Ceftiofur sodium, a third generation cephalosporin, was studied to determine the potential of this drug as an alternative bacterial therapeutic agent for the aquaculture and ornamental fish industry. Grass carp, Ctenopharyngodon idella have been selected as the fish model for this study since they are a good representative for both foodfish and ornamental fish and are one of the major species grown worldwide. Pharmacokinetics of ceftiofur sodium after various routes of administration, histopathologic observations to detect possible toxic effects on the tissues involved in its metabolism and excretion, and the effects on the non-specific immune response were investigated in grass carp. For the pharmacokinetic studies, ceftiofur sodium was administered a single time to grass carp by four different routes : intracardiac (IC), intraperitoneal (IP), intramuscular (IM) and oral (PO) at a dosage of 8 mg/kg body weight. Serial blood samples were obtained and plasma samples were analyzed by high performance liquid chromatography for ceftiofur (as measured its metabolite, desfuroylceftiofur (DFC) and DFC-related metabolite concentrations). Disposition pharmacokinetic data were best described by a two compartment open model for IC and by a non-compartment model with no lag time for IP and IM administrations. Oral absorption of ceftiofur was not observed in this species. Following IC, IP and IM ceftiofur sodium administration, the final elimination half-lives, maximum plasma concentration, time to reach maximum concentration, volume of distribution and plasma clearance were 0.38, 0.45 and 13.86 hours ; 157.09, 31.54 and 8.86 mg/ml ; 0, 0.25 and 0.5 hours ; 0.09, 0.17, 0.53 l/kg ; and 0.21, 0.26, 0.26 ml/min.kg, respectively. Desfuroylceftiofur metabolite was highly bound with plasma protein at pH 7.0 and 8.0. For the histopathological studies, a single intramuscular dose of ceftiofur sodium at three different concentrations, 8 (1X), 40 (5X) and 80 (10X) mg/kg was administered to separate groups of grass carp for evaluation of the potential toxicity to major tissues involved in metabolism and excretion of this drug. These included the anterior kidney, posterior kidney, liver, and spleen. After 48 hours, lesions were seen in the posterior kidney at the highest dose of ceftiofur (10X). Morphological alterations observed microscopically included increased number of renal tubules, tubular necrosis and infiltration of inflammatory cells. No adverse effects on the glomeruli were observed at any concentration of the drug. For the immunotoxicity studies on the non-specific immune response, dosages of either 8 or 40 mg/kg body weight were administered intramuscularly. After 24 and 48 h, leukocyte number, phagocytic ability and H2O2 production were examined in the cells of the pronephros. The results showed that neither dosage had an effect on the number of leukocytes in the pronephros. Phagocytosis was also not significantly altered at either dosage in macrophages from the pronephros. Hydrogen peroxide production was not altered in the pronephros of fish dosed at 8 mg/kg, while at a dosage of 40 mg/kg, H2O2 production was significantly increased. In summary, ceftiofur sodium has potential as an efficacious chemotherapeutic agent for controlling bacterial infection in brood stock and ornamental fish at the recommended dose of 8 mg/kg. A dose as high as 40 mg/kg can be use with careful consideration. This dosage may not directly injure the posterior kidney but it may affect the non-specific immune response of the fish. / Ph. D.

