Single-step selection of drug resistant Acinetobacter baylyi ADP1 mutants reveals a functional redundancy in the recruitment of multidrug efflux systems

Members of the genus Acinetobacter have been the focus recent attention due to both their clinical significance and application to molecular biology. The soil commensal bacterium Acinetobacter baylyi ADP1 has been proposed as a model system for molecular and genetic studies, whereas in a clinical environment, Acinetobacter spp. are of increasing importance due to their propensity to cause serious and intractable systemic infections. Clinically, a major factor in the success of Acinetobacter spp. as opportunistic pathogens can be attributed to their ability to rapidly evolve resistance to common antimicrobial compounds. Whole genome sequencing of clinical and environmental Acinetobacter spp. isolates has revealed the presence of numerous multidrug transporters within the core and accessory genomes, suggesting that efflux is an important host defense response in this genus. In this work, we used the drug-susceptible organism A. baylyi ADP1 as a model for studies into the evolution of efflux mediated resistance in genus Acinetobacter, due to the high level of conservation of efflux determinants across four diverse Acinetobacter strains, including clinical isolates. A single exposure of therapeutic concentrations of chloramphenicol to populations of A. baylyi ADP1 cells produced five individual colonies displaying multidrug resistance. The major facilitator superfamily pump craA was upregulated in one mutant strain, whereas the resistance nodulation division pump adeJ was upregulated in the remaining four. Within the adeJ upregulated population, two different levels of adeJ mRNA transcription were observed, suggesting at least three separate mutations were selected after single-step exposure to chloramphenicol. In the craA upregulated strain, a T to G substitution 12 nt upstream of the craA translation initiation codon was observed. Subsequent mRNA stability analyses using this strain revealed that the half-life of mutant craA mRNA was significantly greater than that of wild-type craA mRNA.This work was supported by National Health and Medical Research Council (NHMRC; http://www.nhmrc.gov.au/) project grant APP1021479 to PJL and by NHMRC project grant 535053 to ITP

Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii

Multidrug efflux pumps provide clinically significant levels of drug resistance in a number of Gram-negative hospital-acquired pathogens. These pathogens frequently carry dozens of genes encoding putative multidrug efflux pumps. However, it can be difficult to determine how many of these pumps actually mediate antimicrobial efflux, and it can be even more challenging to identify the regulatory proteins that control expression of these pumps. In this study, we developed an innovative high-throughput screening method, combining transposon insertion sequencing and cell sorting methods (TraDISort), to identify the genes encoding major multidrug efflux pumps, regulators, and other factors that may affect the permeation of antimicrobials, using the nosocomial pathogen Acinetobacter baumannii. A dense library of more than 100,000 unique transposon insertion mutants was treated with ethidium bromide, a common substrate of multidrug efflux pumps that is differentially fluorescent inside and outside the bacterial cytoplasm. Populations of cells displaying aberrant accumulations of ethidium were physically enriched using fluorescence-activated cell sorting, and the genomic locations of transposon insertions within these strains were determined using transposon-directed insertion sequencing. The relative abundance of mutants in the input pool compared to the selected mutant pools indicated that the AdeABC, AdeIJK, and AmvA efflux pumps are the major ethidium efflux systems in A. baumannii. Furthermore, the method identified a new transcriptional regulator that controls expression of amvA. In addition to the identification of efflux pumps and their regulators, TraDISort identified genes that are likely to control cell division, cell morphology, or aggregation in A. baumannii. IMPORTANCE Transposon-directed insertion sequencing (TraDIS) and related technologies have emerged as powerful methods to identify genes required for bacterial survival or competitive fitness under various selective conditions. We applied fluorescence-activated cell sorting (FACS) to physically enrich for phenotypes of interest within a mutant population prior to TraDIS. To our knowledge, this is the first time that a physical selection method has been applied in parallel with TraDIS rather than a fitness-induced selection. The results demonstrate the feasibility of this combined approach to generate significant results and highlight the major multidrug efflux pumps encoded in an important pathogen. This FACS-based approach, TraDISort, could have a range of future applications, including the characterization of efflux pump inhibitors, the identification of regulatory factors controlling gene or protein expression using fluorescent reporters, and the identification of genes involved in cell replication, morphology, and aggregation

State-building, war and violence : evidence from Latin America

In European history, war has played a major role in state‐building and the state monopoly on violence. But war is a very specific form of organized political violence, and it is decreasing on a global scale. Other patterns of armed violence now dominate, ones that seem to undermine state‐building, thus preventing the replication of European experiences. As a consequence, the main focus of the current state‐building debate is on fragility and a lack of violence control inside these states. Evidence from Latin American history shows that the specific patterns of the termination of both war and violence are more important than the specific patterns of their organization. Hence these patterns can be conceptualized as a critical juncture for state‐building. While military victories in war, the subordination of competing armed actors and the prosecution of perpetrators are conducive for state‐building, negotiated settlements, coexistence, and impunity produce instability due to competing patterns of authority, legitimacy, and social cohesion

