). A number of recent studies have attempted to set up reliable identification schemes based on DNA homology, phenotypic characters, and comparison of cell envelope and outer membrane protein patterns, but such methods yield inconsistent results (1,10,11,12,23). The phenotypic characters of Acinetobacter species are susceptible to the condit...

Bacteria belonging to the genus Acinetobacter are ubiquitous in soil and water. Only a few species, including Acinetobacter baumannii, and the unnamed Acinetobacter genomic species (gen. sp.) 3 and 13TU, which together with the soil organism Acinetobacter calcoaceticus are combined in the A. calcoaceticus-A. baumannii (Acb) complex, have been recognized as important nosocomial infectious agents. The ecology, epidemiology and pathology of most species are not yet well established. Lack of practical and accurate methods limits routine identification of clinical isolates and thus hampers precise identification of those of the Acb complex and other Acinetobacter species of possible clinical significance. We previously identified a 350 bp highly variable zone on the rpoB gene which appeared to be a promising target for rapid molecular identification. In the present study, we validated this method for accuracy on a collection of reference strains belonging to A. calcoaceticus (5 strains), Acinetobacter gen. sp. 3 (29 strains), A. gen. sp. 13TU (18 strains), A. baumannii (30 strains) and one strain each of A. radioresistens, A. gen. sp. 15TU, A. gen. sp. 10, A. gen. sp. 11, A. gen. sp. 'between 1 and 3' and A. gen. sp. 14TU513BJ. This represents the largest analysis to date that compares a large number of well-identified strains of the Acb complex to assess the intra-and interspecies variation within this complex. All were correctly identified with 98.9-100 % intraspecies relatedness based on partial rpoB sequence analysis. We then applied this tool to identify 99 Acinetobacter clinical isolates from four public hospitals in Marseille, France. All isolates could easily be identified to species as they were separated into 13 species sequence types with a sequence variance of 0-2.6 % from their respective type strains. Of these 99 isolates, 10 were A. haemolyticus, 52 were A. baumannii, 27 were A. gen. sp. 3, 5 were A. schindleri, 1 was A. lwoffii, and 1 was A. gen. sp. 13TU. Three were provisionally identified as A. gen. sp. 9. This is the first work to identify all specimens of a set of clinical Acinetobacter isolates at species level using rpoB sequence analysis. Our data emphasize the recognition of A. schindleri as an emerging cause of Acinetobacter-related infection and confirm that A. gen. sp. 3 is the second most commonly isolated Acinetobacter species after A. baumannii in patients.

Background:The anti-TB prodrugs isoxyl (ISO) and thiacetazone (TAC) inhibit mycolic acid biosynthesis. Results: We show that ISO and TAC both target the dehydration step of the FAS-II elongation system. Conclusion: ISO and TAC share the same mode of action. Significance: ISO and TAC are the first antibiotics reported to target the FAS-II dehydratase(s) of Mycobacterium tuberculosis.