O uso excessivo de antimicrobianos em medicina veterinária e humana tem contribuído para o surgimento e seleção de bactérias multirresistentes (MR) em animais de produção, sendo que a presença de resíduos de antibióticos em alimentos derivados constitui uma predisposição para o estabelecimento de reações alérgicas para o consumidor, e altera as características organolépticas dos alimentos. No Brasil, embora a presença de Escherichia coli produtora de β-lactamase de espectro estendido (ESBL) em avicultura, bubalinocultura e suinocultura tenha sido documentada recentemente, não há informações sobre sua prevalência em bovinocultura, que é um setor importante do agronegócio no país. Assim, o presente estudo teve como objetivo investigar a presença de linhagens de Escherichia coli produtoras de ESBLs na microbiota intestinal de bovinos de leite, em uma fazenda comercial com prática de uso rotineiro de cefalosporinas na forma terapêutica. Amostras de suabe retal foram coletadas em 95 ruminantes (lactantes e adultos saudáveis), na presença de um regime de tratamento com ceftiofur terapêutico, em uma fazenda no estado de São Paulo. As amostras foram triadas para a presença de bactérias Gram-negativas produtoras de ESBL, onde os isolados positivos foram identificados por MALDI-TOF, com posterior identificação dos genes codificando variantes ESBL, por PCR e sequenciamento. A presença de cepas de E. coli multirresistentes (resistência a >= 3 classes de antibióticos) foi confirmada em 45 ruminantes (47,3%). Adicionalmente, foram identificadas 68 cepas de E. coli produtoras de ESBL (71,5%), sendo 7 portadoras do gene blaCTX-M-2, 11 portadoras do gene blaCTX-M-8, e 50 portadoras do gene blaCTX-M-15. As cepas de E. coli produtoras de CTX-M-15 foram clonalmente relacionadas por ERIC-PCR e PFGE, confirmando-se a presença de linhagens clonais em diferentes animais, na mesma propriedade. Quatro cepas representativas deste clone tiveram seus genomas sequenciados, demonstrando pertencer ao complexo clonal 23 (ST90), mundialmente reportado em animais e humanos. Estes resultados denotam uma alta prevalência de E. coli produtora de ESBL do tipo CTX-M na microbiota intestinal do gado leiteiro, com predominância de clones de importância clínica humana e veterinária, que hipoteticamente possuem uma genética que favorece sua adaptação (provavelmente favorecida pela pressão seletiva de antibióticos) na interface animal-humana, o que representa um potencial zoonótico de importância para a saúde pública. / The excessive use of antimicrobials in veterinary medicine has contributed to the emergence and selection of multiresistant (MR) bacteria in livestock animals, and the presence of residues of antibiotics in food products is a predisposition for the establishment of allergic reactions to the consumer, changing the organoleptic characteristics of food. In Brazil, although the presence of Escherichia coli-producing extended-spectrum β-lactamase (ESBL) in poultry, buffalo and swine farming has been documented recently, there is no information on its prevalence in cattle breeding, which is an important sector of agribusiness in the country. Thus, the present study aimed to investigate the presence of Escherichia coli strains producing ESBLs in the intestinal microbiota of dairy cattle in a commercial farm with routine practice of cephalosporins in the therapeutic form. Rectal swab samples were collected from 95 ruminants (healthy calf and adults) in the presence of a treatment regimen with ceftiofur therapeutic, on a farm in the state of São Paulo. The samples were screened for the presence of ESBL-producing Gram-negative bacteria, where positive isolates were identified by MALDI-TOF, with subsequent identification of genes encoding ESBL variants, by PCR and sequencing. The presence of multiresistant strains of E. coli (resistance to >= 3 classes of antibiotics) was confirmed in 45 ruminants (47.3%). In addition, 68 ESBL-producing E. coli strains (71.5%) were identified, 7 of which were carriers of the blaCTX-M-2 gene, 11 carriers of the blaCTX-M-8 gene, and 50 carriers of the blaCTX-M-15. The E. coli strains producing CTX-M-15 were clonally related by ERIC-PCR and PFGE, confirming the presence of clonal lineages in different animals, on the same property. Four representative strains of this clone had their genomes sequenced, demonstrating belonging to the clonal complex 23 (ST90), worldwide reported in animals and humans. These results indicate a high prevalence of E. coli producing CTX-M ESBL in the intestinal microbiota of dairy cattle, with a predominance of clones of human and veterinary clinical importance, which hypothetically have a genetics that favors their adaptation (probably favored by the selective pressure of antibiotics) at the animal-human interface, which represents a zoonotic potential of public health importance.

