Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
J Pharm Biomed Anal ; 248: 116259, 2024 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-38870837

RESUMO

BACKGROUND: The investigation of drug disposition in tissues is critical to improving dosing strategy and maximizing treatment effectiveness, yet developing a multi-tissue bioanalytical method could be challenging due to the differences among various matrices. Herein, we developed an LC-MS/MS method tailored for the quantitation of piperacillin (PIP), cefazolin (CFZ), and cefoxitin (CFX) in rat plasma and 12 tissues, accompanied by validation data for each matrix according to the FDA and EMA guidelines. RESULTS: The method required only a small sample volume (5 µL plasma or 50-100 µL tissue homogenates) and a relatively simple protocol for simultaneous quantitation of PIP, CFZ, and CFX within different biological matrices. Mobile phase A was composed of 5 mM ammonium formate and 0.1 % formic acid in water, while mobile phase B contained 0.1 % formic acid in acetonitrile. The mobile phase was pumped through a Synergi Fusion-RP column equipped with a guard column with a gradient elution program at a 0.3 mL/min flow rate. The mass spectrometer was operated in positive ionization mode (ESI+) using multiple reaction monitoring. SIGNIFICANCE: The validated method has been successfully applied to quantify PIP, CFZ, and CFX from the plasma and tissue samples collected in a pilot rat study and will further be used in a large pharmacokinetic study. To our knowledge, this is also the first report presenting long-term, freeze-thaw, and autosampler stability data for PIP, CFZ, and CFX in rat plasma and multiple tissues.


Assuntos
Cefazolina , Cefoxitina , Piperacilina , Espectrometria de Massas em Tandem , Animais , Espectrometria de Massas em Tandem/métodos , Ratos , Cefazolina/sangue , Cefazolina/farmacocinética , Cefazolina/análise , Piperacilina/sangue , Piperacilina/farmacocinética , Piperacilina/análise , Cefoxitina/farmacocinética , Cefoxitina/sangue , Cefoxitina/química , Cefoxitina/análise , Cromatografia Líquida/métodos , Reprodutibilidade dos Testes , Distribuição Tecidual , Ratos Sprague-Dawley , Antibacterianos/sangue , Antibacterianos/farmacocinética , Antibacterianos/análise , Masculino , Espectrometria de Massa com Cromatografia Líquida
2.
Molecules ; 26(18)2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34576958

RESUMO

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some ß-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some ß-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.


Assuntos
beta-Lactamases/química , beta-Lactamases/metabolismo , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Cefazolina/química , Cefazolina/metabolismo , Cefoxitina/química , Cefoxitina/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Imipenem/química , Imipenem/metabolismo , Cinética , Leucina/genética , Meropeném/química , Meropeném/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Fluorescência , beta-Lactamases/genética
3.
Curr Top Med Chem ; 20(25): 2300-2307, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32819244

RESUMO

BACKGROUND: The increasing and inappropriate use of antibiotics has increased the number of multidrug-resistant microorganisms to these drugs, causing the emergence of infections that are difficult to control and manage by health professionals. As an alternative to combat these pathogens, some monoterpenes have harmful effects on the bacterial cell membrane, showing themselves as an alternative in combating microorganisms. Therefore, the positive enantiomer α -pinene becomes an alternative to fight bacteria, since it was able to inhibit the growth of the species Escherichia coli ATCC 25922, demonstrating the possibility of its use as an isolated antimicrobial or associated with other drugs. AIMS: The aim of this study is to evaluate the sensitivity profile of E. coli ATCC 25922 strain against clinical antimicrobials associated with (+) -α-pinene and how it behaves after successive exposures to subinhibitory concentrations of the phytochemicals. METHODS: The minimum inhibitory concentration (MIC) was determined using the microdilution method. The study of the modulating effect of (+) -α-pinene on the activity of antibiotics for clinical use in strains of E. coli and the analysis of the strain's adaptation to the monoterpene were tested using the adapted disk-diffusion method. RESULTS: The results demonstrate that the association of monoterpene with the antimicrobials ceftazidime, amoxicillin, cefepime, cefoxitin and amikacin is positive since it leads to the potentiation of the antibiotic effect of these compounds. It was observed that the monoterpene was able to induce crossresistance only for antimicrobials: cefuroxime, ceftazidime, cefepime and chloramphenicol. CONCLUSION: It is necessary to obtain more concrete data for the safe use of these combinations, paying attention to the existence of some type of existing toxicity reaction related to the herbal medicine and to understand the resistance mechanisms acquired by the microorganism.


