Probiotics and Antimicrobial Proteins

, Volume 11, Issue 3, pp 990–998 | Cite as

Genome Sequencing and Analysis of Bacillus pumilus ICVB403 Isolated from Acartia tonsa Copepod Eggs Revealed Surfactin and Bacteriocin Production: Insights on Anti-Staphylococcus Activity

  • Mahammed Zidour
  • Yanath BelguesmiaEmail author
  • Benoit Cudennec
  • Thierry Grard
  • Christophe Flahaut
  • Sami Souissi
  • Djamel DriderEmail author


Here we show that Bacillus pumilus ICVB403 recently isolated from copepod eggs is able to produce, after 48–72 h of growth in Landy medium, extracellular inhibitory compounds, which are active against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 43300, MRSA-S1, Staphylococcus epidermidis 11EMB, Staphylococcus warneri 27EMB, and Staphylococcus hominis 13EMB. Moreover, these extracellular inhibitory compound(s) were able to potentiate erythromycin against the aforementioned staphylococci. The minimum inhibitory concentration (MIC) of erythromycin was reduced from 32 μg/mL to 8 μg/mL for MRSA ATCC 43300 and MRSA SA-1 strains, and from 32–64 μg/mL to 4 μg/mL for S. epidermidis 11EMB and S. hominis 13EMB strains.

The genome sequencing and analysis of B. pumilus ICVB403 unveiled 3.666.195 nucleotides contained in 22 contigs with a G + C ratio of 42.0%, 3.826 coding sequences, and 73 RNAs. In silico analysis guided identification of two putative genes coding for synthesis of surfactin A, a lipopeptide with 7 amino acids, and for a circular bacteriocin belonging to the circularin A/uberolysin family, respectively.


Copepods Bacillus pumilus Methicillin-resistant Staphylococcus aureus Antagonism Bacteriocin Surfactin 



We would like to thank Dr. Matthieu Duban and Dr. Gabrielle Chataîgnée for their helpful assistance in the genome analysis and mass spectrometry analysis. We thank past and present members of the group of LOG COPEFISH team (SS) for their involvement in maintaining several cultures of copepods and algae and the Communauté d’Agglomération du Boulonnais (CAB) for supporting the implementation of a copepod-rearing pilot project (agreement Lille University-CAB).

Funding Information

This work is partly supported by CPER/FEDER Alibiotech grant (2016-2020) from la Région des Hauts-de-France. This work is a contribution to the project CPER 2014-2020 MARCO funded by the French government and the region Hauts-de-France.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

All authors of this paper have read and approved the final version submitted. The contents of this manuscript have not been copyrighted or published previously. No procedures performed in these studies have been conducted in human participants and/or animals.

Supplementary material

12602_2018_9461_MOESM1_ESM.docx (191 kb)
ESM 1 (DOCX 191 kb)


