1,720,999 research outputs found
Interaction of Ceftaroline with Clinically-Relevant β-Lactamases
No full textBackground: Ceftaroline fosamil is a cephalosporin approved for community-acquired pneumonia and acute bacterial skin and skin structure infections. Ceftaroline, the active metabolite of ceftaroline fosamil, has broad-spectrum in vitro activity against Gram-positive pathogens (including MRSA) and some Gram-negative organisms. To our knowledge, the hydrolysis of ceftaroline by clinically-relevant β-lactamases (BLs) has not been systematically studied. We thus investigated the hydrolytic activity of various BLs, including ESBLs, AmpC-type chromosomal and plasmid-mediated enzymes and serine- and metallo-carbapenemases, on ceftaroline and their potential contribution to ceftaroline resistance.
Methods: Ceftaroline MICs were determined according to CLSI with isogenic laboratory E. coli strains producing representative BLs of class A (TEM-1, TEM-72, TEM-76, SHV-5, SHV-12, CTX-M-2, CTX-M-15, BEL-1, BEL-2, KPC-2), class B (IMP-1, IMP-18, VIM-2, NDM-1), class C (P. aeruginosa AmpC, E. cloacae AmpC, CMY-2, CMY-4) and class D (OXA-10, OXA-23, OXA-40, OXA-46, OXA-48) and compared to those of cephalothin, cefotaxime and ceftazidime. The kinetic parameters for the hydrolysis of these antibiotics by the 23 aforementioned purified BLs were determined by spectrophotometry.
Results: All BLs efficiently hydrolyzed ceftaroline (kcat/Km ≥105 M-1.s-1), except TEM-76, BEL-2 and OXA-48. When compared to other tested cephalosporins, ceftaroline was hydrolyzed with similar (cephalothin and ceftriaxone) or higher (ceftazidime) efficiencies. Large differences in the individual kinetic parameters were observed, with turnover rates ranging 0.5 to 650 s-1. Class C enzymes and NDM-1 exhibited the lowest turnover rates for ceftaroline hydrolysis. Ceftaroline MICs for isogenic BL-producing E. coli strains ranged 0.12 to 256 μg/ml, most being resistant. Interestingly, the strains susceptible or intermediate to ceftaroline (according to CLSI criteria) were those producing AmpC, CMY-2, NDM-1 and OXA-48, characterized by low turnover rates (kcat ≤ 2 s-1).
Conclusions: Although ceftaroline was readily hydrolyzed by most tested BLs, remarkable differences in turnover rates (3 orders of magnitude) were observed. The ceftaroline MIC data correlated with the kinetic parameters and showed that enzymes with low turnover rates were unable to confer ceftaroline resistance in an E. coli laboratory strain
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Microbiological analysis of hospital wastewaters: impact of water treatment on microbial biodiversity and antimicrobial resistance.
No full textBackground. Hospital effluents are considered an important source of both microorganisms and chemical substances (e. g. drug residues, disinfectants) that might impact the environment, often requiring pre-treatment before they can be released to public wastewater treatment plants. In order to investigate how wastewater treatment could affect the microbial biodiversity and its associated antimicrobial resistance, we performed a microbiological and molecular analysis of samples collected at the inlet and outlet of our local wastewater treatment plant.
Methods. Samples were collected at the University Hospital of Siena wastewater treatment plant during Feb and Oct 2012. Microbial aerobic count was determined on Nutrient Agar and McConkey Agar plates at 30 or 37 °C. Identification of microorganisms was carried out using a Vitek MS. Antimicrobial susceptibility testing was performed as recommended by CLSI. Determinants of antimicrobial resistance were analyzed using either phenotypic tests or molecular methods.
Results. A comparative analysis of pre- and post-treatment samples (total bacterial counts in the various samples ranged 5×104 – 2×106 CFU/ml) revealed important differences in both microbial biodiversity and its associated antimicrobial resistance: (a) the percentage of Gram-negative (GN) bacteria in post-treatment samples was significantly lower than that found in pre-treatment samples (5 vs 40% total, respectively) and (b) a higher proportion of antibiotic-resistant GN isolates was found in post-treatment samples, with 59% isolates showing ceftazidime resistance (vs 6% in pre-treatment samples). Approx. 10% of CAZ-resistant GN isolates exhibited resistance to carbapenems, fluoroquinolones and aminoglycosides and belonged to genera Citrobacter, Klebsiella and Shewanella. Molecular analysis revealed the presence of KPC-producing Klebsiella pneumoniae and VIM-2-producing Citrobacter spp. isolates (including C. braaki in which such determinant was not previously reported).
