The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.

(3R,5R)-Clavulanic acid is a clinically important inhibitor of Class A β-lactamases. Progress has been made in to establishing the steps of clavulanic acid biosynthesis leading to (3S,5S)-clavaminic acid. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde remains elusive. It is believed that the products of the later genes (orf10-orf18) of the clavulanic acid biosynthesis gene cluster are probably involved in this conversion. Part I of this thesis describes biochemical and structural studies carried out on OAT2, a member of N-terminal nucleophile (Ntn) hydrolase superfamily of enzymes. OAT2 has been characterised to be an ornithine acetyl transferase (OAT) and is involved in clavulanic acid biosynthesis. OAT2 catalyses the reversible transfer of the acetyl group between N-acetyl-L-ornithine and L-glutamate. It was found that this reaction is catalysed via the formation of an acyl-enzyme intermediate. Subsequent studies including mass spectrometry, 13C NMR spectroscopy, infrared spectroscopy, X-ray crystallography and molecular dynamics simulations, further confirmed the viability of the intermediate. This acyl-enzyme intermediate of OAT2 was found to be exceptionally stable at physiological pH, as compared to the acyl-enzyme intermediates involved in catalysis by hydrolytic enzymes including proteases, Ntn hydrolases and β-lactamses. The X-ray studies revealed possible reason for this unusual stability. The infrared studies revealed two conformations for the acyl-enzyme. Modeling (MDS) studies assigned one of these to the structure observed by X-ray and proposed the other one to result from a hydroxyl hydrogen 'flip' involving the oxyanion hole component Thr-111 resulting in a singly hydrogen bonded acyl-enzyme intermediate. α, β Subunit co-expression studies with OAT2 were used to investigate the autocatalytic cleavage step. In one case an interesting N-acyl enzyme species was observed. Part II of this thesis describes efforts carried out to characterise the ORF10 and ORF15 proteins of clavulanic acid biosynthesis. ORF10 was characterised to be an 'active' cytochrome P450 and ORF10 crystals were obtained in the presence spinach ferredoxin, highlighting the role of the ferredoxin interaction in assisting ORF10 crystallisation. ORF15 was shown to be a probable peptide transporter, which binds bradykinin as observed in the crystal structure.

This research developed validated QSPKR and PK-AS models for predicting human systemic PK properties of three, preselected, pharmacological classes of drugs, namely opioids, β-adrenergic receptor ligands (β-ARL) and β-lactam antibiotics (β-LAs) using pertinent human and animal systemic PK properties (fu,, CLtot, Vdss, fe) and their biologically relevant unbound counterparts from the published literature, followed by an assessment of the effect of different molecular descriptors on these PK properties and on the PK-AS slopes for CLtot and Vdss from two species (rat and dog). Lipophilicity (log (D)7.4) and molecular weight (MW) were found to be the most statistically significant and biologically plausible, molecular properties affecting the biologically relevant, systemic PK properties: For compounds with log (D)7.4 > -2.0 and MW < 350 D (e.g., most opioids and β-ARL), increased log (D)7.4 resulted in decreased fu and increased Vdssu, CLtotu and CLnonrenu, indicating the prevalence of hydrophobic interactions with biological membrane/proteins. As result, the final QSPKR models using log (D)7.4 provided acceptable predictions for fu, Vdssu, CLtotu and CLnonrenu. CLnonrenu and CLtotu. For both the datasets, inclusion of drugs undergoing extrahepatic clearance worsened the QSPKR predictions. For compounds with log (D)7.4 < -2.0 and MW > 350 D (e.g., β-LA), increased MW (leading to more hydrogen bond donors/acceptors) resulted in a decrease in fu, likely indicating hydrogen bonding interactions with plasma proteins. In general, it was more difficult to predict PK parameters for β-LAs, as their Vdssu approached plasma volume and CLrenu and CLnonrenu were low - as a result of their high hydrophilicity and large MW, requiring specific drug transporters for distribution and excretion. The PK-AS analysis showed that animal body size accounted for most of the observed variability (r2> 0.80) in systemic PK variables, with single species methods, particularly those using dog, gave the best predictions. The fu correction of PK variables improved goodness of fit and predictability of human PK. There were no apparent effects of molecular properties on the predictions. CLren, CLrenu, CLnonren, and CLnonrenu were the most difficult variables to predict, possibly due to the associated interspecies differences in the metabolism, renal and hepatobiliary drug transporters.

