SENTHILKUMARAN.AL.KR(Reg. No. 09MBT20)

M. Sc., Marine Biotechnology

Under the guidance ofDr. M. SUNDARARAMAN

Associate ProfessorDEPARTMENT OF MARINE BIOTECHNOLOGY

BHARATHIDASAN UNIVERSITYTIRUCHIRAPPALLI-620 024

• Candida is dimorphic fungus present in normal flora of human and animal’s gastrointestinal and reproductive tract.

• This fungus may change into opportunistic pathogen to immunocompromised patients (HIV, TB and cancer etc.,).

• Globally, C. albicans is a most common invasive fungus than the other non-albicans Candida. Next to the C. albicans (52.7%), the emergence of other non-albicans Candida viz., C. glabrata (14.7%), C. parapsilosis (13.9%), C. tropicalis (11.8%), C. krusei (3.3%), C. lusitaniae (1.2%), C. guilliermondii (1.1%), C. kefyr (0.8%), C. famata (0.4%) and C. pelliculosa (0.2).

• Four important virulent factors -adhesion, hyphal formation, hydrolytic enzymes production (Secreted Aspartyl Proteinase (SAP) and lipases) and phenotype switching.

• Antioxidant-inhibiting oxidation of other molecules

• Neutralize the free radicals.

• Antioxidant – donating an electron to free radicals to convert them to harmless molecule.

• Extractions of compound from certain marine and terrestrial plants for anticandidal lead molecules.

• To screen anticandidal activity and find mode of inhibition analysis for active extracts.

• To investigate antioxidant potential of volatile organic compound(s) from bacteria using a novel method.

• Purification of bioactive crude extracts by column chromatography and thin layer chromatography

• Extraction of volatile organic compound(s) from mangrove associated bacterium Bacillus sp. M12.

• Determination of active principle(s) by Gas Chromatogram-Mass Spectrometry.

Extraction and Screening of plants, algae and cyanobacteria for anticandidal activity.

Anticandidal activity (agar well method, Disc diffusion method and Minimal Inhibitory concentration).

Identification of compounds (Gas Chromatography- Mass Spectroscopy)

Bioassay guided fractionation method (column chromatography and Thin layer chromatography

Mode of action (Germ tube inhibition assay, Proteinase inhibition assay, Phospholipase inhibition assay and Ergosterol

biosynthesis inhibition assay)

Work flow- Anticandidal activity

Inoculation of bacterial cultures

Transfer to Glass vials

Identification of compounds( GC-MS)

Apple exposed to cultures (browning effect)

Extraction of volatile compounds

Antioxidant activity

Materials and Methods

Strains used:

Fungal strains:

Candida albicans (NCIM 3074), C. glabrata (NCIM 3236), C. tropicalis (NCIM 3118) and two clinical isolates such as Candia sp. (159), Candida sp.(S350)

Bacterial strains:

Staphylococcus aureus (NCIM 5021), Escherichia coli (NCIM 2065), Vibrio Cholerae (MTCC 3904) and Mycobacterium smegmatis (TRC, Chennai).

Screening of anticandidal activity:• Agar well method (Kirby-Bauer, 1966)]• Disc diffusion method (Kirby-Bauer, 1966)• Minimal Inhibitory Concentration (NCCLS, 2001)

Mode of Inhibition:• Germ tube inhibition assay.• Proteinase inhibition assay. (GirishKumar et al., 2006)• Phospholipase inhibition assay. (Tsang et al,-2007)• Ergosterol biosynthesis inhibition assay. (Arthington-Skaggs et al.

- 1999).

Bioassay guided fractionation method:• Column chromatography• Thin layer Chromatography

Identification of Compounds:• Gas chromatography- Mass Spectroscopy.

Strains used:• Mangrove associated bacteria such as Pseudomonas aeruginosa

and Bacillus sp. • Inoculated into King’s B broth.• Bacterial cultures were inoculated into King’s B broth and incubated

for 24 hrs.• After incubation, the cultures were transferred to glass vials.• Sliced apple were inserted to the bottle.

