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A collection of laboratory mutants and clinical MRSA strains, additionally exhibiting resistance to glycopeptide antibiotics, was studied in detail. The nature of resistance to glycopeptides was found to be different from that existing in vancomycin resistant (VR) enterococci. The mutants produced abnormal murein in which the level of highly oligomeric muropeptides was drastically reduced. Biochemical and genetic analyses of Penicillin Binding Proteins (PBPs) showed inactivation of PBP4. Changes in other PBPs were not apparent, except for PBP2a that was inactivated in the highly VR mutant VM. Transposon inactivation of the pbpB gene and several other genes involved in synthesis of staphylococcal peptidoglycan all caused dramatic reduction of glycopeptide resistance in the staphylococcal mutants. While inactivation of PBP2a slightly increased the levels of glycopeptide resistance, a combination of vancomycin or teicoplanin with β-lactam inhibitors, chosen on the basis of their relatively selective affinities for individual staphylococcal PBPs completely inhibited the expression of glycopeptide resistance in MRSA. Glycopeptide antibiotics caused a virtually complete inhibition of cell wall turnover and autolysis and massive overgrowth of cell wall material in the glycopeptide resistant mutants. Bacteria were able to remove quantitatively glycopeptide molecules from the growth medium, and sequestered antibiotic could be recovered in biologically active form from the purified cell walls. These observations and the results of the vancomycin binding studies suggest alterations in the structural organization of the mutants' cell wall such that access of glycopeptide molecules to the sites of wall biosynthesis is blocked by steric hindrance.
Pheromone-responsive plasmids constitute a unique group of ~ 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids posses a sophisticated conjugation mechanism based on response to sex pheromones - small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role - in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence of enterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.
The prevalence of glycopeptides, aminoglycosides and erythromycin resistance among Enterococcus faecalis and Enterococcus faecium was investigated. The susceptibility of 326 enterococcal hospital isolates to amikacin, kanamycin, netilmicin and tobramycin were determined using disk diffusion method. The minimum inhibitory concentration (MIC) of vancomycin, teicoplanin, gentamicin, streptomycin, and erythromycin were determined by microbroth dilution method. The genes encoding aminoglycoside modifying enzymes described as AMEs genes, erythromycin-resistant methylase (erm) and vancomycin-resistant were targeted by multiplex-PCR reaction. High level resistance (HLR) to gentamicin and streptomycin among enterococci isolates were 52% and 72% respectively. The most prevalent of AMEs genes were aac (6')-Ie aph (2") (63%) followed by aph (3') -IIIa (37%). The erythromycin resistance was 45% and 41% of isolates were positive for ermB gene. The ermA gene was found in 5% of isolates whereas the ermC gene was not detected in any isolates. The prevalence of vancomycin resistant enterococci (VRE) was 12% consisting of E. faecalis (6%) and E. faecium (22%) and all of them were VanA Phenotype. The results demonstrated that AMEs, erm and van genes are common in enterococci isolated in Tehran. Furthermore our results show an increase in the rate of vancomycin resistance among enterococci isolates in Iran.
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