Yip, Yuen Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 128-146). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Figures --- p.xi / List of Tables --- p.xii / List of Appendix --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1 --- Pseudomonas aeruginosa --- p.1 / Chapter 1.1 --- Microbiology --- p.1 / Chapter 1.1.1 --- Morphology --- p.1 / Chapter 1.1.2 --- Identification --- p.1 / Chapter 1.1.3 --- Pathogenesis and virulence --- p.2 / Chapter 1.1.4 --- Host defenses --- p.2 / Chapter 1.1.5 --- Epidemiology --- p.2 / Chapter 1.1.6 --- Clinical manifestations --- p.3 / Chapter 1.1.7 --- Treatment --- p.3 / Chapter 2 --- β-Lactams --- p.4 / Chapter 2.1 --- Mode of action of β-lactams --- p.6 / Chapter 2.2 --- β-Lactams resistance --- p.7 / Chapter 2.3 --- Resistance mechanisms --- p.7 / Chapter 2.3.1 --- Changes in PBPs --- p.7 / Chapter 2.3.2 --- Impermeability --- p.8 / Chapter 2.3.3 --- β-Lactamases --- p.8 / Chapter 2.3.3.1 --- Extended spectrum β-lactamases --- p.10 / Chapter 2.3.3.2 --- Carbapenemases --- p.11 / Chapter 2.3.4 --- Efflux pump systems --- p.14 / Chapter 2.4 --- Mechanisms of imipenem resistance in P. aeruginosa --- p.16 / Chapter 2.4.1 --- Prevalence of imipenem resistant P. aeruginosa isolates --- p.18 / Chapter 3 --- Integrons --- p.20 / Chapter 3.1 --- Structure and classification --- p.20 / Chapter 3.1.1 --- Class 1 integrons --- p.21 / Chapter 3.1.2 --- Other class of integrons --- p.22 / Chapter 3.2 --- Integrons in imipenem-resistant P. aeruginosa --- p.23 / Chapter 4 --- Objectives --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 1 --- Materials --- p.25 / Chapter 1.1 --- Bacterial strains --- p.25 / Chapter 1.1.1 --- Bacterial strains used in this study --- p.25 / Chapter 1.1.2 --- Reference strains --- p.25 / Chapter 2 --- Methods --- p.26 / Chapter 2.1 --- Subculture of isolates --- p.26 / Chapter 2.2 --- Identification --- p.26 / Chapter 2.3 --- Antibiotic susceptibility testing --- p.26 / Chapter 2.3.1 --- Preparation of antibiotic plates --- p.27 / Chapter 2.3.2 --- Inoculation of antibiotic plates --- p.27 / Chapter 2.3.3 --- Determination of minimum inhibitory concentration (MIC) --- p.28 / Chapter 2.4 --- Phenotypic detection of metallo-beta-lactamase (MBL) production --- p.28 / Chapter 2.4.1 --- Preparation of inoculum --- p.28 / Chapter 2.4.2 --- Imipenem-EDTA disk test --- p.28 / Chapter 2.4.3 --- Determination of MBL strains --- p.29 / Chapter 2.5 --- Extraction of crude β-lactamase --- p.29 / Chapter 2.5.1 --- Detection of β-lactamase production --- p.29 / Chapter 2.6 --- Isoelectric focusing (IEF) --- p.30 / Chapter 2.6.1 --- Set up of electrophoresis equipment --- p.30 / Chapter 2.6.2 --- Sample application and instrument preparation --- p.30 / Chapter 2.6.3 --- Running conditions --- p.30 / Chapter 2.6.4 --- Detection of β-lactamase --- p.31 / Chapter 2.6.5 --- Determination of isoelectric point (pi) --- p.31 / Chapter 2.7 --- Bioassay of imipenem hydrolysis --- p.31 / Chapter 2.7.1 --- Preparation of inoculum and plate --- p.31 / Chapter 2.7.2 --- Preparation and incubation of sample mixtures --- p.32 / Chapter 2.7.3 --- Application of sample mixtures --- p.32 / Chapter 2.7.4 --- Determination of imipenem hydrolysis --- p.32 / Chapter 2.8 --- Detection of β-lactamase genes --- p.33 / Chapter 2.8.1 --- Polymerase chain reaction (PCR) --- p.33 / Chapter 2.8.2 --- Preparation of DNA template --- p.33 / Chapter 2.8.3 --- Preparation of PCR master mix --- p.33 / Chapter 2.8.4 --- PCR running conditions --- p.34 / Chapter 2.8.5 --- Agarose gel electrophoresis --- p.34 / Chapter 2.8.6 --- DNA sequencing --- p.35 / Chapter 2.9 --- Detection and characterization of integrons --- p.35 / Chapter 2.9.1 --- PCR --- p.35 / Chapter 2.9.2 --- DNA sequencing --- p.36 / Chapter 2.10 --- Detection and characterization of gene cassettes --- p.36 / Chapter 2.10.1 --- PCR --- p.36 / Chapter 2.10.2 --- DNA sequencing --- p.37 / Chapter 2.11 --- Investigation of membrane permeability --- p.37 / Chapter 2.11.1 --- Extraction of outer membrane proteins (OMP) --- p.37 / Chapter 2.11.2 --- Quantification of OMP --- p.38 / Chapter 2.11.3 --- Preparation of the albumin standards and working reagents --- p.38 / Chapter 2.11.4 --- Determination of protein concentration --- p.39 / Chapter 2.12 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.39 / Chapter 2.12.1 --- Sample preparation --- p.39 / Chapter 2.12.2 --- Gel preparation and sample application --- p.39 / Chapter 2.12.3 --- Staining and destaining of the gel --- p.40 / Chapter 2.13 --- Expression of the oprD gene --- p.40 / Chapter 2.13.1 --- Extraction of RNA --- p.40 / Chapter 2.13.1.1 --- Inhibition of RNase degradation --- p.41 / Chapter 2.13.1.2 --- Removal of DNA --- p.41 / Chapter 2.13.1.3 --- Quantification of RNA samples --- p.42 / Chapter 2.13.2 --- Real-time RT-PCR --- p.42 / Chapter 2.13.2.1 --- Preparation of real-time RT-PCR mixtures --- p.42 / Chapter 2.13.2.2 --- Real-time RT-PCR running conditions --- p.43 / Chapter 2.13.2.3 --- Construction of relative standard curves --- p.43 / Chapter 2.13.3 --- Analysis of real-time RT-PCR results --- p.43 / Chapter 2.14 --- Characterization of outer membrane protein regulator mexT --- p.44 / Chapter 2.14.1 --- PCR --- p.44 / Chapter 2.14.2 --- DNA sequencing --- p.44 / Chapter Chapter 3 --- Results / Chapter 1 --- Prevalence of imipenem-resistant P. aeruginosa isolated from patients in hospitals of the New Territories East Cluster (NTEC) from 2001 to 2005 --- p.46 / Chapter 1.1 --- Age and sex distribution of patients --- p.46 / Chapter 1.2 --- Antimicrobial susceptibilities --- p.46 / Chapter 1.2.1 --- Susceptibility to carbapenems --- p.46 / Chapter 1.2.2 --- Susceptibility to other β-lactams --- p.47 / Chapter 1.2.3 --- Susceptibility to aminoglycosides and fluoroquinolones --- p.47 / Chapter 1.2.4 --- Resistance patterns --- p.48 / Chapter 2 --- Phenotypic detection of metallo-beta-lactamase (MBL) producing strains --- p.48 / Chapter 2.1 --- Characterization of β-lactamases --- p.49 / Chapter 2.1.1 --- Production of β-lactamases --- p.49 / Chapter 2.1.2 --- Determination of isoelectric points of β-lactamases --- p.49 / Chapter 2.2 --- Imipenem hydrolysis by β-lactamases --- p.50 / Chapter 2.3 --- Detection of β-lactamase genes --- p.50 / Chapter 2.3.1 --- DNA sequence determination --- p.51 / Chapter 3 --- Detection and characterization of integrons --- p.51 / Chapter 3.1 --- Antibiotic susceptibility and resistance patterns of isolates harboring integrons --- p.51 / Chapter 4 --- Detection of gene cassettes --- p.52 / Chapter 5 --- Outer membrane permeability --- p.52 / Chapter 5.1 --- Outer membrane protein profiles --- p.52 / Chapter 5.2 --- mRNA expression of the oprD gene --- p.53 / Chapter 6 --- Regulatory gene studies --- p.53 / Chapter Chapter 4 --- Discussion / Chapter 1 --- Epidemiological characteristics of imipenem-resistant P. aeruginosa --- p.55 / Chapter 1.1 --- Prevalence of P. aeruginosa --- p.55 / Chapter 2 --- Antibiotic susceptibilities of imipenem-resistant P. aeruginosa --- p.56 / Chapter 3 --- Mechanisms of imipenem resistance in P. aeruginosa --- p.59 / Chapter 3.1 --- Production of β-lactamases --- p.59 / Chapter 3.2 --- Outer membrane permeability --- p.63 / Chapter 3.3 --- Effects of regulatory gene mutations --- p.64 / Chapter 4 --- Integrons in imipenem-resistant P. aeruginosa --- p.66 / Chapter 5 --- Conclusions --- p.67 / Chapter 6 --- Areas for further study --- p.67 / Figures --- p.69 / Tables --- p.82 / Appendix --- p.121 / References --- p.128

