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PLOS ONE  2012 

Impairment of the Bacterial Biofilm Stability by Triclosan

DOI: 10.1371/journal.pone.0031183

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The accumulation of the widely-used antibacterial and antifungal compound triclosan (TCS) in freshwaters raises concerns about the impact of this harmful chemical on the biofilms that are the dominant life style of microorganisms in aquatic systems. However, investigations to-date rarely go beyond effects at the cellular, physiological or morphological level. The present paper focuses on bacterial biofilms addressing the possible chemical impairment of their functionality, while also examining their substratum stabilization potential as one example of an important ecosystem service. The development of a bacterial assemblage of natural composition – isolated from sediments of the Eden Estuary (Scotland, UK) – on non-cohesive glass beads (<63 μm) and exposed to a range of triclosan concentrations (control, 2 – 100 μg L?1) was monitored over time by Magnetic Particle Induction (MagPI). In parallel, bacterial cell numbers, division rate, community composition (DGGE) and EPS (extracellular polymeric substances: carbohydrates and proteins) secretion were determined. While the triclosan exposure did not prevent bacterial settlement, biofilm development was increasingly inhibited by increasing TCS levels. The surface binding capacity (MagPI) of the assemblages was positively correlated to the microbial secreted EPS matrix. The EPS concentrations and composition (quantity and quality) were closely linked to bacterial growth, which was affected by enhanced TCS exposure. Furthermore, TCS induced significant changes in bacterial community composition as well as a significant decrease in bacterial diversity. The impairment of the stabilization potential of bacterial biofilm under even low, environmentally relevant TCS levels is of concern since the resistance of sediments to erosive forces has large implications for the dynamics of sediments and associated pollutant dispersal. In addition, the surface adhesive capacity of the biofilm acts as a sensitive measure of ecosystem effects.


[1]  Singer H, Muller S, Tixier C, Pillonel L (2002) Triclosan: Occurrence and fate of a widely used biocide in the aquatic environment: Field measurements in wastewater treatment plants, surface waters, and lake sediments. Environmental Science & Technology 36: 4998–5004.
[2]  Adolfsson-Erici M, Pettersson M, Parkkonen J, Sturve J (2002) Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden. Chemosphere 46: 1485–1489.
[3]  Heidler J, Halden RU (2007) Mass balance assessment of triclosan removal during conventional sewage treatment. Chemosphere 66: 362–369.
[4]  Chalew TEA, Halden RU (2009) Environmental exposure of aquatic and terrestrial biota to triclosan and triclocarban. Journal of the American Water Resources Association 45: 4–13.
[5]  Mezcua M, Gomes MJ, Ferrer I, Aguera A, Hernando MD, et al. (2004) Evidence of 2,7/2,8-dibenzodichloro-p-dioxin a photodegradation product of triclosan in water and wastewater samples; 2004; University A Coruna, Spain. Analytica Chimica Acta. pp. 241–247.
[6]  McAvoy DC, Schatowitz B, Jacob M, Hauk A, Eckhoff WS (2002) Measurement of triclosan in wastewater treatment systems. Environmental Toxicology and Chemistry 21: 1323–1329.
[7]  Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, et al. (2002) Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: A national reconnaissance. Environmental Science & Technology 36: 1202–1211.
[8]  Halden RU, Paull DH (2005) Co-occurrence of triclocarban and triclosan in US water resources. Environmental Science & Technology 39: 1420–1426.
[9]  Lindstrom A, Buerge IJ, Poiger T, Bergqvist PA, Muller MD, et al. (2002) Occurrence and environmental behavior of the bactericide triclosan and its methyl derivative in surface waters and in wastewater. Environmental Science & Technology 36: 2322–2329.
[10]  Aguera A, Fernandez-Alba AR, Piedra L, Mezcua M, Gomez MJ (2003) Evaluation of triclosan and biphenylol in marine sediments and urban wastewaters by pressurized liquid extraction and solid phase extraction followed by gas chromatography mass spectrometry and liquid chromatography mass spectrometry. Analytica Chimica Acta 480: 193–205.
