الفهرس | Only 14 pages are availabe for public view |
Abstract In methicillin resistant Staphylococcus aureus (MRSA), antimicrobial susceptibility testing was performed to choose MRSA sensitive to vancomycin and resistant to ciprofloxacin. We could isolate persister cells with enough amount to carry out the experiments by using a high concentration of vancomycin. Biochemical tests were performed on MRSA local isolate and persister cells and used Standard MRSA ATCC 43300 as a positive control. These biochemical tests showed bacteriologically and biochemically persisters were similar to the clinical isolate and standard MRSA strains. But distinctive differences were observable in the DNase, coagulase ,and catalase activities. Molecular identification confirmed that the local isolate was S.aureus (M10) and submitted in gene bank with accession number (MT982652). DNA fingerprinting showed differences between MRSA original cells and its derivatives persister cells which ensuring the presence of genetic differences between them. Ten different efflux pump systems were detected in MRSA and its persister cells by PCR (NorA, NorB, TolC, MepA, TolC, AcrA, AcrB, MsrA, ErmE, PmrA) where they are specific to extrude different antibiotics specially ciprofloxacin. In this study, we have been used the most common three different efflux pump inhibitors specific to S.aureus, and Ethedium bromide as a substrate of efflux pump, Minimum inhibitory concentrations were determined to each EPI and EtBr. Efflux pump proteins were extracted from MRSA and its persister cells by SDS PAGE which showed the presence of 7 efflux pump protein bands in molecular weight ( 53, 48, 43, 41, 36, 31, 27 Kda. Random mutation was performed by exposer MRSA cells to UV (254 nm) for one minute leading to obtaining many mutants become sensitive to some antibiotics specially ciprofloxacin. We selected two mutants which are more sensitive to ciprofloxacin and examined them biochemically and molecularly which showed the differences between them and the parent organism MRSA (M10) in antimicrobial sensitivity test, the MIC to ciprofloxacin, ability to persisters formation, protein profile. |