The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform computational molecular docking in our efforts to develop new inhibitors against CTX-M. The goal of this project was to determine the hydrogen bonding network and proton transfer process at different stages of the reaction pathway as well as develop novel inhibitors against CTX-M β-lactamases. The results I have obtained from the project have elucidated the catalytic mechanism of CTX-M β-lactamase in unprecedented detail and facilitated the development of novel inhibitors for antibiotic drug discovery. The first aim of the project focused on developing high affinity inhibitors against class A β-lactamase using a structure-based drug discovery approach, which ultimately led to the identification of CTX-M9 inhibitors with nanomolar affinity. Compound design was based on the initial use of computational molecular docking results along with x-ray crystal structures with known inhibitors bound in the active site. In addition, chemical synthesis was used to build and extend the existing inhibitor scaffold to improve affinity to CTX-M9 and related serine β-lactamases. Through a fragment-based screening approach, we recently identified a novel non-covalent tetrazole-containing inhibitor of CTX-M. Structure-based design was used to improve the potency of the original tetrazole lead compound more than 200-fold with the use of small, targeted structural modifications. A series of compounds were used to probe specific binding hotspots present in CTX-M. The designed compounds represent the first nM-affinity non-covalent inhibitors of a class A β-lactamase. The complex structures of these potent compounds have been solved using high resolution x-ray crystallography at ~ 1.2-1.4 Å, which provides valuable insight about ligand binding and future inhibitor design against class A β-lactamases. Specifically, the first aim of the project was to use ultra-high resolution x-ray crystallography to study β-lactamase catalysis. Through the use of ultra-high resolution x-ray crystallography with non-covalent and covalent inhibitors, I was able to structurally characterize the critical stages of the enzyme mechanism. Here we report a series of ultra-high resolution x-ray crystallographic structures that reveal the proton transfer process for the early stages of the class A β-lactamase catalytic mechanism. The structures obtained include an a 0.89 Å crystal structure of CTX-M β-lactamase in complex with a recently-developed 89 nM non-covalent inhibitor, and a 0.80 Å structure in complex with an acylation transition state boronic acid inhibitor. Nearly all the hydrogen atoms in the active site, including those on the ligand, polar protein side chains and catalytic water, can be identified in the unbiased difference electron density map. Most surprisingly, compared with a previously determined 0.88 Å apo structure determined under the same conditions, the hydrogen-bonding network has undergone a series of reshuffling upon the binding of the non-covalent ligand. Two key catalytic residues, Lys73 and Glu166, appear to have both changed from a charged state to being neutral. Interestingly, structural evidence suggests the presence of a low barrier hydrogen bond (LBHB) shared between Lys73 and Ser70. These unprecedented detailed snapshots offer direct evidence that ligand binding can alter the pKa's of polar protein side chains and their affinities for protons. Such effects can be a common mechanism utilized by enzymes to facilitate the proton transfer process of a reaction pathway. They also have important implications for computational modeling of protein-ligand interactions. Ultra-high resolution x-ray crystallography allowed us to determine the hydrogen atom positions for key active site residues involved in catalysis. As a result, the ability to characterize the hydrogen bonding network led to the determination of the specific proton transfer process that occurs during the reaction stages of the CTX-M β-lactamase mechanism. Overall, the results from this project demonstrate the effectiveness of using ultra high resolution x-ray crystallography as a useful tool to study enzyme catalysis as well as develop and discover novel inhibitors.

