1,721,108 research outputs found
Cloning and characterization of AdeMNO RND efflux pump of Acinetobacter baumannii
Get PDFAcinetobacter baumannii is an opportunistic pathogen which has been implicated in a variety of nosocomial infections among immunocompromised patients worldwide. Recently, Multi-drug resistant (MDR) isolates of A. baumannii have been isolated from military personnel returning from service in Iraq and Afghanistan. Antibiotic resistance of A. baumannii has limited the number of active antibacterial, making very difficult to treat these types of infections. This work investigated the role of Resistance-Nodulation-cell Division (RND) efflux pumps in the antibiotic resistance mechanism of A. baumannii. Expression of six different RND pumps was analyzed in clinical isolates of A. baumannii. A novel RND family pump, AdeMNO, was found to be present in a majority of isolates. The adeMNO operon was cloned, sequenced, and characterized using the single copy gene expression system in an efflux sensitized surrogate Pseudomonas aeruginosa strain. Antibiotics, trimethoprim, chloramphenicol, and clindamicin were identified as the substrates of this pump. In order to understand the mechanisms of regulation of adeMNO operon, a putative regulator belonging to the lysR-family was identified, cloned, and sequenced from the upstream region of the operon. Promoter regions of the adeMNO operon were also sequenced from various clinical isolates and sequence polymorphisms identified that could be implicated in the regulation of adeMNO expression.University of Ontario Institute of Technolog
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Characterization of two component systems of Acinetobacter baumannii.
Get PDFAcinetobacter baumannii is an important opportunistic pathogen of hospital acquired infection, particularly in intensive care units. The emergence and rapid spread of multidrug-resistant A. baumannii strains has become a major health threat worldwide which severely limits the treatment options for this pathogen. This work investigated global mechanisms of antibiotic resistance and virulence of the problematic pathogen A. baumannii, in particular those mediated by two component regulatory systems (TCSs), that typically consist of a membrane bound sensor kinase and a cognate response regulator. Bacterial TCSs play an important role in the regulation of adaptation to different environmental conditions. Five TCSs in A. baumannii have been characterized; however, there are a number of putative two component systems encoded in the genome of A. baumannii that await detailed characterization. Differential expressions of six different TCSs was observed in two clinical isolates of A. baumannii AB030 and AB031, and whole-genome sequencing of both clinical isolates was performed. Data obtained from the comparative whole-genome analysis revealed the presence of an insertion element in the orphan TCSs response regulator A1S_2006 in AB030, mutation in the promoter region and an 1189 DNA insertion element were present in AdeRS system in AB031. The whole-genome sequencing analysis of TCSs operons in AB030 and AB031 also identified sequence polymorphisms that could alter the activities of these TCSs in AB030 and AB031. Finally, we identified A1S_3229_30 an excellent candidate that may act as global regulator of antibiotic and virulence in A. baumannii. The sequence of A1S_3229_30 was highly conserved among the wild-type ATCC17978, AB030 and AB031, and showed 73% identity to the amgRS operon that encodes for the well characterized AmgRS system that confer resistance to aminoglycoside antibiotics and required for the virulence of the problematic pathogen P. aeruginosa.University of Ontario Institute of Technolog
Impact of environmental conditions on expression levels of resistance-nodulation-division (RND) efflux pumps and outer membrane porins in Acinetobacter baumannii
Get PDFAcinetobacter baumannii is a globally emerging Gram-negative pathogen accounting for a variety of nosocomial conditions, including pneumonia, meningitis, peritonitis, urinary tract infections and, in particular, wound infections in soldiers. One of the major reasons for A. baumannii success as a human pathogen is attributed to a tremendous potential of this organism for development of the multidrug resistant phenotype (MDR). This organism’s ability to regulate expression of its intrinsic antibiotic resistance determinants, such as Resistance-Nodulation-Cell Division (RND) efflux pumps and outer membrane proteins, is regarded to be amongst the most important strategies in the arsenal of this pathogen. Despite considerable efforts being dedicated in the attempt to understand the underlying mechanisms in the development of the MDR phenotype in A. baumannii, the role of environmental stress factors is often overlooked. Evidence from other Gram-negative pathogens suggests that adaptive responses of the bacterial cell to environmental stress often target the same cellular processes that are exploited by antibiotics, therefore indirectly promoting the emergence of the MDR phenotype. This study shows the impact of a variety of environmental factors, namely iron(III) availability, oxidative stress, salicylate exposure, steady-state osmotic stress, ethanol shock, exposure to different incubation temperatures and exposure to blue light on expression levels of three RND efflux pumps and three outer membrane porins of A. baumannii. In addition, we investigated the difference in expression levels of the selected two-component systems between a type strain of A. baumannii, ATCC 19606, and two clinical isolates with the MDR and biofilm-forming phenotypes. The quantitative-real time polymerase chain reaction analysis revealed that levels of iron(III) in growth medium, oxidative stress and ethanol shock were amongst the most potent factors capable of influencing expression of RND efflux pumps and outer membrane porins.University of Ontario Institute of Technolog
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Acinetobacter nosocomialis Causes as Severe Disease as Acinetobacter baumannii in Northeast Thailand: Underestimated Role of A. nosocomialis in Infection
