RESUMO
OBJECTIVES: The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various ß-lactamase inhibitors. METHODS: A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. RESULTS: In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. CONCLUSIONS: Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.
Assuntos
Carbapenêmicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Cefotaxima/farmacologia , Clonagem Molecular , Interações Medicamentosas , Ativação Enzimática/genética , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/genética , Escherichia coli/genética , Imipenem/farmacologia , Cinética , Mutagênese Sítio-Dirigida , Conformação Proteica , Análise de Sequência de Proteína , beta-Lactamases/genéticaRESUMO
A multidrug-resistant strain of Elizabethkingia miricola was isolated from the urine of a 2-year-old boy hospitalized for severe clinical conditions. The strain produces 2 metallo-ß-lactamases belonging to subclasses B1 and B3: a new BlaB variant (BlaB-15) and a GOB-7-like enzyme.
Assuntos
Infecções por Flavobacteriaceae/microbiologia , Flavobacteriaceae/enzimologia , Flavobacteriaceae/isolamento & purificação , Infecções Urinárias/microbiologia , beta-Lactamases/análise , beta-Lactamases/genética , Sequência de Aminoácidos , Pré-Escolar , Farmacorresistência Bacteriana Múltipla , Flavobacteriaceae/efeitos dos fármacos , Humanos , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Alinhamento de Sequência , Urina/microbiologiaRESUMO
Site-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-ß-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.
Assuntos
Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , beta-Lactamases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Prolina/química , Prolina/metabolismo , Análise de Sequência de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Especificidade por Substrato/fisiologia , Zinco/farmacologia , beta-Lactamases/química , beta-Lactamases/genéticaRESUMO
Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants.
Assuntos
Antibacterianos/química , Cefalosporinas/química , Escherichia coli/efeitos dos fármacos , Isoleucina/química , Resistência beta-Lactâmica/genética , beta-Lactamases/química , Substituição de Aminoácidos , Antibacterianos/farmacologia , Biocatálise , Domínio Catalítico , Cefalosporinas/farmacologia , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Isoleucina/metabolismo , Cinética , Modelos Moleculares , Mutação , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Two new natural CphA metallo-ß-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed kcat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.
Assuntos
Aeromonas hydrophila/enzimologia , Proteínas de Bactérias/genética , Esgotos/microbiologia , beta-Lactamases/genética , Aeromonas hydrophila/efeitos dos fármacos , Aeromonas hydrophila/genética , Sequência de Aminoácidos , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Ertapenem , Imipenem/farmacologia , Itália , Dados de Sequência Molecular , Mutação Puntual/genética , Tienamicinas/farmacologia , beta-Lactamas/farmacologiaRESUMO
The in vitro antimicrobial activities of five compounds isolated from lichens, collected in several Southern regions of Chile (including the Chilean Antarctic Territory), were evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC90, MIC50, as well as MBC90 and MBC50, for the lichen compounds were evaluated. The MIC90 was ranging from 32 µg/ml for perlatolic acid to 128 µg/ml for α-collatolic acid. MBC90 was ranging from onefold up to twofold the MIC90 for each compound. A synergistic action was observed in combination with gentamicin, whilst antagonism was observed for some lichen compounds in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. These could mainly be explained by the failure of FIC approach, being too much subjective and sensitive to experimental errors. These findings suggest, however, that the natural compounds from lichens are good candidates for the individuation of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.
Assuntos
Antibacterianos/farmacologia , Líquens/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Benzoatos/farmacologia , Chile , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Estrutura Molecular , Metabolismo SecundárioRESUMO
In this study 114 extensively drug-resistant Acinetobacter baumannii clinical isolates were characterized. The strains were collected at L'Aquila Hospital after the earthquake in L'Aquila city (central Italy) on the 6th of April 2009. The genes blaOXA-23 and blaOXA-51 were detected in all clinical isolates analyzed, whereas blaTEM-1 allele was detected in 56/114 isolates. The blaOXA-23 gene is located downstream the ISAba region and is under control of a strong promoter. On 42/80 A. baumannii the presence of two class 1 integrons was ascertained on chromosomal DNA. Variable regions show different gene array: (1) aadB and aadA2, (2) aacA4, aac(6')-Ib-cr, and aadA1. Macrorestriction analysis using ApaI restriction endonuclease identifies three clusters (A, B, and C) according to pulsed-field gel electrophoresis profiles. All isolates analyzed belong to the clone A. baumannii sequence type 2.
Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/isolamento & purificação , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , beta-Lactamases/genética , Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Sequência de Bases , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Conjugação Genética , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Genes Bacterianos , Hospitais de Ensino/estatística & dados numéricos , Humanos , Itália/epidemiologia , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , Plasmídeos/genética , Polimorfismo de Fragmento de RestriçãoRESUMO
In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149(H240), and TEM-149(H164-H240) versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149(H240), and TEM-149(H164-H240) ß-lactamases, and the Ki (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k+2 and k+3 (first-order acylation and deacylation constants, respectively), and k+2/K values were calculated.
Assuntos
Histidina/química , beta-Lactamases/química , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia , Carbapenêmicos/farmacologia , Cinética , Penicilinas/farmacologiaRESUMO
The role of RecA protein in bacterial resistance to antibiotics makes this protein attractive from a pharmacological point of view. In this study we demonstrate that curcumin is able to inhibit the SOS response in Escherichia coli induced by levofloxacin. The blaTEM-1 gene has been placed under the control of the LexA-binding box and used as reporter gene. The expression of TEM-1 ß-lactamase enzyme was increased in the presence of ssDNA induced by levofloxacin, while, the presence of curcumin at 8µg/ml, reduced dramatically the expression of the reporter gene. Moreover a simple microplate assay suitable for high-throughput screening has been developed.