In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate. / Chemistry and Chemical Biology

In critically ill patients, the pathophysiology of sepsis can affect the interactions between the antibiotic, the bacteria and the patient, leading to potential therapeutic failure and the development of antibiotic resistance. It is well acknowledged that research that optimises antibiotic exposure will assist improvement of outcomes in this patient group. Although beta-lactam antibiotics, such as piperacillin and meropenem, are commonly selected for empiric therapy of sepsis, dosing is unlikely to be optimal. In patients without renal dysfunction, data suggests that disease-induced alterations to pharmacokinetic parameters result in low trough concentrations for significant periods within a dosing interval. Administration of these time-dependent antibiotics by continuous infusion has been suggested to improve the pharmacokinetic-pharmacodynamic profile. Knowledge of concentrations in the extracellular fluid of human tissue, which is the target site of most pathogens, is particularly instructive. Extracellular fluid concentrations can be determined using techniques such as microdialysis. Therefore, the principal aims of this Thesis were to determine the plasma and subcutaneous tissue pharmacokinetics of piperacillin and meropenem administered by bolus dosing and continuous infusion in critically ill patients with sepsis; and to use Monte Carlo simulations to compare the ability of different dosing strategies to achieve pharmacodynamic endpoints. This Thesis also sought to compare the clinical outcomes of bolus dosing and continuous infusion of a beta-lactam antibiotic, ceftriaxone, in a prospective randomised controlled trial and to perform a meta-analysis on clinical outcomes from other similar published studies. Finally, this Thesis aimed to systematically review the published literature to determine any correlation between antibiotic dosing and the development of antibiotic resistance. The results of the pharmacokinetic studies, using piperacillin and meropenem, indicate that beta-lactam distribution into subcutaneous tissue, in critically ill patients with sepsis, is less than that observed in previous studies in healthy volunteers yet superior to studies in patients with septic shock. This supports the notion that the peripheral concentration of drugs may be inversely related to the level of sickness severity. Administration by continuous infusion was found to maintain statistically significantly higher trough beta-lactam concentrations in both plasma and subcutaneous tissue. Further analysis of the plasma data using population pharmacokinetic modeling and Monte Carlo simulations described significant pharmacodynamic advantages for administering meropenem or piperacillin by continuous infusion to organisms with high minimum inhibitory concentrations. Given the documented pharmacodynamic advantages for administering beta-lactams by continuous infusion, a prospective randomized controlled clinical trial was conducted using the beta-lactam antibiotic ceftriaxone. In 57 critically ill patients, we found equivalence between continuous infusion and bolus dosing in the intention-to-treat analysis. However, our a priori analysis criteria, requiring patients receive at least 4-days antibiotic treatment, found significant clinical and bacteriological advantages for administration by continuous infusion. To further investigate any clinical differences between bolus dosing and continuous infusion of beta-lactam antibiotics, we performed a meta-analysis of all published studies. Our analysis of the 13 published prospective randomized controlled trials (846 hospitalised patients) showed equivalence of continuous infusion and bolus dosing. Possible confounders observed within, and between the studies, make interpretation of these results challenging. However, two large retrospective cohorts not included in the meta-analysis, found definitive clinical and bacteriological advantages suggesting further research may be appropriate. The possible relationship between antibiotic dosing, or exposures, on the development of resistance was investigated using a structured review of the published literature. Our analysis of relevant papers found a wealth of data describing increasing levels of resistance with sub-optimal antibiotic dosing, particularly for fluoroquinolone antibiotics, but also for other classes including beta-lactams. These results demonstrate the importance of optimizing antibiotic dosing to decrease the development of antibiotic susceptibility from sub-optimal dosing, particularly for critically ill patients who are likely to have low drug concentrations. The results of this Thesis, suggest that a large, prospective, multi-centre randomised controlled trial in critically ill patients with sepsis, is required to definitively determine the clinical utility of administration of beta-lactam antibiotics by continuous infusion.

