<p>This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium <i>Finegoldia magna</i>. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule.</p><p>Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.</p>

This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule. Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.

Neste trabalho, foram desenvolvidos e validados métodos qualitativos e quantitativos para o controle de qualidade de cefpiroma, antibiótico β-lactâmico de amplo espectro utilizado, principalmente, no tratamento de infecções graves e episódios febris em pacientes com neutropenia, na forma farmacêutica pó para solução injetável. A substância química de referência utilizada nas análises foi caracterizada por espectrofotometria no infravermelho e por espectroscopia de ressonância magnética nuclear. A análise qualitativa foi realizada por cromatografia em camada delgada, determinação da faixa de fusão, espectrofotometria nas regiões do ultravioleta e do infravermelho e cromatografia líquida de alta eficiência, possibilitando a identificação da amostra. A determinação quantitativa foi realizada através dos métodos: espectrofotometria no ultravioleta, cromatografia líquida de alta eficiência e ensaio microbiológico pelo método de difusão em ágar – cilindros em placas, delineamento 3 x 3. Os métodos propostos foram validados segundo guias oficiais e demonstraram ser específicos, lineares, precisos e exatos. Os resultados obtidos demonstraram não haver diferença significativa entre os métodos quando comparados estatisticamente pela análise de variância (ANOVA). Outro objetivo deste trabalho foi o estudo da estabilidade da cefpiroma na forma farmacêutica reconstituída com água para injetável. Os fatores de degradação avaliados foram temperatura (40 ºC) e luz (UVC – 254 nm). Os estudos acelerados de estabilidade demonstraram que a cinética de degradação térmica e fotoquímica foi de primeira ordem. Um produto de degradação térmica foi isolado e identificado, por ressonância magnética nuclear, espectrofometria no infravermelho e espectroscopia de massa, como sulfato de 6-7-diidro-5H-ciclopenta[b]piridinium. / The aim of this study was the development and validation of analytical methods to the determination of cefpirome in powder for injectable preparation and the stability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Cefpirome is a fourth-generation cephalosporin active against a broad spectrum of gram-negative and gram-positive bacterial infections and its principal indication is in the treatment of patients’ septic shock or several sepsis. The substance used as reference standard in the analysis was characterized by infrared spectroscopy and nuclear magnetic resonance. The qualitative analysis was performed by thin layer chromatography, melting range determination, infrared and ultraviolet spectroscopy and high performance liquid chromatography, allowing the identification of samples. The quantitative determination was performed by ultraviolet spectrophotometry, high performance liquid chromatography and cylinder-plate microbiological assay (3 x 3). The proposed methods were validated following official guides and all of them demonstrated to be specific, linear, precise and accurate. The obtained results were analyzed by the ANOVA method and they are not statistically different. The degradation factors evaluated were the temperature (40 ºC) and light (UVC – 254 nm) and the accelerated stability demonstrated that the degradation kinetics from thermal and photo degradation were first-order reactions. A thermal degradation product was isolated and identified, by nuclear magnetic resonance, infrared spectroscopy and mass spectrometry, as 6-7-dihidro-5Hcyclopenta[ b]pyridinium sulfate.

Neste trabalho, foram desenvolvidos e validados métodos qualitativos e quantitativos para o controle de qualidade de cefpiroma, antibiótico β-lactâmico de amplo espectro utilizado, principalmente, no tratamento de infecções graves e episódios febris em pacientes com neutropenia, na forma farmacêutica pó para solução injetável. A substância química de referência utilizada nas análises foi caracterizada por espectrofotometria no infravermelho e por espectroscopia de ressonância magnética nuclear. A análise qualitativa foi realizada por cromatografia em camada delgada, determinação da faixa de fusão, espectrofotometria nas regiões do ultravioleta e do infravermelho e cromatografia líquida de alta eficiência, possibilitando a identificação da amostra. A determinação quantitativa foi realizada através dos métodos: espectrofotometria no ultravioleta, cromatografia líquida de alta eficiência e ensaio microbiológico pelo método de difusão em ágar – cilindros em placas, delineamento 3 x 3. Os métodos propostos foram validados segundo guias oficiais e demonstraram ser específicos, lineares, precisos e exatos. Os resultados obtidos demonstraram não haver diferença significativa entre os métodos quando comparados estatisticamente pela análise de variância (ANOVA). Outro objetivo deste trabalho foi o estudo da estabilidade da cefpiroma na forma farmacêutica reconstituída com água para injetável. Os fatores de degradação avaliados foram temperatura (40 ºC) e luz (UVC – 254 nm). Os estudos acelerados de estabilidade demonstraram que a cinética de degradação térmica e fotoquímica foi de primeira ordem. Um produto de degradação térmica foi isolado e identificado, por ressonância magnética nuclear, espectrofometria no infravermelho e espectroscopia de massa, como sulfato de 6-7-diidro-5H-ciclopenta[b]piridinium. / The aim of this study was the development and validation of analytical methods to the determination of cefpirome in powder for injectable preparation and the stability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Cefpirome is a fourth-generation cephalosporin active against a broad spectrum of gram-negative and gram-positive bacterial infections and its principal indication is in the treatment of patients’ septic shock or several sepsis. The substance used as reference standard in the analysis was characterized by infrared spectroscopy and nuclear magnetic resonance. The qualitative analysis was performed by thin layer chromatography, melting range determination, infrared and ultraviolet spectroscopy and high performance liquid chromatography, allowing the identification of samples. The quantitative determination was performed by ultraviolet spectrophotometry, high performance liquid chromatography and cylinder-plate microbiological assay (3 x 3). The proposed methods were validated following official guides and all of them demonstrated to be specific, linear, precise and accurate. The obtained results were analyzed by the ANOVA method and they are not statistically different. The degradation factors evaluated were the temperature (40 ºC) and light (UVC – 254 nm) and the accelerated stability demonstrated that the degradation kinetics from thermal and photo degradation were first-order reactions. A thermal degradation product was isolated and identified, by nuclear magnetic resonance, infrared spectroscopy and mass spectrometry, as 6-7-dihidro-5Hcyclopenta[ b]pyridinium sulfate.

