KANNAN.N and SHAJUNA BANU.Z School of Biotechnology

1. STUDIES ON THE WOUND HEALING PROPERTY OF MORINDA CITRIFOLIA ON CELL LINE CAUSED BY BACTERIAL INFECTION KANNAN.N and SHAJUNA BANU.Z School of Biotechnology K.S.Rangasamy College of Technology,Tiruchengode.

2. BURNS Infection is a major cause of morbidity and mortality in hospitalized burn patients. The infection is due to the combined effect of : the impairment of the host natural defense system, colonization of the burn wound site, systemic dissemination of the colonizing organisms. Burn wound infections are largely hospital acquired and infecting pathogens differ from one hospital to another.

3. PREDOMINANT PATHOGENS IN BURN WOUNDS: The most commonly prevailing pathogens in burn wounds are: • Pseudomonas aeruginosa • Staphylococcus aureus • Klebsiella sp. • Escherichia coli • Streptococcus pyogenes Of these pathogenes,P.aeruginosa(36%) and S.aureus(29%) accounted more in the burn wound infection site.

4. Pseudomonas aeruginosa: Characteristics: • Gram negative rod • Motile, ubiquitous in soil and water • Never fermentative • Optimum growth at 37ºc up to 42ºc • Resistant to salts, dyes, weak antiseptics and as many commonly used antibiotics • Produces two types of soluble pigments, the fluorescent pigment Pyoverdin and blue pigment Pyocyanin

5. TOXINOGENESIS: Pseudomonas aeruginosa produces two extracellular protein toxins-Exotoxin A and its subunit Exoenzyme S Exotoxin A catalyses ADP-ribosylation and inactivation of elongation factor 2,leading to inhibiton of protein biosynthesis and cell death. Exoenzyme S, also an ADP-ribosyl transferase. ribosylates GTP binding proteins such as Ras and Rho. Ras and Rho are molecular switches that control numerous cellular processes, also contribute to wound healing processes and tissue regeneration.

6. ANTIBIOTIC SENSITIVITY PATTERNS: Pseudomonas aeruginosa is found to be sensitive to…. • Imepinem & Meropenem-100% • Amikacin-68.01%and • Gentamycin-33% • Chloramphenical-29% Several resistant forms have been developed and it is most resistant to Netilmicin(70.04%)

7. Staphylococcus aureus: Characteristics: • Gram positive, cluster forming coccus • Catalase positive • Golden yellow colony on agar • Pathogens of humans, causes wide range of supprative (pus-forming) infections and toxinoses. • It is a major cause of nosocomial infection in surgical wounds and infections are associated with indwelling medical devices.

8. TOXINOGENESIS: The virulence factors of S. aureus are found to be due to • Surface proteins that promote colonization of host tissues. • Invasins that promote bacterial spread in tissues (leukocidin, hyaluronidase, kinases) • Surface factors that inhibit phagocytic engulfment (capsule, protein A). • Biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production) • Membrane damaging toxins

9. S.aureus produces enterotoxin A, B, leukocidin, Exfoliatins, Heamolysins (α,β,γ,δ) lyse erythrocytes. α-lysin is most important in pathogenicity and is thought to facilitate the tissue destruction associated with staphylococcal growth. Leukocidin kills polynuclear leukocytes and macrophages.

10. ANTIBIOTIC SENSITIVITY PATTERNS: Staphylococcus aureus is found to be resistant to • Ampicillin and Tobramycin -99.3% each • Erythromycin -25.3% • Trimethoprim-sulphamethoxazole-13.9% The most effective antibiotics against S. aureus are, • Chloramphenical-78% • Gentamycin -63% • Tetracycline -57%

11. The treatment against bacterial pathogens in burn patients are often difficult due to antibiotic resistance. The mechanism of resistance to drugs include • Reduced cell wall permeability • Production of chromosomal and plasmid mediated β-lactamase, aminoglycoside modifying enzymes • The active multi-drug efflux mechanism. Several types of vaccines are being tested but none is currently available for general use. So, now the researchers are striving to provide an insight into the use of plant extracts against these pathogens in in vitro studies. The objective of the present work is to study the wound healing property of Morinda citrifolia on mouse cell line infected with pathogens.

12. DEVELOPMENT OF MOUSE CELL LINE: Requirements: • 13.5 day pregnant mouse • Two sets sterile instruments, one containing a pair of curved forceps and a pair of iris scissors and the other containing two pairs of curved forceps, one pair iris scissors and no.3 sized scalpel handle. • Sterile medium sized Petri dishes (tissue culture standard) • 18 gauge needle • Luer lock syringe • No.11 sized flat-edged scalpel blade • Large flasks ( tissue culture standard about 154 sq.cm area)

13. Media and Reagents: • Phosphate buffer saline (PBS) • Trypsin /EDTA • Dulbecco’s modification of Eagles medium (DMEM) • 10% fetal calf serum, • 1% penicillin/ streptomycin, • 1% L-glutamine • 0.2% of 0.1mBME

14. ISOLATION OF PRIMARY MOUSE EMBRYO FIBROBLAST: • Embryo from 13 or 14 day pregnant mouse is removed . • Washed with PBS • Finely minced the tissue with iris scissors or 2 ml Trypsin/EDTA was added and incubated at 37ºc(20 min). • Neutralize the Trypsin/EDTA with 20 ml culture medium.

15. Mix the contents and add to T75 culture flask containing 20 ml culture medium with approximately three embryos per T75. • Incubate the flasks overnight at 37ºc • The next day , change the medium to remove debris and toxic cell death products. • Upon confluence, harvest and freeze the primary fibroblasts at a freezing density of 3.0 x 10^7 cells/ml. • To the developed cell line, infection is induced with the pathogens isolated from burn patients.

16. CELL LINE CHARACTERIZATION: The cell line has to be characterized every 30min from the time of infection to observe : • Changes in the cell morphology • The cell viability by MTT{3-(4,5-Dimethyl thiazol-2-yl)-2-5,-diphenyl Tetrazolium bromide} assay. • The cell count using heamocytometer.

17. TREATMENT OF INFECTED CELL LINE: The active ingredients in plant extracts are the alternatives for pathogens that developed resistance agains conventional and synthetic antibiotics. In this way Morinda citrifolia is found to possess antibacterial and other antimicrobial activities. The compounds responsible for the antibacterial properties are… • Acubin, • L-asperuloside, • Alizarin, and • Other anthroquinone compounds

18. The pure extracts from the various parts (roots, leaves) of the plant Morinda citrifolia can be obtained by solvent extraction. These extracts will be injected to the infected cell line at various concentrations and the subsequent changes/improvement in the cell regeneration process could be noted.

19. THANK YOU….