Fig.1 The cut-apples exposed to empty bottle (C1), the medium alone (C2), the culture of Pseudomonas aeruginosa M6 and Bacillus sp. M12

Results and Discussion

S.No Name of extract(code)

C. albicans C. glabrata C. tropicalis

1 AC +++ ++ +++

2 O ++ ++ ++

3 UK ++ ++ ++

4 Fen - - -

5 Pa - - -

6 Thu - - -

7 Thi - - -

8 Etti - - -

9 Kj - - -

10 P7 - - -

11 3 - - -

12 Ms1 - - -

13 L1 - - -

14 U1 - - -

Table 1. Screening of Anticandidal activty

S. No Compound Code

C. albicans C. glabrata C.tropicalis

15 DI - - -

16 G1 - - -

17 UK1 - - -

18 S1 - - -

19 T1 - - -

20 Sargassum - - -

21 Pedina - - -

22 Dic - - -

23 Cal. - - -

24 I12 - - -

25 MPs - - -

26 I-2 - - -

27 P4-13 - - -

28 I-16 - - -

29 I-9 - - -

30 11Osi - - -

32 Va micro - - -

33 T8 Osi - - -

34 1OSi - - -

35 T1osi - - -

36 Bmosi - - -

37 Heterocyst - - -

38 Nostoc - - -

39 LD5 - - -

40 LM n - - -

41 LM o - - -

42 LSP1 - - -

43 LSP2 - - -

44 OSi - - -

45 MP1 + + +

46 MP2 - - -

Con.(mg) C. albicans C. glabrata C. tropicalis Candida sp. 159

Candida sp. S350

Zone of inhibition (mm)

0.5M + + + + +

C + + + + +

1M 1 + 2 2 1

C 2 1 2 3 1

1.5M 2 2 3 3 2

C 3 3 3 4 3

2M 5 4 6 4 5

C 7 4 6 5 6

+ - static inhibition; M – Methanol; C - Chloroform

Fig 2. a) activity of AC plant extract against C. albicans and b) C. tropicalis

Con. (mg) E. coli S. aureus M. smegmatis V. cholerae

Zone of inhibition (mm)

0.5 + 4 1 +

1 1 6 2 1

1.5 2 8 3 2

2 3 10 5 4

+ - static inhibition

Con.(mg)

C. albicans

C. glabrata

C. tropicalis

Candida sp. 159 E. coli S. aureus M.

smegmatis V. cholerae

Zone of inhibition (mm)

0.5 - - - - - 4 - 7

1 - - - - - 6 - 8

1.5 + - + - - 7 - 9

2 1 + 2 - - 8 - 10

+ = static inhibition; - = absence of inhibition

Compound Conc. (mg) 0.5 1 1.5 2 3 4 5

MIC - - - - + ++ +++

Germ tube inhibition assay

Fig 3.Induction of germ tube 3-4 hours(a)Morphological changes and cell membrane shrinkage occur (b) (arrow headed)

Fig 4: Inhibition of Secreted Aspartyl proteinase ( SAP)

Fig 5. Inhibition of Secreted Aspartyl Phospholipase

Inhibition of Ergosterol biosynthesis

The data results showed that probably active fraction was Cis/trans/B-asarone (MW.208, C12H16O3)

Fig 7.The cut apple pieces showing browning after incubation (C1- exposed to empty bottle, C2- exposed to Kings B broth, M6- Marine Pseudomonas and M12- Bacillus sp.

ESIMS spectrum shows most probably the compound (4S, 5S)-(+)-Muricatacin (MW. 284.434, Mol. C17H32O3) is responsible for anti-oxidant characteristics.

• Extracted compounds from various sources (46)( plants, bacteria, algae and cyanobacteria) and checked anticandidal activity.

• The plant compound AC showed good activity against all the standard and clinical isolates of Candida spp.

• Candidal germ tube inhibition, proteinase inhibition, phospholipase inhibition and ergosterol biosynthesis inhibition assays.

• The concentration of 3mg-4mg/ml it was showing minimal fungicidal concentration (MFC).

• The compound partially purified by Column chromatography(silica gel) and 6 fractions were collected.

• The fraction was further analyzed by GC-MS and found asarone group of compound.

• 2 mangrove associated volatile organic compounds producing bacteria (M6, M12) were screened for anticandidal as well as antioxidant activity .

• The organic volatile compounds by GC-MS and found the compound namely “Muricatacin”.

• Candida infections were mostly found in patients infected by HIV and TB in worldwide

• The available antifungal drugs were showing adverse side effects and high toxicity to host tissues

• We have screened large number of plants and microbes for anticandidal lead molecules.

• One Plant extract (AC) bacteria (M12) proved its potential by anticandidal and antioxidant liberation, thus it can be used as in food packing industries even though it may be used to derive anticancer drugs.

• I sincerely express my gratitude to my guide, Dr. M. Sundararaman, Associate Professor, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli.

• Dr. L. Uma, Head, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli.

• Faculty members, Department of Marine Biotechnology, Bharathidasan University, Tiruchirappalli.

