Journal of Basic Microbiology 2009, 49, 363 – 370 363 © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com Research Paper The role of extracellular polymers on Staphylococcus epidermidis biofilm biomass and metabolic activity Cláudia Sousa, Pilar Teixeira and Rosário Oliveira IBB – Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica, Universidade do Minho, Campus de Gualtar, Braga, Portugal Staphylococcus epidermidis is now well established as a major nosocomial pathogen, associated with indwelling medical devices. Its major virulence factor is related with the ability to adhere to indwelling medical devices and form biofilms. In this study, the biofilm matrix of four S. epidermidis clinical isolates was extracted and the polysaccharides and proteins content was quantified. The results were correlated with the total biofilm biomass (determined by crystal violet assay) and cellular metabolic activity (evaluated with XTT reduction assay). According to the results, the exopolymers studied play an important role not only on structure and biofilm biomass but also on cellular activity. Thus, the strain forming biofilms with the highest level of polysaccharides (S. epidermidis 1457) also formed thicker biofilms but with the lowest metabolic activity. The protein concentration also varied among strains, with the biofilm matrix of S. epidermidis 9142 presenting a higher concentration of proteins comparing to the remaining strains. This fact indicates the different levels of importance that matrix proteins can hold on biofilm composition among strains albeit overall, it is suggested that extracellular protein production it is not a determinative factor for biofilm total biomass. Keywords: Biofilm / Extracellular matrix / Metabolic activity / Polysaccharides / Proteins Received: June 11, 2008; accepted: October 28, 2008 DOI 10.1002/jobm.200800196 Introduction * Staphylococcus epidermidis is a coagulase-negative Staphy- lococcus (CNS) that has emerged in the last years as one of the most important nosocomial and opportunistic pathogens. It is most commonly associated with infec- tions originating from indwelling medical devices such as catheters and prostheses [1]. This is directly related with its capacity to adhere and form thick and multi- layered biofilms on abiotic surfaces, constituted of colonizing bacteria and a self-excreted amorphous exopolymeric matrix in which they are embedded [2]. Generally, the S. epidermidis biofilm matrix comprises several extracellular polymeric substances (EPS) such as polysaccharides [3, 4], proteins [5 – 7], considerable amounts of extracellular teichoic acids [4, 8] and also Correspondence: Dr. Cláudia Sousa, IBB - Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica, Universidade do Min- ho, Campus de Gualtar, 4710-057 Braga, Portugal E-mail: claudiasousa@deb.uminho.pt Phone: +351-253-604409 Fax: +351-253-678986 extracellular DNA [9]. All these compounds seem to play an important role in the biofilm formation process and maintenance. The polysaccharide intercellular adhesin (PIA), a polymer of N-acetyl glucosamine has been described as crucial for the process of cell-to-cell adhesion and biofilm accumulation [10] and as an es- sential component of the extracellular matrix [11]. Dur- ing the process of proliferation and accumulation as multilayered cell clusters, S. epidermidis also secretes a few exoenzymes, aiming to help in the invasiveness of host tissues and host defenses [12]. EPS provide me- chanical stability to such structures, forming a three- dimensional, gel-like, highly hydrated and negatively charged environment in which the cells are immobi- lized [13, 14]. The extracellular matrix is extremely important for intercellular connection during surface colonization [15] and protection against the host im- mune system and resistance to antibiotics [16]. Besides, the matrix-enclosed microcolonies are separated by water channels that provide a nutrient flow system within the biofilm [17]. Moreover, depending on the