Assuntos
Antibacterianos/farmacologia , Monoterpenos Bicíclicos/farmacologia , Escherichia coli/efeitos dos fármacos , Amicacina/química , Amicacina/farmacologia , Amoxicilina/química , Amoxicilina/farmacologia , Antibacterianos/química , Monoterpenos Bicíclicos/química , Cefepima/química , Cefepima/farmacologia , Cefoxitina/química , Cefoxitina/farmacologia , Ceftazidima/química , Ceftazidima/farmacologia , Testes de Sensibilidade Microbiana
4.
Chem Biol Drug Des ; 96(4): 1095-1102, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-29938904

RESUMO

The penicillin-binding proteins (PBPs) are important biological target for new antibacterial drugs development. This study focused on molecular interaction between cefoxitin and the Escherichia coli PBP5 by molecular dynamics (MD) using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations approach, searching to develop a computational simulations prototype method on antimicrobial susceptibility of gram-negative bacteria against antibiotics. Escherichia coliATCC 8739 strain susceptibility for the drugs used in the antimicrobial susceptibility testing and selection of bioactive molecules against resistant strain. The protonation revealed a deprotonate state for His146, His151, His216, and His320 residues. The complex was stabilized after 0.6 ns of MD simulation. The global interaction means for inhibition zone diameters of E. coliATCC8739 strain and cefoxitin were 24.33 mm no showing significant difference between computational and experimental methods. Our computational simulation method can reliably be performed as a molecular modeling prototype for gram-negative antimicrobial susceptibility testing bacteria.


Assuntos
Antibacterianos/química , Cefoxitina/química , Proteínas de Escherichia coli/química , Bactérias Gram-Negativas/efeitos dos fármacos , Antibacterianos/farmacologia , Cefoxitina/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Teoria Quântica
5.
Sci Rep ; 9(1): 4917, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894628

RESUMO

The emergence of antibiotic resistant Gram-negative bacteria has become a serious global health issue. In this study, we have employed the intrinsically resistant opportunistic pathogen Stenotrophomonas maltophilia as a model to study the mechanisms involved in the acquisition of mutation-driven resistance to antibiotics. To this aim, laboratory experimental evolution studies, followed by whole-genome sequencing, were performed in the presence of the third-generation cephalosporin ceftazidime. Using this approach, we determined that exposure to increasing concentrations of ceftazidime selects high-level resistance in S. maltophilia through a novel mechanism: amino acid substitutions in SmeH, the transporter protein of the SmeGH RND efflux pump. The recreation of these mutants in a wild-type background demonstrated that, in addition to ceftazidime, the existence of these substitutions provides bacteria with cross-resistance to other beta-lactam drugs. This acquired resistance does not impose relevant fitness costs when bacteria grow in the absence of antibiotics. Structural prediction of both amino acid residues points that the observed resistance phenotype could be driven by changes in substrate access and recognition.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Ceftazidima/farmacologia , Stenotrophomonas maltophilia/efeitos dos fármacos , Resistência beta-Lactâmica/genética , Substituição de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cefotaxima/química , Cefotaxima/farmacologia , Cefoxitina/química , Cefoxitina/farmacologia , Ceftazidima/química , Expressão Gênica , Genes MDR , Aptidão Genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Fenótipo , Ligação Proteica , Estrutura Secundária de Proteína , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/metabolismo , Sequenciamento Completo do Genoma
6.
Artigo em Inglês | MEDLINE | ID: mdl-30397053

RESUMO

While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, ß-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D ß-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional ß-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural blaOXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the kcat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-ß-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat.


Assuntos
Antibacterianos/química , Enterobacteriaceae/genética , Resistência beta-Lactâmica/genética , beta-Lactamases/química , Substituição de Aminoácidos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sequência de Bases , Domínio Catalítico , Cefoxitina/química , Cefoxitina/metabolismo , Cefoxitina/farmacologia , Clonagem Molecular , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Expressão Gênica , Hospitais , Humanos , Hidrólise , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Meropeném/química , Meropeném/metabolismo , Meropeném/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Oxacilina/química , Oxacilina/metabolismo , Oxacilina/farmacologia , Penicilinas/química , Penicilinas/metabolismo , Penicilinas/farmacologia , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Águas Residuárias/microbiologia , beta-Lactamases/genética , beta-Lactamases/metabolismo
7.
Curr Microbiol ; 73(2): 236-41, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27146506

