The study of purified secondary metabolites extracts of Bacillus subtilis and its chemotaxis effect on biofilm-forming bacteria

Document Type: Original Article

Authors

1 Department of Biotic Evolution- Marine Science Center, Basrah University, Basrah, Iraq

2 Department of Biology, Collage of Education for Pure Science, Basrah University, Basrah, Iraq

Abstract

Chemotaxis is the movement of a single cell organism as a reaction to a chemical stimulation in its surrounding environment. Biofilm-forming bacteria are the cause for numerous major health and environmental problems. Bacterial extracts were proven to induce negative chemotaxis reaction against biofilm-forming bacteria and biofilm development. Therefore, using and enhancing these extracts are considered as promising methods in pharmaceutical production and environmental science. In this study, twenty Bacillus isolates and five biofilm-forming bacteria (targeted bacteria) isolates from different water and sediments samples of different areas in Basra province were biochemically diagnosed. Secondary metabolites of Bacillus isolates were extracted and analysed. Total proteins in the extracts were determined using Biuret method and the highest two isolates (BS8) and (BS14) with 13.78 and 12.02 g/l protein, respectively were chosen for the experiment. GC-MS results showed the existence of compounds with proven high antimicrobial properties such as type D-amino acids, N-cyclopropyl carbonyl-, butyl and esters such as d- proline, N-methoxycarbonyl, and pentyl ester. Afterwards, the chemotaxis nature of the purified extracts was studied. The results showed that both extracts had a negative chemotaxis toward the targeted bacteria represented by transparent halos without bacterial growth around the spot where secondary metabolites extracts of Bacillus subtilis were placed. K. kristinae was the most affected species in regards of growth inhibition zone diameter with 23 and 24 mm for (BS8) and (BS14) extracts respectively, while P. aeruginosa was the least affected with 19 and 18 mm for (BS8) and (BS14) extracts respectively.