• Research scholars and my classmates

• My parents

• Aruna Prakash, PhD, Fred Rigelhof and Eugene Miller, PhD 2003: Antioxidant activity.• Batool Sadeghi Nejad, Ablollah Rafiei1 and Fereshteh Moosanejad 2011: Prevalence of Candida

species in the oral cavity of patients with periodentitis. African Journal of Biotechnology 2987-2990• Beth A.Arthington Skaggs, Hoda Jradi, Tejal Desai and Christine j. Morrison 1999: Quantitation of

Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans. JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1999, p. 3332–3337

• C.P. Girish Kumar, S. Suresh Jothi Kumar & Thangam Menon 2005: Phospholipase and proteinase activities of clinical isolates of Candida from immunocompromised patients. Mycopathologia (2006) 161: 213–218

• David A.katz 1997: Apple browning: A study of oxidation of foods• Farag, M. A., Ryu, C.-M., Sumner, L. W. and Paré, P. W. 2006. GC-MS SPME profiling of rhizobacterial

volatiles reveals prospective inducers of growth promotion and induced systemic resistance in plants. Phytochemistry. 67: 2262-2268.

• Gerald M. Sapers and Robert L. Miller 1999: Browning Inhibition in Fresh-Cut PearsJames B. McAlpine, Brian O. Bachmann, Mahmood Piraee, Steve Tremblay, Anne-Marie Alarco, Emmanuel Zazopoulos, and Chris M. Farnet 2005 : Microbial Genomics as a Guide to Drug Discovery and Structural Elucidation:ECO-02301, a Novel Antifungal Agent, as an Example. J. Nat. Prod. 2005, 68, 493-496

• Julian Naglik, Antje Albrecht, Oliver Baderan ,Bernhard Hube 2004 : Candida albicans proteinases and host/pathogen interaction.

• Julian R. Naglik, Stephen J. Challacombe and Bernhard Hube 2003: Candida albicans Secreted Aspartyl Proteinases in Virulenceand Pathogenesis. 400-425

• Justyna Karkowska-Kuleta, Maria Rapala-Kozik and Andrzej Kozik : Fungi pathogenic to humans: molecular bases of virulence of Candida albicans, Cryptococcus neoformans and Aspergillus fumigates. 211–224

• Kirby –Bauer 1966 Disc diffusion method• Mahmoud A.Ghannoum and Louis B. Rice: Antifungal Agents: Mode of Action, Mechanisms

of Resistance, and Correlation of These Mechanisms with Bacterial Resistance• Marianne Kretschmar, Bernhard Hube, Thomas Bertsch, Domonique Sanglard, Retane

Merker and Thomas Nitcherlein 1999: Germ Tubes and Proteinase Activity Contribute to Virulence ofCandida albicans in Murine Peritonitis, INFECTION AND IMMUNITY, 6637–6642

• McEvily, A J., Iyengar, R., and Otwell, W.S. 1992. Inhibitionof enzymatic browning in foods and beverages.Crit. Rev. Food Sci. & Nutr. 32: 253-273.

• Pfaller, M.A., Diekema, D.J., Jones,R.N.,Sader,H.S., Fluit , A.C., Hollis,R.J., Messer, S.A., The SENTRY participant group .2001. International surveillance of blood strea infection due to Candida sp. frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole, and variconazole of isolates collected from 1997 through 1999 in the SENTRY antimicrobial surveillance program, J Clin Microbiol 39:3254-3259.

• Ryu, C.-M., Farag, M. a., Hu, C.-H., Reddy, M. S., Wei, H.-X., Paré, P. W. and Kloepper, J. W. 2003. Bacterial volatiles promote growth in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America. 100: 4927-32

• Ryu, C.-m., Farag, M. A., Hu, C.-h., Reddy, M. S., Kloepper, J. W. and Pare, P. W. 2004. Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiology. 134: 1017-1026.

• Ryu, C.-M., Farag, M. A., Paré, P. W. and Kloepper, J. W. 2005. Invisible signals from the underground : Bacterial volatiles elicit Plant growth promotion and induce systemic resistance. Molecular Plant-Microbe Interactions. 21: 7-12.//

• Ryu, C.-m., Yi, H.-s., Ahn, Y.-r., Kim, W.-i. and Zhang, H. 2007. Dynamic communication between plants and rhizobacteria via volatile signals. Proceedings of the 13th International Congress on Molecular Plant‐Microbe Interactions at Sorrento (Naples), Italy, St. Paul, Minnesota, USA, International Society for Molecular Plant‐Microbe Interactions.