Antonie van Leeuwenhoek (2010) 98:317–329 DOI 10.1007/s10482-010-9444-2 ORIGINAL PAPER Adhesion and biofilm formation on polystyrene by drinking water-isolated bacteria Lu´cia Chaves Simo˜es • Manuel Simo˜es • Maria Joa˜o Vieira Received: 3 February 2010 / Accepted: 6 April 2010 / Published online: 20 April 2010 Ó Springer Science+Business Media B.V. 2010 Abstract This study was performed in order to were non-adherent to PS. A. calcoaceticus, Methylo- characterize the relationship between adhesion and bacterium sp. and M. mucogenicum were weakly biofilm formation abilities of drinking water-isolated adherent. This adhesion ability was correlated with the bacteria (Acinetobacter calcoaceticus, Burkholderia biofilm formation ability when comparing with the cepacia, Methylobacterium sp., Mycobacterium mu- results of 24 h aged biofilms. Methylobacterium sp. cogenicum, Sphingomonas capsulata and Staphylo- and M. mucogenicum formed large biofilm amounts, coccus sp.). Adhesion was assessed by two distinct regardless the biofilm age. Given time, all the bacteria methods: thermodynamic prediction of adhesion formed biofilms; even those non-adherents produced potential by quantifying hydrophobicity and the free large amounts of matured (72 h aged) biofilms. The energy of adhesion; and by microtiter plate assays. overall results indicate that initial adhesion did not Biofilms were developed in microtiter plates for 24, 48 predict the ability of the tested drinking water-isolated and 72 h. Polystyrene (PS) was used as adhesion bacteria to form a mature biofilm, suggesting that other substratum. The tested bacteria had negative surface events such as phenotypic and genetic switching charge and were hydrophilic. PS had negative surface during biofilm development and the production of charge and was hydrophobic. The free energy of extracellular polymeric substances (EPS), may play a adhesion between the bacteria and PS was[ 0 mJ/m2 significant role on biofilm formation and differentia- (thermodynamic unfavorable adhesion). The thermo- tion. This understanding of the relationship between dynamic approach was inappropriate for modelling adhesion and biofilm formation is important for the adhesion of the tested drinking water bacteria, under- development of control strategies efficient in the early estimating adhesion to PS. Only three (B. cepacia, Sph. stages of biofilm development. capsulata and Staphylococcus sp.) of the six bacteria Keywords Adhesion Á Biofilm formation Á Hydrophobicity Á Opportunistic drinking water L. C. Simo˜es (&) Á M. J. Vieira bacteria Á Surface charge IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal e-mail: [email protected] Introduction M. Simo˜es LEPAE, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, Many problems in drinking water distribution sys- s/n, 4200-465 Porto, Portugal tems (DWDS) are related with the presence of 123 318 Antonie van Leeuwenhoek (2010) 98:317–329 microorganisms, including biofilm growth, nitrifica- bacteria as colloids. However, important biological tion, microbially mediated corrosion, and the occur- factors have been largely ignored in those models. rence and persistence of pathogens (Regan et al. Walker et al. (2004, 2005) have found that the 2003; Camper 2004; Emtiazi et al. 2004; Bauman heterogeneity of active sites from cell surface macro- et al. 2009). DWDS are known to harbour biofilms, molecules, such as proteins and lipopolysaccharide- even though these environments are oligotrophic and associated functional groups, controls the adhesion often contain a disinfectant. By adopting this sessile process. mode of life, biofilm-embedded microorganisms Bacterial adhesion is a complex process that is enjoy a number of advantages over their planktonic affected by many factors, including the physico- counterparts, namely the increased resistance to chemical characteristics of bacteria (hydrophobicity, antimicrobials (Gilbert et al. 2002). Microbial adhe- surface charge), the material surfaces properties sion will initiate biofilm formation, exacerbating (chemical composition, surface charge, hydrophobic- contamination of drinking water, reducing the aes- ity, roughness and texture) and by the environmental thetic quality of potable water, increasing the corro- factors (temperature, pH, time of exposure, bacterial sion rate of pipes and reducing microbiological safety concentration, chemical treatment or the presence of through increased survival of pathogens (Percival and antimicrobials and fluid flow conditions). The bio- Walker 1999; Niquette et al. 2000). The development logical properties of bacteria, such as the presence of of a biofilm is believed to occur in a sequential fimbriae and flagella, and the