In the case of gram-positive bacteria, it should be analyzed more confidently if DNA fragmentation is produced after β-lactam treatment, although more delayed than in
gram-negative. If this is the case, despite of the effect, the thicker cell wall of gram-positive bacteria would also prevent the release of DNA fragments. From the practical point of view, the background of DNA fragments was visualized without the necessity of incubation in lysing solution or any manipulation, so it could be used for a rapid determination of sensitivity or resistance, in liquid cultures. Nevertheless, the presence of the background could be indicative of susceptibility only in gram-negative bacteria, in those here assayed at least. Furthermore, the dilution of the culture modifies the density buy MK-8669 of the background, and different bacteria and different strains may show important differences in the amount of extracellular
DNA fragments. A more confident discrimination between sensitive and resistant strains is achieved when also Selleckchem AZD3965 evaluating the cell wall response to the specific lysing solution. The dose-response study shows that the β-lactam induces a progressive effect with increasing dose on the cell wall. This effect is evident even before the MIC dose, although it is very weak and seems not prevent growth of most of bacteria after removing the antibiotic. The cell wall damage is not homogeneous among cells, although a predominant level is observed for each dose. This level is more intense as dose increases. The heterogeneity in the effect on the cell wall is not mainly dependent on the growing stage since the cultures were exponentially growing NADPH-cytochrome-c2 reductase when exposed to ampicillin. The background of DNA fragments appears to be observed at the MIC dose, and increases as dose increases, within the range of doses assayed. The methodology has been confirmed as a rapid and simple procedure to distinguish susceptible and resistant strains of eight
gram-negative and four gram-positive species, assaying four different β-lactams and vancomycin. The results were reproducible and accurate, in the 46 clinical strains. Although preliminary, the results are encouraging. Expanded work analysing many more strains is in progress. For example, links have been established between glycopeptide resistance and cell wall thickening in vancomycin-intermediate Staphylococcus aureus (VISA), as well as between macrolides and thickened cell walls in S. aureus [20, 21]. These are interesting strains to be tested. Furthermore, the examination of the slides is going to be automated using a microscopy platform coupled with image capture and digital image analysis.
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