Evaluation of Bioactive Secondary Metabolites from Ponyfish Associated Bacteria (Photobacterium leiognathi)


Sangeswari Thirukumar(1*), Uthra Selvaraj(2), Srichandan Rath(3), Kanchana Shankar(4), Arumugam Muthuvel(5)

(1) CAS in Marine Biology, Annamalai University, Parangipettai Tamil nadu, India – 608 502.
(2) CAS in Marine Biology, Annamalai University, Parangipettai Tamil nadu, India – 608 502.
(3) CAS in Marine Biology, Annamalai University, Parangipettai Tamil nadu, India – 608 502.
(4) CAS in Marine Biology, Annamalai University, Parangipettai Tamil nadu, India – 608 502.
(5) CAS in Marine Biology, Annamalai University, Parangipettai Tamil nadu, India – 608 502.
(*) Corresponding Author


The marine environment continues to surprise us by producing novel bioactive substances with a wide range of benefits for humans. Materials and Methods: Marine bioluminescent bacteria Photobacterium leiognathi was isolated from pony fish, Secutorruconius which was confirmed with microscopic and molecular characterization. The secondary metabolite of the isolated bacteria was extracted with dichloromethane. The chemical fingerprinting of the isolated metabolite was analyzed through TLC, FT-IR, and HPLC. The nature of the compound present in the metabolite was identified in the gas chromatography-mass spectrometry analysis (GC - MS). The isolated extract was investigated for its antibacterial property against 10 human pathogenic bacteria and also its antioxidant activity using different assays such as 1, 1-Diphenyl-2-picrylhydrazyl, Phosphomolybdenum, Metal chelating, Hydroxyl radical scavenging and hydrogen peroxide scavenging activity. Results: The Presence of functional groups including phenols, sugars, and amino acids in the extracts were identified by TLC. Totally, nine peaks were obtained for the crude extract through the FTIR spectrum range of 400 to 4000 cm-1 for the active sample. The DCM extract showed a broad spectrum of antibacterial activity against the six human bacterial pathogens. Secondary metabolites from the bioluminescent bacteria, P. leiognathi, have strong antioxidant properties. These results will be instrumental in developing novel products with biosensors and bio-imaging applications using P. leiognathi.



Bioluminescent; TLC; FT-IR; HPLC; GC-MS; secondary metabolite

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Al-Massarani, S.M. et al., 2017. Isolation, biological evaluation, and validated HPTLC quantification of the marker constituent of the edible Saudi plant Sisymbrium irio L. Saudi Pharmaceutical Journal, 25(5), pp.750–759. doi: 10.1016/j.jsps.2016.10.012

Armstrong, E. et al., 2001. The symbiotic role of marine microbes on living surfaces. Hydrobiologia, 461(1), pp.37–40. doi: 10.1023/A:1012756913566

Bakaraki, N. et al., 2016. Development of a sensitive liquid--liquid extraction method for the determination of N-butyryl-l-homoserine lactone produced in a submerged membrane bioreactor by gas chromatography mass spectrometry and deuterated anthracene as the internal standard. Analytical Methods, 8(12), pp.2660–2665. doi: 10.1039/C6AY00317F

Bauer, A.W., 1966. Antibiotic susceptibility testing using a standardized single disc method. Am J clin pathol, 45, pp.149–158. doi: 10.1128/am.13.2.279-280.1965.

Chandra, P., Sharma, R.K. & Arora, D.S., 2020. Antioxidant compounds from microbial sources: A review. Food Research International, 129, p.108849. doi: 10.1016/j.foodres.2019.108849

Dobson, A. et al., 2012. Bacteriocin production: a probiotic trait? Applied and environmental microbiology, 78(1), pp.1–6. doi: 10.1128/AEM.05576-11

Engebrecht, J., Nealson, K. & Silverman, M., 1983. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri. Cell, 32(3), pp.773–781. doi: 10.1016/0092-8674(83)90063-6

Farrelly, V., Rainey, F.A. & Stackebrandt, E., 1995. Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Applied and environmental microbiology, 61(7), pp.2798–2801. doi: 10.1128/aem.61.7.2798-2801.1995