RESUMO

The healing properties of hyaluronic acid (HA) in the recovery of wounds are well known. Cefoxitin (Cef), a cephalosporin antibiotic, is generally used to prevent and treat postoperative infections. In this study, we describe the incorporation of Cef in HA thin films (Cef-HAF) by using electrospraying. Scanning electron microscopy images showed that HA-containing thin films (HAF) were composed of numerous nanoparticles (255 ± 177 nm in diameter) with irregular surfaces, connected to each other with nanofibers of 50 ± 11 nm in diameter. Cef-HAF contained fewer, but larger, particles (551 ± 293 nm) with smooth surfaces and were interconnected with nanofibers of 61 ± 13 nm in diameter. Differences in surface morphology between HAF and Cef-HAF were confirmed by atomic force microscopy. Fourier transform infrared and X-ray diffraction analyses revealed that Cef was not modified when incorporated into Cef-HAF and remained active against Klebsiella pneumoniae Xen 39, Staphylococcus aureus Xen 36 and Listeria monocytogenes EDGe. Nanofiber scaffolds of HA-containing Cef may be used in dressings to control postoperative infections.


Assuntos
Antibacterianos/farmacologia , Cefoxitina/farmacologia , Ácido Hialurônico/farmacologia , Antibacterianos/síntese química , Cefoxitina/química , Técnicas Eletroquímicas , Ácido Hialurônico/química , Klebsiella pneumoniae/efeitos dos fármacos , Nanopartículas/química , Staphylococcus aureus/efeitos dos fármacos
8.
Luminescence ; 31(5): 1054-62, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26678839

RESUMO

The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy-transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub-hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (nH ) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin-cefoxitin sodium system. The donor-to-acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non-radiation energy-transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd.


Assuntos
Cefoxitina/química , Fluorescência , Soroalbumina Bovina/química , Animais , Bovinos , Estrutura Molecular , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Temperatura
9.
Bull Environ Contam Toxicol ; 95(1): 131-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25733448

RESUMO

Aquatic ecosystems represent important vehicles for the dissemination of antibiotic resistant bacteria and antibiotic resistance genes. Of particular interest are methicillin-resistant staphylococci (MRS) harboring mecA gene that confers their resistance to ß-lactams. Therefore, in this study, water samples collected from different locations of a river impacted by surrounding facilities and domestic effluents were analyzed to learn more about the occurrence of MRS and mecA gene. Out of 290, 12 surface water isolates displayed resistance to both cefoxitin and oxacillin antibiotics. Resistant staphylococcal and non-staphylococcal isolates, identified by 16S rRNA sequencing, were found to harbor mecA gene. The phylogenetic tree of partial mecA sequences obtained from staphylococcal and non-staphylococcal isolates showed sequence similarity values of 8 %-100 %. Surface water bodies receive contaminated waters via runoff, effluents from industrial, agricultural, and municipal discharges. Therefore, surface waters are not only hot spots for mecA harboring staphylococcal isolates but also non-staphylococcal isolates and require special scientific consideration.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Resistência a Meticilina , Staphylococcus/genética , Microbiologia da Água , Antibacterianos/farmacologia , Sequência de Bases , Cefoxitina/química , Poluentes Ambientais , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Oxacilina/química , Proteínas de Ligação às Penicilinas , Filogenia , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Rios/microbiologia , Homologia de Sequência de Aminoácidos , Turquia , Poluentes Químicos da Água
10.
Sci Total Environ ; 466-467: 547-55, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23948499

RESUMO

An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence. Hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for three common ß-lactam antibiotics, ampicillin, cefalotin, and cefoxitin. Antibiotic hydrolysis rates at pH4-9 at 25 °C, 50 °C, and 60 °C were quantified, and degradation products were identified. The three antibiotics hydrolyzed under ambient conditions (pH7 and 25 °C); half-lives ranged from 5.3 to 27 d. Base-catalyzed hydrolysis rates were significantly greater than acid-catalyzed and neutral pH hydrolysis rates. Hydrolysis rates increased 2.5- to 3.9-fold for a 10 °C increase in temperature. Based on the degradation product masses found, the likely functional groups that underwent hydrolysis were lactam, ester, carbamate, and amide moieties. Many of the proposed products resulting from the hydrolysis of ampicillin, cefalotin, and cefoxitin likely have reduced antimicrobial activity because many products contained a hydrated lactam ring. The results of this research demonstrate that ß-lactam antibiotics hydrolyze under ambient pH and temperature conditions. Degradation of ß-lactam antibiotics will likely occur over several weeks in most surface waters and over several days in more alkaline systems.


