RESUMO
Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.
Assuntos
Antibacterianos/química , Ceftazidima/química , Imipenem/química , Meropeném/química , Zinco/química , beta-Lactamases/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Domínio Catalítico , Cefepima/química , Cefepima/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Ceftazidima/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imipenem/metabolismo , Cinética , Meropeném/metabolismo , Modelos Moleculares , Piperacilina/química , Piperacilina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Zinco/metabolismo , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Piperacillin-tazobactam is a broad spectrum antimicrobial agent that can cause false-positive results in the commercial Platelia Aspergillus EIA test. So far, no study has been performed in Latin America to evaluate the clinical implication of this finding. Here we studied the potential for galactomannan detection in piperacillin-tazobactam batches commercialized in the Brazilian market. Five batches from distinct laboratories were tested in duplicate in the Platelia Aspergillus EIA according to the manufacturer's instructions. Only one drug showed crossreaction at a cut-off of 0.5. Human serum was spiked with this particular drug aiming to mimic achievable piperacillin-tazobactam concentrations in the serum. Results were all negative for galactomannan detection, even at high drug concentrations. Results from this pilot study suggest that piperacillin-tazobactam might not be a clinically significant cause of false-positive results in the Platelia Aspergillus EIA test in Brazil.
Assuntos
Antibacterianos/química , Aspergillus/imunologia , Técnicas Imunoenzimáticas , Mananas/análise , Reações Falso-Positivas , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/química , Piperacilina/químicaRESUMO
Piperacillin-tazobactam is a broad spectrum antimicrobial agent that can cause false-positive results in the commercial Platelia Aspergillus EIA test. So far, no study has been performed in Latin America to evaluate the clinical implication of this finding. Here we studied the potential for galactomannan detection in piperacillin-tazobactam batches commercialized in the Brazilian market. Five batches from distinct laboratories were tested in duplicate in the Platelia Aspergillus EIA according to the manufacturer's instructions. Only one drug showed crossreaction at a cut-off of 0.5. Human serum was spiked with this particular drug aiming to mimic achievable piperacillin-tazobactam concentrations in the serum. Results were all negative for galactomannan detection, even at high drug concentrations. Results from this pilot study suggest that piperacillin-tazobactam might not be a clinically significant cause of false-positive results in the Platelia Aspergillus EIA test in Brazil.
Assuntos
Antibacterianos/química , Aspergillus/imunologia , Técnicas Imunoenzimáticas , Mananas/análise , Reações Falso-Positivas , Galactose/análogos & derivados , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/química , Piperacilina/química , Combinação Piperacilina e TazobactamRESUMO
O estudo comparativo da atividade da azlocilina, da mezlocilina e da piperacilina sobre 123 cepas de bactérias Gram-negativas isoladas nos hospitais do Recife foi efetuado mediante a determinaçäo das CIM e das CBM pela microtécnica de diluiçäo em caldo. Estas três ureidopenicilinas apresentaram uma boa atividade contra E. coli, Salmonella, Shigella, Proteus indol positivo e Klebsiella. Todas as cepas de Pseudomonas foram sensíveis à Azlocilina e à Piperacilina
Assuntos
Piperacilina/farmacocinética , Azlocilina/farmacocinética , Mezlocilina/farmacocinética , Bactérias Gram-Negativas , Infecções Bacterianas/microbiologia , Piperacilina/química , Azlocilina/química , Testes de Sensibilidade Microbiana , Mezlocilina/química , Bactérias Gram-Negativas/isolamento & purificaçãoRESUMO
Mostra a biodisponibilidade, a farmacinética, a atividade in vitro, os estudos clínicos, a segurança, os efeitos colaterias, a posologia e a relação custo/benfício da junção da pipericilina sódica com a tazobactama sódica