We tested 42 strains of amikacin-resistant gram-negative bacilli with amikacin in combination with six beta-lactam antibiotics using the checkerboard and time kill curve techniques. Synergism was demonstrated with time-killing curve in 43-68% of the strains tested. Ceftazidime plus amikacin was the most active combination by the checkerboard technique, while amikacin-cefoperazone was the most active combination by the time-killing curve technique against Pseudomonas aeruginosa. Discrepancies were found between the results of the two methods used.

No description available.

Yes / Stereoselective biocatalysis by crotonase superfamily enzymes is exemplified by use of engineered 5-carboxymethylproline synthases (CMPSs) for preparation of functionalized 5-carboxymethylproline (5-CMP) derivatives methylated at two positions (i.e. C2/C6, C3/C6 and C5/C6), including products with a quaternary centre, from appropriately-substituted-amino acid aldehydes and C-2 epimeric methylmalonyl-CoA. The enzymatically-produced disubstituted 5-CMPs were converted by carbapenam synthetase into methylated bicyclic Β-lactams, which manifest improved hydrolytic stability compared to the unsubstituted carbapenams. The results highlight the use of modi-fied carbapenem biosynthesis enzymes for production of new carbapenams with improved properties. / Medical Research Council, Biotechnology and Biological Sciences Research Council (BB/L000121/1)

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 1978 / Vita. / Includes bibliographical references. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Chemistry

Carbon-13 nuclear magnetic resonance is a very useful spectroscopic technique in studying organic compounds, especially when the proton NMR does not provide much information. In this study of multicyclic lactams, different NMR techniques were used: broadband decoupled spectra, single-frequency off-resonance decoupling (SFORD), and the attached proton test (APT). Lanthanide shift reagents and lanthanide relaxation reagents were also used. Almost all the carbon-13 resonances of mono-, bi-, and polycyclolactams were unambiguously assigned. The most powerful method in the assignments was based on the use of the lanthanide induced shifts (LIS) which confirmed the assignments qualitatively and quantitatively. The quantitative confirmation comes from the calculations of LIS and their comparison with the observed shifts. The carbon-13 chemical shielding of the studied lactams was analyzed and compared with analogous compounds. The results presented provide a consistent picture and the major influences in the trends of the ¹³C chemical shifts. However, no empirical relationships were derived for this series of compounds. The solution conformations of most of these lactams have been investigated using the shifts induced in their carbon-13 NMR by Yb(dpm)₃. These conformations were compared with structures obtained from X-ray data and MINDO/3 calculations. The structural analyses of ε-caprolactam and 3-azabicyclo[4.3.1]decan-4-one showed that these molecules have at least two conformations in solution. The LIS structural analyses were confirmed by using ¹³C T₁ relaxation times in Gd(dpm)₃ and Gd(fod)₃ relaxation reagents. The different contributions to the ¹³C NMR lanthanide induced shifts (LIS) were studied with emphasis in determining the importance of the ligand pseudocontact contribution. This was found to be important, especially in carbons in proximity to the complexation site.

Enterobactérias produtoras de AmpC cromossômica, especialmente Citrobacter, Serratia, Providencia, Proteus e Morganella, entre outros, são patógenos oportunistas e estão implicados em infecção relacionada a assistência à saúde. Uma vez que mecanismos de resistência adquiridos a antibióticos são cada vez mais frequentemente encontrados nesses micro-organismos, o gerenciamento das infecções causadas por eles tem sido desafio para a escolha da antibioticoterapia, pois existem poucos dados fenotípicos e moleculares sobre essas espécies. O objetivo deste trabalho foi a investigação de genes de resistência adquiridos (mediados por plasmídeos) aos antibióticos beta-lactâmicos de amplo espectro e quinolonas, bem como a determinação da epidemiologia molecular das enterobactérias produtoras de AmpC cromossômica, isoladas de pacientes ambulatoriais e internados em hospital universitário. Foram estudadas e comparadas bactérias isoladas em 2007 e 2016 durante o período de cinco meses em cada ano. Foi investigada fenotipicamente a produção de ESBL e AmpC associadas à resistência aos beta-lactâmicos de amplo espectro. Adicionalmente, também foram pesquisados genes de resistência adquiridos aos beta-lactâmicos de amplo espectro e quinolonas tão bem como plasmídeos carreando tais genes. Foram encontrados dois genes blaSHV-5 e um blaCTX-M-2, além de, um gene qnrS2, dois qnrB6, dezenove genes qnrD1 e vinte e um aac(6\')-Ib, sendo que desses oito apresentaram a variante aac(6\')-Ib-cr. O gene qnrD1 já estava presente no hospital estudado antes do primeiro relato do gene no Brasil. Sequenciamento de plasmídeos carreando gene qnrD1 mostra que pelo menos dois plasmídeos distintos estão envolvidos em sua disseminação. Por PFGE foi possível observar que não houve disseminação clonal dos isolados bacterianos no hospital nos períodos estudados. Foi determinada a epidemiologia molecular comparativa das bactérias do estudo. Este conhecimento torna-se fundamental para que, haja informações consistentes sobre as bactérias do estudo, fornecendo subsídio para o tratamento dos pacientes e contribuindo para o melhor prognóstico e gerenciamento das infecções bacterianas. / Enterobacteria producing chromosomal AmpC, especially Citrobacter, Serratia, Providencia, Proteus and Morganella, among others, are opportunistic pathogens implicated in nosocomial infections. Since antibiotic resistance mechanisms are increasingly found in these microorganisms, the management of infections caused by them has been challenging on choosing the antibiotic therapy, as there are few phenotypic and molecular data on these species. The aim of this study was the investigation of acquired (plasmid-mediated) resistance genes to broad-spectrum beta-lactam antibiotics and quinolones, as well as the determination of the molecular epidemiology of chromosomal AmpC-producing enterobacteria isolated from outpatients and inpatients in a university education hospital. Bacteria isolated in 2007 and 2016 during the five-month period each year were studied and compared. The production of ESBL and AmpC associated with resistance to broad-spectrum beta-lactams was phenotypically investigated. In addition, acquired resistance genes from broad-spectrum beta-lactams and quinolones were also screened as well as plasmids carrying such genes. Two blaSHV-5 genes and one blaCTX-M-2 were found, in addition to one qnrS2, two qnrB6, nineteen genes qnrD1 and twenty-one aac(6\')-Ib, of which eight presented the aac(6\')-Ib-cr variant. qnrD1 gene was already present in the hospital studied before the first report of such gene in Brazil. Sequencing of plasmids carrying qnrD1 gene shows that at least two distinct plasmids are involved in its dissemination. Through PFGE it was possible to observe that there was no clonal dissemination of the bacterial isolates in the hospital during the periods studied. Comparative molecular epidemiology of the bacteria in the study was determined. This knowledge becomes critical for consistent information about the bacteria in the study, providing subsidy for the treatment of patients and contributing to the better prognosis and management of bacterial infections.