  1. 1.
    Huss HH (2004) Fresh fish quality and quality changes. In: FAO Fisheries Technical Paper No. 348. Food and Agriculture Organization of the United Nations, Rome, Italy, pp 103–109Google Scholar
  2. 2.
    Food and Agriculture Organization of the United Nations/World Health Organization (FAO/WHO) (2016) La situation mondiale des pêches et aquaculture.Google Scholar
  3. 3.
    Biji KB, Ravishankar CN, Venkateswarlu R, Mohan CO, Gopal TK (2016) Biogenic amines in seafood: a review. J Food Sci Technol 53(5):2210–2218CrossRefGoogle Scholar
  4. 4.
    Rambla-Alegre M, Miles CO, de la Iglesia P, Fernandez-Tejedor M, Jacobs S, Sioen I, Verbeke W, Samdal IA, Sandvik M, Barbosa V, Tediosi A, Madorran E, Granby K, Kotterman M, Calis T, Diogene J (2018) Occurrence of cyclic imines in European commercial seafood and consumers risk assessment. Environ Res 161:392–398CrossRefGoogle Scholar
  5. 5.
    Davis AR, Rabinson B (2003) Incidence of food borne pathogens on European fish. Food Control 12:67–71CrossRefGoogle Scholar
  6. 6.
    Hosseini H, Misaghi A (2004) Incidence of Vibrio spp. in seafood caught of south coast of Iran. Food Control 8:91–98Google Scholar
  7. 7.
    Chintagari S, Hazard N, Edwards G, Jadeja R, Janes M (2017) Risks associated with fish and seafood. Microbiol Spectr 5(1).
  8. 8.
    Elbashir S, Parveen S, Schwarz J, Rippen T, Jahncke M, DePaola A (2018) Seafood pathogens and information on antimicrobial resistance: a review. Food Microbiol 70:85–93CrossRefGoogle Scholar
  9. 9.
    Nayak SK (2010) Probiotics and immunity: a fish perspective. Fish Shellfish Immunol 29:2–14CrossRefGoogle Scholar
  10. 10.
    Wang AR, Ran C, Ringo E, Zhou ZG (2017) Progress in fish gastrointestinal microbiota research. Rev Aquacul 2017:626–640. Google Scholar
  11. 11.
    Feldhusen F (2004) The role of seafood in bacterial food borne diseases. Microbes Infect 2:1651–1660CrossRefGoogle Scholar
  12. 12.
    Edwards AM, Massey RC, Clarke SR (2012) Molecular mechanisms of Staphylococcus aureus nasopharyngeal colonization. Mol Oral Microbiol 27:1–10CrossRefGoogle Scholar
  13. 13.
    Atyah MA, Zamri-Saad M, Siti-Zahrah A (2010) First report of methicillin-resistant Staphylococcus aureus from cage-cultured tilapia (Oreochromis niloticus). Vet Microbiol 144:502–504CrossRefGoogle Scholar
  14. 14.
    Soliman MK, Ellakany HF, Gaafar AY, Elbialy AK, Zaki MS,Younes AM (2014) Epidemiology and antimicrobial activity of methicillin-resistant Staphylococcus aureus (MRSA) isolated from Nile tilapia (Oreochromis niloticus) during an outbreak in Egypt. Life Sci J 11(10):1245–1252Google Scholar
  15. 15.
    Arfatahery N, Mirshafiey A, Abedimohtasab TP, Zeinolabedinizamani M (2015) Study of the prevalence of Staphylococcus aureus in marine and farmed shrimps in Iran aiming the future development of a prophylactic vaccine. Procedia Vaccinol 9:44–49CrossRefGoogle Scholar
  16. 16.
    Pridgeon J, Klesius PH (2012) Major bacterial diseases in aquaculture and their vaccine development. CAB Reviews 2012(048)Google Scholar
  17. 17.
    Rigos G, Troisi GM (2005) Antibacterial agents in Mediterranean finfish farming: a synopsis of drug pharmacokinetics in important euryhaline fish species and possible environmental implications. Rev Fish Biol Fisher 15(1–2):53–73CrossRefGoogle Scholar
  18. 18.
    Reda RM, Ibrahim RE, Ahmed ENG, El-Bouhy ZM (2013) Effect of oxytetracycline and florfenicol as growth promoters on the health status of cultured Oreochromis niloticus. Egypt J Aquat Res 39(4):241–248CrossRefGoogle Scholar
  19. 19.
    Chanu KV, Thakuria D, Kumar S (2018) Antimicrobial peptides of buffalo and their role in host defenses. Veterinary world 11:192–200CrossRefGoogle Scholar
  20. 20.
    Gomez-Gil B, Roque A, Turnbull JF (2000) The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. Aquaculture 191:259–270CrossRefGoogle Scholar
  21. 21.
    Carlet J, Jarlier V, Harbarth S, Voss A, Goossens H, Pittet D (2012) Ready for a world without antibiotics? The pensières antibiotic resistance call to action. Antimicrob Resist Infect Control 14:1–11Google Scholar
  22. 22.
    Drider D, Rebufat S (2011) Prokarytotic antimicrobial peptides: from genes to applications. Springers Editions NY-USA 2011. 451ppGoogle Scholar
  23. 23.
    Abriouel H, Franz CM, Omar NB, Gálvez A (2011) Diversity and applications of Bacillus bacteriocins. FEMS Microbiol Rev 35:201–232CrossRefGoogle Scholar