O uso excessivo de antimicrobianos em medicina veterinária e humana tem contribuído para o surgimento e seleção de bactérias multirresistentes (MR) em animais de produção, sendo que a presença de resíduos de antibióticos em alimentos derivados constitui uma predisposição para o estabelecimento de reações alérgicas para o consumidor, e altera as características organolépticas dos alimentos. No Brasil, embora a presença de Escherichia coli produtora de β-lactamase de espectro estendido (ESBL) em avicultura, bubalinocultura e suinocultura tenha sido documentada recentemente, não há informações sobre sua prevalência em bovinocultura, que é um setor importante do agronegócio no país. Assim, o presente estudo teve como objetivo investigar a presença de linhagens de Escherichia coli produtoras de ESBLs na microbiota intestinal de bovinos de leite, em uma fazenda comercial com prática de uso rotineiro de cefalosporinas na forma terapêutica. Amostras de suabe retal foram coletadas em 95 ruminantes (lactantes e adultos saudáveis), na presença de um regime de tratamento com ceftiofur terapêutico, em uma fazenda no estado de São Paulo. As amostras foram triadas para a presença de bactérias Gram-negativas produtoras de ESBL, onde os isolados positivos foram identificados por MALDI-TOF, com posterior identificação dos genes codificando variantes ESBL, por PCR e sequenciamento. A presença de cepas de E. coli multirresistentes (resistência a >= 3 classes de antibióticos) foi confirmada em 45 ruminantes (47,3%). Adicionalmente, foram identificadas 68 cepas de E. coli produtoras de ESBL (71,5%), sendo 7 portadoras do gene blaCTX-M-2, 11 portadoras do gene blaCTX-M-8, e 50 portadoras do gene blaCTX-M-15. As cepas de E. coli produtoras de CTX-M-15 foram clonalmente relacionadas por ERIC-PCR e PFGE, confirmando-se a presença de linhagens clonais em diferentes animais, na mesma propriedade. Quatro cepas representativas deste clone tiveram seus genomas sequenciados, demonstrando pertencer ao complexo clonal 23 (ST90), mundialmente reportado em animais e humanos. Estes resultados denotam uma alta prevalência de E. coli produtora de ESBL do tipo CTX-M na microbiota intestinal do gado leiteiro, com predominância de clones de importância clínica humana e veterinária, que hipoteticamente possuem uma genética que favorece sua adaptação (provavelmente favorecida pela pressão seletiva de antibióticos) na interface animal-humana, o que representa um potencial zoonótico de importância para a saúde pública. / The excessive use of antimicrobials in veterinary medicine has contributed to the emergence and selection of multiresistant (MR) bacteria in livestock animals, and the presence of residues of antibiotics in food products is a predisposition for the establishment of allergic reactions to the consumer, changing the organoleptic characteristics of food. In Brazil, although the presence of Escherichia coli-producing extended-spectrum β-lactamase (ESBL) in poultry, buffalo and swine farming has been documented recently, there is no information on its prevalence in cattle breeding, which is an important sector of agribusiness in the country. Thus, the present study aimed to investigate the presence of Escherichia coli strains producing ESBLs in the intestinal microbiota of dairy cattle in a commercial farm with routine practice of cephalosporins in the therapeutic form. Rectal swab samples were collected from 95 ruminants (healthy calf and adults) in the presence of a treatment regimen with ceftiofur therapeutic, on a farm in the state of São Paulo. The samples were screened for the presence of ESBL-producing Gram-negative bacteria, where positive isolates were identified by MALDI-TOF, with subsequent identification of genes encoding ESBL variants, by PCR and sequencing. The presence of multiresistant strains of E. coli (resistance to >= 3 classes of antibiotics) was confirmed in 45 ruminants (47.3%). In addition, 68 ESBL-producing E. coli strains (71.5%) were identified, 7 of which were carriers of the blaCTX-M-2 gene, 11 carriers of the blaCTX-M-8 gene, and 50 carriers of the blaCTX-M-15. The E. coli strains producing CTX-M-15 were clonally related by ERIC-PCR and PFGE, confirming the presence of clonal lineages in different animals, on the same property. Four representative strains of this clone had their genomes sequenced, demonstrating belonging to the clonal complex 23 (ST90), worldwide reported in animals and humans. These results indicate a high prevalence of E. coli producing CTX-M ESBL in the intestinal microbiota of dairy cattle, with a predominance of clones of human and veterinary clinical importance, which hypothetically have a genetics that favors their adaptation (probably favored by the selective pressure of antibiotics) at the animal-human interface, which represents a zoonotic potential of public health importance.