Les hypersensibilités retardées (HSR) cutanées sont hétérogènes, à la fois par la nature des mécanismes impliqués (allergiques versus non allergiques) mais aussi par les différents degrés de sévérité rencontrés. Seules les HSR allergiques sont dues à la présence de lymphocytes T (LT), mal caractérisés chez l'homme. Le but de ce travail est d'étudier la contribution des LT CD8 cytotoxiques dans le développement et la sévérité des HSR cutanées chez l'homme, à partir de deux pathologies fréquentes : les toxidermies aux béta lactamines et l'eczéma allergique de contact à la para-phénylènediamine (PPD). Tout d'abord, la présence de LT spécifiques de médicament au sein des toxidermies aux béta lactamines a été recherchée in vivo et in vitro. Nous avons montré que les HSR sévères étaient plus souvent d'origine allergique que les HSR bénignes. Nous avons ensuite caractérisé le rôle des LT CD8 dans les HSR allergiques. Dans les toxidermies bénignes à l'amoxicilline, l'étude de la cinétique de recrutement des LT au niveau cutané ainsi que l'analyse des LT spécifiques du sang circulant ont permis de mettre en évidence le rôle essentiel des LT CD8 cytotoxiques dans l'initiation de ces réactions. Ensuite, dans l'eczéma allergique de contact à la PPD, un recrutement épidermique précoce des LT CD8 associés à des marqueurs de cytotoxicité, a été retrouvé, corrélé avec la sévérité des lésions. Ces résultats ont été confortés par ceux obtenus dans un modèle pré-clinique d'HSR allergique à la PPD chez la souris. En conclusion, ce travail montre que les LT CD8 cytotoxiques pourraient être les principales cellules effectrices des HSR cutanées allergiques chez l'homme / Skin delayed hypersensitivity (DHS) are heterogeneous, by the nature of the mechanisms involved (allergic versus non allergic) and also by their different degrees of severity. Only allergic DHS is due to T cells, poorly characterized in humans. The aim of this work is to study the contribution of cytotoxic CD8 T cells in the development and severity of skin DHS in humans, induced by two common diseases: cutaneous adverse drug reactions to beta lactam antibiotics and allergic contact dermatitis to para-phenylenediamine (PPD). First, the presence of drug specific T cells in cutaneous adverse drug reactions to beta lactams was investigated in vivo and in vitro. We showed that severe DHS were more often allergic than benign DHS. Then, we characterized the role of CD8 T cells in allergic DHS. In benign cutaneous adverse drug reactions to amoxicillin, the study of the kinetics of skin T cell recruitment as well as the analysis of circulating specific T cells highlight the essential role of cytotoxic CD8 T cells in the initiation phase of these reactions. In allergic contact dermatitis to PPD, early recruitment of epidermal CD8 T cells associated with cytotoxic markers was found, correlated with the severity of lesions. These results were supported by those obtained in a mouse model of allergic contact dermatitis to PPD. In conclusion, this work showed that cytotoxic CD8 T cells could be the main effector cells of allergic skin DHS in humans