  24. 24.
    Mondol MAM, Shin HJ, Islam MT (2013) Diversity of secondary metabolites from marine Bacillus species: chemistry and biological activity. Mar Drugs 11(8):2846–2872CrossRefGoogle Scholar
  25. 25.
    Naghmouchi K, Belguesmia Y, Baah J, Teather R, Drider D (2011) Antibacterial activity of class I and IIa bacteriocins combined with polymyxin E against resistant variants of Listeria monocytogenes and Escherichia coli. Res Microbiol 162(2):99–107CrossRefGoogle Scholar
  26. 26.
    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(6):2719–2725CrossRefGoogle Scholar
  27. 27.
    Zidour M, Chevalier M, Belguesmia Y, Cudennec B, Grard T, Drider D, Souissi S, Flahaut C (2017) Isolation and characterization of bacteria colonizing Acartia tonsa copepod eggs and displaying antagonist effects against Vibrio anguillarum, Vibrio alginolyticus and other pathogenic strains. Front Microbial 8:1919CrossRefGoogle Scholar
  28. 28.
    Drago L, Mattina R, Nicola L, Rodighiero V, De Vecchi E (2011) Macrolide resistance and in vitro selection of resistance to antibiotics in Lactobacillus isolates. J Microbiol 49:651–656. CrossRefGoogle Scholar
  29. 29.
    The European Committee on Antimicrobial Susceptibility Testing (2018) Breakpoint tables for interpretation of MICs and zone diameters, version 8.0, 2018.
  30. 30.
    Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75CrossRefGoogle Scholar
  31. 31.
    Overbeek R, Begley T, Butler RM, Choudhuri JV, Chuang HY, Cohoon M, de Crécy-Lagard V, Diaz N, Disz T, Edwards R, Fonstein M, Frank ED, Gerdes S, Glass EM, Goesmann A, Hanson A, Iwata-Reuyl D, Jensen R, Jamshidi N, Krause L, Kubal M, Larsen N, Linke B, McHardy AC, Meyer F, Neuweger H, Olsen G, Olson R, Osterman A, Portnoy V, Pusch GD, Rodionov DA, Rückert C, Steiner J, Stevens R, Thiele I, Vassieva O, Ye Y, Zagnitko O, Vonstein V (2005) The subsystems approach to genome annotation and its use in the project to annotate 1000 genomes. Nucleic Acids Res 33:5691–5702CrossRefGoogle Scholar
  32. 32.
    Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Bruccoleri R, Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema MH (2015) antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43(W1):W237–W243CrossRefGoogle Scholar
  33. 33.
    Berkeley R, Heyndrickx M, Logan N, De Vos P (2008) Applications and systematics of Bacillus and relatives. Wiley-Blackwell, Hoboken 133 ppGoogle Scholar
  34. 34.
    Liu Y, Lai Q, Dong C, Sun F, Wang L, Li G, Shao Z (2013) Phylogenetic diversity of the Bacillus pumilus group and the marine ecotype revealed by multilocus sequence analysis. PLoS One 8:e80097CrossRefGoogle Scholar
  35. 35.
    Miranda CA, Martins OB, Clementino MM (2008) Species-level identification of Bacillus strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis. Antonie Van Leeuwenhoek 93:297–304CrossRefGoogle Scholar
  36. 36.
    Ettoumi B, Raddadi N, Borin S, Daffonchio D, Boudabous A, Cherif A (2009) Diversity and phylogeny of culturable spore-forming bacilli isolated from marine sediments. J Basic Microbiol 49(Suppl 1):S13CrossRefGoogle Scholar
  37. 37.
    Bhate DS (1955) Pumilin, a new antibiotic from Bacillus pumilus. Nature 175(4462):816–817CrossRefGoogle Scholar
  38. 38.
    Brack C, Mikolasch A, Schlueter R, Otto A, Becher D, Wegner U, Albrecht D, Riedel K, Schauer F (2015) Antibacterial metabolites and bacteriolytic enzymes produced by Bacillus pumilus during bacteriolysis of Arthrobacter citreus. Mar Biotechnol (NY) 17:290–304CrossRefGoogle Scholar
  39. 39.
    Kalinovskaya NI, Kuznetsova TA, Ivanova EP, Romanenko LA, Voinov VG, Huth F, Laatsch H (2002) Characterization of surfactin-like cyclic depsipeptides synthesized by Bacillus pumilus from ascidian Halocynthia aurantium. Mar Biotechnol (NY). 4:179–188CrossRefGoogle Scholar
  40. 40.
    Aunpad R, Na-Bangchang K (2007) Pumilicin 4, a novel bacteriocin with anti-MRSA and anti-VRE activity produced by newly isolated bacteria Bacillus pumilus strainWAPB4. Curr Microbiol 55:308–313CrossRefGoogle Scholar
  41. 41.
    Ismail-Ben Ali A, El Bour M, Ktari L, Bolhuis H, Ahmed M, Boudabbous A, Stal LJ (2012) Jania rubens associated bacteria, molecular identification and antimicrobial activity. J Appl Phycol 24:525–534CrossRefGoogle Scholar
  42. 42.
