Isolation of mrsa isolates
- 2. DISSERTATION ON Prevalence of mecA gene in MRSA from Isolated clinical samples of patients from Rajasthan Submitted to Submitted By Ms Apoorva Rana Oshin Raj Assistant Research Scientist BSc Biotechnology Dr. B Lal Institute of Biotechnology Part III
- 3. Introduction • Staphylococcus aureus is a gram positive, cocci shaped bacterium basically found on the surface of the nose and ear and respiratory tract (Bata et. al.;2014) • They belong to the family of Firmicutes. • S. aureus is not always pathogenic in nature, commonly causes skin infection. • Staphylococcus was first identified in 1880 by Sir Alexander Ogston. • S. aureus is catalase positive (that is it can produce enzyme catalase). • S. aureus was found to have natural genetic transformation but only at low frequency.
- 4. Methicillin resistant Staphylococcus aureus • Methicillin resistant Staphylococcus aureus (MRSA) is bacterium that causes infections in different parts of the body. • Its tougher to treat than most Staphylococcus because it is resistant to some commonly used antibiotics. • The symptoms of MRSA depend on where you’re infected. Most often, it cause mild infections on skin but also cause more serious skin infections in lungs or urinary tract. • Staphylococcus can usually be treated with antibiotics. But over decades, some strains have become resistant to antibiotics that once destroyed it. • It’s now resistant to methicillin, amoxcillin, penicillin, oxacillin and other common antibiotics.
- 5. OBJECTIVES • To analyse the prevalence of mecA gene responsible for methicillin resistance in S. aureus (MRSA)
- 6. Work plan Isolation and identification of Methicillin Resistant Staphylococcus aureus (MRSA) from clinical samples DNA Extraction and its amplification Molecular typing of the gene responsible for the MRSA
- 7. Isolation and identification of Methicillin Resistant Staphylococcus aureus (MRSA) from clinical samples Isolation of bacterial isolates from cinical samples of infected patients by Streaking Method Identification of Staphylococcus aureus by Biochemical Testing Identification of MRSA by Double Disk Synergy Test Molecular typing of the gene responsible for the resistance in Staphylococcus aureus (MRSA) METHODOLOGY Extraction of DNA from MRSA isolates Amplification of DNA by Polymerase Chain Reaction against the targeted primers Molecular Typing by Agarose Gel Electrophoresis to identify the gene present
- 8. Isolation of S. aureus from clinical samples by streaking Material and method
- 9. Biochemical identification of Staphylococcus aureus
- 11. DNA Isolation Culturing of bacteria in nutrient broth medium and incubation at 37°C over night with shaking. Transfer culture to 1.5 ml eppendroff tube and spin down cell culture at high speed for 1 minute at table top centrifuge. Discard the supernatant to remove the liquid completely by mixing upside down onto a piece of paper towel for a few second. Add 100µl of lysis solution (P2 Buffer) and mix by gentle inverting the tubes 5-6 times. The solution should quickly turn transparent and become more viscous indicating bacterial lysis has taken place. Add 100µl of re-suspension solution (P1 Buffer) into each tube and vortex to completely re-suspend cell pellet. Add 150 µl of neutralizing solution (P3 Buffer) and mix by inverting the tubes several times. At this point bacterial chromosomal DNA is usually seen as white precipitate.