Get PDFInfections by Acinetobacter species are recognized as a serious global threat due to causing severe disease and their high levels of antibiotic resistance. Acinetobacter baumannii is the most prevalent pathogen in the genus, but infection by Acinetobacter nosocomialis has been reported widely. Diagnosis of patients with A. baumannii infection is often misdiagnosed with other Acinetobacter species, especially A. nosocomialis. This study investigated whether there were significant differences in clinical outcomes between patients infected with A. baumannii versus A. nosocomialis in Northeast Thailand, and to characterize serological responses to infection with these pathogens. The results show that A. baumannii had higher levels of multidrug resistance. Despite this, clinical outcomes for infection with A. baumannii or A. nosocomialis were similar with mortalities of 33% and 36%, respectively. Both pathogens caused community-acquired infections (A. baumannii 35% and A. nosocomialis 29% of cases). Plasma from uninfected healthy controls contained IgG antibody that recognized both organisms, and infected patients did not show a significantly enhanced antibody response from the first week versus 2 weeks later. Finally, the patterns of antigen recognition for plasma IgG were similar for patients infected with A. baumannii or A. nosocomialis infection, and distinct to the pattern for patients infected with non-Acinetobacter. In conclusion, our data revealed that infection with A. nosocomialis was associated with a similarly high level of mortality as infection with A. baumannii, the high rate of community-acquired infection and antibodies in uninfected individuals suggesting that there is significant community exposure to both pathogens. IMPORTANCE Bacterial infections by Acinetobacter species are global threats due to their severity and high levels of antibiotic resistance. A. baumannii is the most common pathogen in the genus; however, infection by A. nosocomialis has also been widely reported but is thought to be less severe. In this study, we have prospectively investigated 48 reported cases of A. baumannii infection in Northeast Thailand, and characterized the serological responses to infection. We found that 14 (29%) of these infections were actually caused by A. nosocomialis. Furthermore, the incidence of antibiotic resistance among A. nosocomialis strains, APACHE II scores, and mortality for patients infected with A. nosocomialis were much higher than published data. Both A. baumannii and A. nosocomialis had unexpectedly mortality rates of over 30%, and both pathogens caused a high rate of community-acquired infections. Importantly, background antibodies in uninfected individuals suggest significant community exposure to both pathogens in the environment
Single copy gene expression system as a tool for the purification of membrane proteins from Pseudomonas aeruginosa
Get PDFPseudomonas aeruginosa is a Gram-negative opportunistic human pathogen known to cause a variety of infections that are difficult to treat due to extremely high resistance to almost all antibiotics currently in clinical use. One of the major contributors to this resistance is the active efflux of antibiotics from the cell, primarily by action of the Resistance Nodulation Division (RND) family of efflux pumps. These pumps are composed of three proteins; an inner membrane RND pump, a periplasmic membrane fusion adaptor protein, and an outer membrane protein. The mechanism by which the three proteins interact to form a functional complex is largely unknown and the methods currently available for their study involves expression systems geared for high levels of expression. In the case of membrane proteins which play a role in clinically relevant activities, such as multidrug resistance, an expression system which does not always reflect biologically relevant levels of protein in the cell is not ideal for studying their interactions as correlation of conclusions from interaction studies to true interactions may not be possible. In this study a single copy gene expression system was designed and demonstrated to better reflect clinically relevant levels of overexpression compared to a multi-copy expression system. Quantitative-real time PCR analysis of C-terminally hexa-histidine tagged outermembrane protein, OpmH, expression shows approximately 100-fold and 20-fold overexpression from multi-copy and single-copy expression systems respectively. OpmH-H6 was successfully purified from both multi copy and single copy expression systems with proportionate purification schemes indicating the feasibility of single copy expression systems for the study of membrane bound protein complexes.University of Ontario Institute of Technolog
Molecular characterization of the interactions of the Resistance-Nodulation-Division Pump MexJK of Pseudomonas aeruginosa with outer membrane proteins OprM and OpmH.
Get PDFPseudomonas aeruginosa is an important Gram-negative opportunistic pathogen, which has become a significant threat due to wide-spread antimicrobial resistance. Resistance Nodulation Division efflux pumps are important contributers to antimicrobial resistance in P. aeruginosa and other organisms. Resistance nodulation division efflux pumps are composed of three proteins; however, the mechanism by which the three proteins interact to form a functional complex remains, for the most part, unknown. The goal of this project is to further our understanding of how these proteins interact, to better understand how these efflux pumps operate in hopes of identifying novel therapeutic targets. We constructed a single copy expression system to study MexJK in the presence of either OprM or OpmH and confirmed these constructs using qRT, immunoblot and MIC. Chimeric OprM proteins were constructed by swapping α-helical domains from the OpmH protein. Using these chimeric proteins, we were able to identify regions within OprM/OpmH that may be responsible for substrate specificity by generating chimeric proteins. Taken together this data broadens our understanding of how this complex interacts.University of Ontario Institute of Technolog
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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