Firudoz, B. et al., 2020. Molecular variation and phylogenetic status of ponyfish (Perciformes: Leiognathidae) in Karaikal, South India. Notulae Scientia Biologicae, 12(2), pp.251–257. doi: 10.15835/nsb12210661

Floyd, R.A. & Lewis, C.A., 1983. Hydroxyl free radical formation from hydrogen peroxide by ferrous iron-nucleotide complexes. Biochemistry, 22(11), pp.2645–2649. doi: 10.1021/bi00280a008

Gromek, S.M. et al., 2016. Leisingera sp. JC1, a bacterial isolate from Hawaiian bobtail squid eggs, produces indigoidine and differentially inhibits vibrios. Frontiers in microbiology, 7, p.1342. doi: 10.3389/fmicb.2016.0134

Gutteridge, J.M. & Halliwell, B., 1988. The deoxyribose assay: an assay both for’free’hydroxyl radical and for site-specific hydroxyl radical production. Biochemical Journal, 253(3), p.932. doi: 10.1042/bj2530932

Haddock, S.H.D., Moline, M.A. & Case, J.F., 2010. Bioluminescence in the sea. Annual review of marine science, 2, pp.443–493. doi: https://doi.org/10.1146/annurev-marine-120308-081028

He, F., 2011. Laemmli-sds-page. Bio-protocol, e80. doi: 10.21769/BioProtoc.80

Kannahi, M. & Sivasankari, S., 2014. Isolation and identification of bioluminescent bacteria from marine water at Nagapattinam sea shore area. Int. J. Pharm. Sci. Rev. Res, 26(2), pp.346–351.

Kekuda, P.T.R. et al., 2010. Studies on antioxidant and anthelmintic activity of two Streptomyces species isolated from Western Ghat soils of Agumbe, Karnataka. Journal of Pharmacy Research, 3(1), pp.26–29.

Kelecom, A., 2002. Secondary metabolites from marine microorganisms. Anais da Academia Brasileira de Ciências, 74(1), pp.151–170. doi: 10.1590/S0001-37652002000100012.

Kim, S.U. et al., 2018. Adenosine triphosphate bioluminescence-based bacteria detection using targeted photothermal lysis by gold nanorods. Analytical chemistry, 90(17), pp.10171–10178. doi: 10.1021/acs.analchem.8b00254. Epub 2018 Aug 20.

Klöppel, A. et al., 2008. HPTLC coupled with bioluminescence and mass spectrometry for bioactivity-based analysis of secondary metabolites in marine sponges. JPC-Journal of Planar Chromatography-Modern TLC, 21(6), pp.431–436. doi: 10.1556/jpc.21.2008.6.7

Kumagai, M. et al., 2018. Antioxidants from the brown alga Dictyopteris undulata. Molecules, 23(5), p.1214. doi: 10.3390/molecules23051214

Matthäus, B., 2002. Antioxidant activity of extracts obtained from residues of different oilseeds. Journal of Agricultural and Food Chemistry, 50(12), pp.3444–3452. doi: 10.1021/jf011440s

Menz, J., Schneider, M. & Kümmerer, K., 2013. Toxicity testing with luminescent bacteria--characterization of an automated method for the combined assessment of acute and chronic effects. Chemosphere, 93(6), pp.990–996. doi: 10.1016/j.chemosphere.2013.05.067

Morin-Crini, N. et al., 2019. Applications of chitosan in food, pharmaceuticals, medicine, cosmetics, agriculture, textiles, pulp and paper, biotechnology, and environmental chemistry. Environmental Chemistry Letters, 17(4), pp.1667–1692. doi: 10.1007/s10311-019-00904-x

Nair, A. V, Vijayan, K.K. & others, 2021. Antibacterial assay guided isolation of a novel hydroxy-substituted pentacyclo ketonic compound from Pseudomonas aeruginosa MBTDCMFRI Ps04. Brazilian Journal of Microbiology, 52(1), pp.335–347. doi: 10.1007/s11033-021-06146-x

Nogi, Y., Masui, N. & Kato, C., 1998. Photobacterium profundum sp. nov., a new, moderately barophilic bacterial species isolated from a deep-sea sediment. Extremophiles, 2(1), pp.1–8. doi: 10.1007/s007920050036

Nunes-Halldorson, V. da S. & Duran, N.L., 2003. Bioluminescent bacteria: lux genes as environmental biosensors. Brazilian journal of Microbiology, 34, pp.91–96. doi: 10.1590/S1517-83822003000200001

Poongodi, S. et al., 2012. Marine actinobacteria of the coral reef environment of the gulf of mannar biosphere reserve, India: a search for antioxidant property. Int. J. Pharm. Pharm. Sci, 4, pp.316–321.

Purushottama, G.B. et al., 2009. Bioactivities of extracts from the marine sponge Halichondria panicea. Journal of Venomous Animals and Toxins including Tropical Diseases, 15(3), pp.444–459. doi: 10.1590/S1678-91992009000300007

Ramesh, C.H. & Mohanraju, R., 2017. Antibacterial activity of marine bioluminescent bacteria. Indian Journal of Geo Marine Sciences. Vol. 46 (10), pp. 2063-2074

Reysenbach, A.-L. & Pace, N.R., 1995. Reliable amplification of hyperthermophilic archaeal 16S rRNA genes by the polymerase chain reaction. Archaea: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp.101–107. doi: 10.1023/A:1015122926687

Ruch, R.J., Cheng, S. & Klaunig, J.E., 1989. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis, 10(6), pp.1003–1008. doi: 10.1093/carcin/10.6.1003

Saeed, N., Khan, M.R. & Shabbir, M., 2012. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complementary and alternative medicine, 12(1), pp.1–12. doi: 10.1186/1472-6882-12-221

Sarkar, M.K.D., Ahmmed, T. & others, 2019. Antibiotic resistance analysis of Vibrio spp. isolated from different types of water sources of Bangladesh and their characterization. Eur J Med Health Sci, 1, pp.19–29. doi: 10.34104/ejmhs.01929

Sharifian, S. et al., 2018. The emerging use of bioluminescence in medical research. Biomedicine & Pharmacotherapy, 101, pp.74–86. doi: 10.1016/j.biopha.2018.02.065

Singh, R. & Chahal, K.K., 2018. Phytochemical analysis and in vitro antioxidant capacity of different solvent extracts of Saussurea lappa L. roots. J Pharma Phytochem, 7(3), pp.427–432. doi: 10.1016/j.sjbs.2022.01.040

Soler-Rivas, C., Esp’in, J.C. & Wichers, H.J., 2000. Oleuropein and related compounds. Journal of the Science of Food and Agriculture, 80(7), pp.1013–1023. doi: 10.1002/(SICI)1097-0010(20000515)80:7<1013::AID-JSFA571>3.0.CO;2-C

These, A., Scholz, J. & Preiss-Weigert, A., 2009. Sensitive method for the determination of lipophilic marine biotoxins in extracts of mussels and processed shellfish by high-performance liquid chromatography--tandem mass spectrometry based on enrichment by solid-phase extraction. Journal of Chromatography A, 1216(21), pp.4529–4538. doi: 10.1016/j.chroma.2009.03.062. Epub 2009 Mar 27.

Yalla, S.K., Cherian, T. & Mohanraju, R., 2018. Antimicrobial potential of secondary metabolites extracted from Vibrio furnissii, a luminescent bacterium associated with squid, Uroteuthis duvauceli. Int. J. of Pharm. and Biol. Sci, 8(1), pp.530–534.

Yen, G.-C. & Chen, H.-Y., 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of agricultural and food chemistry, 43(1), pp.27–32. doi: 10.1021/jf00049a007

Zheng, L. et al., 2005. Antimicrobial screening and active compound isolation from marine bacterium NJ6-3-1 associated with the sponge Hymeniacidon perleve. World Journal of Microbiology and Biotechnology, 21(2), pp.201–206. doi: 10.1007/s11274-004-3318-6

DOI: https://doi.org/10.22146/jtbb.71758

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