production of EPS also process that includes transport of microorganisms to influence the attachment to surface (An and Friedman surfaces, initial reversible/irreversible adhesion, cell– 1998). Recently, adhesion has been described as a cell communication, formation of microcolonies, two-phase process including an initial, instantaneous, extracellular polymeric substances (EPS) production and reversible physicochemical phase and a time- and biofilm maturation (Doyle 2000; Sauer and dependent and irreversible molecular and cellular Camper 2001; Bryers and Ratner 2004; Dobretsov phase (Pavithra and Doble 2008). In the first phase, et al. 2009). Accordingly, the adhesion of bacteria to planktonic bacteria move or are moved to a surface the surface is one of the prime steps in biofilm through and by the effects of physical forces, such as formation. Brownian motion, van der Waals attraction forces, Several theoretical approaches have been applied to gravitational forces, the effect of surface electrostatic describe bacteria-surface adhesion, such as the classi- charge, and hydrophobic interactions. These physical cal Derjaguin–Landau–Verwey–Overbeek (DLVO) interactions are further classified as long-range theory (Rutter and Vincent 1984; van Loosdrecht (non-specific, distances [ 150 nm) and short-range et al. 1988, 1990), the extended DLVO (XDLVO) interactions (distances \ 3 nm). Bacteria are first theory (van Oss 1989; Meinders et al. 1995), and the transported to the surface by the long-range interac- thermodynamic approach (surface Gibbs energy) tions and at closer proximity the short-range interac- (Absolom et al. 1983; Busscher et al. 1984). When a tions become more important. In the second phase, microorganism and a surface in aqueous solution enter molecular reactions between bacterial surface struc- in direct contact the water film present between the tures and substratum surfaces become predominant. interacting entities has to be removed. This is in This implies a firmer adhesion of bacteria to a surface accordance with the thermodynamic theory of adhe- by the bridging function of bacterial surface poly- sion and is expressed by the Dupre´ equation which meric structures. states that the Gibbs free energy of interaction can be The understanding of the overall biofilm formation calculated assuming that the interfaces between bac- process depends on the deep understanding of the teria/liquid medium and solid/liquid medium are main aspects regulating biofilm development, such as replaced by a bacteria/solid interface (Absolom et al. the initial adhesion. However, there is a lack of 1983). The interaction between a microbial cell and a information regarding the behavior of cells in the solid substratum is only possible from a thermody- earlier stages of biofilm formation, and its relation- namic point of view if it leads to a decrease in the ship with the biofilm development process. This surface Gibbs free energy (Absolom et al. 1983; study was performed in order to characterize the Busscher et al. 1984). Those approaches consider adhesion and biofilm formation abilities of drinking 123 Antonie van Leeuwenhoek (2010) 98:317–329 319 water-isolated bacteria to polystyrene (PS) and to Zeta potential assess the possible relationships between adhesion and biofilm results. Zeta potential experiments were performed with the cells resuspended in sterile tap water at a final concentration of 109 cells/ml. The zeta potential of Materials and methods PS was also assessed. The experiments were deter- mined using a Malvern Zetasizer instrument (Zeta- Bacteria isolation and identification sizer Nano ZS ZEN3600, Malvern). Before measuring the electrostatic values, the zeta potential The microorganisms used throughout this work were cell (DTS1060, Malvern) was rinsed three times with isolated from a model laboratory DWDS, as each suspension using a disposable syringe. All described previously by Simo˜es et al. (2006). Iden- experiments were carried out at room temperature. tification tests, by determination of 16S rDNA gene The zeta potential was derived from the electropho- sequence, were performed for putative bacteria retic mobility using the Smoluchowski approximation according to the method described by Simo˜es et al. (Hunter 1981). The experiments were performed in (2007a). triplicate and repeated three times. Planktonic bacterial growth Surface contact angles Assays were performed with 6 representative (above Bacterial lawns for contact angle measurements were 80% of the total bacterial genera isolated and prepared as described by Busscher et al. (1984). The identified) drinking water bacteria: Acinetobacter surface tension of the bacterial surfaces and of calcoaceticus,