prevelance of mecA gene from MRSA isolates from given isolates

1. DISSERTATION PRESENTATION

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

10. Identification of MRSA

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.

16. Gel electrophoresis

17. RESULTS
Nutrient Broth
• Subculturing

18. Results
DNA Isolates

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