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Adherence and Biofilm Formation

Genetic differences between commensal and disease-causing S. epidermidis are an issue which has already been addressed and intensively investigated in the prege-nomic era. In addition to antibiotic resistance, major differences were found in the capacity to colonize inert surfaces. Thus, the most intriguing feature of clinical S. epidermidis isolates is their ability to form thick multilayered biofilms on polymer and metal surfaces [56]. These biofilms consist of staphylococcal cells embedded in a slimy polysaccharide intercellular adhesin (PIA) matrix connecting the bacteria both to the surface and to each other. However, cell-to-cell and cell-to-surface contacts can also be mediated by polysaccharide-independent protein interactions [109-111]. S. epidermidis RP62A is a biofilm-positive strain originally isolated from a patient suffering from intravenous-catheter-associated septicemia. Because of the extraordinary large amount of biofilm produced by it, RP62A serves as a prototype and reference strain in Staphylococcus biofilm studies. In contrast, S. epidermidis ATCC 12228 is known as a biofilm-negative laboratory strain. Biofilm formation is regarded as a two-step mechanism which involves initial adherence of the bacterium to a surface followed by an accumulative stage. The initial attachment of S. epidermidis is mainly mediated by cell-wall-anchored adhe-sins. S. epidermidis possesses some structures which are involved in the interaction with matrix proteins and in adherence to inert surfaces and eukaryotic cells. Thus, genes encoding cell-wall-associated adhesins like fibronectin-, elastin- and fibrinogen-binding proteins were identified both in S. epidermidis RP62A and in ATCC 12228. Moreover, it was shown that in S. epidermidis autolysins which are actually involved in peptidoglycan synthesis and cell division also exhibit adhesive properties. So far in S. epidermidis AtlE and Aae, two members of this autolysin/ adhesin family of surface-associated proteins, have been described. AtlE is responsible for adhesion to unmodified polymer surfaces and for vitronectin binding, whereas Aae interacts additionally with fibrinogen and fibronectin [112, 113]. The atlE and aae genes are present in both S. epidermidis RP62A and S. epidermidis ATCC 12228. The atlE genes are located at the same chromosomal site in the core part of the chromosome, whereas the aae genes are detectable in the inverted high-recombination region of the S. epidermidis genome (Fig. 9.2). Bhp, a homologous protein to the biofilm-associated protein Bap from S. aureus, is another protein that mediates initial adherence, but is also involved in biofilm accumulation of S. epidermidis [111]. The Bhp-encoding gene is only present in S. epidermidis RP62A and resides also in the inverted chromosomal region (Fig. 9.2). In S. aureus the bap gene is part of a novel transposon-like element integrated into a patho-genicity island which is specific for bovine isolates, suggesting that this gene is mobile and transferable [23]. However, for S. epidermidis it is unknown whether the bhp gene is also part of such a transposon-like structure. Also, larger studies on the distribution of the bhp gene in the S. epidermidis population and its significance as a virulence factor are still pending.

In the majority of biofilm-positive S. epidermidis strains, the second, accumulative stage of biofilm production is mainly characterized by synthesis of the PIA. The enzymes involved in PIA production are encoded by the icaADBC operon [114]. Numerous studies have shown that the icaADBC genes are more prevalent in S. epidermidis strains from device-associated infections than in commensal isolates [57, 115-117]. This genetic information has therefore been regarded as a discriminating factor between pathogenic and nonpathogenic S. epidermidis strains. In agreement with these findings, the ica operon is indeed only detectable in the clinical isolate RP62A, but absent in the biofilm-negative commensal strain ATCC 12228 [102]. Interestingly, the icaADBC genes are also part of the recombination region of the S. epidermidis chromosome and form the border of the inverted genome segment (Fig. 9.2). The origin and evolution of ica-positive S. epidermidis strains is still uncertain. However, detection of ica genes in different coagulase-negative staphylococcal species [118] and in E. coli [119] is a clue that this genetic information might spread by horizontal gene transfer. A recent study employing multilocus sequence typing (MLST) indicates that the ica operon occurs in different S. epidermidis backgrounds (Ziebuhr et al., unpublished data). Among these clones one was identified which represents the majority of ica-positive isolates collected from Northern and Middle Europe as well as from the US during the last 10 years. The data suggest that integration of the ica genes into S. epidermidis resulted in the emergence of highly successful clones which were eventually able to spread worldwide. However, it is also conceivable that the ica genes belonged originally to the Staphylococcus core genome and got lost in the course of genome rearrangements in the high-recombination region. In this respect it is important to note that all S. aureus strains analyzed so far carry the ica operon, and genome comparison revealed insertion of the ica operon at the same site in the S. aureus chromosome as in S. epidermidis (data not shown). However, this might also reflect an insertional hot spot for the integration of the ica genes at this locus. Thus, on the basis of these few facts it is currently not yet possible to decide whether the ica genes are mobile or an integral part of staphylococcal genomes.

Another factor which mediates the accumulative stage of S. epidermidis biofilm formation is the accumulation-associated protein Aap [109, 110]. It is a large polypeptide of 1505 amino acids with an N-terminal signal peptide, an LPXTG cell wall anchor, and an extended repeat region. The full-length Aap protein requires proteolytic processing by staphylococcal or host proteases to exhibit its adhesive properties [109]. The aap gene, which is also located in the inverted high-recombination chromosomal region, is detectable in both sequenced S. epidermidis genomes (Fig. 9.2). However, the recent study by Rohde and coworkers also describes an aap-negative S. epidermidis strain, indicating that this genetic information is not necessarily common to all S. epidermidis isolates [109].

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