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.

A large part of the antibiotics consumed ends up in wastewater, and in the wastewater the antibiotics may exert selective pressure for or maintain resistance among microorganisms. Antibiotic resistant bacteria and genes encoding antibiotic resistance are commonly detected in wastewater, often at higher rates and concentrations compared to surface water. Wastewater can also provide favourable conditions for the growth of a diverse bacterial community, which constitutes a basis for the selection and spread of antibiotic resistance. Therefore, wastewater treatment plants have been suggested to play a role in the dissemination and development of antibiotic resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) is a large problem worldwide as a nosocomial pathogen, but knowledge is limited about occurrence in non-clinical environments, such as wastewater, and what role wastewater plays in dissemination and development of MRSA.   In this thesis we investigated the occurrence of MRSA in a full-scale wastewater treatment plant (WWTP). We also investigated the concentration of genes encoding resistance to aminoglycosides (aac(6’)-Ie+aph(2’’)), β-lactam antibiotics (mecA) and tetracyclines (tetA and tetB) in three wastewater-associated environments: (1) soil from an overland flow area treating landfill leachates, (2) biofilm from a municipal wastewater treatment plant, and (3) sludge from a hospital wastewater pipeline. In addition, concentrations of mecA, tetA and tetB were investigated over the treatment process in the WWTP. These investigations were performed to determine how the prevalence and concentration of MRSA and the antibiotic resistence genes are affected in wastewater and wastewater treatment processes over time. The occurrence of MRSA was investigated by cultivation and a commercially available real-time PCR assay. In order to determine concentrations of the genes aac(6’)-Ie+aph(2’’), mecA, tetA and tetB in wastewater we developed a LUXTM real-time PCR assay for each gene.   Using cultivation and real-time PCR we could for the first time describe the occurrence of MRSA in wastewater and show that it had a stable occurrence over time in a WWTP. MRSA could mainly be detected in the early treatment steps in the WWTP, and the wastewater treatment process reduced the number and diversity of cultivated MRSA. However, our results also indicate that the treatment process selects for strains with more extensive resistance and possibly higher virulence. The isolated wastewater MRSA strains were shown to have a close genetic relationship to clinical isolates, and no specific wastewater lineages could be detected, indicating that they are a reflection of carriage in the community. Taken together, these data indicate that wastewater may be a potential reservoir for MRSA and that MRSA are more prevalent in wastewater than was previously thought.   The real-time PCR assays, for aac(6’)-Ie+aph(2’’), mecA, tetA, and tetB that we developed, were shown to be sensitive, fast, and reproducible methods for detection and quantification of these genes in wastewater environments. The highest concentrations of all genes were observed in the hospital pipeline, and the lowest in the overland flow system, with tetA and aac(6´)-Ie+aph(2´´) detected in all three environments. In the full-scale WWTP, we continuously detected mecA, tetA and tetB over the treatment process and over time. In addition, it was shown that the treatment process reduces concentrations of all three genes. The data presented in this thesis also indicate that the reduction for all three genes may be connected to the removal of biomass, and in the reduction of tetA and tetB, sedimentation and precipitation appear to play an important role.

The Turos laboratory has been working with N-thiolated β-lactams for years trying to understand the mode of action and structural features it needs to have biological activity. Over the years new data has shown promising inhibitory activity against various microbes. In this dissertation, a review of the vast amount of work carried out on N-thiolated β-lactams in Turos laboratory has been done and their novelty, in terms of structure and mechanism has been discussed. A complete outline of our work in the discovery and ongoing development of these compounds, starting from our initial, unexpected finding of antimicrobial activity for one of the lead compounds, to a more complete understanding of their chemical and biological mode of action and potential utility as antibacterial compounds, has been provided. Previous researches by graduate students in the Turos laboratory have shown that N-thiolated β-lactams targets Type II Fatty Acid Synthesis (FAS). In process of understanding this further, other FAS inhibiting antibiotics like Triclosan were compared to our lactams by adding excess of exogenous fatty acids. Results revealed vast differences in the MIC value of triclosan and N-thiolated β-lactams, giving an idea that there might be a different mode of action or a different target altogether. The third chapter discusses the study of attaching hydrophilic C3 side chains like amino acids and carbohydrates on N-thiolated β-lactams while studying the influence of microbiological activity. From the study it was found that the lengthening of the side chain halts the inhibitory activity regardless of whether the side chain contains unsaturation or branching. Results showed that polar groups were not well tolerated and the inhibitory activity goes down regardless of polarity. Finally, research on dual-action antibiotics was discussed. Antibiotics cause continuous bacterial resistance and in this aspect use of two drugs with different mode of action can call for reduction of the resistance. Herein, N-acyl ciprofloxacin and N-thiolated β-lactams were connected together via an ester linkage. Six new hybrid compounds have been synthesized successfully and tested against E. faecium, K. pneumoniae, A. baumannii, P. aeruginosa, and E. cloacae.

