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Biochemical and molecular characterization of GES and NDM engineered variants: interactions with β-lactams and inhibitors
Get PDFAntibiotic resistance occurs when bacteria causing an infection survive after being exposed to a drug that, under normal conditions, would kill it or inhibit its growth. As a result, these surviving strains multiply and spread due to the lack of competition from other strains sensitive to the same drug. Carbapenems and 3rd generation cephalosporins resistant Enterobacteriaceae represent one of the most critical group against which there is an urgent need to develop new antibiotics. These bacteria are common pathogens causing severe infections such as bloodstream infections, pneumonia, complicated urinary tract infections and complicated intra-abdominal infections. According to the Centers for Disease Control and Prevention (CDC) definition, CRE are defined as any Enterobacteriaceae which are resistant to any carbapenem or are documented to produce a carbapenemase. There are three major mechanisms by which Enterobacteriaceae become resistant to carbapenems: enzyme production, efflux pumps and porin mutations. Of these, enzyme production is the main resistance mechanism. Gram-negative bacteria generally develop resistances through the production of β-lactam-hydrolyzing enzymes, called β-lactamases. In the present study, two classes of these enzymes were analyzed and characterized also in association with a new molecule with inhibitory activity. In the first part of this thesis, the role of the Ω-loop (159-182 residues) in GES class A β-lactamase is well investigated. Recent studies report that, in these enzymes, carbapenemase activity is attributed to a single amino acid substitution at position 170. In our study, we decided to investigate other positions of the Ω-loop, in particular residue 174, occupied by a proline residue. This residue is well conserved in class A β-lactamases. Our kinetic and computational data have demonstrated that not only 170 residue is important in the carbapenemase activity, but also residue 174. Being Ω-loop a mobile structure of class A β-lactamases, we suppose that any mutation in this region could modify the substrate hydrolysis of the enzyme, not only toward carbapenems. The second part of the thesis is addressed to the involvement of some luecines located in the Loop 10 of NDM-1 and the effect of a new boronic acid molecule (taniborbactam) as inhibitors of metallo-β-lactamases. We have pointed the attention on residues L218, L221 and L269 that give hydrophobicity to the enzyme. Our results have demonstrated that the replacement of these leucines reduces the enzyme catalytic activity toward β-lactams. In addition, we studied the effect of Y229W substitution in our laboratory variant (L209F) with a drastic reduction in β-lactamase activity. This substitution is able to restore the catalytic activity in the L209F mutant enzyme. Actually, one of the most problem is related to the absence of inhibitors for metallo-β-lactamases in clinical therapy. For this reason, in collaboration with VenatoRx Pharmaceuticals (Malvern, PA, USA), we tested a new boronic acid, VNRX-5133, combinated with cefepime (taniborbactam), in NDM-1 and some engineered NDM-1 mutants able to hydrolyze β-lactams very efficiently. Taniborbactam was also tested in NDM-1-
producing Enterobacteriaceae clinical strains. Our results revealed that the addition of VNRX-5133 restored cefepime activity in both NDM-1 producing recombinant strains of E. coli and clinical isolates of Enterobacteriaceae. The development of new non-β-lactam inhibitors would provide a new opportunity for the treatment of bacterial infections with existing antibiotics
P174E substitution in GES-1 and GES-5 β-lactamases improves catalytic efficiency towards carbapenems.
No full textGES-type beta-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Omega-loop of GES-1 and GES-5 was investigated. GES-1(P174E) and GES-5(P174E) mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1P174E and GES-5P174E mutants exhibited lower k(cat) and k(cat)/K-m values for cephalosporins and penicillins. Concerning carbapenems, GES-1(P174E) shared higher k(cat) values but lower K-m values than those calculated for GES-1. The GES-1(P174E) and GES-5(P174E) mutants showed high hydrolytic efficiency for imipenem, with k(cat)/K-m values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1(P174E) and GES-5(P174E). Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1(P174E), and GES-5(P174)E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Omega-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes
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
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
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
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Identification of CTX-M-15 and CTX-M-27 in Antibiotic-Resistant Gram-Negative Bacteria Isolated from Three Rivers Running in Central Italy
No full textThe main goal of this study was to identify Gram-negative bacteria resistant to antibiotics, in particular β-lactams, in stream waters and effluents from urban wastewater treatment plants draining into Fino, Tavo, and Saline rivers of the Abruzzo region, Italy. Eight sampling sites were selected because they were the most contaminated by coliforms during previous sampling campaign. One sample for each site was collected for the detection of total and fecal coliforms, Escherichia coli and Enterococcus species by Colilert-18 and Enterolert-E Quanti-Tray/2000. Antibiotic-resistant bacteria, selected on ampicillin and cefotaxime-supplemented agar plates, were identified by EnteroPluri test systems and then confirmed by MALDI-TOF. The resistant determinants were identified and characterized by PCR and sequencing. The microbiological analysis allowed to detect E. coli, total coliforms, fecal coliforms, and enterococci with a coefficient of variation of 215.7%, 212.8%, 242.5%, and 188.5%, respectively. Several Gram-negative bacteria were identified: Serratia liquefaciens, E. coli, Enterobacter cloacae, Citrobacter freundii, Raoultella ornithinolytica, Acinetobacter johnsonii, Aeromonas veronii, Aeromonas hydrophila, and Pseudomonas koreensis. All strains possessed class 1 integrons, insertion sequences, and genes encoding for serin- and metallo-β-lactamases. Extended-spectrum β-lactamases, such as CTX-M-15 and CTX-M-27, were found in Enterobacteriaceae, whereas CphA metallo-β-lactamase was found in A. veronii and A. hydrophila. The main resistance's mechanism to β-lactams observed among the analyzed strains is represented by the production of serin β-lactamases (CTX-M-15, CTX-M-27, and SHV-1) and metallo β-lactamase (CphA)
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