ISOLATION AND CHARACTERIZATION OF MDR BACTERIA FROM IN VITRO CULTURE OF BACOPA MONNIERA AND SUPPLEMENTATION OF NATURAL EXTRACTS TO CONTROL BACTERIAL CONTAMINATION
Keywords:Contamination, Bacopa monniera, Gram-negative microorganism, 16S rDNA
Objective: The purpose of the present study was to isolate and characterize bacterial contamination from in vitro culture of Bacopa monniera callus culture.
Methods: The two selected isolates (1 and 2) were identified by morphological, biochemical and molecular (16S rRNA gene sequencing) methods. Beside this antibiotic resistance was also determined.
Results: The isolates were identified as closely related to Enterobacter cloacae (KU350623) (Isolate 1) and Myroides odoratimimusÂ (KU382740) (Isolate 2). The isolate 1 and 2 were found to be resistant to streptomycin (25 mcg), dapsone (10 mcg), erythromycin (15 mcg), chloroamphenicol (25 mcg) and ampicillin (10 mcg). Supplementation of the natural extract was tested to control the contamination due to these multi drug resistant bacteria. Aqueous and alcoholic leaf extracts of Azadirachta indica was added to plant tissue culture media i.e. MS media in order to control contamination.
Conclusion: The present studies suggest using natural extracts supplementation as a promising strategy to control the in vitro bacterial contamination in plant tissue culture.
Sharma Y, Nagar A, Shukla S. Antimicrobial activity, phytochemical screening of Adenium obesum (Desert rose) leaf. Int J Pharma Bio Sci 2015;6:85-91.
Rout GR, Samantaray S, Das P. In vitro manipulation and propagation of medicinal plants. Biotechnol Adv 2000;18:91-120.
Staba EJ, Seabrook JEA. Laboratory culture. Plant tissue culture as a source of biochemical. CRC Press: Boca Raton; 1980. p. 1-20.
Sidhu Y. In vitro micropropagation of medicinal plants by tissue culture. Plymouth Student Scientist 2010;4:432-49.
Bhoite HA, Palshikar GS. Plant tissue culture: a review. World J Pharm Sci 2014;2:565-72.
Kane M. Bacterial and fungal indexing of tissue cultures; 2003. Available from http://www.hos.ufl.edu/mooreweb/Tissue Culture/class1/Bacterial%20and%20fungal%20indexing%20of%20tissue%20cultures.doc. [Last accessed on 10 May 2016].
George EF. Plant propagation by tissue culture. Exergetics Ltd., Edington, England; 1993. p. 574.
Enjalric F, Carron MP, Lardet L. Contamination of primary cultures in tropical areas: the case of hevea brasiliensis. ISHS Acta Horticulture 1988;225:57-66.
Kunisaki JT. In vitro propagation of Anthurium andreanum Lind. Hort Sci 1980;15:508â€“9.
Chen WL, Yeh DM. Elimination of in vitro contamination, shoot multiplication, and Ex vitro rooting of aglaonema. Hortscience 2007;42:629â€“32.
Nadha HK, Salwan R, Kasana RC, Anand M, Sood A. Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth. Pharmacogn Mag 2012;8:93-7.
Mukheijee DG, Dey CD. Clinical trial on Brahmi. Int J Exp Med Sci 1966;10:5-11.
Gohil KJ, Patel JA. A review on Bacopa monniera: current research and future prospects. Int J Green Pharm 2010;4:1-9.
Bhattacharya SK, Ghosal S. Anxiolytic activity of a standardized extract of Bacopa monniera: an experimental study. Phytomed 1998;5:77-82.
Martis G, Rao A, Karanth KS. The neuropharmacological activity of Herpestis monniera. Fitoterapia 1992;63:399-404.
Vijayan VA, Helen A. Protective activity of Bacopa monniera Linn. On nicotine-induced toxicity in mice. Phytother Res 2007;21:378-81.
Gupta Y, Sharma D, Sharma Y. Bactericidal activity of different parts of Azadirachta indica on probiotic microbes. Asian J Pharm Clin Res 2016;9:50-3.
Biswas K, Chadhopadhyay, Banarjee RK, Bandhopadhyay U. Biological activities and medicinal properties of neem (Azadirachta indica), review article. Curr Sci 2002;82:1336-45.
Saeed AK, Junaid A. Study on the effect of neem (Azadirachta indica) leaves smoke in controlling airborne bacteria in residential premises. Curr Res Bacteriol 2008;1:64-6.
Talukdar A. Biosynthesis of total bacosides in the callus culture of Bacopa monnieri. L. Pennel from North-east India. Int J Curr Microbiol Appl Sci 2014;3:140-5.
Cappuccino JG, Sherman N. Techniques for isolation of pure culture In. Microbiology: a laboratory manual. (VI Ed.) Pearson Education, Inc. Singapore. Collee, JG, Duguid, JP, Frasee AG, Marmion BP. Published by Pearson Education (Singapore) Pte. Ltd; 1989. p. 63-6, 149-85, 428-31.
Hoffman, Winston. Genomic DNA extraction. Gene 1987;57:267-72.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215;403-10.
Dereeper A, Guignon V, Blanc G, Audic S. Phylogeny. fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 2008;1:465-9.
Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.
Leifert C, Waites WM, Nicholas JR. Bacterial contaminants of micro propagated plant cultures. J Appl Microbiol 1989;67:353-61.
Leifert C, Waites WM. Bacterial growth in plant tissue culture media. J Appl Bacteriol 1992;72:460-6.
Staba EJ, Seabrook JEA. Laboratory culture. Plant tissue culture as a source of biochemicals, CRC Press: Boca Raton; 1980. p. 1-20.
Odutayo OI, Oso RT, Akinyemi BO, Amusa NA. Microbial contaminants of cultured Hibiscus cannabinus and Telfaria occidentalis tissues. Afr J Biotechnol 2004;3:473-6.
Pierik RL. In vitro cultures of higher plants as a tool in the propagation of horticultural crops. In: Plant propagation by tissue culture. ISHA Acta Horticulturae; 1988. p. 24-8.
Boxus PH, Terzi JM. Control of accidental contaminations during mass propagation. ISHS Acta Horticulturae 1988;225:190-8.
Singh V, Tyagi A, Chauhan PK, Kumari P, Kaushal S. Identification and prevention of bacterial contamination on explant used in plant tissue culture labs. Int J Pharm Pharm Sci 2011;3:160-3.
Barrett C, Cassells AC. Alterations in the growth of tissue-cultured tansy (Tanacetum vulgare L.) treated with antibiotics. Plant Cell Tissue Organ Cult 1994;36:169â€“75.