No description available.

Ph.D.

The threat of antibiotic resistance and the global rise of pan-resistant bacteria is a serious concern at present. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is frequently associated with multi and pan-drug resistant infections. This research delves into the mechanism of resistance to a class of drugs known as the β-lactams. AmpC β-lactamase encoded chromosomally in P. aeruginosa is one of the predominant causes of resistance to many β-lactams. Previous research on this pathway identified the AmpC regulatory protein - AmpR and elaborated on its regulon in P. aeruginosa. In this dissertation, further investigation in the mechanisms associated with AmpR regulation of AmpC and its connection with the cell-wall recycling pathway is explored. Cell-wall recycling, a common phenomenon in both Gram-positive and negative bacteria is investigated in some detail in P. aeruginosa for the first time. The identity of the cell-wall recycling products or muropeptides in P. aeruginosa is elucidated. Around 20 distinct muropeptides were identified through liquid chromatography/mass spectrometry analyses of bacterial extracts. Furthermore, iv the muropeptide effector of AmpR that is instrumental in the activation of this transcription factor is identified. The role of two permeases AmpG and AmpP in antibiotic resistance and cell-wall recycling are also investigated by comparing antibiotic susceptibility and muropeptide profile of the isogenic mutants of ampG and ampP with the wild-type PAO1. Along with investigating permeases, the role of a putative N-acetylglucosaminidase FlgJ is also investigated. Finally, keeping in mind the broad role of AmpR in regulating P. aeruginosa virulence and antibiotic resistance, we try to identify small -molecule inhibitors for AmpR. In our effort to identify inhibitors, a novel reporter-based screening assay is developed. In summary, this dissertation increases our understanding of cell-wall recycling and mechanisms of β-lactam resistance and attempts at establishing novel-antibacterial targets and inhibitors.

Pseudomonas aeruginosa is a versatile Gram-negative opportunistic pathogen notorious for its ability to chronically colonize and deteriorate the pulmonary function of the cystic fibrosis lung. It exhibits high resistance to beta-lactam antibiotics, including cephalosporins and monobactams, via induction of their chromosomally encoded AmpC beta-lactamase. Regulation of ampC expression is coupled to the bacterial cell wall recycling pathway by the activity of NagZ, a glycosidase that produces 1,6-anhydroMurNAc-tri-(or penta-) peptides from internalized peptidoglycan metabolites. During beta-lactam therapy, this tripeptide rapidly concentrates in the bacterial cytosol to levels sufficient for it to bind and activate AmpR, the transcriptional activator of ampC. P. aeruginosa also encodes three ampD genes, each expressing an N-acetylmuramyl-L-amidase that cleaves the peptide stems from 1,6-anhydroMurNAc or GlcNAc 1,6-anhydroMurNAc. AmpD thus suppresses 1,6-anhydroMurNAc-peptide accumulation and moderates ampC induction. Selection of AmpD null mutants during therapy thus causes chronic hyperproduction of beta-lactamase, presumably from an increase in NagZ product, and have been identified in P. aeruginosa strains isolated from chronically infected CF patients. Mutants harboring an inactivated nagZ gene in a wild-type P. aeruginosa background were isolated and were found to have increased antibiotic susceptibility to antipseudomonal beta-lactams. Inactivating nagZ in a triple ampD mutant substantially decreased the expression of ampC and rendered these high-level resistant strains susceptible to antipseudomonal beta-lactams at wild-type strain levels. This brings the susceptibility of the P. aeruginosa strains down to the beta-lactam therapy range accepted by CLSI for use in cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa infections. To assess whether P. aeruginosa expresses more than one N-acetyl-beta-glucosaminidase that could contribute to the production of the activating tripeptide, residual activity assays were conducted on nagZ deficient mutants. Mutants were devoid of activity so it was concluded that P. aeruginosa expresses only the one N-acetyl-beta-glucosaminidase in study, NagZ. Complementation studies using the wild type nagZ gene restored the wild type phenotypes, particularly evident in the triple ampD null mutants. These findings suggest that NagZ activity is required for ampC induction, and that an intricate balance exists between NagZ and AmpD activity to regulate the concentration of the inducer molecule 1,6-anhydroMurNAc-tripeptide.