Keywords


  1. de la Fuente-Núñez C, Korolik V, Bains M, Nguyen U, Breidenstein EB, Horsman S, Lewenza S, Burrows L, Hancock RE. Inhibition of bacterial biofilm formation and swarming motility by a small synthetic cationic peptide. Antimicrob. Agents Chemother. 2012;56(5):2696-704. DOI
  2. Allegrucci M, Hu FZ, Shen K, Hayes J, Ehrlich GD, Post JC, Sauer K. Phenotypic characterization of Streptococcus pneumoniae biofilm development. J. Bacteriol. 2006;188(7):2325-35. DOI
  3. Morgan R, Kohn S, Hwang SH, Hassett DJ, Sauer K. BdlA, a chemotaxis regulator essential for biofilm dispersion in Pseudomonas aeruginosa. J. Bacteriol. 2006;188(21):7335-43. DOI
  4. Khanna MR, Bhavsar SP, Kapadnis BP. Effect of temperature on growth and chemotactic behaviour of Campylobacter jejuni. Letters in applied microbiology. 2006;43(1):84-90. DOI
  5. Zhang X, Si G, Dong Y, Chen K, Ouyang Q, Luo C, Tu Y. Escape band in Escherichia coli chemotaxis in opposing attractant and nutrient gradients. Proc. Natl. Acad. Sci. U.S.A. 2019;116(6):2253-8. DOI
  6. Choudoir M, Pepe-Ranney C, Buckley D. Diversification of secondary metabolite biosynthetic gene clusters coincides with lineage divergence in Streptomyces. Antibiotics. 2018;7(1):12. DOI
  7. Yan Q, Lopes LD, Shaffer BT, Kidarsa TA, Vining O, Philmus B, Song C, Stockwell VO, Raaijmakers JM, McPhail KL, Andreote FD. Secondary metabolism and interspecific competition affect accumulation of spontaneous mutants in the GacS-GacA regulatory system in Pseudomonas protegens. MBio. 2018;9(1):e01845-17. DOI
  8. Mondol MA, Shin HJ, Islam MT. Diversity of secondary metabolites from marine Bacillus species: chemistry and biological activity. Mar Drugs. 2013;11(8):2846-72. DOI
  9. Leifert C, Li H, Chidburee S, Hampson S, Workman S, Sigee D, Epton HA, Harbour A. Antibiotic production and biocontrol activity by Bacillus subtilis CL27 and Bacillus pumilus CL45. J. Appl. Microbiol. 1995;78(2):97-108. DOI
  10. Priest FG, de Muro MA, Kaji DA. Systematics of insect pathogenic bacilli: uses in strain identification and isolation of novel pathogens. In Bacterial Diversity and Systematics 1994 (pp. 275-295). Springer, Boston, MA. DOI
  11. Sirtori LR, Cladera-Olivera F, Lorenzini DM, Tsai SM, Brandelli A. Purification and partial characterization of an antimicrobial peptide produced by Bacillus sp. strain P45, a bacterium from the Amazon basin fish Piaractus mesopotamicus. J. Gen. Appl. Microbiol. 2006;52(6):357-63. DOI
  12. Anju KM, Archana MM, Mohandas C, Nambisan B. Purification and identification of an antibacterial protein from the symbiotic bacteria associated with novel entomopathogenic nematode, Rhabditis (Oscheius) sp.  World J. Microbiol. Biotechnol. 2015;31(4):621-32. DOI
  13. Hassan SW, Abdul-Raouf UM, Ali MA. Antagonistic interactions and phylogenetic diversity of antimicrobial agents producing marine bacteria in Suez Bay. Egypt J Aquat Res. 2015;41(1):57-67. DOI
  14. Bechard J, Eastwell KC, Sholberg PL, Mazza G, Skura B. Isolation and partial chemical characterization of an antimicrobial peptide produced by a strain of Bacillus subtilis. J. Agric. Food Chem. 1998;46(12):5355-61. DOI
  15. Spinosa MR, Braccini T, Ricca E, De Felice M, Morelli L, Pozzi G, Oggioni MR. On the fate of ingested Bacillus spores. Res. Microbiol. 2000;151(5):361-8. DOI
  16. Baird RB, Eaton AD, Clesceri LS. Standard methods for the examination of water and wastewater. Rice EW, editor. Washington, DC: American Public Health Association; 2012.
  17. Sabaté DC, Audisio MC. Inhibitory activity of surfactin, produced by different Bacillus subtilis subsp. subtilis strains, against Listeria monocytogenes sensitive and bacteriocin-resistant strains. Microbiol. Res. 2013;168(3):125-9. DOI
  18. Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni GO, Azevedo V, Bertero MG, Bessieres P, Bolotin A, Borchert S, Borriss R. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature. 1997;390(6657):249. DOI
  19. Dusane DH, Damare SR, Nancharaiah YV, Ramaiah N, Venugopalan VP, Kumar AR, Zinjarde SS. Disruption of microbial biofilms by an extracellular protein isolated from epibiotic tropical marine strain of Bacillus licheniformis. PLoS One. 2013;8(5):e64501. DOI
  20. Amin M, Rakhisi Z, Ahmady AZ. Isolation and identification of Bacillus species from soil and evaluation of their antibacterial properties. Avicenna J Clin Microb Infec. 2015;2(1):e23233.
  21. Al-Saraireh H, Al-Zereini WA, Tarawneh KA. Antimicrobial activity of secondary metabolites from a soil Bacillus sp. 7B1 isolated from south Al-Karak, Jordan. Jordan J Biol Sci. 2015;147(3427):1-6. DOI
  22. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature. 1970;227(5259):680. DOI