Peu d’études ont exploré l’utilisation du traitement prolongé pour les mammites cliniques et aucune ne portait sur l’utilisation du traitement prolongé à base de ceftiofur pour le traitement des mammites cliniques légères à modérées. L’objectif de cette étude était d’évaluer l’efficacité du traitement intra-mammaire prolongé à base de ceftiofur pour les mammites cliniques légères à modérées, en considérant toutes les bactéries responsables ou seulement Staphylococcus aureus (S. aureus) et les streptocoques. Des vaches laitières (n = 241) de 22 élevages du Québec et de l’Ontario ont été inclues. L’étude a été conçue comme un essai clinique à allocation aléatoire. Pour chaque cas de mammite clinique légère à modérée, 125 mg d’hydrochloride de ceftiofur (Spectramast® LC) a été administré par voie intra-mammaire une fois par jour pour 2 ou 8 jours. Le pourcentage de guérison clinique 21 jours après la fin du traitement était de 89% (n = 98/110 pour chaque groupe, p = 0,95). Les pourcentages de guérison bactériologique 21 jours après la fin du traitement pour les groupes 2 jours et 8 jours étaient 32% (n = 15/47) et 61% (n = 25/41) respectivement pour toutes les bactéries (p < 0,01); 64% (n = 9/14) et 82% (n = 9/11) respectivement pour les streptocoques (p = 0,50); et 0% (n = 0/20) et 47% (n = 9/19) pour S. aureus (p < 0,01). Il n'y avait pas de différence entre les 2 groupes pour les nouvelles infections intra-mammaires (p = 0,30). Le traitement prolongé à base de ceftiofur est un choix raisonnable pour le traitement des mammites cliniques légères à modérées, en particulier causées par S. aureus. / Little research has focused on extended therapy in lactating cows with clinical mastitis and none were with ceftiofur. The objective of the present study was to evaluate the efficacy of an intramammary extended ceftiofur treatment for mild to moderate clinical mastitis, and to determine whether it would increase cure rates when considering any bacteria or more particularly Staphylococcus aureus (S. aureus) and streptococci. Holstein dairy cows (n = 241) from 22 herds located in Quebec and Ontario, Canada, were included. The study was designed as a randomized clinical trial. For each case of mild to moderate clinical mastitis diagnosed, 125 mg of ceftiofur hydrochloride (Spectramast® LC) was administered intramammary once a day for 2 or 8 consecutive days. Clinical cure rate 21 days after the last treatment was identical in each group (89%; n = 98/110; p = 0.95). Bacteriological cure rates 21 days after the last treatment for the 2- and 8-day regimens were 32% (n = 15/47) and 61% (n = 25/41), respectively, for all bacteria (p < 0.01); 64% (n = 9/14) and 82% (n = 9/11), respectively, for streptococci (p = 0.50); and 0% (n = 0/20) and 47% (n = 9/19), respectively, for S. aureus (p < 0.01). There were no statistical differences between experimental groups for new intramammary infections. Extended therapy appears to be a reasonable choice for the treatment of mild to moderate clinical mastitis, particularly in cases where S. aureus is the pathogen involved.