- 12. Centrifuge the tubes at high speed for 10 minutes. At 10000 rpm at 3°C. Carefully transfer the supernatant to a new eppendorfs. Add 2.5-3 volume of chilled ethanol to each tube and mix by inverting the tube a few times. Spin down plasmid DNA precipitate at high speed for 10 minutes at 10000 rpm at 3°C. Discard the supernatant and remove the remaining liquid as much as possible by leaving the tube upside down on a piece of paper towel. Then keep the tubes in a tube holder and air dry for 10-20 minutes. To dry faster, keep tubes at 37°C heat blocker. DNA precipitate turns white when dry. Re-suspend the DNA pellet with 50µl TE Buffer. Completely dissolve the pellet by pipetting solution several times
- 13. Qualitative and Quantitative estimation of DNA 1O µl DNA sample was taken in TE Buffer The sample was diluted by the factor of 100 i.e. by taking 10 µl of sample in 990µl of TE Buffer After dilution, the optical density value calculated the total amount of DNA recovered The concentration of DNA was determined by using the formula- A260×50×dilution factor/1000 After finding the concentration of DNA, quality of DNA was calculated by taking the ratio O.D at 260/280
- 14. Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or few copies of a segment of DNA across several orders of magnitude, generating thousands of millions of copies of a particular DNA sequence. It is an easy, cheap and reliable way to repeatedly replicate a focused segment of DNA , a concept which is applicable to numerous field in modern biology and related sciences. Polymerase chain reactions
- 15. Gel Electrophoresis • Gel electrophoresis is a technique commonly used in laboratories to separate charged particles like DNA, RNA, PROTEINS according to their size. • Charged molecules move through a gel when an electric current is passed across it. • An electric current is applied across the gel so that one end of the gel has a positive charge and the other end has a negative charge. • The gel consists of a permeable matrix, like a sieve, through which molecules can travel when an electric current is passed across it. • Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result the molecules are separated by size.
- 19. Gel Electrophoresis results of Isolated DNA S. aureus SHC 120 BHIL 653 MRSA SHC 95 CDC 531 JHC 219 C 644
- 20. • Antibiotic sensitivity test was performed by the commonly used Disc diffusion method which is designed to determine the smallest amount of the antibiotic needed to inhibit the growth of a microorganism. • MRSA isolates was cultured on agar plates using streaking method. • DNA of the isolated sample was extracted using the crude boiling method. Conclusion
- 21. REFERENCES • Aminov RI. A brief history of the antibiotic era: lessons learned and challenges for the future. Frontiers in Microbiology 2010;134(1) • Cowan MM. Plant products as antimicrobial agents. Clinical Microbiology Reviews. 1999;12(4):564-582 • Crumplin GC, Odell M. Development of resistance to Ofloxacin. Drugs 1987;34:1-8 • Davidson PM, Naidu AS. Phytophenols. Natural Food Antimicrobial Systems 2000:265-293 • Golkar Z, Bagazra O, Pace DG. Bacteriophage therapy: a potential solution for the antibiotic resistance crisis. J Infect DevCtries 2014;13;8(2):129-135 • Gould IM, Bal AM. New antibiotic agents in the pipeline and how they can overcome micSpellberg B, Gilbert DN. The future of antibiotics and resistance: a tribute to a career of leadership by John Bartlett. Clin Infect Dis 2014;59 suppl 2:S71-S75 • Sergelidis D, Angelidis AS. Methicillin-Resistant Staphylococcus aureus: A controversial food-borne pathogen. LettApplMicrobiol 2017;64(6):409-418 • Zeng F, Wang W, Wu Y. Two prenylated and Cmethylated flavonoids from Tripterygiumwilfordii.PlantaMedica 2010;76(14):1596-1599
- 22. • Liu J, Chen D, Peters BM, Li L, Li B, Xu Z, Shirliff ME. Staphylococcal chromosomal cassessttes mec (SCCmec): A mobile genetic element in methicillin-resistant Staphylococcus aureus. 2015;37(5):442-451 • RomanaChovamsjin, Maria Mikulasovo, StefaniaVaverhova. Int J Microbiol 2013 • Rudramurthy GR, Swamy MK, Sinniah UR, Ghasemzadeh. Review – Nanoparticles:Aletrnatives against drug resistant pathogenic microbes. Molecules 2016;21:836 • Upadhyay A, Upadhyaya I, Johny AK, Venkitanarayanan. Combating pathogenic microorganisms using plant derived antimicrobials: A minireview of the mechanistic basis. BioMed Research Corporation 2014 • Zeng F, Wang W, Wu Y. Two prenylated and Cmethylated flavonoids from Tripterygiumwilfordii.PlantaMedica 2010;76(14):1596-1599 • Kurek A, GrudniakAM, Dowjat K, Wolska KI. New antibacterial therapeutics and strategies. Polish Journal of Microbiology. 2011;60(1):3-12 • Palmer KL, Kos VN, Gilmore MS. Horizontal gene transfer and the genomics of enterococcal antibiotic resistance. Curr. Opin. Microbiol 2010;13:632-639 • Wright GD. Something new: revisiting natural products in the antibiotic drug discovery. Can J Microbiol 2014;60(3):147-154