Production of Purified MRSAcin from Local Clinical Strain of Methicillin-Resistant Staphylococcus aureus and its Applicability as Antimicrobial

Abstract

Confirmation of S. Aureus:The samples collected on mannitol salt broth media were incubated at 37°C for 24h and then inoculated onto mannitol salt agar media and incubated at 37°C for 24h. Selected isolates with colony morphology, Gram stain reactions and biochemical characteristics (i.e., Coagulase, Catalase reagent, Oxidase reagent).Confirmation of MRSA and antibiotic resistance testing: All isolates of S. aureus that demonstrated any level of Oxacillin (Methicillin) and Cefoxitin (FOX) resistance. All cultures were grown on Mueller-Hinton agar plates at 37°C for 18hours in the presence of the following antibiotics: Penicillin (10μg), Cefotaxiitin (30), Cefoxitin (30μg), Chloramphenicol C (30), Cefotaxime, CTX (30), Methicillin, ME(5) Oxacillin ,OX(1), Erythromycin, E (15) AbstractOne hundred fifty clinical samples (burns, wounds, urine, nasal and ear swabs) were collected from patients of Al-Yarmouk Hospital and Teaching Baghdad Hospital during the period from November/2015 to January/2016. Therefore was aimed to produce of Crude and purified bacteriocins from antibiotic resistant bacteria (G -ve ) MRSA isolated from clinical samples and determine the optimization conditions for production of bacteriocin and their purification and characterization. Cultural and morphological characteristic examination, biochemical tests were conducted and confirmed the diagnosis by API Staph and antibiotics sensitivity test confirmed by Vitek-2 system. The results showed 102(68%) isolates of S. aureus, all of which were Methicillin Resistance S. aureus (MRSA), constituted 50% from the ratio in the burns and wounds samples, while the lowest 13(12.7%) isolates were from ear samples, Vitek 2 system gave confirmation of positive results for MRSA with a probability 98-99%. The MRSA isolate S12 was chosen among five bacterial isolates as a good producer for crude MRSAcin according to their widest inhibition zone by Five methods were used to detect of MRSAcin production by (well diffusion assay WDA, flip-streak, spot on the lawn, Cup disc and paper disc method), against indicator pathogens (G +ve , G -ve bacteria and yeast) The optimum conditions for MRSAcin production were in trypticase soya broth with pH 7, at temperature 37°C for 48hrs and inoculums size of bacterial culture 6×10 8 cell/ml. "Bacteriocin after Purification was made by (two steps) method extraction by ammonium sulphate at 70% next step by DEAE-cellulose ion-exchange chromatography and Sephadex G-50 gel filtration chromatography. Also studied of Physical characteristics and the molecular weight showed of MRSAcin (15) KDa by (SDS-PAGE) Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis.

Gentamicin Upregulates the Gene Expression of hla and nuc in Staphylococcus aureus

“…When using of antibiotic sensitivity test, 40 (69%) S. aureus isolates exhibited resistance to gentamicin. This result was approximately parallel with Ahmed and Al Mossaw [30] who showed that 60% of isolates were gentamicin resistant. In a local study, in Duhok by Abdullah [31] showed that 59 (39.07%) of S. aureus isolates from UTI were gentamicinresistant.…”