Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation

Abstract

Biofilm formation is thought to result from the concerted action of primary attachment to a specific surface and accumulation in multilayered cell clusters. Here we describe the isolation and characterization of transposon (Tn917) mutants of Staphylococcus epidermidis O-47 which were biofilm negative in the polystyrene microtiter plate assay. Among 5,000 Tn917 insertion mutants, 4 biofilm-negative mutants were isolated. Each mutant carried one copy of Tn917. The mutants were divided into two phenotypic classes: class A (mut1 and mut1a) and class B (mut2 and mut2a). Mutants of phenotypic class A lacked four cell surface proteins, were less hydrophobic, and were affected in primary attachment to polystyrene, but were still able to form multilayered cell clusters. They were able to form a biofilm on a glass surface, a trait that was even more pronounced than in the wild-type strain O-47. Loss of several surface proteins might have led to the reduced surface hydrophobicity by unmasking hydrophilic structures, thus favoring primary attachment to a glass surface and leading to subsequent biofilm formation. Mutants of phenotype class B were able to attach to polystyrene but were unable to form multilayered cell clusters, had unchanged cell surface proteins and hydrophobicity, and were unable to form a biofilm on a glass surface. mut1 and mut2 could be complemented by wild-type DNA fragments containing the Tn917 insertion sites of mut1 and mut2, respectively. The complemented biofilmpositive clone mut1(pRC20) produced a 60-kDa protein which is postulated to function as the adhesin for binding to plastic. The traits of binding to polystyrene and the ability to form multilayered cell clusters are phenotypically and genetically distinct.

Influence of batch or fed-batch growth on Staphylococcus epidermidis biofilm formation

“…These findings indicate that attention must be taken in choosing the growth method used to measure biofilm production by S. epidermidis in order to make meaningful comparisons among different investigations. Biofilm formation by medically relevant S. epidermidis is used in many studies, including those looking for molecular and cellular correlates of biofilm formation (Heilmann et al 1996;Cramton et al 1999;Galbart et al 2000), in studies concerning prevention of biofilm formation by antimicrobial agents (Rupp and Hamer 1998) or to evaluate some host immune factors (Mohamed et al 1999). As many of these studies attempt to evaluate the role of PNAG in the pathogenesis of infection and in biofilm formation properties, the use of static-batch systems may not be suitable.…”

“…Biofilm formation is one of the major virulence factors produced by these organisms (Voung and Otto 2002). In S. epidermidis, a polymer of N-acetyl glucosamine, initially defined biologically as the polysaccharide intercellular adhesin (PIA) and chemically as poly-Nacetyl-glucosamine (PNAG) has been identified as the molecule responsible for biofilm formation (Mack et al, 1996;Heilmann et al 1996;McKenney et al 1998;Maira-Litrán et al 2002). PNAG is also involved in the agglutination of erythrocytes, which is a common property of S. epidermidis strains (Mack et al 1999;Joyce et al 2003).…”

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

“…The cell-surface hydrophobicity of cells cultured overnight (18 h, 30 1C, 180 r.p.m.) in wastewater was investigated using the water-xylene method described by Heilmann et al (1996). Results are presented as the percentage of cells in the hydrophobic phase.…”

Characterization of starvation‐induced dispersion in Pseudomonas putida biofilms

“…O'Toole et al ., 2000), and the involvement of cellular motility, adhesins, matrix-forming exopolymers, and cell-to-cell signalling has been demonstrated (e.g. Mack et al ., 1994;Heilmann et al ., 1996;Davies et al ., 1998;O'Toole and Kolter, 1998;Pratt and Kolter, 1998;Prigent-Combaret et al ., 1999;Yildiz and Schoolnik, 1999;Danese et al ., 2000;Espinosa-Urgel et al ., 2000;Whitchurch et al ., 2002;Hinsa et al ., 2003).…”