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

Darbo tikslas: susintetinti 3-amino rodanino pagrindą turinčius junginius ir nustatyti jų antimikrobinį aktyvumą. Uždaviniai: 1) Sintezuoti 3-amiono-2-tiokso-4-tiazolidinono darinius; 2) Įvertinti gautų junginių antimikrobinį aktyvumą, naudojant in silico PASS online. 3) Įvertinti gautų junginių antimikrobinį aktyvumą in vitro standžioje terpėje; 4) Susieti junginių struktūrą su antimikrobiniu aktyvumu. Tyrimo metodai: sintetintų junginių antimikrobinis poveikis buvo prognozuojamas „PASS online“ programos pagalba. Antimikrobinis aktyvumas tirtas in vitro, naudojant Miulerio – Hintono agarą. Atlikti ESC tyrimai junginių grynumui nustatyti ir FT-IR tyrimai junginių struktūrai patvirtinti. Tyrimo rezultatai: 3-aminorodanino pagrindu buvo sintetinti 7 junginiai, turintys Šifo bazės struktūrą ir 1 turintis azetidino funkcinę grupę. Atlikti junginių lydymosi temperatūros nustatymo tyrimai, grynumas nustatytas naudojant ESC, junginių struktūra patvirtinta naudojant FTIR metodiką. PASS online programos pagalba buvo nustatyta, kad dauguma junginių turį potencialų antimikrobinį poveikį. Antimikrobinio poveikio in vitro tyrimo metu buvo nustatyta, kad plačiausią poveikį turi junginys VS-3, turintis p-chlorbenzaldehido pakaitą, ir VS-2, turintis p-brombenzaldehido pakaitą. Taip pat nustatyta, kad ribotu antimikrobiniu aktyvumu pasižymėjo junginiai VS-6, VS-7, VS-8. Įterpus azetidino struktūrą į VS-1 junginį, aktyvumas padidėjo Candida grybelių atžvilgiu. Išvados: iš sintetintų junginių... [toliau žr. visą tekstą] / The aim of the research: Synthesis of 3-aminorhodanine derivatives, antimicrobial evaluation. Methods: Antimicrobial activity of synthesized 3-aminorhodanine derivatives was predicted with the help of PASS online program. Factual antimicrobial activity of compounds was determined using in vitro agar method. The purity and structure of compounds was analyzed with ESC and FT-IR methods accordingly. Results: 7 Schiff base derivatives and one azetidine compound were synthesized on the base of 3-aminorhodonine. The melting points were identified, the purity of compounds were determined using ESC, the compound structure was identified using FTIR. Analyzing the compounds with PASS online showed that most of the compounds possess the potential of antimicrobial activity. In vitro antimicrobial studies showed that the widest spectrum of activity was possessed by compound VS-3, which had the 4-chlorbenzaldehyde moiety, and VS-2 with 4-bromobenzaldehyde moiety. Compounds VS-6, VS-7 and VS-8 showed limited antimicrobial activity. Modifying VS-1 structure by adding an azotidine moiety increased the drugs effect on Candida fungi family. Conclusion: amongst all the synthesized compounds 3-(4-chlorophenyl)methyleneamino]-2-thioxo-thiazolidin-4-one and 3-(4-bromphenyl)methyleneamino]-2-thioxo-thiazolidin-4-one showed most antimicrobial promise.

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.