Neste trabalho, foram desenvolvidos e validados métodos qualitativos e quantitativos para o controle de qualidade de cefpiroma, antibiótico β-lactâmico de amplo espectro utilizado, principalmente, no tratamento de infecções graves e episódios febris em pacientes com neutropenia, na forma farmacêutica pó para solução injetável. A substância química de referência utilizada nas análises foi caracterizada por espectrofotometria no infravermelho e por espectroscopia de ressonância magnética nuclear. A análise qualitativa foi realizada por cromatografia em camada delgada, determinação da faixa de fusão, espectrofotometria nas regiões do ultravioleta e do infravermelho e cromatografia líquida de alta eficiência, possibilitando a identificação da amostra. A determinação quantitativa foi realizada através dos métodos: espectrofotometria no ultravioleta, cromatografia líquida de alta eficiência e ensaio microbiológico pelo método de difusão em ágar – cilindros em placas, delineamento 3 x 3. Os métodos propostos foram validados segundo guias oficiais e demonstraram ser específicos, lineares, precisos e exatos. Os resultados obtidos demonstraram não haver diferença significativa entre os métodos quando comparados estatisticamente pela análise de variância (ANOVA). Outro objetivo deste trabalho foi o estudo da estabilidade da cefpiroma na forma farmacêutica reconstituída com água para injetável. Os fatores de degradação avaliados foram temperatura (40 ºC) e luz (UVC – 254 nm). Os estudos acelerados de estabilidade demonstraram que a cinética de degradação térmica e fotoquímica foi de primeira ordem. Um produto de degradação térmica foi isolado e identificado, por ressonância magnética nuclear, espectrofometria no infravermelho e espectroscopia de massa, como sulfato de 6-7-diidro-5H-ciclopenta[b]piridinium. / The aim of this study was the development and validation of analytical methods to the determination of cefpirome in powder for injectable preparation and the stability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Cefpirome is a fourth-generation cephalosporin active against a broad spectrum of gram-negative and gram-positive bacterial infections and its principal indication is in the treatment of patients’ septic shock or several sepsis. The substance used as reference standard in the analysis was characterized by infrared spectroscopy and nuclear magnetic resonance. The qualitative analysis was performed by thin layer chromatography, melting range determination, infrared and ultraviolet spectroscopy and high performance liquid chromatography, allowing the identification of samples. The quantitative determination was performed by ultraviolet spectrophotometry, high performance liquid chromatography and cylinder-plate microbiological assay (3 x 3). The proposed methods were validated following official guides and all of them demonstrated to be specific, linear, precise and accurate. The obtained results were analyzed by the ANOVA method and they are not statistically different. The degradation factors evaluated were the temperature (40 ºC) and light (UVC – 254 nm) and the accelerated stability demonstrated that the degradation kinetics from thermal and photo degradation were first-order reactions. A thermal degradation product was isolated and identified, by nuclear magnetic resonance, infrared spectroscopy and mass spectrometry, as 6-7-dihidro-5Hcyclopenta[ b]pyridinium sulfate.