Orientadores : Thales Rocha de Mattos Filho, Francisco Carlos Groppo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-06T06:37:26Z (GMT). No. of bitstreams: 1 Junqueira_MarcelodeSouza_D.pdf: 648615 bytes, checksum: 3b46ad75b56af2255d981b942d82fa8f (MD5) Previous issue date: 2006 / Resumo: O uso de antimicrobianos e de antiinflamatórios é prática comum na odontologia, embora haja escassez de trabalhos sobre seu uso simultâneo. Portanto, o objetivo deste trabalho foi avaliar, em ratos, os efeitos do diclofenaco sódico na absorção, concentração sérica e excreção da amoxicilina utilizando a técnica de perfusão tecidual e o método microbiológico. Foram utilizados 108 ratos Wistar machos (12 grupos, n= 9), com peso entre 150 e 200 g. Foram administrados aos animais: amoxicilina 25 mg/Kg (grupos: G1, D1, S1 e R1), diclofenaco sódico 2,5 mg/Kg + amoxicilina 25 mg/Kg (grupos: G2, D2, S2 e R2) e 1,0 mL de solução de cloreto de sódio a 0,9% (grupos: G3, D3, S3 e R3). As drogas foram administradas por injeção intraluminal aos animais dos grupos G1, G2, G3, D1, D2 e D3 e por via oral aos animais dos grupos S1, S2, S3, R1, R2 e R3. Foram avaliadas nos tempos de 15, 30, 45, 60, 120, 180, 240, 300 e 360 minutos as concentrações plasmáticas e urinárias de amoxicilina e a absorção gastrintestinal ¿in vitro¿ do antimicrobiano. O diclofenaco sódico causou uma redução significativa na absorção intestinal e um aumento na excreção renal do antimicrobiano em ratos, com conseqüente diminuição da sua concentração sérica. Portanto, em algumas situações clínicas, a eficácia da amoxicilina poderia ser prejudicada pela sua co-administração com o diclofenaco sódico / Abstract: The prescription of antibiotics associated to anti-inflammatory drugs is common in dentistry; however its effects have not been studied enough. Thus, the aim of this work was to evaluate the influence of sodium diclofenac on absorption, serum concentration and excretion of amoxicillin, in rats, by the microbiologic and tissue perfusion methods. The sample consisted of 108 male Wistar rats (12 groups, 9 rats each), weighing 150¿200 g. The animals were given: amoxicillin 25 mg/Kg (groups G1, D1, S1 and R1), sodium diclofenac 2.5 mg/Kg plus amoxicillin 25 mg/Kg (groups G2, D2, S2 and R2) and 1.0 mL of solution of sodium chloride 0.9% (groups G3, D3, S3 and R3). The animals in the groups G1, G2, G3, D1, D2 and D3 were administered drugs by intra-luminal injection and the animals in the groups S1, S2, S3, R1, R2 and R3 were administered drugs p.o. After 15, 30, 45, 60, 120, 180, 240, 300 and 360 minutes, were evaluated the blood and urine concentrations of amoxicillin and the ¿in vitro¿ absorption of the antibiotic. Sodium diclofenac had decreased the intestinal absorption, increased renal excretion and, consequently, decreased the serum concentration of the amoxicillin. Thus, sodium diclofenac could decrease the efficacy of amoxicillin under some clinical situations / Doutorado / Farmacologia, Anestesiologia e Terapeutica / Doutor em Odontologia

Orientador: Francisco Carlos Groppo / Dissertação (mestrado) Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-12T01:52:09Z (GMT). No. of bitstreams: 1 Simoes_RobertaPessoa_M.pdf: 1951478 bytes, checksum: fa986a3125f61b41fa59a6ea91b73746 (MD5) Previous issue date: 2000 / Resumo: O presente trabalho teve por objetivo observar o efeito da amoxicilina, do diclofenaco sódico e da associação de ambos sobre a infecção estafilocócica induzida em tecidos granulomatosos, em ratos. Também foram avaliadas as concentrações sérica e teciduais da amoxicilina, observando o efeito da associação do diclofenaco sódico sobre as mesmas. Trinta animais receberam implantes de quatro esponjas de poliuretana subcutâneamente no dorso e, após 14 dias, dois dos tecidos resultantes (posicionados caudalmente) receberam 0,5mL de um inóculo de 108 ufcjmL de S. aureus ATCC 25923. Dois dias após, os animais foram divididos em grupos de seis, os quais receberam uma dose única de amoxicilina 50mgjkgjvo (grupo 1), amoxicilina 25 mg/kg/vo (grup02), diclofenaco sódico 2,5mg/kg/im (grupo 3), diclofenaco sódico 2,5mg/kg/im + amoxicilina 