Conclusions. The hospital wastewater samples contained both environmental and clinically-relevant bacterial species. Despite post-treatment samples showed a lower prevalence of GN species, a higher of rate antibiotic resistance was found
Probing the role of R2-loop deletions and substituions on the functional properties of the acquired AmpC-type β-lactamase CMY-2.
No full textBackground: CMY-2 is a plasmid-encoded AmpC-type β-lactamase (pAmpC) belonging to the CIT cluster which shows the broadest dissemination and represents an important mechanism of β-lactam resistance in Enterobacteriaceae. The crystal structure of CMY-10, a pAmpC belonging to the MOX cluster, revealed subtle differences in the R2-loop of the enzyme (due to a 3-AA deletion) that were associated with a higher hydrolytic activity towards imipenem. In this work, we wanted to probe the impact of modifications in the R2-loop of CMY-2 on the functional features of the enzyme.
Methods: Wild-type cmy-2 gene was cloned in vector pLB-II and propagated in E. coli DH5α. Using mutagenic PCR, several CMY-2 laboratory variants were obtained in which (a) a 3-AA deletion was introduced in the R2-loop (CMY-2Δ319-321, CMY-2Δ320-322, etc.) or (b) the whole R2 loop of CMY-2 was substituted by that found in the structurally-equivalent position of CMY-10 (CMY-2loop10). The resulting recombinant vectors were transformed in E. coli. The β-lactam susceptibility of the recombinant E. coli strains carrying the cloned cmy-2 mutants were determined according to CLSI or using E-test. The hydrolytic activity of the various CMY-2 variants were investigated by standard kinetic measurements.
Results: The various E. coli strains producing the CMY-2 laboratory variants were resistant to ampicillin and cephalothin, indicating that these modified proteins were functional. However, the 3-AA deletion in CMY-2 (such as in mutant CMY-2Δ319-321) apparently increased the susceptibility to FOX and CAZ, while imipenem MIC was unchanged. Similar results were observed with E. coli strains producing the CMY-2loop10 variant. Moreover, enzymatic assays did not show significant differences in imipenem hydrolysis between the wild-type and the CMY-2 variants.
Conclusions: Several CMY-2 variants carrying deletions or substitutions in the R2-loop were obtained and characterized. Despite these variants were functional, they did not decrease imipenem susceptibility when produced in E. coli, indicating that other mutations might be required in CMY-2 to enhance its activity towards imipenem
Characterization of a Functional Metallo-β-Lactamase from the Marine Bacterium Haliangium ochraceum
No full textBackground: Acquired metallo-β-lactamases (MBLs) are worrisome resistance determinants conferring broad-spectrum β-lactam resistance in clinically-relevant pathogens such as Enterobacteriaceae, Pseudomonas and Acinetobacter. However, the origin of these acquired determinants is still poorly understood. Exploiting the increasing number of microbial genome sequences available in public databases, we specifically investigated the presence of genes encoding for proteins showing homology with acquired subclass B1 enzymes in the genomes of environmental bacteria belonging to the class of δ-Proteobacteria.
Methods: Using genome data mining (with the protein sequence of VIM-2 as the search template and the tblastn algorithm available at the NCBI web site), a gene encoding a subclass B1 MBL homologue (locus tag, Hoch_6144) was identified in the genome of Haliangium ochraceum DSMZ 14365. The Hoch_6144 ORF was cloned in plasmid vector pET-9a. The resulting plasmid (pET-HAL-1) were electroporated in E. coli BL21(DE3). The MBL was purified by chromatography and its functional properties investigated by means of kinetic analysis.