Pseudomonas aeruginosa is a versatile Gram-negative opportunistic pathogen notorious for its ability to chronically colonize and deteriorate the pulmonary function of the cystic fibrosis lung. It exhibits high resistance to beta-lactam antibiotics, including cephalosporins and monobactams, via induction of their chromosomally encoded AmpC beta-lactamase. Regulation of ampC expression is coupled to the bacterial cell wall recycling pathway by the activity of NagZ, a glycosidase that produces 1,6-anhydroMurNAc-tri-(or penta-) peptides from internalized peptidoglycan metabolites. During beta-lactam therapy, this tripeptide rapidly concentrates in the bacterial cytosol to levels sufficient for it to bind and activate AmpR, the transcriptional activator of ampC. P. aeruginosa also encodes three ampD genes, each expressing an N-acetylmuramyl-L-amidase that cleaves the peptide stems from 1,6-anhydroMurNAc or GlcNAc 1,6-anhydroMurNAc. AmpD thus suppresses 1,6-anhydroMurNAc-peptide accumulation and moderates ampC induction. Selection of AmpD null mutants during therapy thus causes chronic hyperproduction of beta-lactamase, presumably from an increase in NagZ product, and have been identified in P. aeruginosa strains isolated from chronically infected CF patients. Mutants harboring an inactivated nagZ gene in a wild-type P. aeruginosa background were isolated and were found to have increased antibiotic susceptibility to antipseudomonal beta-lactams. Inactivating nagZ in a triple ampD mutant substantially decreased the expression of ampC and rendered these high-level resistant strains susceptible to antipseudomonal beta-lactams at wild-type strain levels. This brings the susceptibility of the P. aeruginosa strains down to the beta-lactam therapy range accepted by CLSI for use in cystic fibrosis patients suffering from chronic Pseudomonas aeruginosa infections. To assess whether P. aeruginosa expresses more than one N-acetyl-beta-glucosaminidase that could contribute to the production of the activating tripeptide, residual activity assays were conducted on nagZ deficient mutants. Mutants were devoid of activity so it was concluded that P. aeruginosa expresses only the one N-acetyl-beta-glucosaminidase in study, NagZ. Complementation studies using the wild type nagZ gene restored the wild type phenotypes, particularly evident in the triple ampD null mutants. These findings suggest that NagZ activity is required for ampC induction, and that an intricate balance exists between NagZ and AmpD activity to regulate the concentration of the inducer molecule 1,6-anhydroMurNAc-tripeptide.