Assuntos
Ampicilina/metabolismo , Antibacterianos/metabolismo , Cefoxitina/metabolismo , Cefalotina/metabolismo , Ampicilina/química , Antibacterianos/química , Cefoxitina/química , Cefalotina/química , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Hidrólise , Temperatura
11.
Biochemistry ; 52(23): 4097-104, 2013 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-23672214

RESUMO

The constitutively expressed, chromosomally encoded ß-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to ß-lactam antibiotics in Mycobacterium tuberculosis. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity [Tremblay, L. W., et al. (2008) Biochemistry 47, 5312-5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal velocity depends on the ionization state of two groups: a general base exhibiting a pK value of 4.5 and a general acid exhibiting a pK value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose kcat values differ by 5.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both V and V/K. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on V of 3.9 but a small SKIE of 1.8 on V/K in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of ß-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation steps of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate-limiting steps for ß-lactam hydrolysis of these substrates are analyzed, and the chemical steps responsible for the observed SKIE are discussed.


Assuntos
Proteínas de Bactérias/química , Cefoxitina/química , Cefalosporinas/química , Mycobacterium tuberculosis/enzimologia , Tienamicinas/química , beta-Lactamases/química , Acilação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Meropeném , Solventes/química , Resistência beta-Lactâmica
12.
J Med Microbiol ; 62(Pt 9): 1423-1427, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23579394

RESUMO

The culture of toxigenic Clostridium difficile from stool specimens is still seen as the gold standard for the laboratory diagnosis of C. difficile infection (CDI). bioMérieux have released ChromID Cdiff chromogenic agar (CDIF) for the isolation and identification of C. difficile in 24 h. In this study, we compared CDIF to pre-reduced cycloserine-cefoxitin-fructose agar with sodium taurocholate (TCCFA) in the examination of glutamate dehydrogenase-positive faecal specimens that were either GeneOhm positive or negative, using direct culture or culture following alcohol shock. Direct culture on CDIF had a sensitivity of 100 % and recovery of 94 % while for TCCFA these were 87 % and 82 %, respectively. For GeneOhm-positive alcohol-shocked faecal samples, sensitivity and recovery on CDIF was similar to direct culture while on TCCFA they were about 10 % higher. For direct culture, there was a significant difference between growth on CDIF at 24 h and TCCFA at 48 h (P = 0.001) and between the two media at 48 h (P<0.001). A total of 142 strains of C. difficile were recovered in pure culture from all GeneOhm-positive samples used in this study and 11 (7.7 %) of these were A(-)B(-)CDT(-) and may represent mixed infections of toxigenic and non-toxigenic C. difficile. The most dominant ribotype was UK 014 (14.7 %) followed by 002 (11.9 %) and 020 (11.9 %), and 36 % of toxigenic isolates, including an A(-)B(+)CDT(-) strain, could not be assigned a UK ribotype. CDIF outperformed pre-reduced TCCFA by negating the need for alcohol shock treatment and by giving a time saving of 24 h in the isolation of C. difficile. CDIF plates were also more selective than TCCFA and C. difficile colonies were easy to identify and subculture prior to strain typing.


Assuntos
Ágar/química , Técnicas Bacteriológicas/métodos , Cefoxitina/química , Clostridioides difficile/isolamento & purificação , Ciclosserina/química , Fezes/microbiologia , Álcoois/química , Meios de Cultura , Frutose/química , Humanos , Reprodutibilidade dos Testes , Ribotipagem , Sensibilidade e Especificidade , Estresse Fisiológico , Ácido Taurocólico
13.
J Biol Chem ; 287(44): 36854-63, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-22977239

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A(LGA), the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the ß-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 µg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 µg/ml). Similar to PBP2A, the protein homolog PBP2A(LGA) was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A(LGA) did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.


Assuntos
Aminoaciltransferases/fisiologia , Proteínas de Bactérias/fisiologia , Staphylococcus aureus/enzimologia , Resistência beta-Lactâmica/genética , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Compostos de Boro/química , Cefoxitina/química , Cefoxitina/farmacologia , Membrana Celular/química , Clonagem Molecular , Estabilidade Enzimática , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Peso Molecular , Oxacilina/química , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas , Penicilinas/química , Fenótipo , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Especificidade por Substrato
14.
J Chromatogr B Analyt Technol Biomed Life Sci ; 878(3-4): 428-34, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20060790

RESUMO

A rapid, selective and sensitive high performance liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher 60 RP Select B column (125 mm x 4 mm i.d., 5 microm particle size) using acetonitrile:5+/-0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0-15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within +/-6.3% of nominal values. No matrix effect was observed in this method. The validated LC-MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.