Results: The MBL homologue encoded by gene Hoch_6144, named HAL-1, shared the closest ancestry with FIM-1, NDM-1 and VIM-2 acquired MBLs (identity range, 41-46 %). Significant β-lactam-hydrolyzing activity was found in crude extracts of E. coli BL21(DE3)/pET-HAL-1, that was inhibited >95% in the presence of 5 mM EDTA. HAL-1 was successfully produced and purified (>95%) using three chromatography steps. Purified HAL-1 exhibited a broad-spectrum of activity, with catalytic efficiencies for the hydrolysis of β-lactams comparable to that of VIM-2. Interestingly, HAL-1 showed low Km values for most tested substrates, at the exception of ceftazidime. Strikingly, doripenem (the latest carbapenem antibiotic to receive FDA approval) was hydrolyzed 45-fold more efficiently by HAL-1 than by the acquired VIM-2 MBL.
Conclusions: In this work, we described a new functional MBL identified in a marine bacterium of the class δ-Proteobacteria, Haliangium ochraceum. HAL-1 exhibits functional properties very close to that of clinically-relevant subclass B1 enzymes and highlights the potential of δ-Proteobacteria as a reservoir of functional subclass B1 MBL determinants
Variations on the Author
Full text link“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
Mimicking the Ala220 Duplication of OXA-146 (an OXA-23 variant) in OXA-40 and OXA-48 Highlights Mechanistic Differences among Class D Carbapenemases.
No full textBackground: Class D carbapenem-hydrolyzing β-lactamases (CHDLs), such as OXA-23, OXA-24/40 and OXA-48 commonly share very poor catalytic activity on expanded-spectrum cephalosporins, especially ceftazidime. OXA-146, an OXA-23 clinical variant differing by a single alanine (A220) duplication exhibits significant activity on ceftazidime. Interestingly, the duplicated amino acid residue is located in the β5-β6 loop, a structural element whose role in enzyme functional properties was previously recognized. In order to understand the impact of such residue duplication in other CHDLs, similar variants of OXA-24/40 and OXA-48 were generated and characterized.
Methods: OXA-24/40 and OXA-48 alanine insertion variants at the structurally-equivalent position to OXA-23 220 (222 and 213 in OXA-24/40 and OXA-48, respectively) were obtained by mutagenesis. Escherichia coli laboratory strains producing the two β-lactamase variants (OXA-24ins222 and OXA-48ins213) were obtained and their in vitro susceptibility profile determined and compared to that of the wild-type CHDL-producing strains. The functional characterization of the variants was carried out using enzyme assays and the carbapenemase plate assay.
Results: As compared to WT OXA-24/40, the OXA-24ins222 insertion variant confers, in an E. coli background, a susceptibility profile similar to that of OXA-146, with a significant increase of the MIC values of ceftriaxone, ceftazidime, cefepime (16-fold) and aztreonam (128-fold). In addition, the Ala222 insertion in OXA-24/40 also determined an apparent decrease of activity towards imipenem. Furthermore, the OXA-24ins222 variant also showed a delayed accumulation in E. coli, as compared to the WT OXA-24/40, which suggests that this modification might also impact on enzyme stability. Strikingly, the insertion of an Ala residue at position 213 of OXA-48 did not confer any of the properties observed with OXA-146 and OXA-24ins222 variants.
Conclusions: These data further highlight the functional and structural importance of the β5-β6 loop of CHDLs and the mechanistic heterogeneity observed between OXA-23/24 and OXA-48 CHDLs
Dissemination of CTX-M-type extended-spectrum β-lactamase genes to unusual hosts
No full textA Citrobacter amalonaticus and a Morganella morganii producing the CTX-M-1 extended-spectrum β-lactamase (ESBL) were isolated from an area where this enzyme is now widespread in Escherichia coli. This is the first report of CTX-M-1 in the former species. In both cases the ESBL determinant was possibly acquired by these unusual hosts in vivo, after coinfection with E. coli strains carrying conjugative plasmids encoding CTX-M-1. Copyright © 2005, American Society for Microbiology. All Rights Reserved
Linear guanidine derivatives, methods of preparation and uses thereof
No full textThe present invention relates to linear guanidine derivatives, methods of preparation, uses and pharmaceutical compositions thereof. The compounds of Formulas 1 or 2 exhibit high antimicrobial activity against Gram positive and Gram negative bacteria
- …
