title: On the influence of the culture conditions in bacterial antifouling bioassays and biofilm properties: Shewanella algae, a case study.
abstract: BACKGROUND
  A variety of conditions (culture media, inocula, incubation temperatures) are employed in antifouling tests with marine bacteria. Shewanella algae was selected as model organism to evaluate the effect of these parameters on: bacterial growth, biofilm formation, the activity of model antifoulants, and the development and nanomechanical properties of the biofilms.The main objectives were: 1) To highlight and quantify the effect of these conditions on relevant parameters for antifouling studies: biofilm morphology, thickness, roughness, surface coverage, elasticity and adhesion forces. 2) To establish and characterise in detail a biofilm model with a relevant marine strain.
  
  
  RESULTS
  Both the medium and the temperature significantly influenced the total cell densities and biofilm biomasses in 24-hour cultures. Likewise, the IC50 of three antifouling standards (TBTO, tralopyril and zinc pyrithione) was significantly affected by the medium and the initial cell density. Four media (Marine Broth, MB; 2% NaCl Mueller-Hinton Broth, MH2; Luria Marine Broth, LMB; and Supplemented Artificial Seawater, SASW) were selected to explore their effect on the morphological and nanomechanical properties of 24-h biofilms. Two biofilm growth patterns were observed: a clear trend to vertical development, with varying thickness and surface coverage in MB, LMB and SASW, and a horizontal, relatively thin film in MH2. The Atomic Force Microscopy analysis showed the lowest Young modulii for MB (0.16 ± 0.10 MPa), followed by SASW (0.19 ± 0.09 MPa), LMB (0.22 ± 0.13 MPa) and MH2 (0.34 ± 0.16 MPa). Adhesion forces followed an inverted trend, being higher in MB (1.33 ± 0.38 nN) and lower in MH2 (0.73 ± 0.29 nN).
  
  
  CONCLUSIONS
  All the parameters significantly affected the ability of S. algae to grow and form biofilms, as well as the activity of antifouling molecules. A detailed study has been carried out in order to establish a biofilm model for further assays. The morphology and nanomechanics of S. algae biofilms were markedly influenced by the nutritional environments in which they were developed. As strategies for biofilm formation inhibition and biofilm detachment are of particular interest in antifouling research, the present findings also highlight the need for a careful selection of the assay conditions.
authors.affiliation: Institute for Bio-Organic Chemistry "Antonio González", Center for Biomedical Research of the Canary Islands (CIBICAN), University of La Laguna, Avenida Astrofísico Francisco Sánchez 2, La Laguna, Tenerife 38206, Spain. ajmartinr@ull.es.
authors.forename: Alberto J
authors.initials: AJ
authors.lastname: Martín-Rodríguez
authors.forename: Alejandro
authors.initials: A
authors.lastname: González-Orive
authors.forename: Alberto
authors.initials: A
authors.lastname: Hernández-Creus
authors.forename: Araceli
authors.initials: A
authors.lastname: Morales
authors.forename: Roberto
authors.initials: R
authors.lastname: Dorta-Guerra
authors.forename: Manuel
authors.initials: M
authors.lastname: Norte
authors.forename: Víctor S
authors.initials: VS
authors.lastname: Martín
authors.forename: José J
authors.initials: JJ
authors.lastname: Fernández
mesh_terms: D018441:Biofilms / Q000187:drug effects / Q000254:growth & development* / Q000528:radiation effects; D003470:Culture Media / Q000737:chemistry; D004202:Disinfectants / Q000378:metabolism*; D009942:Organometallic Compounds / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D011758:Pyrroles / Q000378:metabolism; D020592:Shewanella / Q000187:drug effects / Q000254:growth & development / Q000502:physiology* / Q000528:radiation effects; D013696:Temperature; D014220:Trialkyltin Compounds / Q000378:metabolism
chemical_list: D003470:Culture Media; D004202:Disinfectants; D009942:Organometallic Compounds; D011725:Pyridines; D011758:Pyrroles; D014220:Trialkyltin Compounds; C005961:bis(tri-n-butyltin)oxide; C000595640:4-bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile; C010423:pyrithione zinc
references.citation: J Vis Exp. 2011 Jan 25;(47):
references.pmid: 21304472
references.citation: Environ Microbiol. 2009 Jul;11(7):1792-802
references.pmid: 19508552
references.citation: Biofouling. 2009;25(4):297-311
references.pmid: 19191083
references.citation: Adv Biochem Eng Biotechnol. 2005;97:29-62
references.pmid: 16261805
references.citation: FEMS Microbiol Ecol. 2011 Aug;77(2):333-46
references.pmid: 21507025
references.citation: Biofouling. 2006;22(3-4):133-44
references.pmid: 17290858
references.citation: J Appl Microbiol. 2002;93(3):438-47
references.pmid: 12174042