Assuntos
Cefuroxima/sangue , Cefuroxima/farmacocinética , Cromatografia Líquida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Administração Oral , Adulto , Cefoxitina/química , Cefuroxima/administração & dosagem , Cefuroxima/química , Estabilidade de Medicamentos , Saúde , Humanos , Índia , Masculino , Reprodutibilidade dos Testes , Fatores de Tempo
15.
J Pharm Biomed Anal ; 44(5): 1040-7, 2007 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-17537608

RESUMO

Acidity constants of six cephalosporin antibiotics, cefalexin, cefaclor, cefadroxil, cefotaxim, cefoperazon and cefoxitin are determined using capillary zone electrophoresis (CZE) and pH-potentiometric titrations. Since CZE is a separation method, it is not necessary for the samples to be of high purity and known concentration because only mobilities are measured. The effect on determination of dissociation constants of different matrices (serum, 0.9% NaCl, fermentation matrix) was examined. The advantages of CZE can be utilized in those fields where potentiometry has limitations (sample quantity, solubility, purity, simultaneous determinations), although pK(a) values that are close to each other can be determined by potentiometry with more accuracy.


Assuntos
Antibacterianos/química , Cefalosporinas/química , Eletroforese Capilar/métodos , Antibacterianos/análise , Cefaclor/análise , Cefaclor/sangue , Cefaclor/química , Cefadroxila/análise , Cefadroxila/sangue , Cefadroxila/química , Cefoperazona/análise , Cefoperazona/sangue , Cefoperazona/química , Cefotaxima/análise , Cefotaxima/sangue , Cefotaxima/química , Cefoxitina/análise , Cefoxitina/sangue , Cefoxitina/química , Cefalexina/análise , Cefalexina/sangue , Cefalexina/química , Cefalosporinas/análise , Cefalosporinas/sangue , Eletroforese Capilar/instrumentação , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Potenciometria/instrumentação , Potenciometria/métodos
16.
J Med Microbiol ; 56(Pt 1): 43-46, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17172515

RESUMO

Mannitol salt agar (MSA), CHROMagar Staph aureus (CSA) and CHROMagar MRSA (CSA-MRSA) were evaluated with nasal surveillance specimens for their ability to detect Staphylococcus aureus and meticillin-resistant S. aureus (MRSA). CSA was found to be more sensitive than MSA in detecting S. aureus (98 versus 84.3 %; P=0.03). CSA and CSA-MRSA were equivalent in the ability to detect MRSA at 24 h (89.7 versus 87.2 %) and at 48 h (94.9 versus 94.9 %). When combined with Staphaurex slide confirmation testing, both CSA and CSA-MRSA were highly specific (100 %) media for detecting MRSA from nasal swab specimens.


Assuntos
Resistência a Meticilina , Mucosa Nasal/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/isolamento & purificação , Ágar , Antibacterianos/química , Antibacterianos/farmacologia , Cefoxitina/química , Cefoxitina/farmacologia , Compostos Cromogênicos/química , Meios de Cultura/química , Humanos , Manitol/química , Meticilina/química , Meticilina/farmacologia , Testes de Sensibilidade Microbiana/métodos , Especificidade da Espécie , Infecções Estafilocócicas/microbiologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/crescimento & desenvolvimento , Staphylococcus/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
17.
Pharm Res ; 23(10): 2254-68, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16941232

