1. AUM SRI SAI RAM
DEPARTMENT OF BIOSCIENCES
An INTERIM REPORT on- “Primer Screening and Optimization for RAPD Analysis of Clinically Isolated MDR Acinetobacter baumannii.”
Under the supervision of
Dr. B.E. Pradeep
Submitted By-
AMIT KUMAR SAHOO 15151

2. An offering at thy lotus feet

3. About(Acinetobacter baumannii)
Acinetobacter baumannii is a Gram-negative coccobacillus bacterium.
It is an opportunistic pathogen in humans, affecting people with compromised immune systems, and is becoming increasingly important as a hospital-derived (nosocomial) infection.
Colloquially, A. baumannii is referred to as 'Iraqibacter' due to its seemingly sudden emergence in military treatment facilities during the Iraq War.
A. baumannii survives for prolonged periods under a wide range of environmental conditions. The organism causes outbreaks of infection and health care-associated infections, including bacteremia, pneumonia, meningitis, urinary tract infection, and wound infection.

4. Molecular Typing PFGE rep-PCR AFLP VNTR RAPD SLST
The Molecular typing studies provide information about time-clonal distribution in different places of occurrence and the complexity of outbreaks.
The results derived from these studies confirms the chances of reappearing of clone at different times.
Thus, endemicity of the strain can be determined.
Type of Molecular Typing Methods
PFGE
rep-PCR
AFLP
VNTR
RAPD
SLST
Of these RAPD was found to be most efficient.

5. RAPD(Random Amplification of Polymorphic DNA)
It involves the use of a single arbitrary primer, unlike the normal PCR reaction , where forward and reverse primers are used .
A discrete DNA product is produced through thermocyclic amplification.
If there is a difference in one or both of the primer binding sites, it is visible as presence or absence of a particular RAPD band. Therefore, the polymorphism between individuals can be detected.
RAPD fingerprinting method has been successfully applied to identify the clonal groups. It is more sensitive, detecting differences even among closely related strain that were apparently identical.

6. Objectives Design of RAPD primers.
Screening of RAPD primers for Acinetobacter Baumannii.
Optimization of RAPD PCR condition.

7. Materials and Methods
Fourteen different types of clinically isolated Multi Drug Resistant strains of Acinetobacter baumannii were used.
Total DNA extraction was done by using NucleoSpin kit(Macherey-Nagel).
Ten RAPD primers have been identified labelled OPA1 to 10.
PCR condition for OPA2 primers has been optimized.

8. RAPD With OPA-2 PCR Program For OPA -2 95⁰C- 5mins 95⁰C- 1mins
4⁰C- Infinite HOLD
Cycle
(40 times)

9. OPA II L C
1000 bp

10. PCR Program Protocol Repeated assay with three more primers.
For RAPD 1, RAPD 2, OPA 4
Initial Denaturation: 94 ⁰C -3 mins
Denaturation: 94 ⁰C -1 min
Annealing: 36 ⁰C – 1 min
Extension: 72 ⁰C – 2 min
Final Extension- 72 ⁰C – 15 mins
Hold- 4 ⁰C – Infinity
PCR amplicons were analyzed on 1.5% agarose gel.
Cycle
(40 times)

11. RAPDI and RAPDII RAPD II RAPD I L
C B B
1000 bp
Key:
C-Control
B-Blank

12. RAPDII and OPA4 RAPD II OPA 4 L 1 2 3 4 5 6 7 8 1112 13 14 15 16 C
1000 bp
Key:
C-Control
B-Blank

13. Future Perspectives: To understand Clonality by RAPD.
To see if they belong to same Multi-Drug Resistant (MDR) strains or different strains.
Eight more primers are to be screened to identify which primer will give maximum number of bands and which primer will express across all the strains.

14. References
Kaye KS, Pogue JM. Infections Caused by Resistant Gram-Negative Bacteria: Epidemiology and Management. Pharmacotherapy. 2015;35(10):949–62.
Balkhy HH, Cunningham G, Chew FK, Francis C, Al Nakhli DJ, Almuneef MA, et al. Hospital- and community-acquired infections: a point prevalence and risk factors survey in a tertiary care center in Saudi Arabia. Int J Infect Dis ;10(4):326–33.
Dent LL, Marshall DR, Pratap S, Hulette RB, García-Garmendia J, Ortiz-Leyba C, et al. Multidrug resistant Acinetobacter baumannii: a descriptive study in a city hospital. BMC Infect Dis [Internet]. BioMed Central; 2010 Dec 7 [cited 2016 Sep 21];10(1):196. Available from:
Welsh J, Mcclelland M. genomes using PCR with arbitrary primers *. 1990;18(24):6531–5.
Websites:
en.wikipedia.org
mic.microbiologyresearch.org

15. Thank You! SAI RAM