50mg/kg/vo (grupo 4) e soro fisiológico 1mLjanimal/vo (controle). Após 6h, dois tecidos granulomatosos (posicionados em direção à cabeça) e 10 _L de soro sangüíneo foram obtidos de cada animal, os quais foram dispostos em diferentes placas com MHA semeado com 108 ufcjmL de S. aureus ATCC 25923. Após incubação, foram medidos os halos de inibição proporcionados pelos tecidos granulomatosos e pelo soro sangüíneo. Dez microlitros, provenientes dos tecidos infectados após dispersão com auxílio de sonicador em tubos de ensaio contendo 10m L de NaCI foram distribuídos em placas contendo ágar salt manitol; permitiram a contagem do número de microrganismos em cada grupo. Os resultados mostraram concentrações teciduais de amoxicilina de 6,6 I-Ig/g para o grupo 1; 2,8I-1g/g para o grupo 2 e 0,8 I-Ig/g para o grupo 4. As concentrações séricas do antimicrobiano encontradas foram 11,6 I-Ig/mL para o grupo 1; 5,4I-1g/mL para o grupo 2 e 1,3I-1g/mL para o grupo 4. Não foram observadas diferenças estatisticamente significantes entre o número de microrganismos dos grupos 1, 2 e 4. Entretanto, estes foram significativamente diferentes do controle e do grupo 3, os quais foram diferentes entre si. Foi concluído que, embora o diclofenaco sódico (grupo 4) tenha diminuído a concentração tecidual da amoxicilina, a concentração resultante foi suficiente para permitir uma redução do número de microrganismos viáveis similar à observada nos grupos 1 e 2 / Abstract: The aim of this work was to observe the effect of the amoxicillin, sodium diclofenac and amoxicillin plus sodium diclofenac against staphylococcal infection in granulomatous tissues. Four polyurethane sponges placed under the back skin of thirty rats induced these tissues. After 14 days two tissues (tall position) received 0.5 mL of 108 ufc of S. aureus ATCC 25923/mL. Two days after, the animais were divided into five groups of six each. Group 1 received an only dose of amoxicillin 50mgjkgjpo, group 2 received amoxicillin 25mgjkgjpo, group 3 received sodium diclofenac 2.5mgjkgjim, group 4 received sodium diclofenac 2.5mg/kg/im plus amoxicillin 50mg/kg/po, and group 5 (control group) received NaCI 1mLjpo. After six hours of drug administration, two tissues of each animal were removed. Blood was collected from cervical plexus and centrifuged. Then, 10j.JL of serum were placed on paper disks. These disks and tissues were placed on dishes with Mueller Hinton agar inoculated with 108 ufc of Staphylococcus aureus/mL The dishes were incubated over 18 hours, and the inhibition zones were measured. The infected granulomatous tissues were placed in 10mL of NaCI, and dispersed by sonic system. Ten microliters of this suspension were spread on salt manitol agar dishes. The resulting microorganisms were counted after the incubation. Results showed tissue concentrations of amoxicillin of 6.6 ug/g for group 1; 2.8ug/g for group 2, and 0.8 ug/g for group 4. Serum concentration of amoxicillin showed 11.6 ug/mL for group 1; 5.4ug/mL for group 2, and 1.3ug/mL for group 4. Statistically significant differences among the number of microorganisms groups 1, 2, and 4 were not observed. However, these previous groups were statistically different from groups 3 and 5, which were statistically different from each other. It was concluded that, although sodium diclofenac (group 4) reduced amoxicillin concentration in the tissue, the resulting concentration was enough to reduce the count of microorganism. Also, the number of microorganisms of group 4 was similar to the one observed in groups 1 and 2 / Mestrado / Farmacologia, Anestesiologia e Terapeutica / Mestre em Odontologia