RESUMO

PURPOSE: To test the hypothesis that the molecular motions associated with chemical degradation in glassy amorphous systems are governed by the molecular motions associated with structural relaxation. The extent to which a chemical process is linked to the motions associated with structural relaxation will depend on the nature of the chemical process and molecular motion requirements (e.g., translation of a complete molecule, rotational diffusion of a chemical functional group). In this study the chemical degradation and molecular mobility were measured in model systems to assess the degree of coupling between chemical reactivity and structural relaxation. The model systems included pure amorphous cephalosporin drugs, and amorphous molecular mixtures containing a chemically labile drug and an additive expected to moderate molecular mobility. METHODS: Amorphous drugs and mixtures with additives were prepared by lyophilization from aqueous solution. The physical properties of the model systems were characterized using optical microscopy and differential scanning calorimetry. The chemical degradation of the drugs alone and in mixtures with additives was measured using high-performance liquid chromatography (HPLC). Molecular mobility was measured using isothermal microcalorimetry to measure enthalpy changes associated with structural relaxation below T (g). RESULTS: A weak correlation between the rates of degradation and structural relaxation times in pure amorphous cephalosporins suggests that reactivity in these systems is coupled to molecular motions in the glassy state. However, when sucrose was added to one of the cephalosporin drugs stability improved even though this addition reduced T (g) and the relaxation time constant, tau(D)(beta), suggesting that there was no correlation between reactivity and structural relaxation in the cephalosporin mixtures. In contrast, the rate of ethacrynate sodium dimer formation in mixtures was more strongly coupled to the relaxation time constant, tau(D)(beta). CONCLUSIONS: These studies suggest that the extent to which chemical degradation is coupled to structural relaxation in glasses motions is determined by how closely the motions of the rate controlling step in chemical degradation are associated with structural relaxation. Moderate coupling between the rate of dimer formation for ethacrynate sodium in mixtures with sucrose, trehalose and PVP and structural relaxation constants suggests that chemical changes that require more significant molecular motion, and includes at least some translational diffusion, are more strongly coupled to the molecular motions associated with structural relaxation. The observation that sucrose stabilizes cefoxitin sodium even though it lowers T (g) and reduces the relaxation time constant, tau(D)(beta) is perhaps a result of the importance of other kinds of molecular motions in determining the chemical reactivity in glasses.


Assuntos
Cristalização , Excipientes Farmacêuticos/química , Preparações Farmacêuticas/química , Varredura Diferencial de Calorimetria , Cefamandol/química , Cefoxitina/química , Cefalotina/química , Fenômenos Químicos , Química Farmacêutica , Físico-Química , Cromatografia Líquida de Alta Pressão , Ácido Etacrínico/química , Liofilização , Povidona/química , Soluções , Sacarose/química , Trealose/química
18.
J Am Chem Soc ; 127(15): 5423-34, 2005 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-15826180

RESUMO

CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.


Assuntos
beta-Lactamases/química , beta-Lactamases/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Sítios de Ligação , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Ácidos Borônicos/farmacologia , Catálise , Cefoxitina/química , Cefoxitina/metabolismo , Cefoxitina/farmacologia , Cristalografia por Raios X , Temperatura Alta , Isoenzimas , Cinética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Proteica , Desnaturação Proteica , Relação Estrutura-Atividade , Inibidores de beta-Lactamases
19.
Int J Pharm ; 271(1-2): 31-9, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15129971

RESUMO

To conquer the clinical restriction of relative short half-life and poor tissue retaining activities, liposomes containing cefoxitin were prepared using three methods in this study. The physicochemical properties including cefoxitin encapsulation percentage, vesicle size, stability, as well as the in vivo biodistribution were studied. The highest entrapment percentage was observed by using reverse phase evaporation method, and the molar ratio of cefoxitin to phospholipids was 1:3, DMPC to cholesterol was 2:1, respectively. From the result of stability, the freeze-drying powder and then stored in the frozen condition of cefoxitin-loaded liposome was an ideal storage state. Accordingly, the formulation by reverse-phase evaporation method was selected to investigate the biodistribution of cefoxitin-loaded liposome and compared to free cefoxitin in rats. It was observed that the cefoxitin levels and the duration retained in the liver, spleen, and pancreas of liposome-injected animals were higher and longer than that of free cefoxitin-injected animals. The drug concentrations of bile after post-injection of liposomal cefoxitin at 0.5, 1 and 2 h were all approximately 2.7 times higher than that of free cefoxitin injection group.


Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Cefoxitina/administração & dosagem , Cefoxitina/farmacocinética , Animais , Antibacterianos/química , Bile/metabolismo , Cefoxitina/química , Colesterol/química , Cromatografia Líquida de Alta Pressão , Dimiristoilfosfatidilcolina/química , Estabilidade de Medicamentos , Liofilização , Lipossomos , Masculino , Tamanho da Partícula , Ratos , Ratos Wistar , Tecnologia Farmacêutica , Distribuição Tecidual
20.
Biochemistry ; 41(6): 1877-85, 2002 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-11827533

RESUMO

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.


Assuntos
Bacillus/enzimologia , beta-Lactamases/química , Domínio Catalítico , Cefoxitina/química , Cefamicinas/química , Cristalografia por Raios X , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Secundária de Proteína , Eletricidade Estática
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA