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Bacillus subtilis subsp. subtilis CBMDC3f with antimicrobial activity against Gram-positive foodborne pathogenic bacteria: UV-MALDI-TOF MS analysis of its bioactive compounds

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Abstract

In this work a new Bacillus sp. strain, isolated from honey, was characterized phylogenetically. Its antibacterial activity against three relevant foodborne pathogenic bacteria was studied; the main bioactive metabolites were analyzed using ultraviolet matrix assisted laser desorption-ionization mass spectrometry (UV–MALDI MS). Bacillus CBMDC3f was phylogenetically characterized as Bacillus subtilis subsp. subtilis after rRNA analysis of the 16S subunit and the gyrA gene (access codes Genbank JX120508 and JX120516, respectively). Its antibacterial potential was evaluated against Listeria monocytogenes (9 strains), B. cereus (3 strains) and Staphylococcus aureus ATCC29213. Its cell suspension and cell-free supernatant (CFS) exerted significant anti-Listeria and anti-S. aureus activities, while the lipopeptides fraction (LF) also showed anti-B. cereus effect. The UV-MALDI-MS analysis revealed surfactin, iturin and fengycin in the CFS, whereas surfactin predominated in the LF. The CFS from CBMDC3f contained surfactin, iturin and fengycin with four, two and four homologues per family, respectively, whereas four surfactin, one iturin and one fengycin homologues were identified in the LF. For some surfactin homologues, their UV-MALDI-TOF/TOF (MS/MS; Laser Induced Decomposition method, LID) spectra were also obtained. Mass spectrometry analysis contributed with relevant information about the type of lipopeptides that Bacillus strains can synthesize. From our results, surfactin would be the main metabolite responsible for the antibacterial effect.

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References

  • Aksu H, Bostan K, Ergün Ö (2000) Presence of Bacillus cereus in packaged some spices and herbs sold in Istanbul. Pak J Biol Sci 3:710–712

    Article  Google Scholar 

  • Audisio MC, Terzolo HR, Apella MC (2005) Bacteriocin from honeybee beebread Enterococcus avium active against Listeria monocytogenes. Appl Environ Microbiol 71:3373–3375

    Article  CAS  Google Scholar 

  • Ayed HB, Hmidet N, Béchet M, Chollet M, Chataigné G, Leclère V, Jacques P, Nasri M (2014) Identification and biochemical characteristics of lipopeptides from Bacillus mojavensis A21. Process Biochem 49:1699–1707

    Article  Google Scholar 

  • Bhunia AK (2008) Bacillus cereus and Bacillus anthracis. In: Bhunia AK (ed) Foodborne microbial pathogens: mechanisms and pathogenesis. Springer, New York, pp 135–147

    Google Scholar 

  • Burkard M, Entian KD, Stein T (2007) Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin. J Microbiol Methods 70:179–185

    Article  CAS  Google Scholar 

  • Compaoré CS, Nielsen DC, Ouoba LI, Berner TS, Nielsen KF, Sawadogo-Lingani H, Diawara B, Ouédraogo GA, Jakobsen M, Thorsen L (2013) Co-production of surfactin and a novel bacteriocin by Bacillus subtilis subsp. subtilis H4 isolated from Bikalga, an African alkaline Hibiscus sabdariffa seed fermented condiment. Int J Food Microbiol 162:297–307

    Article  Google Scholar 

  • Daffonchio D, Borin S, Frova G, Manachini P, Sorlini C (1998) PCR fingerprinting of whole genomes: the spacers between the 16S and 23S rRNA genes and of intergenic tRNA gene regions reveals a different intraespecific genomic variability of Bacillus cereus and Bacillus licheniformis. Int J Syst Bacteriol 48:107–116

    Article  CAS  Google Scholar 

  • de Clerck E, Vanhoutte T, Hebb T, Geerinck J, Devos J, De Vos P (2004) Isolation, characterization, and identification of bacterial contaminants in semifinal gelatin extracts. Appl Environ Microbiol 70:3664–3672