[11]  Okumura T, Nishikawa Y (1996) Gas chromatography mass spectrometry determination of triclosans in water, sediment and fish samples via methylation with diazomethane. Analytica Chimica Acta 325: 175–184.
[12]  Heath RJ, Rubin JR, Holland DR, Zhang EL, Snow ME, et al. (1999) Mechanism of triclosan inhibition of bacterial fatty acid synthesis. Journal of Biological Chemistry 274: 11110–11114.
[13]  Levy CW, Roujeinikova A, Sedelnikova S, Baker PJ, Stuitje AR, et al. (1999) Molecular basis of triclosan activity. Nature 398: 383–384.
[14]  McMurry LM, Oethinger M, Levy SB (1998) Triclosan targets lipid synthesis. Nature 394: 531–532.
[15]  Escalada MG, Harwood JL, Maillard JY, Ochs D (2005) Triclosan inhibition of fatty acid synthesis and its effect on growth of Escherichia coli and Pseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy 55: 879–882.
[16]  Tabak M, Scher K, Hartog E, Romling U, Matthews KR, et al. (2007) Effect of triclosan on Salmonella typhimurium at different growth stages and in biofilms. Fems Microbiology Letters 267: 200–206.
[17]  Schweizer HP (2001) Triclosan: a widely used biocide and its link to antibiotics. Fems Microbiology Letters 202: 1–7.
[18]  Yazdankhah SP, Scheie AA, Hoiby EA, Lunestad BT, Heir E, et al. (2006) Triclosan and antimicrobial resistance in bacteria: An overview. Microbial Drug Resistance-Mechanisms Epidemiology and Disease 12: 83–90.
[19]  McMurry LM, McDermott PF, Levy SB (1999) Genetic evidence that InhA of Mycobacterium smegmatis is a target for triclosan. Antimicrobial Agents and Chemotherapy 43: 711–713.
[20]  McMurry LM, Oethinger M, Levy SB (1998) Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli. Fems Microbiology Letters 166: 305–309.
[21]  Russell AD (2004) Whither triclosan? Journal of Antimicrobial Chemotherapy 53: 693–695.
[22]  Lawrence JR, Zhu B, Swerhone GDW, Roy J, Wassenaar LI, et al. (2009) Comparative microscale analysis of the effects of triclosan and triclocarban on the structure and function of river biofilm communities. Science of the Total Environment 407: 3307–3316.
[23]  Escalada MG, Russell AD, Maillard JY, Ochs D (2005) Triclosan-bacteria interactions: single or multiple target sites? Letters in Applied Microbiology 41: 476–481.
[24]  Villalain J, Mateo CR, Aranda FJ, Shapiro S, Micol V (2001) Membranotropic effects of the antibacterial agent triclosan. Archives of Biochemistry and Biophysics 390: 128–136.
[25]  DeLorenzo ME, Keller JM, Arthur CD, Finnegan MC, Harper HE, et al. (2008) Toxicity of the antimicrobial compound triclosan and formation of the metabolite methyl-triclosan in estuarine systems. Environmental Toxicology 23: 224–232.
[26]  Farre M, Asperger D, Kantiani L, Gonzalez S, Petrovic M, et al. (2008) Assessment of the acute toxicity of triclosan and methyl triclosan in wastwater based on the bioluminescence inhibition of Vibrio fischeri. Analytical and Bioanalytical Chemistry 390: 1999–2007.
[27]  Ricart M, Guasch H, Barcelo D, Brix R, Conceicao MH, et al. (2010) Primary and complex stressors in polluted mediterranean rivers: Pesticide effects on biological communities. Journal of Hydrology 383: 52–61.
[28]  Wilson BA, Smith VH, Denoyelles F, Larive CK (2003) Effects of three pharmaceutical and personal care products on natural freshwater algal assemblages. Environmental Science & Technology 37: 1713–1719.
[29]  Orvos DR, Versteeg DJ, Inauen J, Capdevielle M, Rothenstein A, et al. (2002) Aquatic toxicity of triclosan. Environmental Toxicology and Chemistry 21: 1338–1349.
