الفهرس 
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Abstract
Staphylococci are the major cause of nosocomial infections. Staphylococcal food borne diseases resulting from consumption of food contaminated with SEs are the second most common cause of reported food-borne illnesses. Symptoms includes abdominal cramps, nausea, vomiting (emesis) and diarrhoea. The onset of symptoms usually starts between 0.5 and 8 h after ingestion of the contaminated food, and symptoms usually resolve without intervention within 24 h.
Staphylococcal enterotoxin type A (SEA) is the most important which characterized serologically distinct types of staphylococcal enterotoxins. The enterotoxins are the causative agents of staphylococcal food poisoning syndrome. Non-carriers commonly acquire infections through contaminated food or when food handlers who are carriers contaminate food during preparation. This study focused on isolation of Staphylococcus from infected patients and selection of sea producing organisms.
- The sixty four staphylococci isolates had been isolated from specimens ranged from wounds, pus, throat, sputum, conjunctiva, urine in addition to stool of patients plagued by symptoms of infection. All isolates were cultured on selective media specific for Staphylococcus spp. and then identified by biochemical tests. The results showed that 62 isolates belonged to S.aureus and two isolates belonged to S. epidermidis.
- Collected staphylococci isolates had been subjected to the sensitivity to cefoxitin (FOX) as an indicator of methicillin resistance (MR). Fourty one; S. aureus and one; S. epidermidis out of the sixty two isolate were found to be methicillin resistant. The effectiveness of other commonly used antibiotics towards the fourty two MRS was tested, the results indicated that only one isolate (2.4 %) was sensitive to levofloxacin and teicoplanin. Also, 4 (9.5%), 5 (11.9%), 6 (14.2%), 7 (16.6%), 8 (19.0%) and 10 (23.8%) were sensitive to gentamicin, amikacin,, rifampin, chloramphenicol, erythromycin, trimethoprim/sulfamethoxazole respectively. On the other hand, among the 42 MSR, eleven isolate (26.1 %) were found to be resistant to vancomycin (VRSA) (last choice in treatment).
- Ent A gene was detected by PCR using specific primers, PCR was carried out for 64 specimens of DNA extracted from the isolated staphylococci as well as the DNA extracted from reference strain. The target gene was detected in reference strain, nineteen isolates of S.aureus and one isolate of S.epidermidis.
- SDS-PAGE method carried out to evaluate protein pattern and determine the protein band of target toxin (~27 kDa). The band of interest (27 kDa) was detected in only 12 samples with different intensities in addition to the reference strain.
- Similarly DAS-ELISA results showed that ent A protein was detected only in 12 strains of the 20 PCR-positive strains. It is to be noted that results of SDS-PAGE and DAS-ELISA were similar.
- According to Das-ELISA which is reflects the antigen concentration, strain 41 (SA-41) represented the most potent strain for the production of ent A protein, so; it was selected for further studies including cloning and expression alongside the positive reference strain.
- Two additional primers (Ent A-F and Ent A-R) were used for amplification of the gene of ent A gene by PCR for the reference strain and SA-41. The gene of interest (774 bp) of interest was detected in both strains under investigation.
- The purified gene was ligated into pGEM-T- easy vector. After ligation the plasmid was transformed into E.coli strain GC5 and the putative clone was designated pNH1. The white colonies were selected and subsequently digested by BamH1 and Xhol as a confirmative method that established the insertion of the recombinant plasmid including ent A-orf. A segment of ent A-orf of the successfully transformed colonies containing 774 bp in addition to the characteristic band of pGEM-T easy vector (3015 bp) were detected on agarose gel.
- DNA of a recombinant clone pNH1 was sequenced using F and R M13 universal primers. The sequenced clone was digested with BamHI and XhoI restriction endonuclease enzymes and the released fragment was sub-cloned into PGEX-4T-1 plasmid that was previously digested with the same restriction enzymes, and the obtained plasmid was designated pNH2.
- The recombinant plasmid pNH2 was transformed into E.coli BL 21(as an expression host). The expression process was induced by adding two different concentrations of IPTG (0.1 and 1.0 mM for 1, 2 and 3h respectively at 37°C); optimum production was obtained using of 0.1 and 1.0 mM IPTG after 3 hours of incubation.
- Application of 0.1 mM IPTG was carried out at different incubation temperature and incubation period were tested; results indicated that the optimum temperature was at 16 °C and the optimum incubation time was overnight.
- GST-tagged ent A-fusion protein was purified using GST-bulk purification kit; results showed a band of size 53 kDa for reference and SA-41 strain using SDS-PAGE gel, the produced bands were confirmed by western blot which proved that the purified bands were the specific bands especially when reacted with the specific polyclonal antibody.
- The purified protein bands were subjected to the cleavage procedures by thrombin which resulted in two fractions, the first one is glutathione which was adsorbed onto the column beads allowing the second fraction (Ent A protein) to be free, a band size of 27 kDa were shown from the reference and SA-41 strain. The produced bands were confirmed by SDS and western blot as mentioned previously.
- The transformed E. coli with PNH2 was irradiated by gamma radiation at doses (0.25, 0.5, 0.75, 1.0, 10, 20, 30, 40 Gy) to examine possible stimulation of production of recombinant ent A. The result showed that the doses 30 Gy and 40 Gy prevented the growth of bacteria (bactericidal action). While other doses did not increase the production of ent A protein.
- Similarly, the transformed E. coli were exposed to UV at 254 nm for different time intervals (5, 15, 30, 60 and 90 sec.). The highest repression effect had been showed by action of exposure time (15 sec.). Other exposure time did not affect the production of ent A compared to control.
Stimulation effect hadn’t been detected for both UV and gamma radiation on productivity of ent A protein but, repression effect had been observed especially at exposure time (10 sec.) to UV or 0.75 Gy to gamma irradiation.
- Antiserum was raised against ent A proteins by injecting two balb/c mice with the purified ent A. The doses were 10, 10, and 5 μg at weekly intervals. IgGs was purified from polyclonal antiserum using saturated ammonium sulfate. The purified IgGs was conjugated with alkaline phosphatase. The concentration of purified IgGs was determined and adjusted to 1 mg/ml, the IgG titer was 1/20000.
- The specificity of the produced antiserum (ab developed in balb/c mice) was evaluated by western blot analysis and compared with that of the commercially available ent A antibody. Results indicated that the antiserum reacted specifically with a protein of about 27 kDa which appeared as deep dark purple color on PVDF membrane.
The efficiency of the polyclonal antibody (developed in balb/c mice) was tested and compared to that of the commercially imported ent A antibody by testing the twelve ent A producing strains initially recovered during this study using DAS-ELISA. The results obtained from commercially available antibody and the produced polyclonal antibody was found identical.