Introduction :La prévalence de l’obésité, parmi les patients hospitalisés, est en augmentation. Les patients obèses présentent un risque accru de développer des infections nosocomiales par rapport aux individus non-obèses. Parmi les antibiotiques, la classe des β-lactames est la plus utilisée à l’hôpital tant en prophylaxie chirurgicale que pour le traitement des infections. Les recommandations de doses en vigueur sont basées sur des études pharmacocinétiques (PK) effectuées chez des patients non-obèses. Cependant, les patients obèses présentent des altérations physiologiques qui pourraient théoriquement être à l’origine de modifications des paramètres PK de ces antibiotiques. L’utilisation de doses standards d’antibiotiques pourrait avoir comme conséquence des concentrations sériques inadéquates chez ces patients; elles peuvent, dès lors, conduire à des échecs thérapeutiques et/ou favoriser l’émergence de souches résistantes. Objectifs: Ce travail de thèse a pour but d’approfondir les connaissances sur la PK des β-lactames chez les patients obèses présentant des scores de sévérité clinique différents. Patients et Méthodes: Quatre études PK ont été réalisées chez les patients obèses :1) une étude prospective de patients obèses, et non-obèses, ayant reçu de la céfazoline (CFZ) en prophylaxie de chirurgie digestive, 2) une étude préliminaire, prospective incluant uniquement des patients obèses traités pour des infections non sévères par l’un des 4 β-lactames à large spectre (pipéracilline-tazobactam (TZP), ceftazidime/céfépime (CEF), et méropénem (MEM)), 3) une étude prospective, avec groupe contrôle de patients non-obèses, étudiant la PK du TZP donné en traitement d’infections non sévères et 4) une étude rétrospective cas-témoin évaluant la PK de 4 β-lactames à large spectre (TZP, CEF, ou MEM) donnés pour le traitement d’infections sévères aux soins intensifs. Les taux sériques de ces antibiotiques ont été mesurés par chromatographie liquide et corrélés à plusieurs paramètres démographiques, biologiques et hémodynamiques. Résultats: Etude sur CFZ :Soixante-trois patients ont participé à l’étude: parmi ceux-ci, 43 avaient un index de masse corporelle (IMC) ≥ 35 kg/m2, et 22 un poids total ≥ 120 kg. Sur l’ensemble des patients, les concentrations sériques de CFZ étaient adéquates chez 100% des patients à 180 minutes (T180) et chez 64% (40/63) à 240 minutes (T240) après le début de l’infusion de CFZ. Il n’y avait pas de différence significative entre le pourcentage des patients avec des taux sériques adéquats à T240, quel que soit le paramètre utilisé pour la comparaison (IMC ≥ 35 kg/m2 versus IMC < 35 kg/m2, ou poids total ≥ 120 kg versus poids total < 120 kg). Etude préliminaire sur TZP, CEF et MEM: Cinquante-six patients obèses ont été inclus dans l’étude ;14 ont reçu du MEM, 31 du TZP et 11 du CEF. Parmi ces patients, les concentrations sériques adéquates pour traiter une infection à P. aeruginosa ont été atteintes chez 93% des patients traités par MEM, 68% des patients traités par TZP, et 73% des patients traités par CEF. Le volume de distribution (VD) et la clearance totale (CL) des β-lactames étaient augmentés par rapport à des valeurs rapportées dans la littérature chez les patients non-obèses. En effet, plus de 25% des patients présentaient une clearance en créatinine (CrCl) mesurée supérieure à 150 ml/minute, ce facteur étant identifié comme facteur de risque de concentrations sériques insuffisantes. Etude sur TZP: Trente-deux patients (14 non-obèses et 18 obèses) ont été inclus dans l’étude et ont reçu les doses standards de TZP. Une augmentation du VD (25.4 ± 5.6L vs. 21.0 ± 3.9 L, p= 0.018) et de la CL (18.1 ± 4.7 vs. 13.5 ± 40.1, p= 0.007) a été observé chez les patients obèses par rapport aux non-obèses conduisant à des concentrations statistiquement inférieures de TZP libre par rapport aux patients non-obèses. De même, malgré des valeurs de créatinine sérique semblables (0.8 ± 0.3 versus 0.9 ± 0.3 mg/dl, p=0.26), la CrCl mesurée sur des urines de 24h, était significativement plus élévée chez les obèses par rapport aux non-obèses (128 ± 58 vs. 79 ± 26 ml/min, p=0.006). Etude rétrospective cas-témoin sur TZP, CEF, et MEM aux soins intensifs: Quarante-neuf patients obèses et 59 patients non-obèses ont été inclus. Soixante-huit monitoring thérapeutiques ont été effectués chez les patients obèses et comparés à 68 monitoring thérapeutiques effectués chez les patients non-obèses. Après administration des doses standards des β-lactames, 1/3 des patients obèses avait des concentrations sériques insuffisantes pour traiter des infections à P. aeruginosa, et 1/4 de ces patients avait des concentrations sériques excessives, potentiellement toxiques. Par contre, aucune différence statistiquement significative n’a été observée entre les concentrations sériques des β-lactames des patients obèses et non-obèses. Conclusions :Lors de la prophylaxie chirurgicale, l’administration des doses standards de CFZ a montré des concentrations sériques insuffisantes pour assurer une couverture prophylactique adéquate en cas d’intervention chirugicale d’une durée supérieure à 180 minutes. Ni le poids total, ni l’IMC n’a pu identifier les patients qui nécessiteront une dose plus élevée de CFZ. De même, les doses standards des β-lactames à large spectre se sont avérées insuffisantes dans le traitement des infections non sévères et sévères dues à P. aeruginosa chez les patients obèses. En cas d’infections peu sévères, les concentrations sériques, chez ces patients, étaient significativement moindres que chez les patients non-obèses ;ceci étant lié à une augmentation du VD et de la CL des β-lactames. Un facteur de risque majeur retrouvé était une CrCl augmentée chez les patients obèses. Chez les patients avec infections sévères, la PK des β-lactames n’était par contre pas significativement différente entre les patients obèses et non-obèses. Ceci probablement à cause des perturbations déjà importantes de la PK des β-lactames liées à la séverité du sepsis. En conclusion, ces études identifient des perturbations de la PK des β-lactames chez les patients obèses. Parmi celles-ci, l’augmentation de la clearance rénale est un élement déterminant dans l’inadéquation des concentrations sériques. La situation est plus complexe chez les patients obèses aux soins intensifs où d’autres facteurs peuvent encore s’associer et perturber la PK des β-lactames. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

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.

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.

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.