[30]  Gerbersdorf SU, Hollert H, Brinkmann M, Wieprecht S, Schüttrumpf H, et al. (2011) Anthropogenic pollutants affect ecosystem services of freshwater sediments: the need for a “triad plus x” approach. Springer: Journal of Soils and Sediments. pp. 1099–1114.
[31]  Underwood GJC, Paterson DM (2003) The importance of extracellular carbohydrate production by marine epipelic diatoms. Adv Bot Res 40: 183–240.
[32]  Gerbersdorf SU, Bittner R, Lubarsky H, Manz W, Paterson DM (2009) Microbial assemblages as ecosystem engineers of sediment stability. Journal of Soils and Sediments 9: 640–652.
[33]  Lubarsky HV, Hubas C, Chocholek M, Larson F, Manz W, et al. (2010) The stabilization potential of individual and mixed assemblages of natural bacteria and microalgae. PLOS ONE 5: e13794.
[34]  Gerbersdorf SU, Manz W, Paterson DM (2008) The engineering potential of natural benthic bacterial assemblages in terms of the erosion resistance of sediments. Fems Microbiology Ecology 66: 282–294.
[35]  Priester JH, Olson SG, Webb SM, Neu MP, Hersman LE, et al. (2006) Enhanced exopolymer production and chromium stabilization in Pseudomonas putida unsaturated biofilms. Applied and Environmental Microbiology 72: 1988–1996.
[36]  Onbasli D, Aslim B (2009) Effects of some organic pollutants on the exopolysaccharides (EPSs) produced by some Pseudomonas spp. strains. Journal of Hazardous Materials 168: 64–67.
[37]  Larson F, Lubarsky H, Paterson DM, Gerbersdorf SU (2009) Surface adhesion measurements in aquatic biofilms using magnetic particle induction: MagPI. Limnology and Oceanography: Methods 7: 490–497.
[38]  Marzorati M, Wittebolle L, Boon N, Daffonchio D, Verstraete W (2008) How to get more out of molecular fingerprints: practical tools for microbial ecology. Environmental Microbiology 10: 1571–1581.
[39]  Pearson K (1901) On lines and planes of closest fit to systems of points in space. Philosophical Magazine 2: 559–572.
[40]  Shannon CE (1997) The mathematical theory of communication (Reprinted). M D Computing 14: 306–317.
[41]  Simpson EH (1949) Measurement of diversity. Nature 163: 688.
[42]  Schreiber F, Szewzyk U (2008) Environmentally relevant concentrations of pharmaceuticals influence the initial adhesion of bacteria. Aquatic Toxicology 87: 227–233.
[43]  Johnson DR, Czechowska K, Chevre N, van der Meer JR (2009) Toxicity of triclosan, penconazole and metalaxyl on Caulobacter crescentus and a freshwater microbial community as assessed by flow cytometry. Environmental Microbiology 11: 1682–1691.
[44]  Stal LJ (2003) Microphytobenthos, their extracellular polymeric substances, and the morphogenesis of intertidal sediments. Geomicrobiology Journal 20: 463–478.
[45]  Flemming HC, Wingender J (2001) Relevance of microbial extracellular polymeric substances (EPSs) – Part I: Structural and ecological aspects. Water Science and Technology 43: 1–8.
[46]  Dobretsov S, Dahms HU, Huang YL, Wahl M, Qian PY (2007) The effect of quorum-sensing blockers on the formation of marine microbial communities and larval attachment. Fems Microbiology Ecology 60: 177–188.
[47]  Czaczyk K, Myszka K (2007) Biosynthesis of extracellular polymeric substances (EPS) and its role in microbial biofilm formation. Polish Journal of Environmental Studies 16: 799–806.
[48]  Jain A, Nishad KK, Bhosle NB (2007) Effects of DNP on the cell surface properties of marine bacteria and its implication for adhesion to surfaces. Biofouling 23: 171–177.
[49]  Pennisi E (2002) Materials science – Biology reveals new ways to hold on tight. Science 296: 250–251.
[50]  Decho AW (1990) Microbial exopolymer secretions in ocean environments-their role(s) in food webs and marine processes. Oceanography and Marine Biology 28: 73–153.