references.citation: Lett Appl Microbiol. 2002;34(6):402-6
references.pmid: 12028419
references.citation: Clin Infect Dis. 1995 May;20(5):1327-32
references.pmid: 7620019
references.citation: Antimicrob Agents Chemother. 2007 Jan;51(1):394-6
references.pmid: 17060527
references.citation: Clin Infect Dis. 2007 Jan 15;44(2):280-6
references.pmid: 17173232
references.citation: Microbiology (Reading). 2005 Sep;151(Pt 9):2885-2897
references.pmid: 16151201
references.citation: Microbiol Res. 2006;161(4):355-61
references.pmid: 16517137
references.citation: Int J Food Microbiol. 2008 Aug 15;126(1-2):140-52
references.pmid: 18573556
references.citation: Fish Physiol Biochem. 2014 Feb;40(1):295-309
references.pmid: 23933744
references.citation: Langmuir. 2012 Nov 20;28(46):16060-71
references.pmid: 23113608
references.citation: J Clin Microbiol. 2003 May;41(5):2240-1
references.pmid: 12734291
references.citation: J Nat Prod. 2008 Nov;71(11):1806-11
references.pmid: 18980381
references.citation: Mol Microbiol. 1998 May;28(3):449-61
references.pmid: 9632250
references.citation: Microbiology (Reading). 2000 Oct;146 ( Pt 10):2395-2407
references.pmid: 11021916
references.citation: Proc Natl Acad Sci U S A. 2010 Oct 19;107(42):18127-31
references.pmid: 20937892
references.citation: Appl Environ Microbiol. 2011 Aug;77(15):5352-60
references.pmid: 21642407
references.citation: Molecules. 2007 May 15;12(5):1022-34
references.pmid: 17873837
references.citation: J Med Microbiol. 2011 Sep;60(Pt 9):1387-1390
references.pmid: 21546561
references.citation: Int Microbiol. 2012 Mar;15(1):17-32
references.pmid: 22837149
references.citation: Biofouling. 2007;23(3-4):287-94
references.pmid: 17653938
references.citation: Nat Protoc. 2010 Jul;5(7):1236-54
references.pmid: 20595953
references.citation: J Bacteriol. 2004 Feb;186(3):889-93
references.pmid: 14729720
references.citation: Curr Protoc Pharmacol. 2010 Sep;Chapter 13:Unit 13A.8
references.pmid: 22294365
references.citation: Appl Environ Microbiol. 2008 Jun;74(11):3323-7
references.pmid: 18378645
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14357-62
references.pmid: 14614140
references.citation: Appl Environ Microbiol. 1999 Sep;65(9):3896-900
references.pmid: 10473392
references.citation: Rev Sci Instrum. 2007 Jan;78(1):013705
references.pmid: 17503926
references.citation: Chem Rev. 2012 Aug 8;112(8):4347-90
references.pmid: 22578131
references.citation: J Bacteriol. 2005 Jun;187(11):3864-8
references.pmid: 15901713
references.citation: Appl Environ Microbiol. 2005 Jul;71(7):3840-7
references.pmid: 16000796
references.citation: Front Microbiol. 2012 Aug 23;3:292
references.pmid: 22936927
references.citation: PLoS One. 2011 Apr 29;6(4):e18887
references.pmid: 21559483
references.citation: Environ Microbiol. 2003 May;5(5):338-49
references.pmid: 12713460
references.citation: Annu Rev Microbiol. 2007;61:237-58
references.pmid: 18035608
references.citation: Biotechnol Bioeng. 1995 Mar 20;45(6):503-10
references.pmid: 18623250
references.citation: Clin Microbiol Infect. 2005 May;11(5):347-52
references.pmid: 15819859
references.citation: Fish Shellfish Immunol. 2013 Jul;35(1):86-91
references.pmid: 23602848
references.citation: Langmuir. 2012 May 15;28(19):7461-9
references.pmid: 22515332
references.citation: Am J Trop Med Hyg. 2013 Jul;89(1):151-6
references.pmid: 23690548
references.citation: J Phycol. 2012 Feb;48(1):174-85
references.pmid: 27009662
references.citation: Ultramicroscopy. 2007 Oct;107(10-11):869-74
references.pmid: 17544218
references.citation: Emerg Infect Dis. 2013 Jun;19(6):1019-21
references.pmid: 23735117
references.citation: Langmuir. 2012 Dec 11;28(49):16738-44
references.pmid: 23198968
references.citation: J Struct Biol. 2012 Apr;178(1):1-7
references.pmid: 22369932
references.citation: BMC Microbiol. 2010 Nov 16;10:291
references.pmid: 21080927
references.citation: Nat Methods. 2013 Sep;10(9):847-54
references.pmid: 23985731
references.citation: Biofouling. 2013;29(3):247-59
references.pmid: 23452123
references.citation: Appl Environ Microbiol. 2010 Jun;76(12):3842-9
references.pmid: 20418444
references.citation: Photochem Photobiol. 2005 Jul-Aug;81(4):960-9
references.pmid: 15884971
references.citation: Int Microbiol. 2012 Sep;15(3):131-9
references.pmid: 23847817
references.citation: Biofouling. 2008;24(3):201-8
references.pmid: 18373284
references.citation: Environ Microbiol. 2014 Feb;16(2):445-53
references.pmid: 23879807
references.citation: Nat Methods. 2012 Jul;9(7):671-5
references.pmid: 22930834
references.citation: Appl Environ Microbiol. 2005 Dec;71(12):8982-6
references.pmid: 16332905
references.citation: Langmuir. 2007 Nov 20;23(24):11977-9
references.pmid: 17941657
references.citation: Environ Microbiol Rep. 2010 Feb;2(1):140-4
references.pmid: 23766009