RESUMO
Methicillin resistant Staphylococcus aureus (MRSA) is highly prevalent, and its typing plays a crucial role in epidemiology and evolution in both health and community settings. Multiplex PCR and staphylococcal cassette chromosome mec (SCCmec) typing based on mec complexes and cassette chromosome recombinase (ccr) allotypes have been developed for MRSA identification. The first of these procedures can identify 4 mec classes (A, B, C1, and E) and 2 ccr allotypes (B2 and B4) in one tube, and the second can identify mecA, mec class C2, and 3 allotypes (A1, A3, and C). Our method offers a novel means to further differentiate between the main SCCmec types I through XI and is both highly sensitive (detectable up to 0.3ηg DNA) and specific (100%). Several SCCmec types (I, III, IV, V and a non-typeable group) were found in 66 MRSA isolates obtained from Ho Chi Minh City, Vietnam and Nakhon Pathom, Thailand. SCCmec type III was highly predominant in both regions. The designed assay is rapid, convenient, flexible, and reliable. Therefore, this assay is suitable for the high-throughput screening of the main SCCmec types of MRSA isolates.
Assuntos
Proteínas de Bactérias/genética , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Estafilocócicas/microbiologia , Antibacterianos/farmacologia , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase Multiplex/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Infecções Estafilocócicas/epidemiologia , Tailândia , VietnãRESUMO
A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.
Assuntos
Haemophilus ducreyi/enzimologia , beta-Lactamases/isolamento & purificação , Antibacterianos/metabolismo , Sequência de Bases , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Transformação Bacteriana , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-LactamasRESUMO
Two beta-lactamases, penicillinase type I from Bacillus cereus and TEM-1 beta-lactamase from Haemophilus ducreyi, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow-injection analysis of beta-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins. Moreover, Michaelis constants of the TEM-1 beta-lactamase were markedly increased upon immobilization for all substrates, especially carbenicillin, cephaloridine, and cefoperazone.
Assuntos
Cefalosporinas/análise , Cromatografia em Agarose/métodos , Penicilinase/metabolismo , beta-Lactamases/metabolismo , Antibacterianos/análise , Antibacterianos/metabolismo , Bacillus cereus/enzimologia , Calorimetria , Carbenicilina/análise , Carbenicilina/metabolismo , Cefoperazona/análise , Cefoperazona/metabolismo , Cefaloridina/análise , Cefaloridina/metabolismo , Cefalosporinas/metabolismo , Quelantes , Cromatografia de Afinidade , Enzimas Imobilizadas , Haemophilus ducreyi , Níquel , Penicilinas/análise , beta-Lactamas/análise , beta-Lactamas/metabolismoRESUMO
Chancroid caused by Haemophilus ducreyi has been described as a significantly predisposing factor of HIV heterosexual transmission in an endemic region of both diseases. The fastidious, H. ducreyi has been reported world wide with various antimicrobial susceptibility patterns. A high tendency of drug resistances has generally been found among isolates derived in Thailand. In this study, the plasmids of H. ducreyi were isolated and analysed from 63 clinically derived organisms. Twenty-nine out of 63 isolates (46%) revealed the same plasmid profiles. Plasmid DNA was further cloned into Escherichia coli and transformants were selected. A 3.6 kb plasmid (pCb) carrying ampicillin resistance was subsequently identified. The pCb conferred resistance to various beta-lactam antibiotics including penicillin G, carbenicillin, piperacillin, cefazolin, cefoperazone, ampicillin-sulbactam, and amoxicillin-clavulanate but not to cefoxitin. Co-resistance to streptomycin, chloramphenicol and tetracycline was not detected. Beta-lactamase gene was located on the major pCb fragment of EcoRI and AatII cutting.
