Acta Biologica Hungarica

, Volume 68, Issue 2, pp 208–219 | Cite as

Isolation and Preliminary Characterization of A Bacteriocin-Producer Bacillus Strain Inhibiting Methicillin Resistant Staphylococcus Aureus

  • Ankit Kumar Chauhan
  • Dinesh Kumar MaheshwariEmail author
  • Vivek K. BajpaiEmail author


In a multivalent approach to discover new antimicrobial substance, a total of 160 Bacilli were isolated from termitarium soil, characterized on the basis of their morphological and physiological characters and screened for their antimicrobial activity by agar well diffusion method against certain drug resistant pathogenic bacteria such as Staphylococcus aureus, Methicillin resistant Staphylococcus aureus and common food contaminating bacteria Listeria monocytogenes. After preliminary screening, sixteen isolates showed inhibitory activity against test pathogens. Among them Bacillus isolate TSH58 exhibited maximum inhibitory activity against MRSA, Staphylococcus aureus and Listeria monocytogenes. Based on morphological, physiological, biochemical and 16S rDNA characteristics isolate TSH58 was identified as a member of the Bacillus cereus species group. Various nutrient sources and culture conditions were optimized, the partially purified antimicrobial metabolite was subjected to various treatments such as heat, pH and proteolytic enzymes. Complete loss in the activity observed when the crude metabolite was treated with proteolytic enzymes suggesting its proteinaceous nature and termed as bacteriocin like inhibitory substance (BLIS). Minimal inhibitory concentration of the partially purified bacteriocin determined by microtiter plate assay was 80 μg/ml for MRSA and 40 μg/ml for L. monocytogenes. Tricine SDS PAGE analysis revealed that the partially purified bacteriocin produced by the Bacillus strain TSH58 had an apparent molecular weight of about 4.0 KDa.