    Olmos J, Paniagua-Michel J (2014) Bacillus subtilis a potential probiotic bacterium to formulate functional feeds for aquaculture. J Microb Biochem Technol 6:361–365CrossRefGoogle Scholar
  43. 43.
    Sreenivasulu P, Suman Joshi DSD, Narendra K, Venkata Rao G, Krishna Satya A (2016) Bacillus pumilus as a potential probiotic for shrimp culture. Int J Fish Aquat Stud 4:107–110Google Scholar
  44. 44.
    Prieto ML, O’Sullivan L, Tan SP, McLoughlin P, Hughes H, Gutierrez M, Lane JA, Hickey RM, Lawlor PG, Gardiner GE (2014) In vitro assessment of marine Bacillus for use as livestock probiotics. Mar Drugs 12:2422–2445CrossRefGoogle Scholar
  45. 45.
    Landy M, Warren GH, Roseman SB, Golio LG (1948) Bacillomycin, an antibiotic from Bacillus subtilis active against pathogenic fungi. Proc Soc Exp Biol Med 67:539–541CrossRefGoogle Scholar
  46. 46.
    Naruse N, Tenmyo O, Kobaru S, Kamei H, Miyaki T, Konishi M, Oki T (1990) Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity. J Antibiot (Tokyo) 43:267–280CrossRefGoogle Scholar
  47. 47.
    Grangemard I, Peypoux F, Wallach J, Das BC, Labbé H, Caille A, Genest M, Maget-Dana R, Ptak M, Bonmatin JM (1997) Lipopeptides with improved properties: structure by NMR, purification by HPLC and structure-activity relationships of new isoleucyl-rich surfactins. J Pept Sci 3:145–154CrossRefGoogle Scholar
  48. 48.
    Jemil N, Manresa A, Rabanal F, Ben Ayed H, Hmidet N, Nasri M (2017) Structural characterization and identification of cyclic lipopeptides produced by Bacillus methylotrophicus DCS1 strain. J Chromatogr B Analyt Technol Biomed Life Sci 1060:374–386CrossRefGoogle Scholar
  49. 49.
    Arima K, Kakinuma A, Tamura G (1968) Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31:488–494CrossRefGoogle Scholar
  50. 50.
    Chen H, Wang L, Su CX, Gong GH, Wang P, Yu ZL (2008) Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis. Lett Appl Microbiol 47:180–186CrossRefGoogle Scholar
  51. 51.
    Ndlovu T, Rautenbach M, Vosloo JA, Khan S, Khan W (2017) Characterisation and antimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant. AMB Express 7:108CrossRefGoogle Scholar
  52. 52.
    Perez KJ, Viana J, dos S, Lopes FC, Pereira JQ, dos Santos DM, Oliveira JS, Velho RV, Crispim SM, Nicoli JR, Brandelli A, Nardi RMD (2017) Bacillus spp. isolated from Puba as a source of biosurfactants and antimicrobial lipopeptides. Front Microbiol 8:61Google Scholar
  53. 53.
    Kawai Y, Kemperman R, Kok J, Saito T (2004) The circular bacteriocins gassericin a and circularin a. Curr Protein Pept Sci 5:393–398CrossRefGoogle Scholar
  54. 54.
    Wirawan RE, Swanson KM, Kleffmann T, Jack RW, Tagg JR (2007) Uberolysin: a novel cyclic bacteriocin produced by Streptococcus uberis. Microbiology 153:1619–1630CrossRefGoogle Scholar
  55. 55.
    Gálvez A, Maqueda M, Valdivia E, Quesada A, Montoya E (1986) Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis. Can J Microbiol 32:765–771CrossRefGoogle Scholar
  56. 56.
    Martin-Visscher LA, van Belkum MJ, Garneau-Tsodikova S, Whittal RM, Zheng J, McMullen LM, Vederas JC (2008) Isolation and characterization of carnocyclin A, a novel circular bacteriocin produced by Carnobacterium maltaromaticum UAL307. Appl Environ Microbiol 74:4756–4763CrossRefGoogle Scholar
  57. 57.
    Ananou S, Valdivia E, Martínez Bueno M, Gálvez A, Maqueda M (2004) Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain. J Appl Microbiol 97:48–56CrossRefGoogle Scholar
  58. 58.
    Arslan S, Özdemir F (2017) Molecular characterization and detection of enterotoxins, methicillin resistance genes and antimicrobial resistance of Staphylococcus aureus from fish and ground beef. Pol J Vet Sci 20:85–94CrossRefGoogle Scholar
  59. 59.
    Rios AC, Moutinho CG, Pinto FC, Del Fiol FS, Jozala A, Chaud MV, Vila MM, Teixeira JA, Balcão VM (2016) Alternatives to overcoming bacterial resistances: state-of-the-art. Microbiol Res 191:51–80CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Mahammed Zidour
    • 1
  • Yanath Belguesmia
    • 1
    Email author
  • Benoit Cudennec
    • 1
  • Thierry Grard
    • 1
  • Christophe Flahaut
    • 1
  • Sami Souissi
    • 2
  • Djamel Drider
    • 1
    Email author
  1. 1.Université de Lille, INRA, ISA, Université d’Artois, Université du Littoral-Côte d’OpaleLilleFrance
  2. 2.Université de Lille, CNRS, Université du Littoral Côte d’Opale, Laboratoire d’Océanologie et de GéosciencesWimereuxFrance

Personalised recommendations