Peu d’études ont exploré l’utilisation du traitement prolongé pour les mammites cliniques et aucune ne portait sur l’utilisation du traitement prolongé à base de ceftiofur pour le traitement des mammites cliniques légères à modérées. L’objectif de cette étude était d’évaluer l’efficacité du traitement intra-mammaire prolongé à base de ceftiofur pour les mammites cliniques légères à modérées, en considérant toutes les bactéries responsables ou seulement Staphylococcus aureus (S. aureus) et les streptocoques. Des vaches laitières (n = 241) de 22 élevages du Québec et de l’Ontario ont été inclues. L’étude a été conçue comme un essai clinique à allocation aléatoire. Pour chaque cas de mammite clinique légère à modérée, 125 mg d’hydrochloride de ceftiofur (Spectramast® LC) a été administré par voie intra-mammaire une fois par jour pour 2 ou 8 jours. Le pourcentage de guérison clinique 21 jours après la fin du traitement était de 89% (n = 98/110 pour chaque groupe, p = 0,95). Les pourcentages de guérison bactériologique 21 jours après la fin du traitement pour les groupes 2 jours et 8 jours étaient 32% (n = 15/47) et 61% (n = 25/41) respectivement pour toutes les bactéries (p < 0,01); 64% (n = 9/14) et 82% (n = 9/11) respectivement pour les streptocoques (p = 0,50); et 0% (n = 0/20) et 47% (n = 9/19) pour S. aureus (p < 0,01). Il n'y avait pas de différence entre les 2 groupes pour les nouvelles infections intra-mammaires (p = 0,30). Le traitement prolongé à base de ceftiofur est un choix raisonnable pour le traitement des mammites cliniques légères à modérées, en particulier causées par S. aureus. / Little research has focused on extended therapy in lactating cows with clinical mastitis and none were with ceftiofur. The objective of the present study was to evaluate the efficacy of an intramammary extended ceftiofur treatment for mild to moderate clinical mastitis, and to determine whether it would increase cure rates when considering any bacteria or more particularly Staphylococcus aureus (S. aureus) and streptococci. Holstein dairy cows (n = 241) from 22 herds located in Quebec and Ontario, Canada, were included. The study was designed as a randomized clinical trial. For each case of mild to moderate clinical mastitis diagnosed, 125 mg of ceftiofur hydrochloride (Spectramast® LC) was administered intramammary once a day for 2 or 8 consecutive days. Clinical cure rate 21 days after the last treatment was identical in each group (89%; n = 98/110; p = 0.95). Bacteriological cure rates 21 days after the last treatment for the 2- and 8-day regimens were 32% (n = 15/47) and 61% (n = 25/41), respectively, for all bacteria (p < 0.01); 64% (n = 9/14) and 82% (n = 9/11), respectively, for streptococci (p = 0.50); and 0% (n = 0/20) and 47% (n = 9/19), respectively, for S. aureus (p < 0.01). There were no statistical differences between experimental groups for new intramammary infections. Extended therapy appears to be a reasonable choice for the treatment of mild to moderate clinical mastitis, particularly in cases where S. aureus is the pathogen involved.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Este trabalho objetivou o desenvolvimento e validação de métodos analíticos para determinação de ceftiofur pó para solução injetável e o estudo da fotoestabilidade do fármaco. Os métodos por cromatografia em camada delgada, cromatografia líquida de alta eficiência (CLAE), espectrofotometria no ultravioleta (UV) foram utilizados para análise qualitativa do fármaco na forma farmacêutica. A determinação quantitativa foi realizada através dos métodos cromatografia líquida de alta eficiência, espectrofotometria no ultravioleta e ensaio microbiológico método de difusão em ágar - cillindros em placas, delineamento 3x3, avaliando-se os parâmetros descritos pelas guias de validação. Os resultados obtidos através destes métodos foram comparados estatisticamente por ANOVA, que indicou não haver diferenças estatisticamente significativas entre os mesmos. Estudo preliminar da estabilidade do ceftiofur frente a degradação alcalina, ácida, oxidativa e fotolítica mostrou a oxidação, a temperatura, a luz e a condição alcalina como fatores importantes da degradação. O fármaco é instável às radiações UV-C, em maior grau, e UV-A (degradação menos intensa), em solução. A cinética de fotodegradação do ceftiofur sódico em solução aquosa demonstrou cinética de primeira ordem de reação. / The aim of this study was the development and validation of analytical methods to the determination of ceftiofur sodium in powder for injectable preparation and the photostability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and ultraviolet spectrophotometry (UV), methods were employed to the qualitative analysis of the drug in pharmaceutical formulation. The quantitative determination was performed through the validation of HPLC, UV spectrophotometry and microbiological assay 3x3 using the cylinder plate, evaluating the guidances validation parameters. The results obtained by three methods were compared by ANOVA, which indicated that they are equivalent. Preliminary study of ceftiofur through alkaline, acid, oxidative and fotolitic degradation shows sensibility to oxidation, light and alkaline medium. The drug is unstable to UV-C and UV-A radiations, both in solution, being the degradation on UV-C more intense. The photodegradation kinetics of ceftiofur sodium solutions show first-order kinetic of reaction.

