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Culturing microorganisms under different conditions can have a great influence on their characteristics such as antibiotic resistance and production of virulence factors (Florian Brill, 2005). The development of stress responses in bacteria has received much attention in recent years. From the point of view of food safety, the main reason for investigating bacterial stress responses lies in the fact that traditional methods of food preservation and distillation systems rely on imposing physical and chemical stresses upon cells to limit their growth and survival. However, nowadays, it is known that bacteria may adapt to stress (Cebrián G, 2010).
Staphylococcus aureus is an opportunistic human pathogen that causes major problems in the food area and in the medical field. S. aureus is a very adaptable organism and can live in a wide variety of environments by having different kinds of plasmid that contribute them many resistance factors.
The staphylococcal genome consists of a circular chromosome with prophages, plasmids, and transposons. Genes leading virulence and resistance to antibiotics are found on the chromosome and extrachromosomal elements. These genes are transported between staphylococcal strains, species, or other gram-positive bacterial (Franklin D, 2010).
Plasmid DNAs replicate independently of the bacterial chromosome, and many plasmids can also be transferred naturally among their bacterial hosts. Plasmid genes code proteins that maybe essential or not for their host in its normal environment. Generally, plasmids are nonessential to an organism under most conditions, they give the cell great potential for rapid evolution without endangering its viability as occurs with many chromosomal mutations( Lacey R.W,1975). Plasmid genes can encode different toxins and resistance factor to variety of aspects such as antimicrobial agent, antibiotic, toxic metal ions and etc.
The aim of this study is toâ€¦â€¦â€¦
Other works and importance
Method & Materials
Bacterial Strains and culture media
A total of 150 samples, 80 from clinical sources, 70 from nose swabs and food samples (vegetables, diary, meet, cookies) were collected. All of the clinical samples were derived from patients at different hospital in Tehran over 4 months. S. aureus strains were cultivated on BHI broth, N.A, B.A and Manitol salt agar, incubated at 37Â°C for 24 h and identified by standard microbiological methods.
Plasmid was purified as previously described by Andrup et al (2008). The bacteria were grown in 4 ml of Luria-Bertani (Sambrook et al., 1989) broth for 12-16h at 37Â°C with shaking. Then, the samples harvested by centrifugation at 13,000Â for 2 min at room temperature. The cell pellets was separated by removing the aqueous phase completely, then the pellets suspended by adding 100 of E buffer containing (15% [wt/vol] sucrose, 40 mM Trishydroxide, 2 mM EDTA, pH 7.9) and mixing completely by pipetting and inverting up and down. Digested by Adding 200 of lysis solution containing (3% [wt/vol] sodium dodecyl sulfate, 50 mM Tris, pH 12.5). The lysate was heated at 60 Â°C for 45 min. Added 20 (20 mg/ml) of proteinase k (Fermentase). After that mixed the solution by inverting up and down for 20 times then incubated at 37 Â°C for 60 min. Added 1.0 ml of equal volume of : phenol-chloroform- isoamyl alcohol (25:24:1, cinnagen). The mixture was inverted up and down for 40 times. Centrifuge at 9000g for 7 min, remove the upper aqueous phase to a new tube, being careful not to transfer any of the protein at the phase interface. The upper phase used for gel electrophoresis (Andrup L, 2008).
To study the effect of factors, we used different media and conditions. Three temperature (4, 10, 25, 35 and 40), three congo red concentrations (0.003, 0â€¦â€¦â€¦â€¦..%) and three calcium chloride concentrations (1, 3 and 5 %) were used. Congo red and calcium chloride bought from
Effect of congo red
â€¦â€¦.agar containing 0.003 % congo red ,â€¦â€¦.. was used to study the influence of congo red on plasmids of S. aureus. The plate incubated at 37 for 24h. Then transfer 5 colonies to LB broth and incubated for 12-16h at 37 and finally the plasmid was extracted ( Andrup L, 2008).
Effect of temperature
Heat treatment was carried out by cultured bacteria on nutrient agar and let them to grow over night at 37. Then place the plates on conditions with different temperature.
Effect of calcium chloride
Nutrient agar containing 1%, 3% and 5% cacl2 was used. After that place the plates in 37for 24h and transfer 5 colonies of bacteria to LB broth and incubated at 37for 12-16h.
Effect of ethanol
The isolated plasmids in each condition were collected in new tube and 8 Î¼l of aliquot was run on 1% agarose gel at 80 V, then the gel stained with etidium bromide and documented with a camera system.