    Article  Google Scholar 

  • De Faria AF, Teodoro-Martinez DS, de Oliveira Barbosa GN, Vaz BG, Serrano Silva Í, Simone Garcia J, Tótolac MR, Eberlinb MN, Grossmand M, Alvesb OL, Durranta LR (2011) Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from biodiesel industry. Process Biochem 46:1951–1957

    Article  Google Scholar 

  • Foster TJ (2005) Immune evasion by staphylococci. Nat Rev Microbiol 3:948–958

    Article  CAS  Google Scholar 

  • Freyre-González JA, Manjarrez-Casas AM, Merino E, Martinez-Nuñez M, Perez-Rueda E, Gutiérrez-Ríos RM (2013) Lessons from the modular organization of the transcriptional regulatory network of Bacillus subtilis. BMC Syst Biol 7:127–141

    Article  Google Scholar 

  • Fuchs SW, Jaskolla TW, Bochmann S, Kötter P, Wichelhaus T, Karas M, Stein T, Entian KD (2011) Entianin, a novel subtilin-like lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029T with high antimicrobial activity. Appl Environ Microbiol 77:1698–1707

    Article  CAS  Google Scholar 

  • Gandhi M, Chikindas ML (2007) Listeria: a foodborne pathogen that knows how to survive. Int J Food Microbiol 113:1–15

    Article  Google Scholar 

  • Gomaa EZ (2013) Antimicrobial activity of a biosurfactant produced by Bacillus licheniformis strain M104 grown on whey. Braz Arch Biol Technol 56:259–268

    Article  CAS  Google Scholar 

  • Hiradate S, Yoshida S, Sugie H, Yada H, Fujii Y (2002) Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2. Phytochemistry 61:693–698

    Article  CAS  Google Scholar 

  • Huang X, Wei Z, Zhao G, Gao X, Yang S, Cui Y (2008) Optimization of sterilization of Escherichia coli in milk by surfactin and fengycin using a response surface method. Curr Microbiol 56:376–381

    Article  CAS  Google Scholar 

  • Hue N, Serani L, Laprévote O (2001) Structural investigation of cyclic peptidolipids from Bacillus subtilis by high-energy tándem mass spectrometry. Rapid Commun Mass Spectrom 15:203–209

    Article  CAS  Google Scholar 

  • Imran M, Revol-Junelles AM, Paris C, Guedon E, Linder M, Desobry S (2014) Liposomal nanodelivery systems using soy and marine lecithin to encapsulate food biopreservative nisin. LWT-Food Sci Technol. doi:10.1016/j.lwt.2014.12.046

    Google Scholar 

  • Kaewklom S, Lumlert S, Kraikulb W, Aunpada R (2013) Control of Listeria monocytogenes on sliced bologna sausage using a novel bacteriocin, amysin, produced by Bacillus amyloliquefaciens isolated from Thai shrimp paste (Kapi). Food Control 32:552–557

    Article  CAS  Google Scholar 

  • Khochamit N, Siripornadulsil S, Sukon P, Siripornadulsil W (2015) Antibacterial activity and genotypic-phenotypic characteristics of bacteriocin-producing Bacillus subtilis KKU213: potential as a probiotic strain. Microbiol Res 170:36–50

    Article  CAS  Google Scholar 

  • Kim P, Ryu J, Kim Y, ChI Y-T (2010) Production of biosurfactant lipopeptides Iturin A, fengycin and surfactin A from Bacillus Subtilis CMB32 for control of Colletotrichum gloeosporioides. J Microbiol Biotechnol 20:138–145

    CAS  Google Scholar 

  • Lee NK, Yeo IC, Park JW, Kang BS, Hahm YT (2010) Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus. J Biosci Bioeng 110:298–303