Introdução: estudos de farmacocinética/farmacodinâmica (FC/FD) observaram que a probabilidade de alcançar o alvo FC/FD é maior quando a dose do beta-lactâmico é otimizada. Porém, poucos estudos demonstraram que a otimização de dose resulta em melhores desfechos clínicos. Métodos: Fatores associados com mortalidade em 30 dias foram avaliados em 100 pacientes com bacteremia por enterobactérias tratados com cefepime, piperacilina-tazobactam ou meropenem em um estudo de coorte prospectivo. Posologia dos antibióticos foi classificada em otimizada, apropriada e potencialmente inapropriada. Resultados: Cinquenta e dois (52%) episódios foram causados por E. coli, seguidos por K. pneumoniae (10%). Dezesseis (16%) episódios foram causados por isolados resistentes à cefepime e não houve nenhum caso de resistência à carbapenêmicos. A maioria dos isolados apresentou concentrações inibitórias mínimas (CIMs) baixas para as drogas prescritas (≤0.5, ≤1.0, ≤1/4 mg/L, para meropenem, cefepime e piperacilina-tazobactam respectivamente). Cefepime foi o antimicrobiano mais frequentemente prescrito para tratamento empírico e definitivo. Terapia otimizada foi observada em 42% dos pacientes e terapia adequada em 58%. A mortalidade em 30 dias foi 37%. Escore de Pitt, Charlson e apresentação com sepse severa foram independentemente associados à mortalidade. Não houve diferença em mortalidade entre os pacientes que receberam terapia otimizada e terapia adequada. Conclusões: os resultados mostram que a otimização das doses de cefepime, piperacilina-tazobactam e meropenem não teve impacto em mortalidade Em pacientes recebendo terapia empírica apropriada para bacteremias por Enterobacteriaceae. Este achado pode ser devido aos baixos valores de CIM apresentados pelas bactéria. Comorbidades e a severidade da apresentação são fatores associados à pior evolução. / Background: Pharmacokinetic/pharmacodynamic (PK/PD) studies have shown that the probability of PK/PD target attainment is higher when optimized dosage regimes of beta-lactams are employed, but few studies have shown clinical benefit of such strategy. Methods: We investigated the effect of dosage regimes in 30-day mortality in 100 patients with Enterobacteriaceae bloodstream infections (BSIs) receiving appropriate empirical therapy with cefepime, piperacillin-tazobactam or meropenem. Posology of antibiotic was classified as optimized, adequate and possibly inadequate. Results: Most isolates presented relatively low MIC for the prescribed drugs (≤0.5, ≤1.0, ≤1/4 μg/mL, for meropenem, cefepime and piperacillin-tazobactam respectively). Cefepime was the most common prescribed drug for empirical and main therapy. Optimized posology was prescribed in 42% of patients and adequate in 58%. The overall 30-day mortality was 27.0%. Charlson score, Pitt score and presentation with severe sepsis were independently associated with the 30-day mortality. Patients receiving optimized dosage regime presented no distinct 30-day mortality of those with adequate ones (25.0% versus 28.3%, P=0.89), even after inclusion in multivariate model. Conclusion: Our results suggest that dosage regime optimization of cefepime, piperacillin-tazobactam and meropenem may have no effect on mortality when infecting bacteria with low MICs for these drugs. In patients receiving appropriate empirical therapy for Enterobacteriaceae BSI, baseline comorbidity is an independent predictor of death and the severity of BSI presentation is also significantly associated with this outcome is such patients. Studies in population with higher MIC heterogeneity are required to evaluate the role of optimized doses in clinical setting.