Bacillus Staphylococcus aureus MRSA Listeria monocytogenes bacteriocin 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Abada, E. A. E. M. (2008) Isolation and characterization of an antimicrobial compound fro. Bacillus coagulans. Anim. Cells Sys. 12, 41–46.CrossRefGoogle Scholar
  2. 2.
    Allen, H. K., Trachsel, J., Looft, T., Casey, T. A. (2014) Finding alternatives to antibiotics. Ann. New York Acad. Sci. 1323, 91–100.CrossRefGoogle Scholar
  3. 3.
    Aunpad, R., Na- Bangchang, K. (2007) Pumilicin 4, a novel bacteriocin with anti-MRSA and anti- VRE activity produced by newly isolated bacteri. Bacillus pumilus strain WAPB4. Curr. Microbiol. 55, 308–313.CrossRefGoogle Scholar
  4. 4.
    Aunpad, R., Sripotong, N., Khamlak, K., Inchidjuy, S., Rattanasinganchan, P., Pipatsatitpong, D. (2011) Isolation and characterization of bacteriocin with anti-listeria and anti-MRSA activity produced by food and soil isolated bacteria. Afr. J. Microbiol. 5, 5297–5303.Google Scholar
  5. 5.
    Baindara, P., Mandal, S. M., Chawla, N., Singh, P. K., Pinnaka, A. K., Korpole, S. (2013) Characterization of two antimicrobial peptides produced by a halotoleran. Bacillus subtilis strain SK.DU.4 isolated from a rhizosphere soil sample. AMB Exp. 3, 1–11.CrossRefGoogle Scholar
  6. 6.
    Batdorj, B., Dalgalarrondo, M., Choiset, Y., Pedroche, J., Métro, F., Prévost, H., Chober, J. M., Haertlé, T. (2006) Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag. J. Appl. Microbiol. 101, 837–848.CrossRefGoogle Scholar
  7. 7.
    Bizani, D., Morrissy, J. A. C., Domingue, A. P. M., Brandelli, A. (2008) Inhibition o. Listeria monocytogenes in dairy products using the bacteriocin-like peptide cerein 8A. Int. J. Food Microbiol. 121, 229–233.CrossRefGoogle Scholar
  8. 8.
    Blair, J. M., Webber, M. A., Baylay, A. J., Ogbolu, D. O., Piddock, L. J. (2015) Molecular mechanisms of antibiotic resistance. Nat. Rev. Microbiol. 13, 42–51.CrossRefGoogle Scholar
  9. 9.
    Cotter, P. D., Ross, R. P., Hill, C. (2013) Bacteriocins–a viable alternative to antibiotics. Nat. Rev. Microbiol. 11, 95–105.CrossRefGoogle Scholar
  10. 10.
    Dubey, R. C., Maheshwari, D. K. (2012). Practical Microbiology. S. Chand & Co., New Delhi, India.Google Scholar
  11. 11.
    Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T., Williams, S. T. (1994). Bergey’s Manual of Determinative Bacteriology. Williams and Wilkins Press, Baltimore, USA.Google Scholar
  12. 12.
    Joerger, R. D. (2003) Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poul. Sci. 82, 640–647.CrossRefGoogle Scholar
  13. 13.
    Jiang, H., Dong, H., Zhang, G., Yu, B., Chapman, L. R., Fields, M. W. (2006) Microbial diversity in water and sediment of Lake Chaka, an athalassohaline lake in north-western China. Appl. Environ. Microbiol. 72, 3832–3845.CrossRefGoogle Scholar
  14. 14.
    Kumar, S., Stecher, G., Tamura, K. (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 8, 54–59.Google Scholar
  15. 15.
    Manjula, A., Sathyavathi, S., Pushpanathan, M., Gunasekaran, P., Rajendhran, J. (2014) Microbial diversity in termite nest. Curr. Sci. 106, 1430–1434.Google Scholar
  16. 16.
    Naclerio, G., Ricca, E., Sacco, M., de Felice, M. (1993) Antimicrobial activity of a newly identified bacteriocin o. Bacillus cereus. Appl. Environ. Microbiol. 59, 4313–4316.PubMedPubMedCentralGoogle Scholar
  17. 17.
    Naghmouchi, K., Baah, J., Hober, D., Jouy, E., Rubrecht, C., Sané, F., Drider, D. (2013) Synergistic effect between colistin and bacteriocins in controlling Gram-negative pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial cells. Antimicrob. Agents Chemother. 57, 2719–2725.CrossRefGoogle Scholar
  18. 18.
    Oscariz, J. C., Cintas, L., Holo, H., Lasa, I., Nes, I. F., Pisabarro, A. G. (2006) Purification and sequencing of cerein 7B, a novel bacteriocin produced b. Bacillus cereus Bc7. FEMS Microbiol. Lett. 254, 108–115.CrossRefGoogle Scholar
  19. 19.
    Oscariz, J. C., Lasa, I., Pisabarro, A. G. (1999) Detection and characterization of cerecin 7, a new bacteriocin produced b. Bacillus cereus with a broad spectrum of activity. FEMS Microbiol. Lett. 178, 337–341.CrossRefGoogle Scholar
  20. 20.
    Risoen, P. A., Ronning, P., Hegna, I. K., Kolsto, A. B. (2004) Characterization of a broad range antimicrobial substance fro. Bacillus cereus. J. Appl. Microbiol. 96, 648–655.CrossRefGoogle Scholar
  21. 21.
    Rossolini, G. M., Arena, F., Pecile, P., Pollini, S. (2014) Update on the antibiotic resistance crisis. Curr. Opin. Pharmacol. 18, 56–60.CrossRefGoogle Scholar
  22. 22.
    Saleem, F., Ahmad, S., Yaqoob, Z., Rasool, S. A. (2009) Comparative study of two bacteriocins produced by representative indigenous soil bacteria. Pak. J. Pharma Sci. 22, 252–258.Google Scholar
  23. 23.
    Sambrook, J., Russel, D. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.Google Scholar
  24. 24.
    Sansinenea, E., Ortiz, A. (2011) Secondary metabolites of soi. Bacillus spp. Biotechnol. Lett. 33, 1523–1538.CrossRefGoogle Scholar
  25. 25.
    Schagger, H., Von Jagow, G. (1987) Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.CrossRefGoogle Scholar
  26. 26.
    Sebei, S., Zendo, T., Boudabous, A., Nakayama, J., Sonomoto, K. (2007) Characterization, N-terminal sequencing and classification of cerein MRX1, a novel bacteriocin purified from a newly isolated bacterium. Bacillus cereus MRX1. J. Appl. Microbiol. 103, 1621–1631.CrossRefGoogle Scholar
  27. 27.
    Senbagam, D., Gurusamy, R., Senthilkumar, B. (2013) Physical chemical and biological characterization of a new bacteriocin produced b. Bacillus cereus NS02. Asian Pac. J. Trop. Med. 6, 934–941.CrossRefGoogle Scholar
  28. 28.
    Shokri, D., Zaghian, S., Khodabakhsh, F., Fazeli, H., Mobasherizadeh, S., Ataei, B. (2014) Antimicrobial activity of a UV-stable bacteriocin-like inhibitory substance (BLIS) produced b. Enterococcus faecium strain DSH20 against vancomycin-resistan. Enterococcus (VRE) strains. J. Microbiol. Immunol. Infect. 47, 371–376.CrossRefGoogle Scholar
  29. 29.
    Stein, T., Heinzmann, S., Düsterhus, S., Borchert, S., Entian, K. D. (2005) Expression and functional analysis of the subtilin immunity genes spaIFEG in the subtilin-sensitive hos. Bacillus subtilis MO1099. J. Bacteriol. 187, 822–828.CrossRefGoogle Scholar
  30. 30.
    Tabbene, O., Slimene, I. B., Bouabdallah, F., Mangoni, M. L., Urdaci, M. C., Limam, F. (2009) Production of anti-methicillin-resistant Staphylococcus activity fro. Bacillus subtilis sp. strain B38 newly isolated from soil. Appl. Biochem. Biotechnol. 157, 407–419.CrossRefGoogle Scholar
  31. 31.
    Tagg, J., McGiven, A. R. (1971) Assay system for bacteriocins. Appl. Microbiol. 21, 943.PubMedPubMedCentralGoogle Scholar
  32. 32.
    Wu, W. J., Park, S. M., Ahn, B. Y. (2013) Isolation and characterization of an antimicrobial substance fro. Bacillus subtilis BY08 antagonistic t. Bacillus cereus an. Listeria monocytogenes. Food Sci. Biotechnol. 22, 433–440.CrossRefGoogle Scholar

Copyright information

© Akadémiai Kiadó, Budapest 2017

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Department of Botany and MicrobiologyGurukul Kangri UniversityHaridwarIndia
  2. 2.Department of Applied Microbiology and BiotechnologyYeungnam UniversityGyeongsan, GyeongbukRepublic of Korea

Personalised recommendations