    Article  CAS  Google Scholar 

  • Leenders F, Stein TH, Kablitz B, Franke P, Vater J (1999) Rapid typing of Bacillus subtilis strains by their secondary metabolites using matrix-assisted laser desorption/ionization mass spectrometry of intact cells. Rapid Commun Mass Spectrom 13:943–949

    Article  CAS  Google Scholar 

  • Lunden J, Autio T, Markkula A, Hellstrom S, Korkeala H (2003) Adaptive and cross-adaptive responses of persistent and non-persistent Listeria monocytogenes strains to disinfectants. Int J Food Microbiol 82:265–272

    Article  CAS  Google Scholar 

  • Maria-Neto S, Candido Ede S, Rodrigues DR, de Sousa DA, da Silva EM, de Moraes LM, Otero-Gonzalez Ade J, Magalhães BS, Dias SC, Franco OL (2012) Deciphering the magainin resistance process of Escherichia coli strains in light of the cytosolic proteome. Antimicrob Agents Chemother 56:1714–1724

    Article  CAS  Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Cold Spring Harbor, New York

    Google Scholar 

  • Mukherjee AK, Das K (2005) Correlation between diverse cyclic lipopeptides production and regulation of growth and substrate utilization by Bacillus subtilis strains in a particular habitat. FEMS Microbiol Ecol 54:479–489

    Article  CAS  Google Scholar 

  • Nakamura LK, Roberts MS, Cohan FM (1999) Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov. Int J Syst Evol Microbiol 49:1211–1215

    CAS  Google Scholar 

  • Nonami H, Fukui S, Erra-Balsells R (1997) β-Carboline alkaloids as matrices for matrix-assisted ultraviolet laser desorption time-of flight mass spectrometry of proteins and sulfated oligosaccharides: a comparative study using phenylcarbonyl compounds, carbazoles and classical matrices. J Mass Spectrom 32:287–296

    Article  CAS  Google Scholar 

  • Oka K, Hirano T, Homma M, Ishii H, Murakami K, Mogami S, Motizuki A, Morita H, Takeya K, Itokawa H (1993) Satisfactory separation and MS-MS spectrometry of six surfactins isolated from Bacillus subtilis natto. Chem Pharm Bull 41:1000–1002

    Article  CAS  Google Scholar 

  • Ongena M, Jacques P (2008) Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol 16:115–125

    Article  CAS  Google Scholar 

  • Ouoba LI, Diawara B, Jespersen L, Jakobsen M (2007) Antimicrobial activity of Bacillus subtilis and Bacillus pumilus during the fermentation of African locust bean (Parkia biglobosa) for Soumbala production. J Appl Microbiol 102:963–970

    CAS  Google Scholar 

  • Pandey H, Kumar V, Roy BK (2014) Assessment of genotoxicity of some common food preservatives using Allium cepa L. as a test plant. Toxicol Rep 1:300–308

    Article  CAS  Google Scholar 

  • Patel H, Tscheka C, Edwards K, Karlsson G, Heerklotz H (2011) All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713. Biochim Biophys Acta 1808:2000–2008

    Article  CAS  Google Scholar 

  • Pathak KV, Keharia H (2014) Identification of surfactins and iturins produced by potent fungal antagonist, Bacillus subtilis K1 isolated from aerial roots of banyan (Ficus benghalensis) tree using mass spectrometry. 3. Biotech 4:283–295

    Google Scholar 

  • Pedersen PB, Bjørnvad ME, Rasmussen MD, Petersen JN (2002) Cytotoxic potential of industrial strains of Bacillus sp. Regul Toxicol Pharmacol 36:155–161

    Article  CAS  Google Scholar 

  • Porwal S, Lal S, Cheema S, Kalia VC (2009) Phylogeny in aid of the present and novel microbial lineages: diversity in Bacillus. PLoS One 4:e4438. doi:10.1371/journal.pone.0004438

    Article  Google Scholar 

  • Price NP, Rooney AP, Swezey JL, Perry E, Cohan FM (2007) Mass spectrometric analysis of lipopeptides from Bacillus strains isolated from diverse geographical locations. FEMS Microbiol Lett 271:83–89