Objetivos: Avaliar a adequabilidade do modelo farmacocinético-farmacodinâmico (PK-PD) (NOLTING et al., 1996b) para modelar o efeito bactericida da piperacilina (PIP) em ratos Wistar infectados experimentalmente com Escherichia coli ATCC 25922. Metodologia: Experimentos de Farmacocinética: Determinou-se as concentrações plasmáticas totais e livres teciduais de PIP, através de microdiálise (MD), após administração de 240 mg/kg i.v. bolus a ratos Wistar granulocitopênicos (ciclofosfamida) infectados no músculo esquelético (105 UFC/mL) com E. coli. As amostras de plasma e de MD foram analisadas por CLAE. As sondas de MD foram calibradas por retrodiálise. Experimentos de Farmacodinâmica: Os animais imunodeprimidos e infectados foram tratados com PIP nas doses de 120 ou 240 mg/kg, em intervalos de 4/4, 6/6 e 8/8 horas por 24 h. Em tempos pré-determinados, os animais foram sacrificados (n = 3/tempo), o músculo infectado foi retirado, homogeneizado e o número de UFC/mL foi determinado em placas de ágar-sangue, após diluições sucessivas. Um grupo não tratado foi utilizado como controle. Modelagem PK-PD: A partir dos dados farmacocinéticos e farmacodinâmicos obtidos, avaliou-se efeito de morte bacteriana em função do tempo com o auxílio do programa de regressão não-linear SCIENTIST® v.2.0. Resultados e Discussão: Os parâmetros farmacocinéticos após a administração de PIP (240 mg/kg) foram t½ de 40 ± 8 min; CL de 0,46 ± 0,021 (L/h/kg) e um Vdss de 0,30 ± 0,06 L/kg. O perfil de PIP livre tecidual foi previsto a partir dos parâmetros plasmáticos utilizando ajuste simultâneo dos dados de plasma e tecido e um fator de proporcionalidade de 0,342 ± 0,101. Os parâmetros do modelo PK-PD obtidos foram: EC50 de 1,31 ± 0,27 μg/mL e kmax 1,39 ± 0,20 h-1. Os valores dos parâmetros da modelagem PK-PD obtidos in vivo diferiram dos descritos na literatura para o mesmo antibiótico e bactéria quando simulados in vitro. Conclusões: O modelo Emax-modificado descreveu os perfis de crescimento e morte bacteriana em função do tempo obtidos nas diferentes posologias testadas sendo adequado para modelagem PK-PD da piperacilina nas condições experimentais investigadas. / Purpose: The objective of this study was to model the killing effect of a β-lactam antibiotic, piperacillin (PIP), in neutropenic and E. coli ATCC 25922 infected rats after different dosing regimens using a modified Emax PK-PD model. Methodology: Pharmacokinetic studies: Total plasma and free tissue concentrations of PIP, determined by microdialysis, were investigated after i.v. bolus of 240 mg/kg of the drug to immunecompromised (cyclophosphamide) and E. coli infected (107 CFU) Wistar rats. Microdialysis probes recoveries were determined by retrodialysis. Plasma and tissue samples were analyzed by HPLC. Pharmacodynamic studies: The infected rats were treated with iv bolus PIP 120 mg/kg or 240 mg/kg q8h, q6h, q4h. Three animals were sacrificed at predetermined times up to 24 hours. The infected muscle was removed, homogenized and the number of CFU/mL was determined by plate counting after 24 hours of incubation at 37ºC. A control group without treatment was used. PK-PD modeling: PIP killing effect as a function of time was fitted using the Emax-modified model with the aid of a non-linear regression computer program SCIENTIST® v.2.0. Results and Discussion: The pharmacokinetic parameters determined for PIP 240 mg/kg iv bolus were: t½ of 40 ± 8 min; CL of 0.46 ± 0.021 (L/h/kg) and Vdss of 0.30 ± 0.06 L/kg. Piperacillin free tissue levels were predicted using plasma data ina a simultaneous fitting with a proportionality factor of 0.342 ± 0.101. The parameters derived from PK-PD modeling were: bacterial killing rate (kmax) of 1.39 ± 0.20 h-1 concentration to produce 50% of de maximum effect (EC50) of 1.31 ± 0.27 μg/mL. The PK-PD parameters determined in vivo were different from those reported for the same bacteria and drug in vitro. Conclusions: The Emax model adequately described PIP antibacterial effect in animals for the different dosing regimens investigated.