Quantitative PCR for detection of mRNA and gDNA in environmental isolates

Quantitative PCR is used to gauge the abundance of specific nucleic acid species within purified samples. Due to its high sensitivity and minimal operation costs, this method is routinely applied in modern molecular bioscience laboratories. Nonetheless, all quantitative PCR experiments must include several carefully designed, yet simple, controls to ensure the reliability of the analyses. The aim of this chapter is to provide basic quantitative PCR methods, from primer design through data analysis, that are generally applicable to studies in microbiology. These methods allow the abundance of targeted RNA or DNA molecules to be determined in nucleic acid samples purified from a variety of biological sources.18 page(s

Biology of the staphylococcal conjugative multiresistance plasmid pSK41

Plasmid pSK41 is a large, low-copy-number, conjugative plasmid from Staphylococcus aureus that is representative of a family of staphylococcal plasmids that confer multiple resistances to a wide range of antimicrobial agents. The plasmid consists of a conserved plasmid backbone containing the genes for plasmid housekeeping functions, which is punctuated by copies of IS257 that flank a Tn4001-hybrid structure and cointegrated plasmids that harbour resistance genes. This review summarises the current understanding of the biology of pSK41, focussing on the systems responsible for its replication, maintenance and transmission, and their regulation

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp.

Summary: Within the last 15 years, members of the bacterial genus Acinetobacter have risen from relative obscurity to be among the most important sources of hospital-acquired infections. The driving force for this has been the remarkable ability of these organisms to acquire antibiotic resistance determinants, with some strains now showing resistance to every antibiotic in clinical use. There is an urgent need for new antibacterial compounds to combat the threat imposed by Acinetobacter spp. and other intractable bacterial pathogens. The essential processes of chromosomal DNA replication, transcription, and cell division are attractive targets for the rational design of antimicrobial drugs. The goal of this review is to examine the wealth of genome sequence and gene knockout data now available for Acinetobacter spp., highlighting those aspects of essential systems that are most suitable as drug targets. Acinetobacter spp. show several key differences from other pathogenic gammaproteobacteria, particularly in global stress response pathways. The involvement of these pathways in short- and long-term antibiotic survival suggests that Acinetobacter spp. cope with antibiotic-induced stress differently from other microorganisms

Roles of DHA2 family transporters in drug resistance and iron homeostasis in Acinetobacter spp

Background: Acinetobacterbaumannii is a major cause of nosocomial infections worldwide due to its fitness within clinical settings and recalcitrance to conventional therapies. The drug: H⁺ antiporter 2 (DHA2) family export systems encoded by A. baumannii were investigated for their roles in promoting the success ofthis organism as a human pathogen. Methods: Bioinformatic tools were used to identify the DHA2 family transporters encoded by Acinetobacter spp. and establish their phylogenetic relationships. The drug resistance phenotypes conferred by the transporters were tested using both heterologously expressed proteins in Escherichia coli and Acinetobacter deletion mutants. The transcriptional responses of DHA2 family transporter genes to their substrates were established by qRT-PCR. Results: Six highly conserved DHA2 family proteins were identified in A. baumannii. Drug resistance phenotypes were established for two DHA2 family transporters. The expression of a third DHA2 family protein is highly responsive to the availability of iron. The gene encoding this protein is located within a putative siderophore biosynthesis locus, suggesting a physiological role in iron uptake, possibly via the export of a siderophore. Conclusions: These results highlight functions for DHA2 family proteins in both drug resistance and the maintenance of stable cellular physiology, emphasizing their importance in A. baumannii infections.9 page(s

Bacterial strains, plasmids and primers.

a<p>Cm: chloramphenicol.</p>b<p>Kan^R: kanamycin resistant.</p

Segrosome structure revealed by a complex of ParR with centromere DNA

The stable inheritance of genetic material depends on accurate DNA partition. Plasmids serve as tractable model systems to study DNA segregation because they require only a DNA centromere, a centromere-binding protein and a force-generating ATPase. The centromeres of partition (par) systems typically consist of a tandem arrangement of direct repeats. The best-characterized par system contains a centromere-binding protein called ParR and an ATPase called ParM. In the first step of segregation, multiple ParR proteins interact with the centromere repeats to form a large nucleoprotein complex of unknown structure called the segrosome, which binds ParM filaments. pSK41 ParR binds a centromere consisting of multiple 20-base-pair (bp) tandem repeats to mediate both transcription autoregulation and segregation. Here we report the structure of the pSK41 segrosome revealed in the crystal structure of a ParR-DNA complex. In the crystals, the 20-mer tandem repeats stack pseudo-continuously to generate the full-length centromere with the ribbon-helix-helix (RHH) fold of ParR binding successive DNA repeats as dimer-of-dimers. Remarkably, the dimer-of-dimers assemble in a continuous protein super-helical array, wrapping the DNA about its positive convex surface to form a large segrosome with an open, solenoid-shaped structure, suggesting a mechanism for ParM capture and subsequent plasmid segregation