    Article  CAS  Google Scholar 

  • Romero D, de Vicente A, Rakotoaly RH, Dufour SE, Veening JW, Arrebola E, Cazorla FM, Kuipers OP, Paquot M, Pérez-García A (2007) The itulin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant Microbe Interact 20:430–440

    Article  CAS  Google Scholar 

  • Rooney AP, Price NPJ, Ehrhardt C, Swezey JL, Bannan JD (2009) Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. Int J Syst Evol Microbiol 59:2429–2436

    Article  CAS  Google Scholar 

  • Sabaté DC, Audisio MC (2013) Inhibitory activity of surfactin, produced by different Bacillus subtilis subsp. subtilis strains, against Listeria monocytogenes sensitive and bacteriocin-resistant strains. Microbiol Res 168:125–129

    Article  Google Scholar 

  • Sabaté DC, Carrillo L, Audisio MC (2009) Inhibition of Paenibacillus larvae and Ascosphaera apis by Bacillus subtilis isolated from honeybee gut and honey samples. Res Microbiol 160:193–199

    Article  Google Scholar 

  • Savadogo A, Tapi A, Chollet M, Wathelet B, Traore AS, Jacques P (2011) Identification of surfactin producing strains in Soumbala and Bikalga fermented condiments using polymerase chain reaction and matrix assisted laser desorption/ionization-mass spectrometry methods. Int J Food Microbiol 151:299–306

    Article  CAS  Google Scholar 

  • Seydlova G, Svobodova J (2008) Review of surfactin chemical properties and the potential biomedical applications. Cent Eur J Med 3:123–133

    Article  CAS  Google Scholar 

  • Sun L, Lu Z, Bie X, Lu F, Yang S (2006) Isolation and characterization of a co-producer of fengycins and surfactins, endophytic Bacillus amyloliquefaciens ES-2, from Scutellaria baicalensis Georgi. World J Microbiol Biotechnol 22:1259–1266

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  Google Scholar 

  • Tang J-S, Zhao F, Gao H, Dai Y, Yao Z-H, Hong K, Li J, Ye W-C, Yao X-S (2010) Characterization and online detection of surfactins isomers based on HPLC-MS analyses and their inhibitory effects on the overproduction of nitric oxide and the release of TNF-α and IL-6 in LPS-induces macrophages. Mar Drugs 8:2605–2618

    Article  CAS  Google Scholar 

  • Teixeira ML, Dalla Rosa A, Brandelli A (2013) Characterization of an antimicrobial peptide produced by Bacillus subtilis subsp. spizezinii showing inhibitory activity towards Haemophilus parasuis. Microbiology 159:980–988

    Article  CAS  Google Scholar 

  • Thasana N, Prapagdee B, Rangkadilok N, Sallabhan R, Aye SL, Ruchirawat S, Loprasert S (2010) Bacillus subtilis SSE4 produces subtulene A, a new lipopeptide antibiotic possessing an unusual C15 unsaturated beta-amino acid. FEBS Lett 584:3209–3214

    Article  CAS  Google Scholar 

  • Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS (2002) Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68:6210–6219

    Article  CAS  Google Scholar 

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Acknowledgments

This work had the financial support of CIUNSa (PI 1974), UBA (0055BA) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) of Argentina (PICT2011-07/67 and PICT 2012-0888) and CONICET-Argentina (PIP 0072CO and PIP 00019). M.C. Audisio, R. Erra-Balsells and G. Petroselli are Research Members of CONICET, Argentina.

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Torres, M.J., Petroselli, G., Daz, M. et al. Bacillus subtilis subsp. subtilis CBMDC3f with antimicrobial activity against Gram-positive foodborne pathogenic bacteria: UV-MALDI-TOF MS analysis of its bioactive compounds. World J Microbiol Biotechnol 31, 929–940 (2015). https://doi.org/10.1007/s11274-015-1847-9

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