Objetivos: Avaliar a adequabilidade do modelo farmacocinético-farmacodinâmico (PK-PD) (NOLTING et al., 1996b) para modelar o efeito bactericida da piperacilina (PIP) em ratos Wistar infectados experimentalmente com Escherichia coli ATCC 25922. Metodologia: Experimentos de Farmacocinética: Determinou-se as concentrações plasmáticas totais e livres teciduais de PIP, através de microdiálise (MD), após administração de 240 mg/kg i.v. bolus a ratos Wistar granulocitopênicos (ciclofosfamida) infectados no músculo esquelético (105 UFC/mL) com E. coli. As amostras de plasma e de MD foram analisadas por CLAE. As sondas de MD foram calibradas por retrodiálise. Experimentos de Farmacodinâmica: Os animais imunodeprimidos e infectados foram tratados com PIP nas doses de 120 ou 240 mg/kg, em intervalos de 4/4, 6/6 e 8/8 horas por 24 h. Em tempos pré-determinados, os animais foram sacrificados (n = 3/tempo), o músculo infectado foi retirado, homogeneizado e o número de UFC/mL foi determinado em placas de ágar-sangue, após diluições sucessivas. Um grupo não tratado foi utilizado como controle. Modelagem PK-PD: A partir dos dados farmacocinéticos e farmacodinâmicos obtidos, avaliou-se efeito de morte bacteriana em função do tempo com o auxílio do programa de regressão não-linear SCIENTIST® v.2.0. Resultados e Discussão: Os parâmetros farmacocinéticos após a administração de PIP (240 mg/kg) foram t½ de 40 ± 8 min; CL de 0,46 ± 0,021 (L/h/kg) e um Vdss de 0,30 ± 0,06 L/kg. O perfil de PIP livre tecidual foi previsto a partir dos parâmetros plasmáticos utilizando ajuste simultâneo dos dados de plasma e tecido e um fator de proporcionalidade de 0,342 ± 0,101. Os parâmetros do modelo PK-PD obtidos foram: EC50 de 1,31 ± 0,27 μg/mL e kmax 1,39 ± 0,20 h-1. Os valores dos parâmetros da modelagem PK-PD obtidos in vivo diferiram dos descritos na literatura para o mesmo antibiótico e bactéria quando simulados in vitro. Conclusões: O modelo Emax-modificado descreveu os perfis de crescimento e morte bacteriana em função do tempo obtidos nas diferentes posologias testadas sendo adequado para modelagem PK-PD da piperacilina nas condições experimentais investigadas. / Purpose: The objective of this study was to model the killing effect of a β-lactam antibiotic, piperacillin (PIP), in neutropenic and E. coli ATCC 25922 infected rats after different dosing regimens using a modified Emax PK-PD model. Methodology: Pharmacokinetic studies: Total plasma and free tissue concentrations of PIP, determined by microdialysis, were investigated after i.v. bolus of 240 mg/kg of the drug to immunecompromised (cyclophosphamide) and E. coli infected (107 CFU) Wistar rats. Microdialysis probes recoveries were determined by retrodialysis. Plasma and tissue samples were analyzed by HPLC. Pharmacodynamic studies: The infected rats were treated with iv bolus PIP 120 mg/kg or 240 mg/kg q8h, q6h, q4h. Three animals were sacrificed at predetermined times up to 24 hours. The infected muscle was removed, homogenized and the number of CFU/mL was determined by plate counting after 24 hours of incubation at 37ºC. A control group without treatment was used. PK-PD modeling: PIP killing effect as a function of time was fitted using the Emax-modified model with the aid of a non-linear regression computer program SCIENTIST® v.2.0. Results and Discussion: The pharmacokinetic parameters determined for PIP 240 mg/kg iv bolus were: t½ of 40 ± 8 min; CL of 0.46 ± 0.021 (L/h/kg) and Vdss of 0.30 ± 0.06 L/kg. Piperacillin free tissue levels were predicted using plasma data ina a simultaneous fitting with a proportionality factor of 0.342 ± 0.101. The parameters derived from PK-PD modeling were: bacterial killing rate (kmax) of 1.39 ± 0.20 h-1 concentration to produce 50% of de maximum effect (EC50) of 1.31 ± 0.27 μg/mL. The PK-PD parameters determined in vivo were different from those reported for the same bacteria and drug in vitro. Conclusions: The Emax model adequately described PIP antibacterial effect in animals for the different dosing regimens investigated.