Este trabalho objetivou o desenvolvimento e validação de métodos analíticos para determinação de ceftiofur pó para solução injetável e o estudo da fotoestabilidade do fármaco. Os métodos por cromatografia em camada delgada, cromatografia líquida de alta eficiência (CLAE), espectrofotometria no ultravioleta (UV) foram utilizados para análise qualitativa do fármaco na forma farmacêutica. A determinação quantitativa foi realizada através dos métodos cromatografia líquida de alta eficiência, espectrofotometria no ultravioleta e ensaio microbiológico método de difusão em ágar - cillindros em placas, delineamento 3x3, avaliando-se os parâmetros descritos pelas guias de validação. Os resultados obtidos através destes métodos foram comparados estatisticamente por ANOVA, que indicou não haver diferenças estatisticamente significativas entre os mesmos. Estudo preliminar da estabilidade do ceftiofur frente a degradação alcalina, ácida, oxidativa e fotolítica mostrou a oxidação, a temperatura, a luz e a condição alcalina como fatores importantes da degradação. O fármaco é instável às radiações UV-C, em maior grau, e UV-A (degradação menos intensa), em solução. A cinética de fotodegradação do ceftiofur sódico em solução aquosa demonstrou cinética de primeira ordem de reação. / The aim of this study was the development and validation of analytical methods to the determination of ceftiofur sodium in powder for injectable preparation and the photostability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and ultraviolet spectrophotometry (UV), methods were employed to the qualitative analysis of the drug in pharmaceutical formulation. The quantitative determination was performed through the validation of HPLC, UV spectrophotometry and microbiological assay 3x3 using the cylinder plate, evaluating the guidances validation parameters. The results obtained by three methods were compared by ANOVA, which indicated that they are equivalent. Preliminary study of ceftiofur through alkaline, acid, oxidative and fotolitic degradation shows sensibility to oxidation, light and alkaline medium. The drug is unstable to UV-C and UV-A radiations, both in solution, being the degradation on UV-C more intense. The photodegradation kinetics of ceftiofur sodium solutions show first-order kinetic of reaction.

Este trabalho objetivou o desenvolvimento e validação de métodos analíticos para determinação de ceftiofur pó para solução injetável e o estudo da fotoestabilidade do fármaco. Os métodos por cromatografia em camada delgada, cromatografia líquida de alta eficiência (CLAE), espectrofotometria no ultravioleta (UV) foram utilizados para análise qualitativa do fármaco na forma farmacêutica. A determinação quantitativa foi realizada através dos métodos cromatografia líquida de alta eficiência, espectrofotometria no ultravioleta e ensaio microbiológico método de difusão em ágar - cillindros em placas, delineamento 3x3, avaliando-se os parâmetros descritos pelas guias de validação. Os resultados obtidos através destes métodos foram comparados estatisticamente por ANOVA, que indicou não haver diferenças estatisticamente significativas entre os mesmos. Estudo preliminar da estabilidade do ceftiofur frente a degradação alcalina, ácida, oxidativa e fotolítica mostrou a oxidação, a temperatura, a luz e a condição alcalina como fatores importantes da degradação. O fármaco é instável às radiações UV-C, em maior grau, e UV-A (degradação menos intensa), em solução. A cinética de fotodegradação do ceftiofur sódico em solução aquosa demonstrou cinética de primeira ordem de reação. / The aim of this study was the development and validation of analytical methods to the determination of ceftiofur sodium in powder for injectable preparation and the photostability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and ultraviolet spectrophotometry (UV), methods were employed to the qualitative analysis of the drug in pharmaceutical formulation. The quantitative determination was performed through the validation of HPLC, UV spectrophotometry and microbiological assay 3x3 using the cylinder plate, evaluating the guidances validation parameters. The results obtained by three methods were compared by ANOVA, which indicated that they are equivalent. Preliminary study of ceftiofur through alkaline, acid, oxidative and fotolitic degradation shows sensibility to oxidation, light and alkaline medium. The drug is unstable to UV-C and UV-A radiations, both in solution, being the degradation on UV-C more intense. The photodegradation kinetics of ceftiofur sodium solutions show first-order kinetic of reaction.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.