Bacteriological and Genetic Studies on Oxacillin Resistant Staphylococcus aureus Isolated from Some Hospital in Baghdad City

Two hundred and six clinical specimens were collected from four different hospitals in Baghdad during the period (February to October 2010); these specimens included swabs from anterior nares, hands and ear swabs that were collected from visitors, hospitalized patients and the health care workers in these hospitals. Diagnostic results show that 154 out of the 206 specimens gave positive bacterial cultures and 61 isolates are characterized as Staphylococcus aureus depending on the cultural and biochemical examinations. In addition, the antibiotic susceptibility profile for these isolates was examined against fifteen types of antibiotics of the following groups (β-lactam antibiotics, fluoroquinolones, aminoglycosides, macrolides, glycopeptide, phenicoles and tetracycline). The results showed that (77%) of the isolates were multiresistant
as determined by the resistance of such isolates to more than three types of antibiotics. Highest resistance was recorded against ticarcillinclavulanic acid, penicillin, oxacillin, cefepime, cefoxitin, cephalothin and
erythromycin with a percentage of (93.4%, 78.7%, 77.1%, 68.9%, 65.6%, 55.7% and 47.5%), respectively. Followed by moderate resistance against tetracyclin, gentamycin and amikacin (with the same percentage of resistance) then ciprofloxacin and chloramphenicol. On the other hand the tested isolates showed a high degree of susceptibility towards vancomycin, tobramycin and imipenem. The isolates that exhibit oxacillin resistance in addition of being resistant to five or more of the antibiotics were selected to determine their MICs values against oxacillin using a concentrations ranging from (0.25μg/ml –1280 μg/ml) and as a result (23) isolates are chosen as their MICs values were (≥ 4.0) to test their hemolytic activity (hemolysis assay) to determine their ability to produce (alpha-toxin). The results indicate that all the isolates except two of them were able to produce the toxin with variable degree of activity. A confirmatory test was carried out for the selected isolates using PCR technique for further characterization up to the species level by the amplification of (nuc) gene (Staphylococcus aureus specific gene) and all the isolates are found to be positive for the presence of (nuc+) gene as their agarose gel revealed the presence of DNA band of nuc gene with a molecular size of (300 bp.). The gel electrophoresis of the plasmid DNA purified from these isolates was carried out; the results revealed that (11) of these isolates had a plasmid and (5) had two plasmids with a molecular size of (5200 bp.) and (10,000 bp.), while (5) of these isolates were found to have a plasmid with large molecular size (out of the maximum size of the leader) and one isolate shows three band of DNA plasmid with molecular size (4000 bp., 10,000 bp.) and the last band is out of the maximum size of the leader. Detection of oxacillin-resistance gene represented by (mecA+) revealed the presence of (19) positive isolates with the appearance of DNA band of about (200 pp.); A correlation between positivity of the isolates for cefoxitin resistant test and the molecular aspect for the presence of (mecA). Results of the detecting the alpha-hemolysin specific gene (hla+) showed that it was positive in (21) of the isolates as they appear to have a band with a molecular size of about (550 bp.), and these results were used to confirm the results of the (hemolysis assay). These results were almost considered as the first sign for the correlation between the presence of alpha-hemolysin gene and oxacillin- resistant trait. The enhanced effect of zinc oxide nanoparticles on the antibacterial activity of oxacillin was studied and the effect was determined against specific selected isolate. A positive and induced effect for zinc oxide nanoparticle was detected upon the antibacterial activity of oxacillin through increasing the diameter of inhibition zone for the antibiotic as it recorded (15mm) for the mixed activity to the antibiotic with nanoparticles compared with that of (10mm) for the zinc oxide nanoparticle alone and the disappearance of the inhibition zone for the antibiotic alone. These results might be considered as the first report in Iraq for the possibility to use the
synergistic effect of nanoparticles with antibiotics to stop the progress resistance of bacteria against antibiotics used in the present time. The antibacterial activity of the crude ethanolic olive leaf extract was
evaluated alone or in combination with either oxacillin or zinc oxide nanoparticles against oxacillin resistant Staphylococcus aureus using disc diffusion method. The results revealed that olive leaf extracts (2.25-9
mg/disc) neither exhibited antibacterial activity when used alone, nor potentiated the antibacterial activity of zinc oxide nanoparticles (100μg/disc). However, in presence of the highest concentration of crude olive leaf extract (9 mg/disc), the antibacterial activity of oxacillin (1μg/disc) was improved when the diameter of inhibition zone increased from zero to a maximum of 14 mm, suggesting a potentiation of the antimicrobial activity of oxacillin against the already existing oxacillin-resistant strain of Staphylococcus aureus. If this
unexpected finding can be reproduce by further studies using various concentrations of olive leaf extracts, then it may represent the first report in the available literature that signifies the potential utilization of olive leaf extracts in combination therapy against oxacillin-resistant Staphylococcus aureus.