  23. Mohan G, Thangappanpillai AK, Ramasamy B. Antimicrobial activities of secondary metabolites and phylogenetic study of sponge endosymbiotic bacteria, Bacillus sp. at Agatti Island, Lakshadweep Archipelago. Biotechnol Rep. 2016;11:44-52. DOI
  24. Dusane DH, Pawar VS, Nancharaiah YV, Venugopalan VP, Kumar AR, Zinjarde SS. Anti-biofilm potential of a glycolipid surfactant produced by a tropical marine strain of Serratia marcescens. Biofouling. 2011; 27(6):645-54. DOI
  25. Naves P, Del Prado G, Huelves L, Rodriguez-Cerrato V, Ruiz V, Ponte MC, Soriano F. Effects of human serum albumin, ibuprofen and N-acetyl-L-cysteine against biofilm formation by pathogenic Escherichia coli strains. J. Hosp. Infect. 2010;76(2):165-70. DOI
  26. Valle J, Da Re S, Henry N, Fontaine T, Balestrino D, Latour-Lambert P, Ghigo JM. Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide. Proc. Natl. Acad. Sci. U.S.A. 2006;103(33):12558-63. DOI
  27. Teasdale ME, Liu J, Wallace J, Akhlaghi F, Rowley DC. Secondary metabolites produced by the marine bacterium Halobacillus salinus that inhibit quorum sensing-controlled phenotypes in gram-negative bacteria. Appl. Environ. Microbiol.. 2009;75(3):567-72. DOI
  28. Sharma PK, Goel M, Dureja P, Uniyal PL. Isolation and identification of secondary metabolites from hexane extract of culture filtrate of Bacillus licheniformis MTCC 7445. Arch. Phytopathol. Pflanzenschutz 2010;43(16):1636-42.  DOI
  29. Barrios-Gonzalez J, Fernandez FJ, Tomasini A. Microbial secondary metabolites production and strain improvement. Indian J. Biotechnol. 2003;2(3):322-33.
  30. Motta AS, Cannavan FS, Tsai SM, Brandelli A. Characterization of a broad range antibacterial substance from a new Bacillus species isolated from Amazon basin. Arch. Microbiol. 2007;188(4):367-75. DOI
  31. Marhaeni B, Radjasa OK, Khoeri MM, Sabdono A, Bengen DG, Sudoyo H. Antifouling activity of bacterial symbionts of seagrasses against marine biofilm-forming bacteria. J Environ Prot. 2011;2(9):1245. DOI
  32. Sharon FB, Kalidass S, Daniel RR. Qualitative analysis of antimicrobial compound by high performance thin layer chromatography method. Asian J Pharm Clin Res. 2013;6(4):117-20.
  33. Gordillo A, Maldonado MC. Purification of peptides from Bacillus strains with biological activity. Chromatography and Its Applications. 2012;11:201-25.
  34. Teixeira ML, Dalla Rosa A, Brandelli A. Characterization of an antimicrobial peptide produced by Bacillus subtilis subsp. spizezinii showing inhibitory activity towards Haemophilus parasuis. Microbiology. 2013;159(5):980-8. DOI
  35. Shai Y. From innate immunity to de-novo designed antimicrobial peptides. Curr. Pharm. Des. 2002;8(9):715-25. DOI
  36. Fernandes PB, Chu DT. . Quinolone Antibacterial Agents. In Annual Reports in Medicinal Chemistry. Academic Press. 1988;23,:.133-40. DOI
  37. Kalinovskaya NI, Romanenko LA, Kalinovsky AI. Antibacterial low-molecular-weight compounds produced by the marine bacterium Rheinheimera japonica KMM 9513T. Antonie Van Leeuwenhoek. 2017;110(5):719-26. DOI
  38. Zhang W, Hu JF, Lv WW, Zhao QC, Shi GB. Antibacterial, antifungal and cytotoxic isoquinoline alkaloids from Litsea cubeba. Molecules. 2012;17(11):12950-60. DOI
  39. Sharma PC, Jain SA. Synthesis and in vitro antibacterial activity of some novel N-nicotinoyl-1-ethyl-6-fluoro-1, 4-dihydro-7-piperazin-1-yl-4-oxoquinoline-3-carboxylates. Acta Pol Pharm. 2008;65:551-6.
  40. Ramyabharathi SA, Raguchander T. Efficacy of Secondary Metabolites Produced by EPCO16 against Tomato Wilt Pathogen f. sp. J Mycol Plant Pathol. 2014;44(2):148.
  41. Leiman SA, May JM, Lebar MD, Kahne D, Kolter R, Losick R. D-amino acids indirectly inhibit biofilm formation in Bacillus subtilis by interfering with protein synthesis. J. Bacteriol. 2013;195(23):5391-5. DOI
  42. Skariyachan S, G. Rao A, Patil MR, Saikia B, Bharadwaj Kn V, Rao Gs J. Antimicrobial potential of metabolites extracted from bacterial symbionts associated with marine sponges in coastal area of Gulf of Mannar Biosphere, India. Lett. Appl. Microbiol. 2014;58(3):231-41. DOI
  43. Sharma T, Kaul S, Dhar MK. Diversity of culturable bacterial endophytes of saffron in Kashmir, India. SpringerPlus. 2015;4(1):661. DOI
  44. Suarez CA, Montano ID, Nucci ER, Iemma MR, Giordano RD, Giordano RD. Assessment of the metabolism of different strains of Bacillus megaterium. Braz. arch. biol. technol. 2012;55(4):485-90. DOI
  45. Li H, Zhu J. Targeted metabolic profiling rapidly differentiates Escherichia coli and Staphylococcus aureus at species and strain level. Rapid Commun. Mass Spectrom. 2017;31(19):1669-76. DOI
  46. Phillips DA, Fox TC, King MD, Bhuvaneswari TV, Teuber LR. Microbial products trigger amino acid exudation from plant roots. Plant Physiol. 2004;136(1):2887-94. DOI