RESUMO
In order to assess the performance of two detection methods, a set of 93 recent clinical isolates of Staphylococcus aureus, including a large number of strains that demonstrated low-level methicillin-resistance were evaluated using the MRSA-Screen (Denka Seiken, Japan), a commercial latex agglutination test to detect penicillin-binding protein 2' (PBP2'), and a polymerase chain reaction assay using the LightCycler Instrument (Roche Diagnostics, Switzerland). The results show that the latex agglutination test is highly sensitive if performed after induction by cefoxitin. Inconclusive results can be rapidly confirmed on the same day by real-time polymerase chain reaction used to detect mecA and femA genes.
Assuntos
Hexosiltransferases , Resistência a Meticilina , Peptidil Transferases , Staphylococcus aureus/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Humanos , Muramilpentapeptídeo Carboxipeptidase/genética , Proteínas de Ligação às Penicilinas , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
A novel teicoplanin-associated operon termed tcaR-tcaA-tcaB was identified by Tn917-mediated insertional mutagenesis. Resistance to teicoplanin rose 4-fold by insertional inactivation of tcaA or by deletion of the entire operon. tcaA encodes a hypothetical transmembrane protein with a metal-binding motif, possibly a sensor-transducer. tcaB codes for a membrane-associated protein, which has sequence homologies to a bicyclomycin resistance protein. The two genes are preceded by tcaR encoding a putative regulator with sequence homologies to the transcriptional regulator MarR. The fact that tcaA inactivation as well as deletion of tcaRAB produced the same increase in teicoplanin resistance confirmed the association of tcaRAB with teicoplanin susceptibility. Cotransductional crosses showed that the level of teicoplanin resistance produced by these insertions was strain-dependent and that in the methicillin-resistant strain COL, it was paired with a remarkable decrease in methicillin resistance. This allowed to postulate that tcaRAB may be involved in some way in cell wall biosynthesis, and that teicoplanin may interact with TcaA and/or TcaB either directly or indirectly.
Assuntos
Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Óperon , Staphylococcus aureus/genética , Teicoplanina/farmacologia , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Deleção de Genes , Genótipo , Dados de Sequência Molecular , Mutagênese Insercional , Staphylococcus aureus/efeitos dos fármacosRESUMO
The FemAB-like factors Lif and Epr confer resistance to glycylglycine endopeptidases lysostaphin and Ale-1, respectively, by incorporating serine residues into the staphylococcal peptidoglycan interpeptide bridges specifically at positions 3 and 5. This required the presence of FemA and/or FemB, in contrast to earlier postulations.
Assuntos
Proteínas de Bactérias/metabolismo , Resistência Microbiana a Medicamentos , Peptidoglicano/metabolismo , Serina/metabolismo , Proteínas de Bactérias/genética , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Serina/genética , Staphylococcus/genética , Staphylococcus/metabolismoRESUMO
The factor catalyzing the first step in the synthesis of the characteristic pentaglycine interpeptide in Staphylococcus aureus peptidoglycan was found to be encoded by the essential gene fmhB. We have analyzed murein composition and structure synthesized when fmhB expression is reduced. The endogenous fmhB promoter was substituted with the xylose regulon from Staphylococcus xylosus, which allowed glucose-controlled repression of fmhB transcription. Repression of fmhB reduced growth and triggered a drastic accumulation of uncrosslinked, unmodified muropeptide monomer precursors at the expense of the oligomeric fraction, leading to a substantial decrease in overall peptidoglycan crosslinking. The composition of the predominant muropeptide was confirmed by MS to be N-acetylglucosamine-(beta-1,4)-N-acetylmuramic acid(-L-Ala-D-iGln-L-Lys-D-Ala-D-Ala), proving that FmhB is involved in the attachment of the first glycine to the pentaglycine interpeptide. This interpeptide plays an important role in crosslinking and stability of the S. aureus cell wall, acts as an anchor for cell wall-associated proteins, determinants of pathogenicity, and is essential for the expression of methicillin resistance. Any shortening of the pentaglycine side chain reduces or even abolishes methicillin resistance, as occurred with fmhB repression. Because of its key role FmhB is a potential target for novel antibacterial agents that could control the threat of emerging multiresistant S. aureus.
Assuntos
Genes Bacterianos , Peptidoglicano/genética , Staphylococcus aureus/genética , Transcrição Gênica , Sequência de Carboidratos , Parede Celular/química , Genes Essenciais , Glucose/metabolismo , Lisostafina/farmacologia , Meticilina/farmacologia , Resistência a Meticilina , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mapeamento de Peptídeos , Peptidoglicano/biossíntese , Peptidoglicano/química , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Regulon , Staphylococcus/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Xilose/metabolismoRESUMO
Three new proteins, FmhA, FmhB and FmhC, with significant identities to FemA and FemB were identified in the Staphylococcus aureus (ATCC 55748) genome database. They were mapped to the SmaI-C, SmaI-H and SmaI-A fragments of the S. aureus 8325 chromosome, respectively. Whereas insertional inactivation of fmhA and fmhC had no effects on growth, antibiotic susceptibility, lysostaphin resistance, or peptidoglycan composition of the strains, fmhB could not be inactivated, strongly suggesting that fmhB may be an essential gene. As deduced from the functions of FemA and FemB which are involved in the synthesis of the peptidoglycan pentaglycine interpeptide, FmhB may be a candidate for the postulated FemX thought to add the first glycine to the nascent interpeptide.
Assuntos
Proteínas de Bactérias/genética , Fases de Leitura Aberta/genética , Staphylococcus aureus/genética , Parede Celular/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Sondas de DNA , Genoma Bacteriano , Resistência a Meticilina/genética , Mutagênese , Proteoglicanas/metabolismo , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , TemperaturaRESUMO
Methicillin resistance in Staphylococcus aureus is primarily due to the acquisition of an additional penicillin-binding protein, PBP2' (also known as PBP2a), that confers resistance to virtually all beta-lactam antibiotics. This foreign PBP2' has strict requirements on peptidoglycan precursor formation and composition in order to function optimally. The level of methicillin resistance is governed by genomic factors which are involved in cell wall metabolism and/or are constituents of the cytoplasmic membrane. The formation of the pentaglycine interpeptide bridge of peptidoglycan plays a key function and depends on at least three factors, FemX, FemA and FemB.
RESUMO
The formation of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide chain needs FemA and FemB for the incorporation of glycines Gly2-Gly3, and Gly4-Gly5, respectively. The lysostaphin immunity factor Lif was able to complement FemB, as could be shown by serine incorporation and by an increase in lysostaphin resistance in the wild-type as well as in a femB mutant. However, Lif could not substitute for FemA in femA or in femAB-null mutants. Methicillin resistance, which is dependent on functional FemA and FemB, was not complemented by Lif, suggesting that serine-substituted side chains are a lesser substrate for penicillin-binding protein PBP2' in methicillin resistance.
