Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Abstract

BACKGROUND: Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Psychrobacter celer, Psychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

METHODS: Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

RESULTS: Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Psychrobacter celer, Psychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

CONCLUSION: Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

Key words： biofilm; MTP assay; extracellular polymeric substances; 16S rRNA gene sequencing; Psychrobacter; Kocuria

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgements

The authors gratefully acknowledge Vellore Institute of Technology, Vellore for the support in extending the necessary facilities.

References

1 Aiassa V, Barnes A I, Albesa I (2010). Resistance to ciprofloxacin by enhancement of antioxidant defenses in biofilm and planktonic Proteus mirabilis. Biochem Biophys Res Commun, 393(1): 84–88

2 An Y H, Friedman R J (2000). Handbook of bacterial adhesion: principles, methods, and applications. Totowa, N.J. Humana Press.

3 Anwar H, Strap J L, Costerton J W (1992). Eradication of biofilm cells of Staphylococcus aureus with tobramycin and cephalexin. Can J Microbiol, 38(7): 618–625

4 Beech I B, Sunner J (2004). Biocorrosion: towards understanding interactions between biofilms and metals. Curr Opin Biotechnol, 15(3): 181–186

5 Beveridge T J, Makin S A, Kadurugamuwa J L, Li Z (1997). Interactions between biofilms and the environment. FEMS Microbiol Rev, 20(3-4): 291–303

6 Chaudhary A, Gupta L K, Gupta J K, Banerjee U C (1997). Studies on slime-forming organisms of a paper mill-slime production and its control. J Ind Microbiol Biotechnol, 18(5): 348–352

7 Costerton J W, Lewandowski Z, Caldwell D E, Korber D R, Lappin-Scott H M (1995). Microbial biofilms. Annu Rev Microbiol, 49(1): 711–745

8 Costerton J W, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G (1994). Biofilms, the customized microniche. J Bacteriol, 176(8): 2137–2142

9 Cottingham K L, Chiavelli D A, Taylor R K (2003). Environmental microbe and human pathogen: the ecology and microbiology of Vibrio cholerae. Front Ecol Environ, 1(2): 80–86

10 Dang H Y, Chen R P, Wang L, Shao S D, Dai L Q, Ye Y, Guo L Z, Huang G Q, Klotz M G (2011). Molecular characterization of putative bio corroding microbiota with a novel niche detection of Epsilon- and Zetaproteobacteria in Pacific Ocean coastal seawaters. Environ Microbiol, 13(11): 3059–3074

11 Dang H Y, Lovell C R (2000). Bacterial primary colonization and early succession on surfaces in marine waters as determined by amplified rRNA gene restriction analysis and sequence analysis of 16S rRNA genes. Appl Environ Microbiol, 66(2): 467–475

12 Davey M E, O’Toole G A (2000). Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev, 64(4): 847–867

13 Decho A W (2000). Microbial biofilms in intertidal systems: an overview. Cont Shelf Res, 20(10-11): 1257–1273

14 Eighmyi T T, Maratea D, Bishop P L (1983). Electron microscopic examination of wastewater biofilm formation and structured components. Appl Environ Microbiol, 45: 1921–1931

15 Feron D, Dupont I (1995). Marine biofilms on stainless steels: effects on the corrosion behaviour in: Developments in marine corrosion Ed. Campbell, S. A., Campbell, N., Walsh F. C. Publishers-Royal Society of Chemistry, Cambridge.

16 Flemming H C, Neu T R, Wozniak D J (2007). The EPS matrix: the “house of biofilm cells. J Bacteriol, 189(22): 7945–7947

17 Hall-Stoodley L, Costerton J W, Stoodley P (2004). Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol, 2(2): 95–108

18 Huq A, Whitehouse C A, Grim C J, Alam M, Colwell R R (2008). Biofilms in water, its role and impact in human disease transmission. Curr Opin Biotechnol, 19(3): 244–247

19 Jordjevic D, Wiedmann M, McLandsborough L A (2002). Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl Environ Microbiol, 68(6): 2950–2958

20 Li Y H, Tang N, Aspiras M B, Lau P C, Lee J H, Ellen R P, Cvitkovitch D G (2002). A quorum-sensing signaling system essential for genetic competence in Streptococcus mutans is involved in biofilm formation. J Bacteriol, 184(10): 2699–2708

21 Manca M C, Lama L, Improta R, Esposito E, Gambacorta A, Nicolaus B (1996). Chemical composition of two exopolysaccharides from Bacillus thermoantarcticus. Appl Environ Microbiol, 62: 3265–3269

22 Newman D K, Banfield J F (2002). Geomicrobiology: how molecular-scale interactions underpin biogeochemical systems. Science, 296(5570): 1071–1077

23 Pandit S, Kim J E, Jung K H, Chang K W, Jeon J G (2011). Effect of sodium fluoride on the virulence factors and composition of Streptococcus mutans biofilms. Arch Oral Biol, 56(7): 643–649

24 Pitt T L, Malnick H, Shah J, Chattaway M A, Keys C J, Cooke F J, Shah H N (2007). Characterisation of Exiguobacterium aurantiacum isolates from blood cultures of six patients. Clin Microbiol Infect, 13(9): 946–948

25 Priest F G (1993). Systematics and ecology of Bacillus. In: Losick R, Hoch J A, Sonenshein A L, editors. Bacillus subtilis and other gram-positive bacteria: biochemistry physiology and molecular genetics. Washington (DC): ASM Press. p. 3–16

26 Sakurai K, Yoshikawa H (2012). Isolation and identification of bacteria able to form biofilms from deep subsurface environments. J Nucl Sci Technol, 49(3): 287–292

27 Salta M, Wharton J A, Blache Y, Stokes K R, Briand J F (2013). Marine biofilms on artificial surfaces: structure and dynamics. Environ Microbiol, 15: 2879–2893

28 Sass A M, Sass H, Coolen M J L, Cypionka H, Overmann J (2001). Microbial communities in the chemocline of a hypersaline deep-sea basin (Urania Basin, Mediterranean Sea). Appl Environ Microbiol, 67(12): 5392–5402

29 Sollankorva S P, Neubauer P, Azeredo J (2008). Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A. BMC Biotechnol, 8: 1–2

30 Sonak S, Bhosle N (1995). Observations on biofilm bacteria isolated from aluminum panels immersed in estuarine waters. Biofouling, 8(3): 243–254

31 Steinberg P D, De Nys R, Kjelleberg S (2002). Chemical cues for surface colonization. J Chem Ecol, 28(10): 1935–1951

32 Trevors J T (2011). Hypothesized origin of microbial life in a prebiotic gel and the transition to a living biofilm and microbial mats. C R Biol, 334(4): 269–272

33 Verhoef R, Waard P, de, Schols H A (2002). Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine. Carbohydr Res, 337(20): 1821–1831

34 Watnick P, Kolter R (2000). Biofilm, city of microbes. J Bacteriol, 182(10): 2675–2679

35 Wrangstadh M, Szewzyk U, Ostling J S, Kjelleberg S (1990). Starvation-specific formation of a peripheral exopolysaccharide by a marine Pseudomonas sp., strain S9. Appl Environ Microbiol, 56: 2065–2072

36 Xu M, Wang P, Wang F, Xiao X (2005). Microbial diversity at a deep-sea station of the Pacific nodule province. Biodivers Conserv, 14(14): 3363–3380

37 Yang S, Sugawara S, Monodane T, Nishijima M, Adachi Y, Akashi S, Miyake K, Hase S, Takada H (2001). Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner. Infect Immun, 69(4): 2025–2030