[51]  Solan M, Batty P, Bulling MT, Godbold JA (2008) How biodiversity affects ecosystem processes: implications for ecological revolutions and benthic ecosystem function. Aquatic Biology 2: 289–301.
[52]  Brusetti L, Borin S, Mora D, Rizzi A, Raddadi N, et al. (2006) Usefulness of length heterogeneity-PCR for monitoring lactic acid bacteria succession during maize ensiling. Fems Microbiology Ecology 56: 154–164.
[53]  Wittebolle L, Verstraete W, Boon N (2009) The inoculum effect on the ammonia-oxidizing bacterial communities in parallel sequential batch reactors. Water Research 43: 4149–4158.
[54]  Lawrence JR, Swerhone GDW, Topp E, Korber DR, Neu TR, et al. (2007) Structural and functional responses of river biofilm communities to the nonsteroidal anti-inflammatory diclofenac. Environmental Toxicology and Chemistry 26: 573–582.
[55]  Brummer IHM, Felske A, Wagner-Dobler I (2003) Diversity and seasonal variability of beta-proteobacteria in biofilms of polluted rivers: Analysis by temperature gradient gel electrophoresis and cloning. Applied and Environmental Microbiology 69: 4463–4473.
[56]  Fernandez AS, Hashsham SA, Dollhopf SL, Raskin L, Glagoleva O, et al. (2000) Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose. Applied and Environmental Microbiology 66: 4058–4067.
[57]  Hubas C, Lamy D, Artigas LF, Davoult D (2007) Seasonal variability of intertidal bacterial metabolism and growth efficiency in an exposed sandy beach during low tide Marine Biology 151: 41–52.
[58]  Hubas C, Artigas LF, Davoult D (2007) Role of the bacterial community in the annual benthic metabolism of two contrasted temperate intertidal sites (Roscoff Aber Bay, France). Marine Ecology-Progress Series 344: 39–48.
[59]  Fuhrman JA, Azam F (1982) Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters-evaluation and field result. Marine Biology 66: 109–120.
[60]  Garet MJ, Moriarty DJW (1996) Acid extraction of tritium label from bacterial DNA in clay sediment. Journal of Microbiological Methods 25: 1–4.
[61]  Cho BC, Azam F (1990) Biogeochemical significance of bacterial biomass in the oceans euphotic zone. Marine Ecology-Progress Series 63: 253–259.
[62]  Lee S, Fuhrman JA (1987) Relationships between biovolume and biomass of naturally derived marine bacterioplankton. Applied and Environmental Microbiology 53: 1298–1303.
[63]  Muyzer G, Dewaal EC, Uitterlinden AG (1993) Profiling of complex microbial-populations by denaturing gradient gel-electrophoresis analysis of polymerase chain reaction-amplified genes-coding for 16S ribosomal-RNA. Applied and Environmental Microbiology 59: 695–700.
[64]  Muyzer G, Ramsing NB (1995) Molecular methods to study the organization of microbial communities. Water Science and Technology 32: 1–9.
[65]  Bassam BJ, Caetanoanolles G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry 196: 80–83.
[66]  Ludwig W, Strunk O, Westram R, Richter L, Meier H, et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Research 32: 1363–1371.
[67]  Peplies J, Kottmann R, Ludwig W, Glockner FO (2008) A standard operating procedure for phylogenetic inference (SOPPI) using (rRNA) marker genes. Systematic and Applied Microbiology 31: 251–257.
[68]  Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig WG, et al. (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Research 35: 7188–7196.
[69]  Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009) Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities. Applied and Environmental Microbiology 75: 7537–7541.
[70]  Dubois M, Gilles KA, Hemilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28: 380–356.
[71]  Raunkjaer K, Hvitvedjacobsen T, Nielsen PH (1994) Measurement of pools of protein, carbohydrate and lipid in domestic waste- water. Water Research 28: 251–262.
[72]  Oksanen J, Kindt R, Legendre P, O’Hara B, Simpson GL, et al. (2009) vegan: Community Ecology Package. R package version 117–4. Available:
[73]  Dray S, Dufour AB (2007) The ade4 package: Implementing the duality diagram for ecologists. Journal of Statistical Software 22: 1–20.


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