No description available.

N-sec -butylthioloated β-lactam (NsβL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsβL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsβL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsβL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that Nsβ L has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.

The work described in this thesis focused on the use of nuclear magnetic resonance spectroscopy (NMR) to study two classes of metallo enzymes - the Fe(II)- and 2oxoglutarate (2OG)-dependent dioxygenases and the metallo β-lactamases (MBLs). These enzymes are involved in clinically important biological processes, i.e. the hypoxic response and antimicrobial resistance, respectively. Both protein systems are interesting from an NMR perspective because they have dynamic regions involved in catalysis and ligand interactions. The work included mechanistic studies, protein-ligand interaction studies, and method development for inhibitor discovery. NMR was applied to study the human prolyl hydroxylase domain-containing protein 2 (PHD2), which is crucially involved in the chronic hypoxic response. The results reveal that binding of the C- and the N-terminus of the oxygen dependent degradation domains CODD and NODD, respectively, induce different interactions with PHD2. The substitution of a single amino acid, as occurs with PHD2 variants linked to erythrocytosis and breast cancer, can alter the selectivity of PHD2 towards its ODD substrates. Studies with the Trichoplax adhaerens PHD provide insights into the evolutionary substrate preference of the PHDs. Using ¹³C-labelled peptidyl-substrates; NMR was applied to investigate proposed 'alternative' PHD2 substrates/interaction partners. The product release mechanism of PHD2 was investigated using NMR; the results reveal that the presence of 2OG strongly discriminates between the binding of CODD and hydroxylated CODD to PHD2. NMR was also applied to monitor PHD2 kinetics and inhibition. Competition and displacement assays were designed and applied to investigate PHD inhibitor binding modes. Comparative studies on the activities and selectivities of PHD inhibitors in clinical trials should aid in the work on the therapeutic manipulation of the natural hypoxic response. Protein-observe ¹9F-NMR was used to study the São Paolo MBL (SPM-1). The results provide new structural insights into SPM-1 catalysis and the requirements for inhibitor development. They also reveal that the hydrolysed β-amino acid products of MBL catalysis can bind to SPM-1. They illustrate the utility of ¹⁹F-NMR for detecting metal chelation, which is not always readily tractable in studies on metallo enzyme inhibition, new binding modes, and stereoisomer binding/epimerisation in solution. The interaction of a cyclobutanone analogue, a broad-spectrum MBL inhibitor, with SPM-1 was investigated. A combination of ¹H, ¹⁹F, ¹³C-NMR and crystallographic analyses reveal that cyclobutanone binding may mimic formation of the oxyanion tetrahedral intermediate in β-lactam hydrolysis. The susceptibility of avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, to MBL-catalysed hydrolysis was studied. The results reveal that avibactam is not an MBL inhibitor and a poor substrate of most members of all three clinically relevant subclasses of MBLs. In some cases, avibactam undergoes slow hydrolysis in a process different from that observed with serine β-lactamases. Overall, the results illustrate the utility of NMR for studying dynamic aspects of enzyme catalysis and inhibitor binding.

Carbonyl compounds are of central importance to organic chemistry and their reactions have been described as the ‘backbone of organic synthesis’. Over recent decades, palladium-catalysed C–H carbonylation reactions have emerged as a powerful means of introducing carbonyl motifs to organic molecules. This thesis describes the development of a general C–H carbonylation reaction of secondary aliphatic amines, which provides facile access to synthetically useful β-lactam products. The first part of the thesis explores the scope and limitations of this reaction. Whilst previous C(sp3)–H carbonylation methodologies were restricted to ‘Type F’ secondary aliphatic amines, the reaction described in this thesis was found to be broadly applicable all structural sub-classes of secondary aliphatic amine. Furthermore, the reaction was found to be remarkably tolerant of functional groups, even those that commonly cause issues in palladium-catalysed C–H activation reactions such as heteroaromatics and thioethers. The second part of this thesis investigates the mechanism of this C–H carbonylation reaction. Interestingly, the reaction was found not to proceed via a traditional C–H carbonylation mechanism comprising of C–H activation, 1,1-migratory carbon monoxide insertion and reductive elimination. Instead, a new mechanistic paradigm for palladium-catalysed C–H carbonylation is proposed, which invokes a putative ‘palladium anhydride’ intermediate. A series of DFT calculations and experiments were conducted in order to support this mechanistic proposal. The work described within this PhD thesis was published in Science.