La résistance aux antimicrobiens (RAM) est un problème de santé publique mondial avec des répercussions importantes en médecine vétérinaire et humaine. Elle est classiquement associée à une surutilisation des antimicrobiens. Les bactéries commensales des animaux et des humains, tel Escherichia coli, sont souvent utilisées comme bactéries indicatrices pour surveiller la RAM. Parmi les mécanismes de résistance de E. coli, la production de β-lactamases à spectre étendue (BLSE) ou de type AmpC est particulièrement inquiétante. Ces enzymes peuvent inactiver une classe d’antimicrobien de très haute importance en santé humaine également utilisée en médecine vétérinaire : les céphalosporines de 3e génération. La prévalence générale de la RAM ainsi que la présence de E. coli producteur de BLSE/AmpC dans les troupeaux laitiers québécois sont inconnues. De plus, la transmission de ces bactéries résistantes et les facteurs de risque associés à leurs excrétions par les bovins laitiers sont actuellement peu documentés. L’objectif général de cette thèse était d’explorer la RAM par une étude transversale observationnelle sur des fermes québécoises de bovins laitiers sélectionnées aléatoirement (n = 101). La première étape du projet constituait à décrire la prévalence de cette RAM pour la bactérie E. coli isolée des matières fécales des animaux (vaches en lactation, veaux pré-sevrage) et de l’environnement (fosse à fumier). La prévalence de RAM observée pour les E. coli indicateurs était faible (<5%) pour les antimicrobiens de très haute importance en médecine humaine (céphalosporines de 3e génération et fluoroquinolones). Cependant, il y avait une prévalence élevée (85%) de fermes avec la présence d’au moins un E. coli producteur de BLSE/AmpC. La RAM était particulièrement importante pour les E. coli isolés chez les veaux pré-sevrage. Pour la deuxième étape, cette RAM était analysée au niveau génétique par le séquençage du génome entier des E. coli les plus résistants. La grande majorité de la RAM (>95%) était expliquée par des mutations ou des gènes de résistance. Certains de ceux-ci étaient à proximité l’un de l’autre et leur configuration laissait supposer qu’une partie de ces gènes étaient présents sur des éléments génétiques mobiles. De plus, il y avait une dissémination clonale de E. coli résistant entre les fermes. La dernière étape constituait à déterminer des facteurs de risque (utilisation des antimicrobiens ou pratiques à la ferme) de la RAM. Grâce à des analyses multivariées utilisant l’intelligence artificielle, il a été possible d’observer des facteurs de risque significatifs associés à la taille de la ferme et à la santé des animaux. Bref, un portrait de la situation de la RAM est maintenant établi dans les troupeaux de bovins laitiers du Québec pour 2017. Il s’agit d’un phénomène complexe qui ne se limite pas simplement à un lien direct avec l’utilisation des antimicrobiens. Ces travaux serviront de bases pour suivre l’évolution temporelle de la RAM. De plus, des études prospectives pourraient être réalisées afin de confirmer les impacts des observations notées dans cette thèse. L’ensemble de ces informations seront déterminantes en vue d’établir des mesures concrètes pour tenter de limiter cette importante problématique. / Antimicrobial resistance (AMR) is a global public health problem with major repercussions in both veterinary and human medicine. It is classically associated with the overuse of antimicrobials. Animal and human commensal bacteria, such as Escherichia coli, are often used as indicator bacteria to monitor AMR. Among the resistance mechanisms of E. coli, the production of an extended-spectrum β-lactamase (ESBL) or AmpC-type is of particular concern. These enzymes can inactivate a class of antimicrobials of great importance in human health also used in veterinary medicine: third-generation cephalosporins. The overall prevalence of AMR and the presence of ESBL/AmpC-producing E. coli are unknown in Québec dairy herds. Furthermore, the transmission of these resistant bacteria and the risk factors associated with their excretion by dairy cattle are currently poorly documented. The overall objective of this thesis was to explore AMR through an observational cross-sectional study on randomly selected Québec dairy farms (n = 101). The first step of the project was to describe the prevalence of AMR for E. coli isolated from animal feces (lactating cows, pre-weaned calves) and the environment (manure pit). The prevalence of AMR observed for indicator E. coli was low (<5%) for antimicrobials of very high importance in human medicine (third-generation cephalosporins and fluoroquinolones). However, there was a high prevalence (85%) of farms with at least one ESBL/AmpC-producing E. coli. AMR was particularly high for E. coli isolated from pre-weaned calves. In the second step, AMR was analyzed at the genetic level by whole genome sequencing of the most resistant E. coli isolates. The vast majority of AMR (>95%) was explained by mutations or resistance genes. Some of these were in close proximity to each other, and their configuration suggested that some of these genes were present on mobile genetic elements. In addition, there was clonal dissemination of resistant E. coli between farms. The final step was to identify risk factors (antimicrobial use or farm practices) for AMR. Using artificial intelligence methods for multivariate analyses, it was possible to identify significant risk factors associated with farm size and the health of animals. In summary, our results provide a portrait of the AMR situation in Québec dairy herds. This complex phenomenon is not simply limited to a direct link with antimicrobial use. This work will serve as a baseline for monitoring the temporal evolution of AMR. In addition, prospective studies could be carried out to confirm the impacts of the observations noted in this thesis. All this information will be decisive in establishing concrete measures to try and limit this major problem.